Galectin-1, an endogenous lectin expressed in lymphoid organs and immune-privileged sites, induces death of human and murine thymocytes and T cells. Galectin-1 binds to several glycoproteins on the T cell surface, including CD7. However, the T cell surface glycoprotein receptors responsible for delivering the galectin-1 death signal have not been identified. We show that CD7 is required for galectin-1-mediated death. This demonstrates a novel function for CD7 as a death trigger and identifies galectin-1/CD7 as a new biologic death signaling pair.

Galectin-1 induces death of human and murine immature thymocytes and activated T cells (1, 2, 3). In the immune system, galectin-1 is expressed in thymus, spleen, lymph nodes, bone marrow, liver, and immune-privileged sites (4, 5). Galectin-1 may be involved in death of negatively selected and nonselected immature thymocytes (1, 3) and may modulate TCR-mediated responses of peripheral T cells (2, 3). Galectin-1 has potent immunomodulatory effects in vivo, suppressing disease in animal models of experimental autoimmune encephalomyelitis, myasthenia gravis, rheumatoid arthritis, hepatitis, and graft-vs-host disease (6, 7, 8, 9, 10). Galectin-1 administration resulted in T cell death in vitro (8), and Ag-specific T cells were not detected in lymph nodes of galectin-1-treated animals (6). Galectin-1 therapy also resulted in a Th1 to Th2 shift (8, 9).

Galectin-1 does not trigger death via known T cell surface apoptotic triggers such as Fas or CD3 (11). Although CD2, CD3, CD4, CD7, CD43, and CD45 have been identified as specific T cell surface receptors for galectin-1 (11, 12), it is not known which receptor(s) is required for galectin-1-induced cell death. Of the galectin-1 receptors, CD7 appears to have immunomodulatory activity, yet no ligand for CD7 has previously been identified (13). We noted that all galectin-1-susceptible T cells, including human and murine immature thymocytes, activated T cells, and T cell lines expressed CD7. Therefore, we examined the role of CD7 in human T cell death induced by galectin-1.

ARR, CEM, HUT78, and MOLT-4 (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640, 10% FBS, and 10 mM HEPES (complete media). Human CD7 cDNA was provided by Dr. Brian Seed (14). MT910 (CD2), UCHT1 (CD3), MT310 (CD4), DF-T1 (CD43), PD7/26/16&2B11 (CD45), PE-conjugated goat anti-mouse IgG1 and IgG2a, and mouse IgG1- and IgG2a-negative controls were obtained from Dako (Carpinteria, CA); LT-7 (CD7) was obtained from Advanced Immunochemical (Long Beach, CA); and Indo-1 acetoxymethyl ester was obtained from Molecular Probes (Eugene, OR). Human recombinant galectin-1 was prepared as described (2).

A total of 2 × 105 cells were treated with 20 μM galectin-1, 1.0 mM DTT, or 1.0 mM DTT buffer control and rocked for 3 h at 37°C in 5% CO2. Death was measured by annexin V binding and propidium iodide permeability and analyzed using a Becton Dickinson FACScan and CellQuest software (11). Percent annexin V+ cells was determined as follows: (annexin V+ (galectin-1) − annexin V+ (control)/100 − annexin V+ (control)) × 100. Percent cell loss was determined as follows: 100 − ((viable annexin V (galectin-1)/viable annexin V (control)) × 100).

Human CD7 cDNA was subcloned into the pcDNA3.1/Zeo+ vector (Invitrogen, Carlsbad, CA). HUT78 cells were electroporated with 10 μg of linearized vector containing CD7 cDNA or with vector only (15). Cells were pulsed for 10 s at 280V/960 mF in an Invitrogen Electroporator II (15). Cells were cultured for 48 h in complete media and transferred to complete media containing 300 μg/ml zeocin (Invitrogen). Individual clones were isolated by limiting dilution, and three CD7+ clones were identified by flow cytometry and used for galectin-1 death assays.

