T cell burst size is regulated by the duration of TCR engagement and balanced control of Ag-induced activation, expansion, and apoptosis. We found that galectin-1-deficient CD8 T cells undergo greater cell division in response to TCR stimulation, with fewer dividing cells undergoing apoptosis. TCR-induced ERK signaling was sustained in activated galectin-1-deficient CD8 T cells and antagonized by recombinant galectin-1, indicating galectin-1 modulates TCR feed-forward/feedback loops involved in signal discrimination and procession. Furthermore, recombinant galectin-1 antagonized binding of agonist tetramers to the TCR on activated OT-1 T cells. Finally, galectin-1 produced by activated Ag-specific CD8 T cells negatively regulated burst size and TCR avidity in vivo. Therefore, galectin-1, inducibly expressed by activated CD8 T cells, functions as an autocrine negative regulator of peripheral CD8 T cell TCR binding, signal transduction, and burst size. Together with recent findings demonstrating that gal-1 promotes binding of agonist tetramers to the TCR of OT-1 thymocytes, these studies identify galectin-1 as a tuner of TCR binding, signaling, and functional fate determination that can differentially specify outcome, depending on the developmental and activation stage of the T cell.

Antigen-specific CD8 T cells respond to TCR engagement by dividing and differentiating into effector CTLs, which participate in mediating adaptive immunity. Whereas only brief encounter with Ag is required for programming expansion and development of primary CTL effectors (1, 2), sustained TCR engagement and signaling are required to achieve maximal CD8 burst size (3, 4). Indeed, experiments designed to selectively interrupt continued antigenic stimulation or disrupt Lck signaling after initial programming demonstrated a reduction in the magnitude of CD8 T cell expansion (3, 4). Furthermore, imaging studies have detected durable and continued CD8/APC interactions in vivo well beyond the minimum time frame required for CTL programming (5). Together, these findings have led to the suggestion that CD8 burst size depends on the duration of uninterrupted TCR engagement and Lck-dependent signaling over several days. However, the molecules that regulate sustained TCR engagement, signaling, and CD8 burst size in vivo have yet to be elucidated.

Galectin-1 (gal-1)3 is a member of a family of endogenous β-galactose-binding proteins secreted by activated effector CD8 T cells and other immune and nonimmune cell types (6, 7). It has potent immunomodulatory activity and has recently been implicated in regulating thymocyte and CD4 Th development and activity (8, 9). Previous reports indicate that rgal-1 can inhibit proliferation (6) and induce apoptosis in human PHA blasts (10). However, a role for endogenous gal-1 in regulating CD8 responses in vivo has yet to be elucidated. Moreover, the mechanism by which endogenous gal-1 regulates T cell activity in vivo has not been established for any T cell subtype.

Although gal-1 has minimal specificity for N-acetyllactosamine (11), it preferentially binds O- and N-linked glycans bearing repeating units of N-acetyllactosamine (11, 12). Despite the abundance of such structures, gal-1 binds to a discrete subset of T cell surface glycoproteins, presumably because they decorate their protein backbones with repeating lactosamine units spaced to favor multivalent and high avidity bivalent gal-1 binding and packing. Indeed, properly glycosylated CD45, CD43, CD3, CD2, CD4, and CD7 have been identified as primary gal-1 T cell counterreceptors (7, 13, 14, 15). TCR α and β chains and GM1 have been proposed as additional counterreceptors (16, 17, 18, 19). Furthermore, rgal-1 drives the assembly of glycolattices that selectively segregate gal-1 ligands into distinct CD45/CD3 and CD43/CD7 microdomains on some T cell surfaces (14). Expression of the glycosyltransferases responsible for creating gal-1 ligands and/or blocking galectin binding is developmentally regulated (17, 20, 21, 22, 23), thus potentially endowing T cells the differential capacity to bind and be regulated by gal-1 at distinct stages of development and activation.

The TCR transmits signals in the context of an organized immune synapse at the T cell/APC interface, which has been proposed to regulate TCR signal specificity and functional fate determination (24, 25, 26). The synapse is generated by precise recruitment, organization, and exclusion of cell surface proteins in response to antigenic stimulation (25, 26). Remnants of this organized synapse and the oppositely polarized distal pole complex persist for days after initial antigenic stimulation and T cell division (24, 27, 28). Alternate synaptic organization is found in T cells at different stages of development and with distinct effector functions, implicating a potential mechanism for specifying TCR signal transduction in distinct T cell subpopulations and T cell activation states (25).

Because the gal-1 receptors, such as CD3, CD4, CD2, CD45, GM1, and CD43, are known to impact TCR signal transduction and be reorganized to opposite poles during synaptogenesis and distal pole complex formation (7, 14, 19, 27, 29), gal-1 immunoregulatory activity may result from its ability to modulate TCR signal transduction, T cell synaptic organization, and/or T cell polarity. In support of this suggestion, rgal-1 can induce partial TCR ζ-chain phosphorylation and antagonize processive ζ-chain phosphorylation (30), a signaling signature associated with TCR antagonist-ligand activity (31, 32). The rgal-1 also antagonizes polarized synaptic lipid raft clustering (30) and can selectively influence TCR functional outcome (8, 26, 30, 33) in some CD4/class II-restricted cells and hybridomas. Although CD8 T cells have been reported to secrete and bind gal-1, the ability of rgal-1 or endogenous gal-1 to modulate CD8 TCR signal transduction, functional outcome, or in vivo dynamics has yet to be elucidated.

To investigate a role for endogenous gal-1 in regulating primary CD8 T cell responses, we examined the effects of gal-1 deficiency on CD8 T cell survival and function. We found that gal-1-deficient cells hyperproliferated in response to TCR engagement. Fewer dividing gal-1-deficient CD8 cells underwent apoptosis, suggesting gal-1 may limit burst size by promoting apoptosis. Coinciding with induced expression of gal-1 and its receptors within 48 h of activation, TCR-induced signaling was sustained in gal-1-deficient CD8 cells during that time frame. Indeed, assessment of the effects of gal-1 ablation or reconstitution with rgal-1 on sustained TCR signal transduction indicated that gal-1 regulates signal procession by antagonizing ERK-positive feed forward and/or facilitating Lck/Src homology region 2 domain-containing phosphatase 1 (SHP-1)-negative feedback pathways. Furthermore, gal-1 antagonized persistent TCR agonist binding in activated peripheral CD8 T cells, as determined in vitro by a tetramer decay assay and in vivo by diminished Ag-induced tetramer dulling. Together with recent findings demonstrating that gal-1 promotes agonist TCR binding and signaling during thymocyte development (8), these studies highlight how a single lectin can differentially modulate TCR binding and functional outcome, depending on the developmental and activation stage of the T cell (34). These data also demonstrate that induced gal-1 expression by activated CD8 T cells regulates T cell proliferation vs apoptosis by opposing continued agonist TCR binding and signal transduction, identifying a novel mechanism by which TCR avidity can be modulated in peripheral CD8 cells to affect functional outcome.

Gal-1−/− mice on 129S/V background were backcrossed 13 generations with C57BL/6 mice to produce gal-1−/− C57BL/6 mice. Gal-1−/− OT-1 mice were generated by crossing OT-1 mice with gal-1−/− mice. Gal-1−/− H-Y mice were generated by crossing H-Y TCR transgenic with gal-1−/− mice. All studies were done with C57BL/6 mice. All experiments involving mice followed an approved protocol of the University of California Animal Research Committee. Mice were genotyped as described previously (8).

Splenocytes from female H-Y or OT-1 mice were isolated, labeled with CFSE (Invitrogen), and incubated with syngeneic APCs presenting the H-Y(KCSRNRQYL) or OVA257–274(SIINFEKL) peptide (Invitrogen), respectively, for 72 h. Cells were stained with anti-Vβ8.1–8.2 (H-Y) or anti-Vα2 (OT-1) Abs and analyzed using a FACSCalibur (BD Biosciences). For C57BL/6 mice, splenocytes were enriched for CD8 cells using CD8 isolation kit (Miltenyi Biotec) and magnetic cell sorting (AutoMACS). Purity ranged from 80 to 90% CD8 T cells. CD8 cells were labeled with CFSE and stimulated in six-well plates coated with anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) Abs for 72 h. Cells were stained with anti-CD8 allophycocyanin. All flow cytometry samples were acquired on a FACSCalibur, and analyzed using CellQuest software (BD Biosciences).

Average stage of division was calculated by analyzing the CFSE histograms gated on live CD8 cells. Percentage of cells in each CFSE division was then inserted into the following equation:

\[\mathrm{Average\ stage}{=}\ \frac{((\%\ \mathrm{of\ undivided}){+}(2\ {\cdot}\ \%\ \mathrm{div}\ 1){+}(3\ {\cdot}\ \%\ \mathrm{div}\ 2){+}{\cdot}\ {\cdot}\ {\cdot}\ {\cdot})}{100}{-}1\]

CD8 cells were stimulated with Abs for 48 h. mRNA and cDNA were prepared per manufacturer’s protocols (Invitrogen). Gal-1 expression was measured using quantitative PCR with the following primers: gal-1, 5′-TGAACCTGGGAAAAGACAGC, and gal-1, 3′-TCAGCCTGGTCAAAGGTGAT; L32, 5′-AAGCGAAACTGGCGGAAAC, and L32, 3′-TAACCGATGTTGGGCATCAG, with the following conditions: 1) 94°C, 3 min; 2) 94°C, 45 s; 3) 55°C, 45 s; 4) 72°C, 30 s (steps 2–5, 40 cycles); and 5) 72°C, 10 min.

CD8 cells were stimulated for 48 h and stained with anti-CD8 and the 1B11 clone PE Ab (BD Pharmingen).

CFSE-labeled CD8 cells were stimulated with Abs for 48 h and stained with annexin V PE, per manufacturer’s protocol (BD Pharmingen). Analysis was performed on live (based on forward and side scatter), dividing (based on CFSE dilution) cells.

CD8 cells were stimulated with Abs for 72 h. For TCR disengagement studies, stimulated CD8 cells were transferred to a clean well with no stimulus after 36 h of initial stimulation. For Lck inhibition proliferation studies, 50 μM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (Calbiochem) was added 36 h poststimulation. Cells were cultured for additional 36 h. After 72 h, cells were stained with anti-CD8 and acquired.

