In this study, we suggest that CD8 levels on T cells are not static, but can change and, as a result, modulate CD8+ T cell responses. We describe three models of CD8 modulation using novel weak-agonist (K1A) and super-agonist (C2A) altered peptide ligands of the HY smcy peptide. First, we used peripheral nonresponsive CD8low T cells produced after peripheral HY-Db MHC class I tetramer stimulation of female HY TCR transgenic and wild-type mice. Second, we used genetically lowered CD8int T cells from heterozygote CD8+/0 mice. Finally, we used pre-existing nonresponsive CD8low T cells from male HY TCR transgenic mice. In CD8low and CD8high mice, presence of a lower level of CD8 greatly decreased the avidity of the peptide-MHC for HY TCR as reflected by avidity (KD) and dissociation constant (T1/2) measurements. All three models demonstrated that lowering CD8 levels resulted in the requirement for a higher avidity peptide-MHC interaction with the TCR to respond equivalently to unmanipulated CD8high T cells of the same specificity. Additionally, direct injections of wild-type HY-Db and C2A-Db tetramers into female HY TCR or female B6 mice induced a high frequency of peripheral nonresponsive CD8low T cells, yet C2A-Db was superior in inducing a primed CD8+CD44+ memory population. The ability to dynamically modulate the size and responsiveness of an Ag-specific T cell pool by “CD8 tuning” of the T cell during the early phases of an immune response has important implications for the balance of responsiveness, memory, and tolerance.
The importance of CD8 in T cell function has been known for some time. Blocking CD8 during the interaction between a CD8+ T cell and an APC can inhibit the ability of most CD8+ T cells to lyse target cells; it has been demonstrated that CD8 binding depends mostly on the invariant α3 domain of the MHC class I molecule (1). In addition, transfection experiments demonstrated that the CD8 must bind to the same MHC as TCR to be functional (2). Recently our laboratory, using soluble mutant α3 MHC class I tetramers, has shown a complex relationship between peptide-MHC (pMHC)4/TCR avidity and the requirement of CD8 engagement; we demonstrated that removing the ability of CD8 to engage cognate pMHC results in poor stimulation of CD8+ T cells to moderate avidity ligands (3). Indeed, even high avidity pMHC ligands require longer Ag exposure for effective CD8+ T cell response in a CD8-binding deficient pMHC/TCR interaction compared with CD8-competent MHC class I tetramers (3). Other studies have supported the notion that CD8 is required to form a stable complex between MHC class I and TCR (4, 5, 6). Thus, regulation of CD8 levels may be a means for CD8+ T cells to regulate their effective avidity window and their resultant effector function (7, 8). For instance, it was recently demonstrated (4) that the contribution of CD8 at lower pMHC/TCR affinities is very significant; dose response of T cell hybrids can be greater than a million-fold improved with CD8 present than without. Obviously, there is a need to further define the functional effects of CD8 modulation of a low avidity pMHC/TCR interaction.
Minor H Ags are naturally processed peptide fragments derived from normal cellular proteins presented by MHC molecules (9, 10, 11). A representative and well-characterized H Ag is HY, encoded by genes specific to the Y chromosome, reviewed in Ref.12 . In male mice, HY immunodominant epitopes presented by murine MHC class I are encoded by the ubiquitously expressed Smcy and Uty genes (13). This is a natural “congenic” response mediated by CD8+ T cells that can be analyzed both in vitro and in vivo.
von Boehmer and colleagues (16) produced a TCRαβ transgenic mouse specific for the HY-encoded Smcy, Db-restricted epitope KCSRNRQYL (HY TCR mice). We have previously described the presence of long-lived HY-specific T cells with a TCR+CD4−CD8lowCD44high phenotype (CD8low T cells) (14) in female HY TCR mice after three doses of HY-Db class I tetramer in both HY TCR and wild-type B6 females. CD8low T cells recovered from such mice proliferated poorly in vitro in response to stimulation by male Ag (splenic cells, peptide and HY-Db). In addition, such treated female B6 mice also reject male skin grafts more slowly.