A total of 2 × 105 cells were incubated with 0.2 μg of primary mAb for 45 min at 4°C, followed by the appropriate PE-conjugated secondary Ab for 45 min at 4°C. Flow cytometry data was acquired and analyzed as above.

A total of 8 × 106 cells at 2 × 106 cells/ml were loaded with 2 μg/ml Indo-1 acetoxymethyl for 40 min at 37°C. Cells were resuspended in 1 ml 0.5% BSA and HBSS and transferred to cuvettes. Fluorescence was monitored with a Photon Technology International spectrofluorometer (Princeton, NJ) at 37°C (excitation, 350 nm; emission, 400 and 485 nm).

The galectin-1 receptor phenotypes of several human T cell lines are shown in Table I. Because cell lines lacking CD2, CD3, CD4, and CD45 are susceptible to galectin-1, these receptors may modulate, but must not be essential for, galectin-1 death (2). Importantly, all of the galectin-1-susceptible human T cell lines that we have identified expressed CD7. We also found that the HUT78 cell line, which was CD7 (Table I), was resistant to galectin-1-induced death. Galectin-1 treatment of HUT78 cells resulted in minimal cell death detected by either annexin V/propidium iodide staining or cell loss (range 0–11%, average 4.5%) in five representative experiments, while galectin-1 treatment of CD7+ MOLT-4 cells resulted in death of 85% of the cells using either method (Fig. 1). This suggested that CD7 was required for galectin-1 cell death.

Table I.

Galectin-1 receptor expression on human T cell lines

CD7aCD2CD3CD4CD43CD45% Deathb
HUT78 <1 90 96 10 11 
HUT78.7 97 96 95 48 
ARR 89 <1 <1 <1 97 99 33 
CEM 95 80 86 99 99 99 73 
MOLT-4 91 54 89 75 99 98 85 
CD7aCD2CD3CD4CD43CD45% Deathb
HUT78 <1 90 96 10 11 
HUT78.7 97 96 95 48 
ARR 89 <1 <1 <1 97 99 33 
CEM 95 80 86 99 99 99 73 
MOLT-4 91 54 89 75 99 98 85 
a

Percent positive.

b

Percent death expressed as percent annexin V+ or percent cell loss (see Materials and Methods).

FIGURE 1.

CD7 HUT78 cells were not susceptible to galectin-1. Cell death was measured by annexin V binding (A) and cell loss (B). CD7+ MOLT-4 cells were very susceptible to galectin-1 death, while galectin-1 treatment of HUT78 cells resulted in minimal annexin V binding or cell loss.

FIGURE 1.

CD7 HUT78 cells were not susceptible to galectin-1. Cell death was measured by annexin V binding (A) and cell loss (B). CD7+ MOLT-4 cells were very susceptible to galectin-1 death, while galectin-1 treatment of HUT78 cells resulted in minimal annexin V binding or cell loss.

Close modal

To determine whether CD7 is necessary for galectin-1 death, we expressed human CD7 in HUT78 cells (Fig. 2,A). Importantly, the expression of other galectin-1 receptors was not altered by CD7 expression (Table I). Specifically, both HUT78 and HUT78.7 cells expressed no detectable CD45, indicating that CD45 is not essential for galectin-1 death. As shown in Fig. 2, B and C, CD7 expression rendered the HUT78 cells susceptible to galectin-1, determined by both cell loss and phosphatidylserine exposure. Galectin-1 induced death of ∼50% of CD7+ HUT78 cells, while only 12% of mock transfectants died (Fig. 2,B). Galectin-1 treatment of CD7+ HUT78 cells also resulted in phosphatidylserine exposure as indicated by a shift in annexin V staining intensity of the entire cell population (Δpfc 68) (Fig. 2 C).

FIGURE 2.