Gal-1−/− OT-1 cells were stimulated for 48 h with anti-CD3/CD28, and incubated with 0.1 μM rgal-1 for 5 min, washed gently, and stained with anti-CD4, anti-CD8, and OVA257–274/Kb tetramers (Immunomics) for 45 min at room temperature. Anti-Kb Ab was added, and aliquots were taken 0 and 10 min later. Thirty thousand events per sample were acquired. Tetramer decay for CD4 CD8 thymocytes was performed similarly, but without stimulation. Analysis was performed, as described previously (8, 35). Mean fluorescent intensity (MFI) of OVA257–274/Kb tetramer-positive cells for each sample at the 10-min timepoint was normalized relative to the MFI of OVA257–274/Kb tetramer-positive cells for the corresponding sample at time 0. Average of the natural log of normalized values was plotted against time. Negative slope of decay plots was determined by ln (Fa/Fb)/t, where Fa is MFI at the beginning of interval, Fb is MFI at the end of interval, and t is time, in hours, of interval. Analysis was performed on the largest blasts by electronically gating on this population using forward and right angle parameters.

CD8 cells were stimulated with Abs for 48 h. For rgal-1 studies, 1 μM rgal-1 was added for last 10 min of 48-h stimulation. Phosphorylated ERK (pERK) staining was performed, as described previously (8). Specificity of pERK staining was confirmed by abrogation of signal using MEK inhibitor and negative staining in isotype-stained activated T cell controls (data not shown).

For 5-min stimulations, magnetic bead-enriched CD8 cells were incubated with anti-CD3 (10 μg/ml)/CD28 (20 μg/ml) Ab mixture for 30 min on ice and washed once with ice-cold serum-free medium. Cells were resuspended in prewarmed 50 μg/ml secondary Ab (Jackson ImmunoResearch Laboratories) and incubated at 37°C for 5 min. Reaction was stopped with ice-cold medium. Cells were lysed with 50 mM Tris, 1% Nonidet P-40, 2 mM EDTA, pH 8.0 (TNE) buffer containing protease and phosphatase inhibitors and immunoprecipitated with Lck Ab (Santa Cruz Biotechnology) overnight at 4°C. Products were separated on 10% SDS-PAGE gel and immunoblotted with a polyclonal anti-Lck rabbit Ab. For 48-h stimulations, CD8 cells were stimulated for 48 h and lysed with TNE buffer containing protease and phosphatase inhibitors. Sample was split to immunoprecipitate with Lck, and other was left untreated for total cell lysate. Samples were immunoprecipitated overnight at 4°C with anti-Lck Ab (Santa Cruz Biotechnology). Products were separated on 10% SDS-PAGE gel, and immunoblotted with a polyclonal anti-Lck rabbit Ab for the IP samples, or with anti-pERK Ab (Cell Signaling Technology) for total cell lysates. Blots were stripped and reimmunoblotted with anti-SHP-1 Ab (Santa Cruz Biotechnology) for Lck IP blots and anti-ERK Ab (Cell Signaling Technology) for total cell lysate blots.

Mice were inoculated with 2 × 105 PFU of Armstrong strain of lymphocytic choriomeningitis virus (LCMV). At day 9 postinfection, splenocytes were obtained, counted, and stained with anti-CD8 and gp33 (KAVYNFATC) tetramer (Immunomics).

Wild-type or gal-1−/− OT-1 cells were enriched (80–90%), as described above. A total of 1 × 106 cells was tail vein injected into C57BL/6 recipients. After 2 days, mice were immunized with 5 mg of OVA (Sigma-Aldrich). Splenocytes and lymph nodes were obtained, counted, and stained with anti-CD8 and OVA257–274 tetramer (Immunomics).

A total of 1 × 106 cells from recipient spleens was incubated with 25 pM OVA257–274 (Invitrogen) peptide and 1 μl of Golgi-plug (BD Pharmingen) per well. Cells were stimulated for 5 h and stained with OVA257–274 tetramer (Immunomics) and IFN-γ allophycocyanin (eBiosciences) with the intracellular cytokine-staining kit (BD Pharmingen).

To determine whether endogenous gal-1 expression modulates TCR responsiveness to Ag in CD8 T cells, mature CD8 cells from OT-1 and H-Y (female) TCR-transgenic mice lacking the gal-1 gene (gal-1−/−) were assessed for their ability to proliferate in response to specific Ag. CFSE-loaded wild-type and gal-1−/− CD8 cells from TCR-transgenic mice were stimulated with syngeneic irradiated APCs presenting cognate Ag. Seventy-two hours later, we found a higher percentage of gal-1−/− OT-1 (Fig. 1, A–C) and H-Y (Fig. 1, D–F) TCR-transgenic CD8 cells residing in later divisions relative to their wild-type counterparts.

FIGURE 1.

Gal-1−/− TCR-transgenic CD8 T cells underwent a greater number of divisions in response to antigenic stimulation. A and D, CFSE dilution flow profiles of wild-type (left panels) and gal-1−/− (right panels) OT-1 (A) or H-Y (D) TCR transgenic CD8 T cells stimulated for 48 h with APCs presenting specific Ag (25 pM OVA257–274 for A or 0.1 μM H-Y peptide for D). Shaded, unstimulated; solid lines, stimulated. The vertical broken line in A distinguishes cells having undergone five or more divisions (to the left) from those having undergone four or fewer divisions (to the right), or cells having undergone three or more divisions (to the left) from those having undergone two or fewer divisions (to the right) in D. B and E, The percentages of wild-type (□) or gal-1−/− (▪) CD8 T cells having undergone the indicated number of divisions in response to specific Ag as determined from CFSE profiles of OT-1 CD8 T cells stimulated with 25 pM OVA257–274 (B) or H-Y CD8 T cells stimulated with 0.1 μM H-Y peptide (E). C and F, The average stage of division of stimulated wild-type (□) and gal-1−/− (▪) OT-1 (C) or H-Y (F) CD8 T cells after stimulation with indicated Ag peptide concentrations as calculated based on CFSE dilution profiles. Asterisks represent statistical significance based on two-tailed unpaired Student’s t test (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001) or p values are listed in C and F. Bars represent the SD of duplicate samples. Results are representative of at least three independent experiments per TCR transgenic system.

FIGURE 1.

Gal-1−/− TCR-transgenic CD8 T cells underwent a greater number of divisions in response to antigenic stimulation. A and D, CFSE dilution flow profiles of wild-type (left panels) and gal-1−/− (right panels) OT-1 (A) or H-Y (D) TCR transgenic CD8 T cells stimulated for 48 h with APCs presenting specific Ag (25 pM OVA257–274 for A or 0.1 μM H-Y peptide for D). Shaded, unstimulated; solid lines, stimulated. The vertical broken line in A distinguishes cells having undergone five or more divisions (to the left) from those having undergone four or fewer divisions (to the right), or cells having undergone three or more divisions (to the left) from those having undergone two or fewer divisions (to the right) in D. B and E, The percentages of wild-type (□) or gal-1−/− (▪) CD8 T cells having undergone the indicated number of divisions in response to specific Ag as determined from CFSE profiles of OT-1 CD8 T cells stimulated with 25 pM OVA257–274 (B) or H-Y CD8 T cells stimulated with 0.1 μM H-Y peptide (E). C and F, The average stage of division of stimulated wild-type (□) and gal-1−/− (▪) OT-1 (C) or H-Y (F) CD8 T cells after stimulation with indicated Ag peptide concentrations as calculated based on CFSE dilution profiles. Asterisks represent statistical significance based on two-tailed unpaired Student’s t test (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001) or p values are listed in C and F. Bars represent the SD of duplicate samples. Results are representative of at least three independent experiments per TCR transgenic system.

Close modal

Similarly, in the absence of TCR transgene, CD8 T cells from gal-1−/− mice hyperproliferated in response to TCR/CD28 engagement, indicating that gal-1 ablation influences responsiveness to TCR/CD28 engagement in the absence of APCs (Fig. 2, A–D). Indeed, hyperproliferation was evident at 48 and 72 h poststimulation (Fig. 2,D). Consistent with these findings, more CD8 cells accumulated in gal-1−/− cultures following 48–72 h of continuous TCR/CD28 stimulation (Fig. 2 E). Because hyperproliferation occurs in response to TCR stimulation by either Ag/APCs or anti-CD3/CD28 Abs, this phenomenon is not secondary to changes within the repertoire of TCR affinities expressed in gal-1−/− peripheral CD8 cells.

FIGURE 2.

Purified CD8 T cells from gal-1−/− C57BL/6 mice were hyperresponsive to stimulation with Abs directed against CD3 and CD28. A, CFSE dilution flow profiles of anti-CD3/anti-CD28 Ab-stimulated CD8 cells from wild-type (left) and gal-1−/− (right) mice. Shaded, unstimulated; solid lines, stimulated. The vertical broken line distinguishes cells having undergone three or more divisions (to the left) from those having undergone two or fewer divisions (to the right). B and C, The percentage of cells in each division (B) or average stage of division (C) based on CFSE dilution flow profiles of purified CD8 T cells from wild-type (□) or gal-1−/− (▪) mice stimulated with anti-CD3/CD28 Abs for 72 h. D, The average stage of division from purified wild-type (□) and gal-1−/− (▪) CD8 T cells stimulated with anti-CD3/CD28 Abs for 24, 48, or 72 h. Results from A–D are representative of four independent experiments. E and F, The total number of wild-type (□) and gal-1−/− (▪) CD8 cells in culture (E) and the amount of IL-2 detected in the supernatants (F) after 12, 24, 36, 48, 60, and 72 h of stimulation with anti-CD3/CD28 Abs. Asterisks (B, C, D, and F) represent statistical significance based on two-tailed unpaired Student’s t test (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001) or p values are listed in C and D. Bars in B, C, D, and F represent the SD of triplicate cultures. ND, nondetectable. G, Side scatter of wild-type (♦, solid line) and gal-1−/− (▪, broken line) CD8 cells stimulated with anti-CD3/CD28 Abs for indicated times. Values were obtained by electronically gating out dead cells to focus on live cells. Results from E–G are representative of three independent experiments. H, Lck activation of wild-type and gal-1−/− CD8 cells after 5 min of stimulation in the presence or absence of rgal-1 was assessed by blotting Lck immunoprecipitates with an Ab specific for Lck. The relative ratio of 59:56-kDa Lck was calculated based on densitometry and listed under each lane. Results are representative of two independent experiments.