Interestingly, in the original descriptions of the HY TCR transgenic mouse (15, 16), when T cells were examined in the periphery of the male, there was only about a 50% reduction in the number of CD8+ T cells in male mice compared with female HY TCR mice. However, these T cells expressed lower levels of CD8 than females, may develop via a thymus-independent pathway in males (17, 18, 19), and were found to be unresponsive to male tissue (20, 21). It has been reported that they can proliferate and produce IFN-γ after anti-TCR Ab stimulation (19, 21) which suggests that these self-reactive T cells are not completely anergic and can express effector functions outside the male host, but are not autoaggressive in vivo because they have an increased threshold of T cell activation (22). Curiously, these cells express high levels of CD44, they are refractory to stimulation by male splenic cells (in both proliferation and IFN-γ secretion), yet respond to IL-15 (22). We and others have hypothesized that the higher threshold of activation is mediated by the low level of CD8 expression decreasing the effective avidity of pMHC for HY TCR (22). Such cells may serve as analogous cells to the peripheral nonresponsive CD8low T cells that appear after MHC class I tetramer injection into female mice and provide a model for peripheral dynamic down-regulation of CD8 expression.
We have investigated the presence of a pMHC/TCR-CD8 “avidity window” for TCR in both the dynamic induction of allospecific CD8+ T cell tolerance to HY peptide by reduction of CD8 levels in the periphery of female HY TCR and wild-type B6 mice. We have defined novel weak-agonist (K1A) and super-agonist (C2A) altered peptide ligands (APL)4 of the Smcy HY epitope. We found that the response of HY TCR transgenic mice to HY was highly dependent on the level of the CD8 coreceptor. Presence of a lower CD8 level greatly decreased the avidity of the HY TCR for peptide-MHC complex in both female and male HY TCR mice. Direct injection of soluble MHC class I tetramers bearing wild-type HY peptide induced tolerance in female mice to HY Ag and male graft. This is mediated by an increase in the numbers of nonresponsive naive CD8low T cells in the female recipient mouse after tetramer injection. In contrast, direct injection of Db tetramers bound with a novel super-agonist APL induces greater responsiveness (priming) to HY Ag in vitro and male grafts in female mice. We also demonstrate that high avidity peptide can overcome CD8low T cell nonresponsiveness on in vitro restimulation and result in the formation of competent CD8+ effector cells.
Materials and Methods
HY TCRαβ transgenic mice (16) that carry a transgene specific for male HY Ag were obtained from the National Institute of Allergy and Infectious Diseases via Taconic Laboratories (C57BL/6-TgN(TcrHY)). These mice are referred to as HY TCR mice. HY-CD8int mice were obtained as the offspring of HY TCR mice with CD80/0 mice (C57BL/6-Cd8atm1Mak; The Jackson Laboratory) matings. Resultant HY TCR+ mice were CD8+/0 heterozygotes and expressed intermediate levels of CD8 compared with HY TCR+ homozygote CD8+/+ mice. Normal B6 mice were purchased from The Jackson Laboratory or Charles River Laboratories. All animals used in this study were maintained under specific pathogen-free conditions in the American Association of Laboratory Animal Care-accredited University of North Carolina Department of Laboratory Animal Medicine Facilities and were routinely used at 8–12 wk of age.
HY peptide (KCSRNRQYL) (23), HY APL C2A (KASRNRQYL), K1A (ACSRNRQYL), and irrelevant lymphocytic choriomeningitis virus gp33 (KAVYNFATC) and influenza peptide (ASNENMETM) were synthesized by the University of North Carolina Microchemical facility (Chapel Hill, NC), purified by HPLC, and purity and identity were confirmed by mass spectroscopy.
Tetramer preparation and injection
Recombinant protein was prepared and folded with peptide to form soluble pMHC class I tetramers as previously described by Wang et al. (24). To drive optimal formation of tetrameric complexes, avidin (with or without conjugated fluorophore, where indicated) was added to the pMHC monomer at a ratio of 1:6. Quality of tetramer was assured by gel shift analysis and flow cytometry. Monomer was routinely tested for endotoxin contamination (Pyrochrome kit; Cape Cod), and was found to be within normal limits. For in vivo tetramer injection experiments, HY-Db or gp33-Db was prepared in sterile PBS and 30 μg per mouse in 150 μl were injected directly into the peritoneal cavity.