CD7 expression rendered HUT78 cells susceptible to galectin-1. A, CD7 was highly expressed on the surface of HUT78.7 cells transfected with CD7 cDNA. CD7 expression was not detectable on parental HUT78 cells or HUT78 cells transfected with vector alone (mock). B, CD7+ HUT78.7 cells demonstrated a 5-fold increase in galectin-1-induced death, compared with CD7 parental HUT78 and mock-transfectedcells. C, Galectin-1 treatment of CD7+ HUT cells resulted in phosphatidylserine exposure as indicated by a shift in the annexin V staining of the cell population (Δpfc 68). Peak channel fluorescence of the population is indicated in the upper right of each histogram. Results are mean ± SD and represent five independent experiments. Similar results were obtained with three independent CD7 transfectants of HUT78 cells.

FIGURE 2.

CD7 expression rendered HUT78 cells susceptible to galectin-1. A, CD7 was highly expressed on the surface of HUT78.7 cells transfected with CD7 cDNA. CD7 expression was not detectable on parental HUT78 cells or HUT78 cells transfected with vector alone (mock). B, CD7+ HUT78.7 cells demonstrated a 5-fold increase in galectin-1-induced death, compared with CD7 parental HUT78 and mock-transfectedcells. C, Galectin-1 treatment of CD7+ HUT cells resulted in phosphatidylserine exposure as indicated by a shift in the annexin V staining of the cell population (Δpfc 68). Peak channel fluorescence of the population is indicated in the upper right of each histogram. Results are mean ± SD and represent five independent experiments. Similar results were obtained with three independent CD7 transfectants of HUT78 cells.

Close modal

Galectin-1 binding has been shown to result in an increase in intracellular calcium concentration ([Ca2+]i)3 (12, 16, 17). Increased [Ca2+]i is required for some apoptotic mechanisms (18) and occurs after Ab cross-linking of CD7 (13). We observed that galectin-1 treatment of CD7 HUT78 cells did not result in a significant increase in [Ca2+]i. Expression of CD7 in HUT78 cells imparted galectin-1 susceptibility, but did not result in increased [Ca2+]i after galectin-1 binding (Fig. 3). We have previously shown that blocking the galectin-1-induced rise in [Ca2+]i with EGTA did not block cell death, implying that increased [Ca2+]i was not required for death (H. P. Hahn and L. G. Baum, unpublished observations). Taken together, these data show that CD7 is necessary for galectin-1-induced death via a Ca2+-independent pathway.

FIGURE 3.

Galectin-1-induced death does not require an increase in [Ca2+]i. MOLT-4 cells demonstrated a significant increase in [Ca2+]i after galectin-1 binding. However, there was no galectin-1-induced increase in [Ca2+]i in CD7+ HUT78.7 cells, compared with the parental HUT78 or mock-transfected cells.

FIGURE 3.

Galectin-1-induced death does not require an increase in [Ca2+]i. MOLT-4 cells demonstrated a significant increase in [Ca2+]i after galectin-1 binding. However, there was no galectin-1-induced increase in [Ca2+]i in CD7+ HUT78.7 cells, compared with the parental HUT78 or mock-transfected cells.

Close modal

Cell death is crucial for the proper functioning of the immune system (19, 20). Although many T cell surface receptors capable of transducing apoptotic signals have been discovered, very few biologic ligands for these receptors have been identified (20). Known apoptotic signaling pairs include members of the TNF/TNFR family and Ag/TCR (20). We have demonstrated a novel function for CD7 as a death trigger and identified galectin-1/CD7 as a new biologic signaling pair. The data in Table I also suggest that additional T cell surface glycoproteins may contribute to galectin-1-induced T cell death. Thus, CD7 may be an essential component of a pro-apoptotic complex of receptors that send the galectin-1 death signal (11).