FIGURE 2.

Purified CD8 T cells from gal-1−/− C57BL/6 mice were hyperresponsive to stimulation with Abs directed against CD3 and CD28. A, CFSE dilution flow profiles of anti-CD3/anti-CD28 Ab-stimulated CD8 cells from wild-type (left) and gal-1−/− (right) mice. Shaded, unstimulated; solid lines, stimulated. The vertical broken line distinguishes cells having undergone three or more divisions (to the left) from those having undergone two or fewer divisions (to the right). B and C, The percentage of cells in each division (B) or average stage of division (C) based on CFSE dilution flow profiles of purified CD8 T cells from wild-type (□) or gal-1−/− (▪) mice stimulated with anti-CD3/CD28 Abs for 72 h. D, The average stage of division from purified wild-type (□) and gal-1−/− (▪) CD8 T cells stimulated with anti-CD3/CD28 Abs for 24, 48, or 72 h. Results from A–D are representative of four independent experiments. E and F, The total number of wild-type (□) and gal-1−/− (▪) CD8 cells in culture (E) and the amount of IL-2 detected in the supernatants (F) after 12, 24, 36, 48, 60, and 72 h of stimulation with anti-CD3/CD28 Abs. Asterisks (B, C, D, and F) represent statistical significance based on two-tailed unpaired Student’s t test (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001) or p values are listed in C and D. Bars in B, C, D, and F represent the SD of triplicate cultures. ND, nondetectable. G, Side scatter of wild-type (♦, solid line) and gal-1−/− (▪, broken line) CD8 cells stimulated with anti-CD3/CD28 Abs for indicated times. Values were obtained by electronically gating out dead cells to focus on live cells. Results from E–G are representative of three independent experiments. H, Lck activation of wild-type and gal-1−/− CD8 cells after 5 min of stimulation in the presence or absence of rgal-1 was assessed by blotting Lck immunoprecipitates with an Ab specific for Lck. The relative ratio of 59:56-kDa Lck was calculated based on densitometry and listed under each lane. Results are representative of two independent experiments.

Close modal

To determine whether the effects of gal-1 expression could be appreciated before cell division, we analyzed the effects of gal-1 deficiency on IL-2 production and T cell blasting (Fig. 2, F and G). More IL-2 was detected in the supernatants from TCR/CD28-stimulated gal-1−/− cultures as early as 12 h poststimulation, although differences between wild-type and gal-1−/− CD8 cells were maximally apparent after 36–72 h of activation (Fig. 2,F). By 24 h poststimulation, we observed increased blasting, as measured by side scatter, in the population of gal-1−/− CD8 T cells. Again, differences between wild-type and gal-1−/− T cells grew even greater by 48–60 h poststimulation (Fig. 2 G). Taken together, these findings indicate that endogenous gal-1 antagonizes T cell activation after initial TCR engagement, but before programming of maximal IL-2 production, and T cell blasting, expansion, and proliferation.

We found that the earliest measures of cell activation were relatively unaffected by gal-1 gene deletion, whereas the effects of gal-1 expression were increasingly appreciated after 24 h of T cell activation. Consistently, freshly isolated gal-1−/− and wild-type CD8 cells showed no difference in their ability to induce Lck-serine-59 phosphorylation 5 min after stimulating with anti-CD3/CD28 Abs (Fig. 2,H). However, the addition of exogenous rgal-1 was able to antagonize Lck activation (Fig. 2 H), implying some minimal degree of gal-1 ligand binding on nonblasting T cells when provided exogenously. Taken together, these findings indicate that endogenous gal-1 may act after initial T cell activation.

This suggestion is consistent with the published kinetics of the expression of gal-1 and its ligands during the course of CD8 T cell activation (6) and our own findings that TCR/CD28 engagement increasingly induced gal-1 mRNA expression between 24 and 72 h poststimulation (Fig. 3,A). Furthermore, reactivity with the Ab 1B11, which specifically binds an activation-induced core 2 O-glycosylated isoform of murine CD43 (and CD45) recognized by gal-1 (21, 36, 37), was similarly up-regulated in both wild-type and gal-1−/− CD8 cells after 48 h of TCR/CD28 engagement (Fig. 3 B). Low levels of 1B11 staining were detected in resting CD8 T cells, consistent with gal-1 binding at the 0 timepoint. Equivalent binding of 1B11 Ab to wild-type and gal-1−/− T cells suggests the presence of potential ligands on gal-1−/− CD8 cells, ruling out potential secondary effects due to compensatory modulation of gal-1 ligands in the absence of its binding. Together, these findings point to a role for gal-1 as a negative regulator of CD8 T cell activation that functions with increased activity as its expression and the expression of its glycan ligands rise during the course of T cell activation.

FIGURE 3.

Both gal-1 expression and gal-1 ligands were up-regulated in response to TCR/CD28 engagement. A, Gal-1 mRNA expression in wild-type CD8 cells stimulated with anti-CD3/CD28 Abs for 24, 48, and 72 h was quantitated using quantitative PCR and normalized relative to the L32 housekeeping gene. Values are expressed as the ratio of gal-1 relative fluorescence units to L32 relative fluorescence units (RFU/RFU). B, Mean fluorescent intensity of glycoreactive 1B11 Ab staining on wild-type (□) and gal-1−/− (▪) CD8 cells 48 h after anti-CD3/CD28 stimulation. Results are representative of three independent experiments.

FIGURE 3.

Both gal-1 expression and gal-1 ligands were up-regulated in response to TCR/CD28 engagement. A, Gal-1 mRNA expression in wild-type CD8 cells stimulated with anti-CD3/CD28 Abs for 24, 48, and 72 h was quantitated using quantitative PCR and normalized relative to the L32 housekeeping gene. Values are expressed as the ratio of gal-1 relative fluorescence units to L32 relative fluorescence units (RFU/RFU). B, Mean fluorescent intensity of glycoreactive 1B11 Ab staining on wild-type (□) and gal-1−/− (▪) CD8 cells 48 h after anti-CD3/CD28 stimulation. Results are representative of three independent experiments.

Close modal

Based on reports that rgal-1 can induce apoptosis in activated T cells (10) and T cell lines (30) and skew the outcome of TCR engagement toward apoptosis (30, 33), we hypothesized that TCR-induced endogenous gal-1 expression might control CD8 T cell proliferation by enhancing apoptosis in activated CD8 cells. To address this question, CFSE-loaded CD8 cells were stimulated with anti-CD3/CD28 for 48 h and assessed for apoptosis using annexin V staining. CFSE profiles were used to distinguish divided from undivided cells. Approximately 33% of the gal-1−/− CD8 cells that underwent one or more divisions were annexin V positive/apoptotic (Fig. 4,A). In contrast, 82% of wild-type T cells undergoing one or more divisions were annexin V positive/apoptotic (Fig. 4,A), providing one explanation as to why fewer cells were able to divide further. No differences between wild-type and gal-1−/− CD8 cells were observed in the ratio of apoptotic to nonapoptotic T cells in the undivided population (Fig. 4 B). Similar conclusions were reached by analyzing the ratio of dead to live cells as defined by forward and right angle scatter gates (data not shown). As predicted, rgal-1-induced death in T cells activated for 48 h was inhibited by the gal-1 competitive inhibitors, L2hmda (38) and lactose (data not shown), demonstrating that gal-1-induced death is a carbohydrate-binding dependent process. These findings demonstrate that gal-1 produced by activated CD8 T cells promotes their apoptosis, elucidating a novel mechanism by which CD8 cells negatively autoregulate their continued expansion.

FIGURE 4.

Endogenous gal-1 promoted apoptosis in anti-CD3/CD28-stimulated dividing CD8 T cells. A, Annexin V staining of dividing wild-type (left) and gal-1−/− (right) CD8 cells stimulated with anti-CD3/CD28 Abs for 48 h. CFSE division profiling was used to enable electronic gating on dividing (A and B) or undivided (B) cell populations. M1 and M2 markers delineate annexin V-positive (apoptotic) and annexin V-negative populations, respectively, and the percentage of cells in each population is indicated above the markers. B, The ratio of annexin V-positive (apoptotic) to annexin V-negative (live) cells in undivided and divided wild-type (□) and gal-1−/− (▪) CD8 populations stimulated with anti-CD3/CD28 for 48 h. These results are representative of three independent experiments.

FIGURE 4.

Endogenous gal-1 promoted apoptosis in anti-CD3/CD28-stimulated dividing CD8 T cells. A, Annexin V staining of dividing wild-type (left) and gal-1−/− (right) CD8 cells stimulated with anti-CD3/CD28 Abs for 48 h. CFSE division profiling was used to enable electronic gating on dividing (A and B) or undivided (B) cell populations. M1 and M2 markers delineate annexin V-positive (apoptotic) and annexin V-negative populations, respectively, and the percentage of cells in each population is indicated above the markers. B, The ratio of annexin V-positive (apoptotic) to annexin V-negative (live) cells in undivided and divided wild-type (□) and gal-1−/− (▪) CD8 populations stimulated with anti-CD3/CD28 for 48 h. These results are representative of three independent experiments.

Close modal

In addition to inducing T cell apoptosis (10, 30), we have reported that rgal-1 can antagonize processive TCR signal transduction, IL-2 production, and proliferation in class II-restricted T cell lines and hybridomas (30). Because endogenous gal-1 is only up-regulated and abundantly expressed in CD8 cells after TCR engagement (Fig. 3,A) (6), we hypothesize that gal-1 might antagonize the continued TCR signal transduction required for optimal CD8 T cell expansion. To address this issue, we examined the consequences of disrupting TCR signal transduction in wild-type and gal-1−/− CD8 cells. Wild-type or gal-1−/− CD8 cells were stimulated with anti-CD3/CD28 Abs for a 72-h culture period or for only 36 h, followed by 36 h of culture in the absence of TCR engagement. We found that wild-type and gal-1−/− CD8 cells stimulated continually for 72 h underwent more divisions than those stimulated only for the first 36 h (Fig. 5, and vs □, and data not shown). Similarly, pharmacologically blocking the most proximal TCR signal transducer, Lck, 36 h after TCR engagement also impaired maximal proliferation over a 72-h culture period (Fig. 5, vs ). Therefore, continued TCR signal transduction beyond 36 h was required for maximal T cell division in both wild-type and gal-1−/− CD8 cells.