Flow cytometric analysis
The directly conjugated anti-mouse Abs used for cell surface staining in this study were anti-CD8α (53-6.7), anti-CD3 (145-2C11), anti-CD44 (Pgp-1, IM7), anti-CD62L (MEL-14), anti-CD25 (PC61), and anti-CD43 (1B11) and were purchased from BD Pharmingen. Two- and three-color staining was performed using standard methods. List mode data were collected on a FACScan (BD Biosciences) and analyzed using Summit software (Cytomation).
Peptide-binding assays were performed as described previously (25). Briefly, TAP-deficient T2/Db cells (174 × CEM T2 (26), transfected with a cDNA for H-2Db) were incubated with the indicated concentrations of peptides at 37°C in 5% CO2 overnight. Cells were washed and incubated with either 28.14.8s (27) or 28.8.6s (28) supernatants on ice. The binding of mAbs was examined by staining with PE-labeled anti-mouse IgG Ab (BD Pharmingen) and analyzed by flow cytometry using Cyclops software (Cytomation). Fluorescence due to isotype-matched control staining was subtracted from the fluorescence for each concentration of peptide as background.
Purification of CD8+ TCR transgenic T cells from spleen
Cell suspensions were prepared from the spleens of TCR transgenic mice. Cells were incubated at 37°C for 1 h in tissue culture dishes (Nunc) to remove adherent cells before column purification. CD8+ T cells were negatively selected by depletion of CD4+, MHC class II+, and CD11b+ cells (24) using the MACS magnetic separation system according to the manufacturer’s instructions (Miltenyi Biotec).
Preparation of irradiated splenocytes
Splenocytes were prepared from male B6 mice and resuspended at 2 × 106 cells/ml in RPMI 1640 medium plus 10% FCS. Cells were then irradiated by exposure to 3000 rad (Gammacell 40; Atomic Energy of Canada).
Purified CD8+ T cells (4 × 105 per well) were stimulated with peptide or tetramer at appropriate concentrations in 200 μl of complete RPMI 1640 in 96-well flat-bottom plates. The cultures were incubated for 48 h, and 1 μCi of [3H]thymidine was added to each well for the final 10 h of culture. Cells were harvested using a multiple sample harvester (Inotech), and incorporation of [3H]thymidine was measured by scintillation counting using a Beckman LS5000 counter. All data represent the average cpm of triplicate determinations. All proliferation experiments were repeated at least three times.
HY TCR splenocytes at 1 × 105/well were stimulated with 10 μM wild-type HY or APL peptide. After 48 h, the cells from each treatment were pooled, washed, and used as effector cells. EL4 cells were labeled with 51Cr, then pulsed with 10 μM of either HY or irrelevant (gp33) peptide and used as targets in a standard CTL assay (previously described in Ref.24). Samples were assayed in triplicate, and the mean 51Cr release plotted.
Orthotopic tail skin grafts were performed as previously described (29). Each female recipient mouse received a male allograft and a female isograft as a control. Glass tubes were placed over the grafted area for 3 days to prevent removal of the graft by the mouse. Grafts that had failed to vascularize properly with apparent rejection at 3 days were classed as “technical failures” and removed from the analysis. Remaining grafts were scored daily. Fully intact grafts were scored as 100% and when <30% of the graft remained, it was considered rejected.
Analysis of tetramer binding to living T cells
For Scatchard (KD) analysis, splenocytes from HY TCR mice were stained with anti-CD8α mAb (clone 53.6.7) (BD Pharmingen) and increasing concentrations of PE-conjugated tetramer for 1 h at 4°C, washed twice with FACS buffer, and staining was measured by FACS. Median fluorescence intensity (MFI) of tetramer staining was calculated using Summit software (Cytomation), and background fluorescence (from Db tetramer containing irrelevant gp33 peptide) was subtracted to determine MFI values of specific tetramer staining. Data were normalized to the saturation concentration of HY-Db tetramer and standard Scatchard analysis performed as we have previously described (3).