CD7 is present on human immature thymocytes and is up-regulated on activated T cells, two T cell populations that are susceptible to galectin-1 (1, 2). CD7+ T cells may bind galectin-1 expressed by stromal or dendritic cells in tissues where T cells die, such as the thymus during T cell development (19, 21) or peripheral lymphoid organs following an immune response (4, 20). Galectin-1 may mediate interactions between T cells and stromal cells directly (1, 2, 8), or galectin-1 may modulate T cell adhesion by cross-linking CD7 on T cells, thereby up-regulating integrin-mediated adhesion (22, 23). We have previously demonstrated that galectin-1 expressed on human endothelial cells induced carbohydrate-dependent death of bound human T cells (2). Thus, galectin-1 expression by stromal cells may trigger death of susceptible CD7+ T cells that enter different tissues.

Little is known about the biologic functions of CD7 in vivo; however, many potential immunomodulatory functions have been ascribed to CD7 (13). Ab cross-linking of CD7 resulted in phosphatidylinositol 3-kinase and protein kinase C activity and led to tyrosine phosphorylation of several proteins including two proteins of 97 and 120 kDa (13, 24). Interestingly, these proteins are similar to the size of tyrosine-phosphorylated proteins seen after galectin-1 treatment (3). However, the role of tyrosine phosphorylation in galectin-1 death is not yet understood. Treatment of rheumatoid arthritis patients with anti-CD7 mAbs resulted in amelioration of disease and a 50% decrease in peripheral T cells (25). Anti-CD7 Ab treatment of renal transplant patients was also immunosuppressive and increased allograft survival (26). The decrease in T cell numbers and the immunosuppressive effects caused by administration of CD7 Abs may have resulted from the triggering of death via CD7 cross-linking, mimicking the cross-linking of CD7 by galectin-1 (11).

CD7 expression is lost in a number of T cell-mediated inflammatory diseases, as well as in T cell neoplasms. CD7 expression is lost from neoplastic T cells in the cutaneous T cell lymphomas Sezary syndrome and mycosis fungoides (27). Galectin-1 is expressed in both the dermis and epidermis (28), suggesting that loss of CD7 expression would enhance survival of CD7 neoplastic T cells in the skin. Interestingly, the HUT78 cell line was derived from a patient with Sezary syndrome (29). Peripheral T cells are CD7 in some patients with rheumatoid arthritis (30); loss of CD7 expression may enhance the survival of these autoreactive T cells. CD7 is not present on human CD4+CD45RACD45R0+ memory T cells (31). CD7+ effector T cells may be eliminated by galectin-1 following an immune response, while CD7 memory T cells may escape galectin-1-mediated death. Although no major phenotypic abnormalities were identified in CD7−/− mice (32, 33), Lee et al. did note a transient increase in CD4+CD8+ thymocytes in 3-mo-old mice. Importantly, the CD4+CD8+ thymocyte population is most susceptible to galectin-1-mediated death (1, 3, 34).

Expression of both the CD7 polypeptide and specific saccharide ligands on CD7 will be critical for regulating susceptibility to galectin-1. Galectin-1 preferentially binds to clustered lactosamine (Gal-GlcNAc) sequences on N- or O-linked glycans (35). Human CD7 possesses 2 N-linked glycans that are clustered on either side of an IgG fold and numerous O-linked glycans arranged closely in a mucin-like domain (13). The arrangement of these glycans may present the saccharides in a clustered patch, creating optimal ligands for cross-linking by galectin-1 (36). We have found that both N- and O-linked glycans are important for galectin-1 death and that CD7+ T cells must express required glycosyltransferases to receive the death signal (2, 34). We are currently examining specific glycoforms of CD7 to determine the precise requirements for CD7 signaling in galectin-1 T cell death.

We thank M. Galvan, M. Donnell, and B. Premack for technical assistance, B. Premack and M. C. Miceli for critical reading of the manuscript, and O. Witte, R. Ahmed, and P. Cresswell for helpful discussions.

1

This work was supported by National Institutes of Health Grant AI40118 and American Cancer Society Research Project Grant 9704901IM (to L.G.B.) and National Institutes of Health Grant CA16042 (to the Jonsson Comprehensive Cancer Center).

3

Abbreviation used in this paper: [Ca2+]i, intracellular calcium concentration.

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