FIGURE 5.

Gal-1 expression phenocopied premature truncation of sustained TCR/Lck signaling required for maximal CD8 T cell division. A and B, Wild-type or gal-1−/− CD8 cells were as follows: stimulated continuously with anti-CD3/CD28 for 72 h (TCR engaged, and ); stimulated with anti-CD3/CD28 for the first 36 h of culture, followed by an additional 36 h of culture without stimulus (TCR disengaged, □); or stimulated with anti-CD3/CD28 for 36 h before the addition of the Src family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, for the final 36 h of the 72-h culture (Lck inhibited, ). CFSE dilution profiles were used to calculate the average stage of division (A) and percentage of cells having undergone greater than two divisions (B). Results are representative of three independent experiments.

FIGURE 5.

Gal-1 expression phenocopied premature truncation of sustained TCR/Lck signaling required for maximal CD8 T cell division. A and B, Wild-type or gal-1−/− CD8 cells were as follows: stimulated continuously with anti-CD3/CD28 for 72 h (TCR engaged, and ); stimulated with anti-CD3/CD28 for the first 36 h of culture, followed by an additional 36 h of culture without stimulus (TCR disengaged, □); or stimulated with anti-CD3/CD28 for 36 h before the addition of the Src family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, for the final 36 h of the 72-h culture (Lck inhibited, ). CFSE dilution profiles were used to calculate the average stage of division (A) and percentage of cells having undergone greater than two divisions (B). Results are representative of three independent experiments.

Close modal

More importantly, gal-1 up-regulation and expression in wild-type CD8 T cells phenocopied the effects of truncating TCR signaling in gal-1−/− CD8 cells. Indeed, CFSE dilution of gal-1−/− T cells in which the TCR signals have been disrupted or TCR disengaged after 36 h recapitulates those observed in wild-type T cells stimulated continuously for 72 h (Fig. 5, □ and vs ). Furthermore, the dead to live cell ratio (based on forward and side scatter gates) was higher in cultures in which the TCR had only been stimulated for the first 36 of 72 h, relative to those receiving continual stimulation (data not shown), reminiscent of the increased apoptotic to live cell ratio observed in activated wild-type vs gal-1−/− CD8 cells (Fig. 4). These data demonstrate a requirement for continued TCR engagement and signal transduction for optimal activation, coincident with the kinetics of gal-1 expression. Furthermore, they highlight similarities between the behavior of fully stimulated gal-1-expressing T cells and gal-1−/− T cells that had TCR signaling or engagement disrupted after 36 h, prompting us to directly investigate whether gal-1 antagonizes persistent TCR binding or signaling required for continued T cell activation.

We next considered the possibility that gal-1 might antagonize proliferation by diminishing specific peptide/MHC binding to TCRs on activated T cells. Using a tetramer decay assay, we measured the effect of rgal-1 on the persistent binding of fluorescently labeled agonist OVA257–274/Kb tetramers to large activated gal-1−/− OT-1 T cell blasts over time. Linear decay plots of the natural log of fluorescence vs time were constructed for rgal-1-treated and untreated samples (Fig. 6,A). Quantitative analysis revealed that plots from rgal-1-treated T cells had a larger negative slope than plots from untreated cells over the 0- to 10-min time interval (Fig. 6 B), indicating that gal-1 antagonized TCR ligand binding. Before the addition of competing anti-Kb Ab and allowing time for decay, gal-1−/− T cells had comparable levels of CD3, CD8, and tetramer binding regardless of pretreatment with rgal-1 (data not shown). These findings rule out increased TCR levels on activated gal-1−/− T cells or direct gal-1 steric interference as a mechanism of TCR tetramer dulling.

FIGURE 6.

Recombinant gal-1 antagonized persistent TCR binding to agonist peptide/MHC tetramers. OVA257–274/Kb tetramer decay kinetics for 48 h activated gal-1−/− OT-1 CD8 T cell blasts and freshly isolated double-positive thymocytes from the same mice preincubated with or without 0.1 μM rgal-1 before tetramer addition. A, Plot of the natural logarithm of normalized fluorescence vs time after anti-Kb addition to rgal-1-treated (▪, broken line) and untreated (♦, solid line) activated gal-1−/− OT-1 CD8 T cells. This plot represents the average of normalized values obtained from 10 individual mice. B, Negative slope of tetramer-staining decay plots at the 0- to 10-min interval between rgal-1-treated (0.1 μM rgal-1, n = 10) and untreated (none, n = 10) activated CD8 cells. The slope was determined as described in Materials and Methods. Each dot represents analysis of T cells from an independent mouse. Analysis was restricted to large blasting cells as determined by forward and right angle scatter for activated CD8 cells. C, Plot of the average natural logarithm of normalized fluorescence vs time after anti-Kb addition to rgal-1-treated (▪, broken line) or untreated (♦, solid line) gal-1−/− OT-1 CD4 CD8 double-positive thymocytes obtained from seven of the mice represented in A in a side-by-side analysis. Values of p determined by paired Student’s t test demonstrated that differences were statistically significant. SD at the 10-min timepoint for A and C are ±0.025 and ±0.032, respectively.

FIGURE 6.

Recombinant gal-1 antagonized persistent TCR binding to agonist peptide/MHC tetramers. OVA257–274/Kb tetramer decay kinetics for 48 h activated gal-1−/− OT-1 CD8 T cell blasts and freshly isolated double-positive thymocytes from the same mice preincubated with or without 0.1 μM rgal-1 before tetramer addition. A, Plot of the natural logarithm of normalized fluorescence vs time after anti-Kb addition to rgal-1-treated (▪, broken line) and untreated (♦, solid line) activated gal-1−/− OT-1 CD8 T cells. This plot represents the average of normalized values obtained from 10 individual mice. B, Negative slope of tetramer-staining decay plots at the 0- to 10-min interval between rgal-1-treated (0.1 μM rgal-1, n = 10) and untreated (none, n = 10) activated CD8 cells. The slope was determined as described in Materials and Methods. Each dot represents analysis of T cells from an independent mouse. Analysis was restricted to large blasting cells as determined by forward and right angle scatter for activated CD8 cells. C, Plot of the average natural logarithm of normalized fluorescence vs time after anti-Kb addition to rgal-1-treated (▪, broken line) or untreated (♦, solid line) gal-1−/− OT-1 CD4 CD8 double-positive thymocytes obtained from seven of the mice represented in A in a side-by-side analysis. Values of p determined by paired Student’s t test demonstrated that differences were statistically significant. SD at the 10-min timepoint for A and C are ±0.025 and ±0.032, respectively.

Close modal

We recently demonstrated that rgal-1 can promote TCR avidity on OT-1 double-positive thymocytes during the course of a 20-min tetramer decay assay (8). Consistent with our previous findings, OT-1 thymocytes from the same mice analyzed in Fig. 6,A bound agonist tetramers stronger in the presence of rgal-1 in side-by-side assays over the 10-min interval (Fig. 6 C). The effect of gal-1 on thymocyte TCR binding is in stark contrast to the antagonistic effect of TCR binding on activated CD8 cells in the presence of rgal-1. Together, these data demonstrate that gal-1 has the capacity to alternatively modulate TCR avidity depending on the developmental stage of the T cell.

Studies describing the molecular basis of TCR antagonism have led to the identification of feed forward/feedback loops that function in signal discrimination by enforcing signals crossing a critical threshold and quenching those that do not (39). In the presence of an agonist ligand, TCR-induced ERK activation induces Lck-serine-59 phosphorylation to prevent Lck from associating with the SHP-1 phosphatase, resulting in activation (39). Alternatively, suboptimal TCR stimulation provided by partial agonist/antagonist peptide TCR ligands is insufficient for optimal ERK activation (39). In the absence of Lck-serine-59 phosphorylation, SHP-1 binds Lck to down-regulate continued TCR signal transduction and to abort T cell activation (39). We have previously noted similarities between the TCR signaling signatures induced by rgal-1 and TCR antagonist/partial agonist ligands (30, 31, 32). Thus, we next examined the effects of gal-1 expression in signal discrimination regulated by the ERK-positive and SHP-1-negative feedback pathways (39).

To this end, we examined ERK phosphorylation, Lck-serine-59 phosphorylation status, and Lck:SHP-1 coassociation in wild-type and gal-1−/− CD8 cells 48 h after TCR/CD28 engagement. Using an Ab directed against activated/pERK and flow cytometry, we found that sustained pERK activity could be detected in a subset of CD8 cells after 48 h of continued TCR engagement (Fig. 7,A). Furthermore, stimulated gal-1−/− T cells had a greater percentage of cells expressing pERK than stimulated wild-type T cells (Fig. 7,A), suggesting that endogenous gal-1 antagonized sustained ERK activation. Similarly, activated gal-1−/− CD8 T cells demonstrated increased pERK levels relative to wild-type CD8 T cells when analyzed by Western blot (Fig. 7,C). Finally, exposure of gal-1−/− cells to rgal-1 48 h poststimulation reduced the percentage of CD8 cells expressing pERK (Fig. 7 B). Antagonism of ERK activity was detected with significant and increasing titratable effects over the 0.1–10 μM rgal concentrations (data not shown).

FIGURE 7.

Endogenous gal-1 or exogenously added rgal-l antagonized TCR/CD28-induced ERK activation to promote the Lck/SHP-1-negative feedback loop. A, pERK-staining intensity in unstimulated wild-type (dotted line) and 48-h anti-CD3/CD28-stimulated wild-type (thin line) or gal-1−/− (shaded) CD8 cells. B, pERK-staining intensity of 48-h stimulated gal-1−/− CD8 cells in the absence (shaded) or presence of 1 μM (thin line) rgal-1 for the last 10 min of the culture period. In both A and B, the M1 marker was used to delineate pERK-positive cells. The percentages of pERK-positive cells are indicated. Results are representative of three independent experiments. C, Wild-type or gal-1−/− CD8 cells were stimulated with anti-CD3/CD28 Abs for 48 h, and TNE lysates were separated on SDS-PAGE gels and immunoblotted with anti-pERK or anti-ERK2 Ab. The pERK1:ERK2 ratio was calculated based on densitometry and is indicated below each lane. D, Lck immunoprecipitates were immunoblotted with Abs directed against Lck or SHP-1, and the relative ratios of 59:56-kDa Lck or SHP-1:59-kDa Lck were calculated based on densitometry. E, The average relative ratios of 59:56-kDa Lck (left) and SHP-1:59-kDa Lck (right) from six independent experiments comparing wild-type (circles) or gal-1−/− (triangles) CD8 cells stimulated as described above. Values of p determined by paired Student’s t test are listed in E.