For tetramer dissociation (T1/2) measurements, splenocytes from HY TCR mice were stained with anti-CD8α mAb (clone 53.6.7) (BD Pharmingen) and a concentration of PE-conjugated tetramer (HY wild-type, APL, and control gp33-Db) known to give optimal binding (typically 1 μg/ml) for 1 h at 4°C. The cells were then washed to remove unbound tetramer and Ab, and then placed at 37°C in the presence of anti-MHC class I Ab (clone 20-8-45) to capture dissociated Db tetramer and prevent rebinding. After 0, 5, 10, 15, 30, 45, 60, 120, and 240 min of incubation, a cell sample was fixed with 0.1% (v/v) paraformaldehyde. Flow cytometric analysis determined the MFI of tetramer staining which was calculated using Summit software (Cytomation), and background fluorescence (from Db tetramer containing irrelevant gp33 peptide) was subtracted to determine MFI values of specific tetramer staining at each time point. The percentage of MFI due to tetramer binding was normalized to 100% MFI at t = 0 min and log (% MFI) was plotted against time. Linear fitting (GraphPad Software) was performed and the time taken for 50% maximal tetramer dissociation was calculated.
HY-Db tetramer identifies HY-reactive CD8high T cells in HY TCR female and CD8low T cells in HY TCR male mice
To investigate the presence of HY-Db-reactive T cells, we stained HY TCR splenocytes from female or male HY TCR mice with PE-labeled HY-Db tetramer. Fig. 1 shows staining of CD8high HY-reactive T cells in splenocytes from female or male HY TCR mice compared with splenocytes stained with irrelevant gp33-Db tetramer. In the male mice, HY-Db-specific T cells express lower levels of the CD8 coreceptor (CD8low) than the female mice. Staining with HY-Db tetramer can be blocked by addition of T3.70 Ab in both males and females (15 ; kindly provided by Dr. R. Welsh, Department of Molecular Genetics and Microbiology, University of Massachusetts, North Worcester, MA), which binds to a clonotypic determinant provided by transgenic TCRαβ heterodimers (data not shown). This confirms the earlier results that male, but not female, HY TCR mice accumulate a population of TCR transgenic CD8low T cells in the periphery (20, 21, 30). Strikingly, the level of tetramer staining is also significantly lower in the majority of male CD8+ T cells when compared with the female equivalent (in a representative experiment, MFI of female CD8 = 95.2, compared with male CD8 = 22.4). In contrast, the level of TCRαβ expression is equivalent between males and females as determined by surface staining using T3.70 (data not shown) and Northern blot analyses (15). Together, these data suggest that either the avidity of TCR in males is lower than the females or that the level of expression of CD8 contributes to pMHC-TCR binding, or both. This is consistent with other studies that show that concurrent CD8 binding by MHC class I molecules plays an important quantitative role in the avidity of TCR for pMHC (3, 4, 5, 6).
These observations led us to the idea that CD8low T cells induced in female mice after wild-type MHC class I tetramer injection (14) might resemble male-nonresponsive CD8low T cells from the male HY TCR mouse. Use of such cells enabled us to have a constant source of well-defined CD8low T cells to perform immunological analyses on and to evaluate the contribution of CD8 levels in HY pMHC/TCR interactions.
Altered HY peptide ligands change HY TCR T cell responses in vitro
To investigate the role of pMHC/TCR avidity in HY CD8+ T cell responses, we constructed a panel of 20 singly substituted peptides (APL). The substitutions of Smcy HY epitope were based on canonical MHC/TCR binding residues (31). These peptides were used to stimulate purified CD8+ T cells from HY TCR female splenocytes in an in vitro proliferation assay (data not shown) and for a cytolytic assay where peptide stimulated cells were harvested and tested with EL4 cells pulsed with HY wild-type Smcy peptide in a standard 4-h 51Cr release assay. Cells initially stimulated with C2A peptide gave a better CTL response than wild-type peptide, which was in turn better than K1A (Fig. 2 A). Lytic ability was assayed in all cases with wild-type peptide-pulsed target cells. From these data, we have defined a weak-agonist (K1A) and super-agonist (C2A) APL.