FIGURE 7.

Endogenous gal-1 or exogenously added rgal-l antagonized TCR/CD28-induced ERK activation to promote the Lck/SHP-1-negative feedback loop. A, pERK-staining intensity in unstimulated wild-type (dotted line) and 48-h anti-CD3/CD28-stimulated wild-type (thin line) or gal-1−/− (shaded) CD8 cells. B, pERK-staining intensity of 48-h stimulated gal-1−/− CD8 cells in the absence (shaded) or presence of 1 μM (thin line) rgal-1 for the last 10 min of the culture period. In both A and B, the M1 marker was used to delineate pERK-positive cells. The percentages of pERK-positive cells are indicated. Results are representative of three independent experiments. C, Wild-type or gal-1−/− CD8 cells were stimulated with anti-CD3/CD28 Abs for 48 h, and TNE lysates were separated on SDS-PAGE gels and immunoblotted with anti-pERK or anti-ERK2 Ab. The pERK1:ERK2 ratio was calculated based on densitometry and is indicated below each lane. D, Lck immunoprecipitates were immunoblotted with Abs directed against Lck or SHP-1, and the relative ratios of 59:56-kDa Lck or SHP-1:59-kDa Lck were calculated based on densitometry. E, The average relative ratios of 59:56-kDa Lck (left) and SHP-1:59-kDa Lck (right) from six independent experiments comparing wild-type (circles) or gal-1−/− (triangles) CD8 cells stimulated as described above. Values of p determined by paired Student’s t test are listed in E.

Close modal

We next determined whether increased ERK activation was correlated with increased levels of Lck-serine-59 phosphorylation and decreased Lck-associated-SHP-1. We found higher levels of the slower migrating Lck-serine-59-phosphorylated (59-kDa) isoform relative to the faster migrating Lck (56-kDa) isoform in gal-1−/− CD8 cells vs wild-type cells after 48 h of continual TCR engagement (Fig. 7, D and E). Consistent with these findings, less SHP-1 coimmunoprecipitated with Lck in activated gal-1−/− CD8 T cells relative to wild-type cells (Fig. 7, D and E). Taken together, these data support the suggestion that endogenous gal-1 antagonizes persistent TCR signal transduction by promoting the SHP-1-negative feedback pathway and antagonizing the ERK-positive feedback pathway.

To determine whether endogenous gal-1 regulates CD8 T cell responsiveness and expansion in vivo, wild-type and gal-1−/− mice were infected with LCMV and assessed for expansion of virus-specific CD8 cells. LCMV-specific CD8 cells were tracked 9 days postinfection using GP33–41/Db tetramers. We found that gal-1−/− mice had both higher percentages and absolute numbers of total and tetramer-positive CD8 T cells in the spleen relative to wild-type mice (Fig. 8), indicating enhanced expansion of gal-1−/− LCMV-specific CD8 cells in response to infection. These findings demonstrate a role for endogenous gal-1 in limiting CD8 T cell burst size in vivo. However, these experiments do not address the cellular source of gal-1, given that several potentially relevant cell types including CD4 T cells, APCs, and other stromal cells all lack gal-1 in gal-1−/− mice (40).

FIGURE 8.

Gal-1 expression limited CD8 burst size in response to infection with LCMV in vivo. A, Flow cytometry profiles of splenocytes from day 9 postinfected wild-type (left) and gal-1−/− (right) mice stained with anti-CD8 Ab and LCMV-specific tetramers (GP33–41/Db). The percentage of tetramer-positive CD8 T cells is indicated in the upper right quadrant. B and C, The total numbers of CD8 (B) or tetramer-positive CD8 (C) cells from wild-type (□) or gal-1−/− (▪) mice at day 9 postinfection were determined by multiplying the total number of splenocytes by the percentage of CD8- or CD8 GP33–41/Db-positive cells. Bars represent SDs of T cell responses from three independent wild-type and two gal-1−/− mice 9 days after infection with LCMV. Values of p determined by paired Student’s t test are listed in B and C.

FIGURE 8.

Gal-1 expression limited CD8 burst size in response to infection with LCMV in vivo. A, Flow cytometry profiles of splenocytes from day 9 postinfected wild-type (left) and gal-1−/− (right) mice stained with anti-CD8 Ab and LCMV-specific tetramers (GP33–41/Db). The percentage of tetramer-positive CD8 T cells is indicated in the upper right quadrant. B and C, The total numbers of CD8 (B) or tetramer-positive CD8 (C) cells from wild-type (□) or gal-1−/− (▪) mice at day 9 postinfection were determined by multiplying the total number of splenocytes by the percentage of CD8- or CD8 GP33–41/Db-positive cells. Bars represent SDs of T cell responses from three independent wild-type and two gal-1−/− mice 9 days after infection with LCMV. Values of p determined by paired Student’s t test are listed in B and C.

Close modal

To address this issue, wild-type or gal-1−/− OT-1 TCR-transgenic CD8 T cells were transferred into B6 mice that were immunized with OVA 2 days later. OVA257–274/Kb tetramers were used to track the expansion of Ag-specific OT-1 CD8 cells at days 2, 3, and 4 postimmunization. Cell surface levels of tetramer binding were diminished in wild-type vs gal-1−/− T cells 3 days after in vivo activation (Fig. 9, A and B) at the peak of the response, validating our findings that gal-1 can antagonize TCR/tetramer binding in vitro. Tetramer dulling in wild-type CD8 T cells could begin to be appreciated days 2 postimmunization (approaching statistical significance), but was no longer apparent by day 4 (Fig. 9 B), providing a window for gal-1 TCR antagonist activity in response to OVA challenge.

FIGURE 9.

Endogenous expression of gal-1 by Ag-specific CD8 T cells controlled CD8 T cell expansion, tetramer binding, and burst size in vivo. Wild-type or gal-1−/− OT-1 CD8 cells were adoptively transferred into wild-type C57BL/6 mice that were immunized with OVA. A, Flow profiles (OVA257–274/Kb tetramer vs CD8) of splenocytes from mice that had received wild-type (left) or gal-1−/− (right) OT-1 donor CD8 cells 3 days after immunizing with OVA. The boxed region indicates percentage of tetramer-positive CD8 cells. B, The relative MFI of tetramer staining of wild-type (□) or gal-1−/− (▪) donor OT-1 CD8 cells at days 2, 3, and 4 postimmunization. Each sample was normalized relative to the average tetramer MFI of wild-type OT-1 CD8 cells at each day. C, CFSE profiles of CD8 tetramer-negative (thin line) and tetramer-positive (shaded) populations from mice that received wild-type (left) or gal-1−/− (right) donor cells 3 days postimmunization. D, The total numbers of wild-type (□) or gal-1−/− (▪) donor OT-1 CD8 cells in days 2, 3, and 4 postimmunized spleens were determined by multiplying the total number of splenocytes by the percentage of tetramer-positive CD8 cells. E, The total number of CD8 tetramer-positive cells in unimmunized and immunized recipient mice, and CD8 tetramer-negative cells in unimmunized and immunized recipient mice that received wild-type (□) or gal-1−/− (▪) OT-1 CD8 donor cells at day 3 postimmunization. F, The percentage of wild-type (□) or gal-1−/− (▪) tetramer-positive CD8 splenocytes making IFN-γ after restimulation with OVA257–274 peptide. G, The percentage of wild-type (♦, solid line) or gal-1−/− (▪, broken line) OT-1 CD8 cells in days 2, 3, and 4 postimmunized recipient lymph nodes. Values of p determined by paired Student’s t test are listed in B–G. Bars represent SDs of three mice at each timepoint, and data are representative of three independent experiments.

FIGURE 9.

Endogenous expression of gal-1 by Ag-specific CD8 T cells controlled CD8 T cell expansion, tetramer binding, and burst size in vivo. Wild-type or gal-1−/− OT-1 CD8 cells were adoptively transferred into wild-type C57BL/6 mice that were immunized with OVA. A, Flow profiles (OVA257–274/Kb tetramer vs CD8) of splenocytes from mice that had received wild-type (left) or gal-1−/− (right) OT-1 donor CD8 cells 3 days after immunizing with OVA. The boxed region indicates percentage of tetramer-positive CD8 cells. B, The relative MFI of tetramer staining of wild-type (□) or gal-1−/− (▪) donor OT-1 CD8 cells at days 2, 3, and 4 postimmunization. Each sample was normalized relative to the average tetramer MFI of wild-type OT-1 CD8 cells at each day. C, CFSE profiles of CD8 tetramer-negative (thin line) and tetramer-positive (shaded) populations from mice that received wild-type (left) or gal-1−/− (right) donor cells 3 days postimmunization. D, The total numbers of wild-type (□) or gal-1−/− (▪) donor OT-1 CD8 cells in days 2, 3, and 4 postimmunized spleens were determined by multiplying the total number of splenocytes by the percentage of tetramer-positive CD8 cells. E, The total number of CD8 tetramer-positive cells in unimmunized and immunized recipient mice, and CD8 tetramer-negative cells in unimmunized and immunized recipient mice that received wild-type (□) or gal-1−/− (▪) OT-1 CD8 donor cells at day 3 postimmunization. F, The percentage of wild-type (□) or gal-1−/− (▪) tetramer-positive CD8 splenocytes making IFN-γ after restimulation with OVA257–274 peptide. G, The percentage of wild-type (♦, solid line) or gal-1−/− (▪, broken line) OT-1 CD8 cells in days 2, 3, and 4 postimmunized recipient lymph nodes. Values of p determined by paired Student’s t test are listed in B–G. Bars represent SDs of three mice at each timepoint, and data are representative of three independent experiments.