To demonstrate equivalent binding of the peptides to Db, we tested all peptides in a T2 surface stabilization assay and found that all gave a consistent pattern. C2A stabilizes Db equally as well as wild-type HY peptide, yet the weak agonist K1A stabilizes Db to a greater extent (data not shown). We have evidence that this is most likely due to a change in the P1 anchor residue leading to a conformational change in Db which results in poor TCR binding (data not shown) as we have described for the P14 TCR (25).
We used the APL bound to Db to measure binding avidity of the HY pMHC interaction with HY TCR and to define the contribution of the CD8 coreceptor. Using flow cytometry, we measured the binding by Scatchard analysis (KD, Fig. 2,B) and dissociation time (T1/2, Fig. 2,C) for wild-type, C2A, and K1A pMHC tetrameric complexes for the transgenic HY TCR on both male (CD8low) and female (CD8high) purified CD8+ T cells. Representative Scatchard and dissociation data are shown in Fig. 2, B and C, for the female HY TCR CD8+ T cells; data from both female and male mice are summarized in Table I.
|.||Female .||.||.||Male .||.||.|
|.||Wild type .||C2A .||K1A .||Wild type .||C2A .||K1A .|
|KD (nM)||171 ± 21||221 ± 73||1159 ± 280∗∗||807 ± 87||197 ± 8∗∗||n/d|
|T1/2 (min)||50 ± 18||124 ± 26∗∗||63 ± 5||17 ± 3||31 ± 10||10 ± 5|
|.||Female .||.||.||Male .||.||.|
|.||Wild type .||C2A .||K1A .||Wild type .||C2A .||K1A .|
|KD (nM)||171 ± 21||221 ± 73||1159 ± 280∗∗||807 ± 87||197 ± 8∗∗||n/d|
|T1/2 (min)||50 ± 18||124 ± 26∗∗||63 ± 5||17 ± 3||31 ± 10||10 ± 5|
Using flow cytometry (FACS), we measured the avidity (KD) and dissociation time (T1/2) for wild-type, C2A, and K1A pMHC tetrameric complexes for the transgenic HY TCR expressed on both male (tolerant CD8low) and female (reactive CD8high) purified CD8+ T cells. This table shows the average KD and T1/2 data for both female and male mice and each APL (n > 8 for each experiment; ∗∗ represents significance (p < 0.005) after Student’s t test compared to wild-type HY-Db stained cells; n/d represents undetectable staining).
Wild-type HY peptide-Db tetramer binding for HY TCR was dramatically reduced in the male CD8low HY TCR compared with the female CD8high HY TCR (Fig. 2,B, top), but surprisingly not in the case of C2A-Db (Fig. 2,B, middle) which showed equivalent binding to both female CD8high and male CD8low T cells (Fig. 2, B and C, and Table I). In contrast, C2A-Db has a significantly slower off-rate from HY TCR than either wild-type or K1A-Db on CD8high T cells (Fig. 2,C). Taken with the observation that C2A-Db is 100,000-fold better in lytic ability but only 6-fold different in KD than K1A- or wild-type HY-Db (Table I), this suggests that at nonlimiting levels of CD8 expression, disassociation time is a crucial factor in determining the degree of agonism by APL. Further, it is clear that lowered expression of the CD8 coreceptor results in a significant reduction on the overall avidity of a specific pMHC for the same TCR on cells. This is possibly mediated by stabilization of the pMHC/TCR complex as evident by the decreased off-rates of pMHC from CD8low T cells with the higher avidity epitopes. This stabilization becomes critical for efficient T cell signaling by ligands such as wild-type HY peptide/Db, which suggests that reduced CD8 expression can act to significantly alter T cell responses to suboptimal ligands.
Genetic reduction of CD8 expression reduces T cell proliferation
To confirm the influence of reduced CD8 expression on T cells on their ability to respond to HY APL-Db ligands, we generated HY TCR mice with lower CD8 expression (HY-CD8int) by crossing HY mice with HY-CD80/0. Expression levels of CD8 in heterozygous female mice were ∼30% of normal female CD8+/+ and 60% of normal male CD8+/+ homozygotic levels (Fig. 3,A). TCR levels were equivalent between intermediates and CD8+/+ mice. Purified splenic CD8+ T cells from male or female HY and HY-CD8int mice were stimulated with either wild-type HY-Db, low avidity K1A-Db, or high avidity C2A-Db tetramer. The proliferative response to wild-type and K1A-Db was much reduced in T cells from male and female HY-CD8int mice (Fig. 3,B) compared with wild-type CD8+/+. Responses to C2A-Db were much unaffected, except with male HY-CD8int T cells in which responses were somewhat reduced (Fig. 3 B). These data highlight the ability of CD8 to modulate CD8+ T cell responses resulting from moderately high avidity pMHC/TCR interactions, not just low avidity interactions. These data also suggest the ability of C2A-Db to overcome low levels of CD8 on T cells even on conditions of very limiting CD8 expression, such as the male CD8int mouse. We observed a similar effect of CD8 engagement in lymphocytic choriomeningitis virus P14 TCR transgenic mice (3).
Induction of CD8low T cells in female mice is dependent on pMHC/TCR avidity
We previously showed that injection of female HY TCR mice with three doses of wild-type tetramer in vivo induces a CD8low T cells population. This correlates with decreased responsiveness to male spleen cells in vitro (14). To examine the effect of increasing avidity for TCR we injected HY TCR female mice with HY wild-type, C2A-Db, K1A-Db tetramer as described previously (14). Fig. 4 shows the number of CD8low T cells induced 14 days after the final tetramer injection. Wild-type HY-Db induced a substantial population (mean = 15%) of CD8low T cells in comparison to higher avidity C2A-Db that induced a smaller, yet statistically significantly increased, population of CD8low T cells (mean = 12%) compared with controls (Fig. 4). Mice which received irrelevant gp33-Db or K1A-Db were indistinguishable from mice treated with saline alone. Strikingly, the CD8high T cell population was biased toward a memory-phenotype (CD3+CD44+CD62L+CD25−CD43−CD69−CD8high) population in both wild-type and C2A-Db treated mice, with a higher CD44 expression in the C2A-Db groups (Table II). Fig. 5 summarizes these data with representative CD44 profiles of CD8+ T cells 14 days after three doses of each tetramer. This suggests that a pMHC/TCR interaction of increased avidity generates a larger percentage of memory-like CD8+ T cells.
|Cell Frequency .||CD62L .||CD69 .||CD25 .||CD44 .||CD43 .|
|Cell Frequency .||CD62L .||CD69 .||CD25 .||CD44 .||CD43 .|
Female HY TCR mice were injected with three doses of 30 μg of wild-type HY, K1A, or C2A-Db MHC class I tetramer separated by 2-day intervals. Spleens were harvested 14 days after final tetramer injection, and CD8+ T cells purified by magnetic-bead negative selection. Cells were surface-stained with fluorophore-conjugated HY-Db MHC class I tetramer (allophycocyanin), anti-CD3 (FITC), anti-CD8 (PerCP), and either anti-CD44, CD62L, CD25, or CD43 (PE) Abs. Cell frequencies with each phenotype are indicated, after gating on CD3+ HY-Db MHC class I tetramer+ cells.
We then compared the increase in CD44+ T cells as a measure of a memory response to numbers of tetramer doses and found that there is an earlier and stronger induction of CD8highCD44+ cells with C2A-Db treatment compared with wild-type HY-Db (Fig. 6). C2A-Db provided a much stronger CD44+ response after a single tetramer dose. Taken together, these data suggest that use of a higher avidity pMHC/TCR ligand drove the HY T cell response toward a memory phenotype rather than a nonresponsive CD8low phenotype as observed with wild-type HY-Db tetramer.
Presence of CD8low T cells correlates with nonresponsiveness after HY wild-type-Db injections, yet injection of C2A-Db results in a priming
Considering the important role of the CD8 coreceptor in pMHC/HY TCR binding as revealed by the avidity measurements, we expected that the appearance of CD8low T cells would be linked to the level of resultant T cell response to HY Ag in female HY TCR mice. We used HY-Db tetramer to in vitro restimulate CD8+ T cells purified from naive HY TCR female mice or those previously injected with three doses of wild-type HY, C2A, or K1A-Db tetramers. Cells from mice treated with K1A-Db were similar to naive cells in their response to relevant HY-Db restimulation (Fig. 7 A). As expected, mice previously treated with wild-type HY-Db tetramers were inhibited and proliferated only at 10- to 100-fold higher doses of tetramer than naive CD8+ T cells, as we reported previously (14).