Close modal

Cells were labeled with CFSE before adoptive transfer to provide a second mechanism for tracking donor-specific TCR-transgenic T cells. Indeed, tetramer-positive CD8 cell population as a whole stains brighter in the CFSE channel than tetramer-negative CD8 cells from mice receiving wild-type or gal-1−/− donor cells (Fig. 9,C), indicating that the majority of tetramer-positive cells were donor cells. A greater percentage and total number of tetramer-positive CD8 cells were observed in the spleen of mice that received gal-1−/− CD8 donor cells relative to mice that received wild-type donor cells (Fig. 9, A and D). These findings demonstrate that gal-1−/− OT-1 CD8 donor cells are hyperresponsive to antigenic challenge in vivo and that gal-1 effect is intrinsic to CD8 T cells. Additionally, no expansion in the total number of tetramer-negative, CFSE-negative CD8 T cells was observed at day 3, regardless of whether the donor population expressed gal-1 or not (Fig. 9,E). Comparable numbers of tetramer-negative T cells were observed in unimmunized mice (Fig. 9 E), establishing that the expansion observed is due to specific Ag-induced TCR stimulation.

We also found a greater percentage of transferred gal-1−/− T cells producing IFN-γ upon in vitro Ag restimulation than transferred wild-type T cells (Fig. 9,F). These findings are in agreement with our findings that gal-1−/− CD8 cells undergo more cell division (Figs. 1 and 2) and published reports demonstrating that IFN-γ effector function is greater in cells that have undergone several divisions (1, 41, 42, 43). Similarly, a higher percentage of tetramer-positive cells was observed in the lymph nodes of mice that received gal-1−/− OT-1 CD8 donor cells (Fig. 9 G). That gal-1−/− CD8 donor cells expanded to a greater extent than wild-type T cells, even in the context of a gal-1-expressing host, demonstrates a role for CD8-derived gal-1 in controlling CD8 burst size and function.

The investigation of regulators of TCR signal transduction has traditionally been focused on initiation of T cell activation. However, recent data point to a role for continued TCR engagement in shaping T cell responses (3, 4). In this study, we demonstrate that gal-1 secreted by CD8 T cells antagonizes persistent TCR agonist ligand binding and signal transduction throughout T cell activation to control CD8 hyperresponsiveness and burst size.

We find that TCR engagement up-regulates gal-1 mRNA expression as early as 24 h, with levels continuing to increase for the 72 h following CD8 T cell activation. Our findings complement and extend previous reports that gal-1 protein and mRNA levels increase within CD8 cells after activation and CTL differentiation (6, 44). Over this same time period, CTL effectors are known to up-regulate the expression of glycosyltransferases and neuraminidases and alter their glycosylation profile in ways predicted to increase gal-1 binding to glycans (17, 22, 36, 45, 46). We found that T cells lacking gal-1 demonstrated enhanced T cell blasting and IL-2 production as early as 12–30 h after TCR engagement. However, differences between gal-1-deficient and wild-type cells and their impact on cell division became most apparent later (42–72 h), when gal-1 and its ligands began to accumulate.

Previous studies by Mescher and colleagues (47, 48, 49) have identified a period of activation-induced nonresponsiveness (AINR) in CD8 T cells 2–4 days after initial TCR engagement, wherein CD8 T cells become unable to produce IL-2 or proliferate in response to secondary TCR engagement. The addition of exogenous IL-2 can reverse AINR, which has been characterized to result from impaired TCR-induced MAPK activation (including ERK) and IL-2 production (47, 48, 49). We found that cultures of stimulated gal-1-deficient CD8 cells produce more IL-2 than their wild-type counterparts. It is possible that hyperproliferation observed in CD8 cells from gal-1-deficient mice results from increased TCR-induced IL-2 production and prevention of AINR. Thus, our findings elucidate a potential role for gal-1 in mediating AINR.

We found that gal-1 expression phenocopied truncation of continued TCR engagement and Lck signaling, limiting cell division and increasing apoptosis. In light of our previous findings highlighting similarities between TCR antagonist peptides and gal-1 activity (30, 33, 50, 51), we explored the possibility that gal-1 might function as a TCR antagonist to dampen sustained TCR signaling to oppose proliferation and promote apoptosis. We measured the effect of the addition of rgal-1 on cognate tetramer decay on activated gal-1-deficient T cells and found that gal-1 antagonizes durable TCR binding of tetramer in the presence of a Kb competitor. We measured a 300% change in slope as a result of gal-1 binding. Published studies demonstrating a 50–100% change in slope from tetramer decay assays have been interpreted to provide explanations for TCR avidity threshold changes accompanying negative vs positive thymocyte selection (52) and thymocyte vs naive T cell activation and primary vs secondary T cell activation (35). Therefore, the changes we observe in TCR binding are most likely sufficient to account for the biological phenomenon observed. In vivo tetramer dulling in response to antigenic activation in wild-type vs gal-1-deficient T cells recapitulates our in vitro findings and identifies a window wherein endogenous gal-1 antagonizes TCR avidity. For OT-1 T cell response to OVA, gal-1-induced tetramer dulling became apparent just before the peak of the T cell response and was no longer measurable during the contraction phase.

Recent studies have demonstrated that high input frequency of donor cells in adoptive transfer models may alter the quality and character of the immune response (53). Planned experiments will assess the effects of gal-1 on primary and memory T cell expansion in the context of lower numbers of adoptively transferred input T cells. Although our input frequency was higher relative to these recent studies, they are in keeping with many other published studies that have identified key roles for T cell regulators (54, 55, 56, 57, 58, 59). Furthermore, the findings from our LCMV experiments in the absence of T cell transfer serve to validate our conclusion that gal-1 regulates primary CD8 response in vivo.

Gal-1-deficient CD8 T cells displayed sustained ERK activation and diminished SHP-1/Lck association, a signaling signature previously associated with TCR antagonism and in setting thresholds for TCR responsiveness (39). The addition of rgal-1 reversed ERK hyperactivation in gal-1-deficient T cells. Together, these findings support the suggestion that the gal-1 acts as a TCR antagonist to limit sustained TCR signaling during continued CD8 T cell activation. Furthermore, they identify a role for the ERK/SHP-1 feed forward/feedback loops in regulating sustained signaling and CD8 burst size.

Gal-1 is well characterized as a lectin that can regulate cellular activity by binding specific glycoproteins on the cell surface (60, 61). More recently, a role for intracellular gal-1 in regulating Ras membrane localization and downstream signaling has been reported in non-T cells (62, 63). Because the effects of gal-1 ablation on ERK activation can be reversed through the addition of rgal-1 to the extracellular medium and because we find that rgal-1-induced apoptosis in CD8 cells can be blocked by addition of competitive inhibitors lactose or L2hmda (data not shown), we favor a model whereby gal-1 acts extracellularly as a lectin to modulate surface glycoprotein organization, signaling, and ligand binding in the context of TCR/CD28 engagement.

During antigenic stimulation, the T cell:APC interface differentiates into a specialized contact, referred to as the immune synapse, remnants of which persist even after cell division (24, 27, 28). This synapse provides context for TCR engagement and tunes downstream signal transduction and functional outcome through the juxtaposition and organization of T cell surface glycoproteins and intracellular signaling molecules. We have yet to establish which gal-1 glycoprotein receptors are responsible for gal-1 activity on activated CD8 cells or how gal-1 binding affects immune synapse formation or duration. However, the gal-1 receptors CD45 and CD43 have been reported to oppose TCR binding and processive signaling and have been implicated in regulating gal-1-induced apoptosis, T cell synaptic organization, and function (14, 37, 46, 64, 65, 66, 67, 68). Therefore, CD45 and CD43 represent excellent candidate gal-1 effectors. CD3, CD4, CD7, and GM1 represent alternate known gal-1 receptors with the potential to influence TCR binding and signal transduction and synaptic dynamics (13, 15, 19, 29, 60). A direct assessment of gal-1 effects on glycoprotein counterreceptor organization on CD8 T cells is necessary, because it is becoming increasingly clear that galectins can have alternate activities on distinct T cell subpopulations (8, 9, 69).

Accumulating data support the concept that galectin-driven glycolattices organize T cell surfaces to regulate activation and functional outcome. Indeed, T cells from mice deficient in the enzyme Mgat5, which modifies N-glycans to facilitate galectin binding, show increase TCR mobility and lower activation thresholds (18). In another recent study, anergy in cultured human T cells, and potentially T cells responding to tumors, resulted from defective CD8/TCR colocalization that was reversed by addition of galectin disaccharide ligands (70). In both instances, galectin-3 was implicated, but not directly proven to be the endogenous lectin responsible for organizing T cell regulatory glycolattices to regulate T cell activity. Together with previous studies establishing gal-1 as a regulator of TCR signal transduction, binding, and fate determination during thymocyte selection and in CD4 T cell activation (8, 30), the findings presented in this study point to a role for galectin-driven glycolattices as regulators of TCR signal specificity and functional outcome. However, gal-1 and galectin-3 have been demonstrated to elicit distinct functions in T cell subpopulations (6, 33, 71, 72, 73, 74). Furthermore, gal-1 and galectin-3 have different structures (75, 76, 77), valencies (77, 78, 79), cell surface glycoprotein receptor-binding specificities (13, 14, 73, 80, 81), and T cell expression patterns in resting, activated, anergic, and memory T cell populations (44, 82). Therefore, it is unlikely that they function through identical mechanisms. Rather, we propose that galectin family members function through nonredundant mechanisms to differentially tune TCR signals at distinct stages of T cell development.

Gal-1 TCR antagonist activity in CD8 cells is in sharp contrast to its activity in developing thymocytes undergoing selection, in which endogenous gal-1 enforces fate decisions by promoting TCR agonist binding and signaling (8). In both thymus and periphery, gal-1 tuning of TCR binding and signaling impacts ERK pathways involved in signal procession and discrimination. How gal-1 alternately promotes or opposes TCR avidity and downstream signals depending on the developmental context of TCR binding remains to be determined, although alternate glycosylation and synaptic organization within developing and activated T cell subpopulations have been noted (25, 28).