Considering the induction of a CD8low T cell population (Fig. 4), one would expect both HY and C2A-Db treatment to generate T cell nonresponsiveness. Surprisingly, when T cells from C2A-Db-treated mice were tested, they proliferated at a 10- to 100-fold lower concentration of HY-Db tetramer (Fig. 7,A, right panel). We compared the half maximal concentrations of HY-Db required for proliferation (Fig. 7,B) and demonstrate that C2A-Db and HY-Db treatments did indeed shift the CD8+ T cell restimulation dose responses in opposite directions. Both treatments induced CD8low T cells (Fig. 4), but these data suggest that a highly responsive memory response has been induced in the C2A-Db-treated mice which overrode the nonresponsive CD8low T cells possibly mediated by the increased CD8high CD44+ populations after C2A-Db treatment (Fig. 5).
Graft prolongation depends on pMHC/TCR avidity
We have previously shown prolongation of B6 male skin graft survival on B6 female recipients after injection of wild-type HY-Db tetramer (14). To determine the effect of increasing or decreasing the avidity of the injected pMHC for HY TCR, we grafted female wild-type B6 mice after tetramer injection of high and low avidity ligands. Three days after the last tetramer injection, tail skin grafts from unmanipulated male B6 donors were orthotopically grafted onto the tetramer-treated mice. Male skin grafts had a median survival time (MST) of 18 days on naive and saline-treated B6 female recipients (Fig. 8). Wild-type HY-Db-treated mice rejected their grafts more slowly (MST = 22 days, Wilcox rank order, p < 0.005), as shown previously (14). As predicted by the HY TCR data in which memory T cells were induced, C2A-Db tetramer caused significantly more rapid graft rejection than wild-type (MST = 12 days, Wilcox rank order, p < 0.001), comparable with recipient mice previously primed with male splenocytes (Fig. 8). K1A-Db-treated recipient mice did not reject male grafts with any difference to naive recipients.
Induction of CD8low T cells in wild-type B6 mice by APL tetramers has an optimum pMHC/TCR avidity
To determine the impact on CD8 levels, we examined splenocytes from the recipient mice after all grafts were rejected (by day 30). Fig. 9 shows that there was an increase in the proportion of CD8low T cells relative to CD8high T cells in B6 mice after treatment with wild-type HY-Db (Fig. 9,A) even after graft rejection. Likewise, this was reflected in the MFI of CD8 staining on the HY-Db+ CD3+ alloreactive splenocytes (Fig. 9 B). Male-reactive splenic CD8+ T cells from grafted B6 female mice which had received three doses of any of the HY-derived pMHC tetramer complexes expressed a significantly lower level of CD8 expression compared with saline or gp33-Db-treated recipients. This was unexpected for K1A-Db because it had no other obvious effects on the cell phenotype and subsequent responses of HY TCR transgenic mice. Control tetramers and grafting alone did not have this effect.
High avidity C2A-HY peptide overcomes the inability of self-reactive CD8low T cells to proliferate in vitro
Lower levels of CD8 expression by CD8+ T cells has been postulated to result in a higher activation threshold (22). We have demonstrated dynamic down-regulation of CD8 levels and decreased T cell responses in the periphery of female HY TCR and B6 mice after suboptimal wild-type HY peptide-MHC class I tetramer treatment. Therefore, we wanted to investigate whether strong agonist C2A HY peptide could cause proliferation in male CD8low T cells, acting as a model for peripheral female induction of CD8low T cells by tetramer injection.