By infecting gal-1-deficient mice with LCMV and tracking Ag-specific CD8 T cells, we determined that gal-1 expression leads to decreased TCR avidity and regulates CD8 burst size in vivo. Adoptive transfer of gal-1-deficient TCR-transgenic CD8 cells into wild-type hosts demonstrated that Ag-induced gal-1 production by activated CD8 cells is responsible for limiting their expansion and effector activity. These findings identify gal-1 as a novel autocrine negative regulator of CD8 burst size and provide the first definitive identification of an endogenous lectin that modulates TCR ligand binding during the course of T cell activation in vivo. Based on our findings, we propose a model whereby gal-1 secreted and bound by activated CD8 T cells imposes alternate cell surface TCR packing and organization, qualitatively changing TCR binding and signal specificity to promote apoptosis over proliferation, thus controlling CD8 burst size.

We thank members of the Miceli Lab and Linda Baum for critical reading of the manuscript. We also thank Dr. Valeri V. Mossine (University of Missouri, Columbia, MO) for the gal-1-specific inhibitor. Cell enrichment was performed at the University of California Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Grant NIH R01A1056155 (to M.C.M.). S.D.L. was supported by the Microbial Pathogenesis Training Grant T32 AI07323-15, Clinical and Fundamental Training Grant AI07126-30, and Warsaw Fellowship. T.T. is a recipient of the Microbial Pathogenesis Training Grant 2-T32-AI-07323. F.P. received financial support from Association pour la Recherche sur le Cancer and Ligue Contre le Cancer. University of California Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry Core Facility were supported by National Institutes of Health Awards CA-16042 and AI-28697.

3

Abbreviations used in this paper: gal-1, galectin-1; AINR, activation-induced nonresponsiveness; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescent intensity; pERK, phosphorylated ERK; SHP-1, Src homology region 2 domain-containing phosphatase 1; TNE, 50 mM Tris, 1% Nonidet P-40, 2 mM EDTA, pH 8.0.

1
Kaech, S. M., R. Ahmed.
2001
. Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naive cells.
Nat. Immunol.
2
:
415
-422.
2
Van Stipdonk, M. J., G. Hardenberg, M. S. Bijker, E. E. Lemmens, N. M. Droin, D. R. Green, S. P. Schoenberger.
2003
. Dynamic programming of CD8+ T lymphocyte responses.
Nat. Immunol.
4
:
361
-365.
3
Prlic, M., G. Hernandez-Hoyos, M. J. Bevan.
2006
. Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response.
J. Exp. Med.
203
:
2135
-2143.
4
Tewari, K., J. Walent, J. Svaren, R. Zamoyska, M. Suresh.
2006
. Differential requirement for Lck during primary and memory CD8+ T cell responses.
Proc. Natl. Acad. Sci. USA
103
:
16388
-16393.
5
Mempel, T. R., S. E. Henrickson, U. H. Von Andrian.
2004
. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.
Nature
427
:
154
-159.
6
Blaser, C., M. Kaufmann, C. Müller, C. Zimmermann, V. Wells, L. Mallucci, H. Pircher.
1998
. β-Galactoside-binding protein secreted by activated T cells inhibits antigen-induced proliferation of T cells.
Eur. J. Immunol.
28
:
2311
-2319.
7
Rabinovich, G. A., N. Rubinstein, M. A. Toscano.
2002
. Role of galectins in inflammatory and immunomodulatory processes.
Biochim. Biophys. Acta
1572
:
274
-284.
8
Liu, S. D., C. C. Whiting, T. Tomassian, M. Pang, S. J. Bissel, L. G. Baum, V. V. Mossine, F. Poirier, M. E. Huflejt, M. C. Miceli.
2008
. Endogenous galectin-1 enforces class I-restricted TCR functional fate decisions in thymocytes.
Blood
112
:
120
-130.
9
Toscano, M. A., G. A. Bianco, J. M. Ilarregui, D. O. Croci, J. Correale, J. D. Hernandez, N. W. Zwirner, F. Poirier, E. M. Riley, L. G. Baum, G. A. Rabinovich.
2007
. Differential glycosylation of TH1, TH2 and TH17 effector cells selectively regulates susceptibility to cell death.
Nat. Immunol.
8
:
825
-834.
10
Perillo, N. L., K. E. Pace, J. J. Seilhamer, L. G. Baum.
1995
. Apoptosis of T cells mediated by galectin-1.
Nature
378
:
736
-739.
11
Leffler, H., S. Carlsson, M. Hedlund, Y. Qian, F. Poirier.
2004
. Introduction to galectins.
Glycoconj. J.
19
:
433
-440.
12
Lau, K. S., E. A. Partridge, A. Grigorian, C. I. Silvescu, V. N. Reinhold, M. Demetriou, J. W. Dennis.
2007
. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation.
Cell
129
:
123
-134.
13
Pace, K. E., H. P. Hahn, M. Pang, J. T. Nguyen, L. G. Baum.
2000
. CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death.
J. Immunol.
165
:
2331
-2334.
14
Pace, K. E., C. Lee, P. L. Stewart, L. G. Baum.
1999
. Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1.
J. Immunol.
163
:
3801
-3811.
15
Rappl, G., H. Abken, J. M. Muche, W. Sterry, W. Tilgen, S. Andre, H. Kaltner, S. Ugurel, H. J. Gabius, U. Reinhold.
2002
. CD4+CD7 leukemic T cells from patients with Sezary syndrome are protected from galectin-1-triggered T cell death.
Leukemia
16
:
840
-845.
16
Baum, L. G..
2002
. Developing a taste for sweets.
Immunity
16
:
5
-8.
17
Daniels, M. A., K. A. Hogquist, S. C. Jameson.
2002
. Sweet ‘n’ sour: the impact of differential glycosylation on T cell responses.
Nat. Immunol.
3
:
903
-910.
18
Demetriou, M., M. Granovsky, S. Quaggin, J. W. Dennis.
2001
. Negative regulation of T-cell activation and autoimmunity by Mgat5 N-glycosylation.
Nature
409
:
733
-739.
19
Kopitz, J., C. von Reitzenstein, M. Burchert, M. Cantz, H. J. Gabius.
1998
. Galectin-1 is a major receptor for ganglioside GM1, a product of the growth-controlling activity of a cell surface ganglioside sialidase, on human neuroblastoma cells in culture.
J. Biol. Chem.
273
:
11205
-11211.
20
Amano, M., M. Galvan, J. He, L. G. Baum.
2003
. The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering, phosphatase modulation, and T cell death.
J. Biol. Chem.
278
:
7469
-7475.
21
Galvan, M., S. Tsuboi, M. Fukada, L. G. Baum.
2000
. Expression of a specific glycosyltransferase enzyme regulates T cell death mediated by galectin-1.
J. Biol. Chem.
275
:
16730
-16730.
22
Lowe, J. B..
2001
. Glycosylation, immunity, and autoimmunity.
Cell
104
:
809
-812.
23
Priatel, J. J., D. Chui, N. Hiraoka, C. J. T. Simmons, K. B. Richardson, D. M. Page, M. Fukuda, N. M. Varki, J. M. Marth.
2000
. The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis.
Immunity
12
:
273
-283.
24
Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer, A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, et al
2007
. Asymmetric T lymphocyte division in the initiation of adaptive immune responses.
Science
315
:
1687
-1691.
25
Friedl, P., A. T. den Boer, M. Gunzer.
2005
. Tuning immune responses: diversity and adaptation of the immunological synapse.
Nat. Rev. Immunol.
5
:
532
-545.
26
Miceli, M. C., M. Moran, C. D. Chung, V. P. Patel, T. Low, W. Zinnanti.
2001
. Costimulation and counter-stimulation: lipid raft clustering controls TCR signaling and functional outcomes.
Semin. Immunol.
13
:
1
-14.
27
Cullinan, P., A. I. Sperling, J. K. Burkhardt.
2002
. The distal pole complex: a novel membrane domain distal to the immunological synapse.
Immunol. Rev.
189
:
111
-122.
28
Yeh, J. H., S. S. Sidhu, A. C. Chan.
2008
. Regulation of a late phase of T cell polarity and effector functions by Crtam.
Cell
132
:
846
-859.
29
Walzel, H., M. Blach, J. Hirabayashi, K. I. Kasai, J. Brock.
2000
. Involvement of CD2 and CD3 in galectin-1 induced signaling in human Jurkat T-cells.
Glycobiology
10
:
131
-140.
30
Chung, C. D., V. P. Patel, M. Moran, L. A. Lewis, M. C. Miceli.
2000
. Galectin-1 induces partial TCR ζ-chain phosphorylation and antagonizes processive TCR signal transduction.
J. Immunol.
165
:
3722
-3729.
31
Madrenas, J., R. L. Wange, J. L. Wang, N. Isakov, L. E. Samelson, R. N. Germain.
1995
. ζ Phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists.
Science
267
:
515
-518.
32
Reis e Sousa, C., E. H. Levine, R. N. Germain.
1996
. Partial signaling by CD8+ T cells in response to antagonist ligands.
J. Exp. Med.
184
:
149
-157.
33
Vespa, G. N., L. A. Lewis, K. R. Kozak, M. Moran, J. T. Nguyen, L. G. Baum, M. C. Miceli.
1999
. Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL-2 production and proliferation.
J. Immunol.
162
:
799
-806.
34
Perone, M. J., A. T. Larregina, W. J. Shufesky, G. D. Papworth, M. L. Sullivan, A. F. Zahorchak, D. B. Stolz, L. G. Baum, S. C. Watkins, A. W. Thomson, A. E. Morelli.
2006
. Transgenic galectin-1 induces maturation of dendritic cells that elicit contrasting responses in naive and activated T cells.
J. Immunol.
176
:
7207
-7220.
35
Savage, P. A., J. J. Boniface, M. M. Davis.
1999
. A kinetic basis for T cell receptor repertoire selection during an immune response.
Immunity
10
:
485
-492.
36
Harrington, L. E., M. Galvan, L. G. Baum, J. D. Altman, R. Ahmed.
2000
. Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans.
J. Exp. Med.
191
:
1241
-1246.
37
Onami, T. M., L. E. Harrington, M. A. Williams, M. Galvan, C. P. Larsen, T. C. Pearson, N. Manjunath, L. G. Baum, B. D. Pearce, R. Ahmed.
2002
. Dynamic regulation of T cell immunity by CD43.
J. Immunol.
168
:
6022
-6031.
38
Huflejt, M. E., V. V. Mossin, O. Naidenko, M. Jazayeri, P. Rogers, N. Tinari, S. Iacobelli, M. Elliot, J. Lustgarten, and M. Croft. 2001. Synthetic lactulose amines bind tumor-promoting galectins-1 and -4 and inhibit breast cancers in Her-2/neu transgenic mice. 24th Annual San Antonio Breast Cancer Symposium.
39
Stefanova, I., B. Hemmer, M. Vergelli, R. Martin, W. E. Biddison, R. N. Germain.
2003
. TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways.
Nat. Immunol.
4
:
248
-254.
40
Poirier, F., E. J. Robertson.
1993
. Normal development of mice carrying a null mutation in the gene encoding the L14 S-type lectin.
Development
119
:
1229
-1236.
41
Agarwal, S., A. Rao.
1998
. Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation.
Immunity
9
:
765
-775.
42
Murali-Krishna, K., R. Ahmed.
2000
. Cutting edge: naive T cells masquerading as memory cells.
J. Immunol.
165
:
1733
-1737.
43
Oehen, S., K. Brduscha-Riem.
1998
. Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division.
J. Immunol.
161
:
5338
-5346.
44
Wherry, E. J., S. J. Ha, S. M. Kaech, W. N. Haining, S. Sarkar, V. Kalia, S. Subramaniam, J. N. Blattman, D. L. Barber, R. Ahmed.
2007
. Molecular signature of CD8+ T cell exhaustion during chronic viral infection.
Immunity
27
:
670
-684.
45
Galvan, M., K. Murali-Krishna, L. L. Ming, L. Baum, R. Ahmed.
1998
. Alterations in cell surface carbohydrates on T cells from virally infected mice can distinguish effector/memory CD8+ T cells from naive cells.
J. Immunol.
161
:
641
-648.
46
Nguyen, J. T., D. P. Evans, M. Galvan, K. E. Pace, D. Leitenberg, T. N. Bui, L. G. Baum.
2001
. CD45 modulates galectin-1-induced T cell death: regulation by expression of core 2 O-glycans.
J. Immunol.
167
:
5697
-5707.
47
Deeths, M. J., R. M. Kedl, M. F. Mescher.
1999
. CD8+ T cells become nonresponsive (anergic) following activation in the presence of costimulation.
J. Immunol.
163
:
102
-110.
48
Tham, E. L., M. F. Mescher.
2001
. Signaling alterations in activation-induced nonresponsive CD8 T cells.
J. Immunol.
167
:
2040
-2048.
49
Tham, E. L., P. Shrikant, M. F. Mescher.
2002
. Activation-induced nonresponsiveness: a Th-dependent regulatory checkpoint in the CTL response.
J. Immunol.
168
:
1190
-1197.
50
Combadière, B., C. R. e Sousa, R. N. Germain, M. J. Lenardo.
1998
. Selective induction of apoptosis in mature T lymphocytes by variant T cell receptor ligands.
J. Exp. Med.
187
:
349
-355.
51
Sloan-Lancaster, J., B. D. Evavold, P. M. Allen.
1993
. Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells.
Nature
363
:
156
-159.
52
Savage, P. A., M. M. Davis.
2001
. A kinetic window constricts the T cell receptor repertoire in the thymus.
Immunity
14
:
243
-252.
53
Badovinac, V. P., J. S. Haring, J. T. Harty.
2007
. Initial T cell receptor transgenic cell precursor frequency dictates critical aspects of the CD8+ T cell response to infection.
Immunity
26
:
827
-841.
54
Ch'en, I. L., D. R. Beisner, A. Degterev, C. Lynch, J. Yuan, A. Hoffmann, S. M. Hedrick.
2008
. Antigen-mediated T cell expansion regulated by parallel pathways of death.
Proc. Natl. Acad. Sci. USA
105
:
17463
-17468.
55
Kearney, E. R., K. A. Pape, D. Y. Loh, M. K. Jenkins.
1994
. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo.
Immunity
1
:
327
-339.
56
Lefrancois, L., J. D. Altman, K. Williams, S. Olson.
2000
. Soluble antigen and CD40 triggering are sufficient to induce primary and memory cytotoxic T cells.
J. Immunol.
164
:
725
-732.
57
Lefrancois, L., A. Marzo, K. Williams.
2003
. Sustained response initiation is required for T cell clonal expansion but not for effector or memory development in vivo.
J. Immunol.
171
:
2832
-2839.
58
Prlic, M., B. R. Blazar, A. Khoruts, T. Zell, S. C. Jameson.
2001
. Homeostatic expansion occurs independently of costimulatory signals.
J. Immunol.
167
:
5664
-5668.
59
Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois.
2000
. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo.
Nat. Immunol.
1
:
426
-432.
60
Elola, M. T., M. E. Chiesa, A. F. Alberti, J. Mordoh, N. E. Fink.
2005
. Galectin-1 receptors in different cell types.
J. Biomed. Sci.
12
:
13
-29.
61
Rabinovich, G. A., L. G. Baum, N. Tinari, R. Paganelli, C. Natoli, F. T. Liu, S. Iacobelli.
2002
. Galectins and their ligands: amplifiers, silencers or tuners of the inflammatory response?.
Trends Immunol.
23
:
313
-320.
62
Elad-Sfadia, G., R. Haklai, E. Ballan, H. J. Gabius, Y. Kloog.
2002
. Galectin-1 augments Ras activation and diverts Ras signals to Raf-1 at the expense of phosphoinositide 3-kinase.
J. Biol. Chem.
277
:
37169
-37175.
63
Paz, A., R. Haklai, G. Elad-Sfadia, E. Ballan, Y. Kloog.
2001
. Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation.
Oncogene
20
:
7486
-7493.
64
Hernandez, J. D., J. T. Nguyen, J. He, W. Wang, B. Ardman, J. M. Green, M. Fukuda, L. G. Baum.
2006
. Galectin-1 binds different CD43 glycoforms to cluster CD43 and regulate T cell death.
J. Immunol.
177
:
5328
-5336.
65
Fierro, N. A., G. Pedraza-Alva, Y. Rosenstein.
2006
. TCR-dependent cell response is modulated by the timing of CD43 engagement.
J. Immunol.
176
:
7346
-7353.
66
Tong, J., E. J. Allenspach, S. M. Takahashi, P. D. Mody, C. Park, J. K. Burkhardt, A. I. Sperling.
2004
. CD43 regulation of T cell activation is not through steric inhibition of T cell-APC interactions but through an intracellular mechanism.
J. Exp. Med.
199
:
1277
-1283.
67
Chen, I. J., H. L. Chen, M. Demetriou.
2007
. Lateral compartmentalization of T cell receptor versus CD45 by galectin-N-glycan binding and microfilaments coordinate basal and activation signaling.
J. Biol. Chem.
282
:
35361
-35372.
68
Zhang, M., M. Moran, J. Round, T. A. Low, V. P. Patel, T. Tomassian, J. D. Hernandez, M. C. Miceli.
2005
. CD45 signals outside of lipid rafts to promote ERK activation, synaptic raft clustering, and IL-2 production.
J. Immunol.
174
:
1479
-1490.
69
Motran, C., K. Molinder, S. Liu, F. Poirier, M. C. Miceli.
2008
. Galectin-1 functions as a Th2 cytokine that selectively induces Th1 apoptosis and promotes Th2 function.
Eur. J. Immunol.
38
:
3015
-3027.
70
Demotte, N., V. Stroobant, P. J. Courtoy, P. Van Der Smissen, D. Colau, I. F. Luescher, C. Hivroz, J. Nicaise, J. L. Squifflet, M. Mourad, et al
2008
. Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes.
Immunity
28
:
414
-424.
71
Joo, H. G., P. S. Goedegebuure, N. Sadanaga, M. Nagoshi, W. von Bernstorff, T. J. Eberlein.
2001
. Expression and function of galectin-3, a β-galactoside-binding protein in activated T lymphocytes.
J. Leukocyte Biol.
69
:
555
-564.
72
Perillo, N., C. Uittenbogaart, J. Nguyen, L. Baum.
1997
. Galectin-1, an endogenous lectin produced by thymic epithelial cells, induces apoptosis of human thymocytes.
J. Exp. Med.
185
:
1851
-1858.
73
Stillman, B. N., D. K. Hsu, M. Pang, C. F. Brewer, P. Johnson, F. T. Liu, L. G. Baum.
2006
. Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death.
J. Immunol.
176
:
778
-789.
74
Stowell, S. R., Y. Qian, S. Karmakar, N. S. Koyama, M. Dias-Baruffi, H. Leffler, R. P. McEver, R. D. Cummings.
2008
. Differential roles of galectin-1 and galectin-3 in regulating leukocyte viability and cytokine secretion.
J. Immunol.
180
:
3091
-3102.
75
Ahmad, N., H. J. Gabius, S. Andre, H. Kaltner, S. Sabesan, R. Roy, B. Liu, F. Macaluso, C. F. Brewer.
2004
. Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes.
J. Biol. Chem.
279
:
10841
-10847.
76
Hirabayashi, J., K. Kasai.
1993
. The family of metazoan metal-independent β-galactoside-binding lectins: structure, function and molecular evolution.
Glycobiology
3
:
297
-304.
77
Rabinovich, G. A., F. T. Liu, M. Hirashima, A. Anderson.
2007
. An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer.
Scand. J. Immunol.
66
:
143
-158.
78
Brewer, C. F..
2002
. Binding and cross-linking properties of galectins.
Biochim. Biophys. Acta
1572
:
255
-262.
79
Brewer, C. F., M. C. Miceli, L. G. Baum.
2002
. Clusters, bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions.
Curr. Opin. Struct. Biol.
12
:
616
-623.
80
Fukumori, T., Y. Takenaka, T. Yoshii, H. R. Kim, V. Hogan, H. Inohara, S. Kagawa, A. Raz.
2003
. CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis.
Cancer Res.
63
:
8302
-8311.
81
Walzel, H., U. Schulz, P. Neels, J. Brock.
1999
. Galectin-1, a natural ligand for the receptor-type protein tyrosine phosphatase CD45.
Immunol. Lett.
67
:
193
-202.
82
McHugh, R. S., M. J. Whitters, C. A. Piccirillo, D. A. Young, E. M. Shevach, M. Collins, M. C. Byrne.
2002
. CD4+CD25+ immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.
Immunity
16
:
311
-323.