Although splenocytes derived from male HY TCR mice are unable to proliferate when stimulated with irradiated male splenocytes in vitro (16), it is thought that they are not intrinsically anergic as they were able to respond vigorously to anti-TCR T3.70 Ab in vitro (20, 21, 22). We stimulated male HY TCR CD8low T cells with wild-type, K1A, or C2A HY peptide and measured proliferation. In all cases male HY TCR CD8low T cells responded, but required higher doses of peptide compared with female CD8high T cells (Fig. 10). Equivalent proliferation occurred at the highest concentration of HY wild-type peptide (10 μM), reinforcing the notion that male CD8low T cells can respond and are not intrinsically anergic to male Ag. Interestingly, the lower avidity K1A peptide caused proliferation in female CD8high T cells but not in male CD8low T cells (Fig. 10). In contrast, high avidity C2A peptide caused a proliferative response in both CD8low male and CD8high female T cells, although the response in CD8low T cells required a 10-fold higher peptide concentration. Presence of anti-CD8α Abs abolished the weak response of CD8low T cells to wild-type HY peptide and reduced their response to C2A by ∼4-fold (data not shown). To confirm that CD8low T cells from males were indeed capable of cell division, we CFSE-labeled spleen cells and after in vitro stimulation with HY peptide noted a reduction in CFSE intensity in CD8low T cells (data not shown). We were also able to demonstrate that CD8low T cells stimulated with C2A peptide were able to differentiate into competent HY-specific CTL effector cells (data not shown). Taken together, these data confirm that reduction of CD8 levels does indeed increase the activation threshold of CD8+ T cells to proliferate and become effector cells, yet the threshold can be exceeded by the use of stronger agonist peptide ligands.
In the experiments described here, we have investigated the idea of “CD8 tuning” of the CD8+ T cell response. We suggest that post-thymic CD8 levels are not static, but can be reduced peripherally and as a result decrease T cell responses. We used three models of CD8 modulation to describe this. First, we used CD8low T cells produced after peripheral MHC class I tetramer stimulation of female HY TCR transgenic and wild-type mice. Second, we used genetically lowered CD8int T cells from heterozygote CD8+/0 mice. Finally, we used male CD8low T cells from HY TCR transgenic mice. All models gave a consistent picture; lowered levels of CD8 result in the requirement for a higher avidity interaction with the TCR to respond equivalently to CD8high T cells.
It is clear that the initial phase of HY tolerance induction by soluble wild-type smcy-derived pMHC tetrameric MHC class I complexes results in a nonproliferative population of HY- specific peripheral TCR+CD44+CD8low T cells. We have shown here that a reduction in CD8 levels causes a much reduced binding of the HY-TCR for wild-type HY-Db pMHC (increased KD and reduced T1/2) and a corresponding decrease in Ag response. This is in agreement with our published findings (3) and others (4, 5, 6, 7, 8, 32) that the CD8 coreceptor increases the avidity of pMHC/TCR interactions. Importantly, down-regulation of CD8 levels has been implicated as a means of fine tuning T cell avidity to self-Ags during thymic selection (33). We hypothesize that the level of CD8 expression can be reduced in the periphery and in turn set the threshold of T cell responsiveness. Using HY APL, we have provided evidence that suggests that the avidity of pMHC for specific TCR impacts the balance of resultant nonresponsive CD8low T cells: responsive CD8high T cells after repeated Ag encounter.
Studies of CD8 down-regulation in Ag-specific tolerance induction have been sparse; injection of female transgenic mice with syngeneic B6 male (expressing the HY Ag) activated CD8+ lymphocytes found a similar initial expansion of female HY-reactive CD8+ T cells followed by a decline to below the starting number (30). Transfer of female B6 CD8+ T cells did not mediate this effect, suggesting an Ag-specific effect. Remaining recipient transgenic CD8high T cells were responsive, but there was a large increase in the number of recipient transgenic CD8low T cells that were nonresponsive to both male Ag and anti-CD3 Abs (30). No accompanying TCR down-regulation was observed. Similarly, other groups have also found that activated CD8+ CTL lines can inactivate T cells that recognize them, again possibly mediated by CD8 down-regulation on the recognizing T cell (34, 35, 36). Earlier studies have postulated that immune recognition of HLA molecules specifically down-modulates CD8 expression on CD8+ T cells (35). A recent study demonstrated a regulatory CD8+ T cell from patients that had received HA-1-mismatched grafts. Such cells had reduced avidity for HA-1 MHC class I tetramer and suppressed anti-HA-1 CD8+ T cell responses by a TGF-β, IL-10, and CTLA4-dependent mechanism (37).
The origin of the nonresponsive CD8low T cells is currently unclear. We have previously shown that for naive CD8+ T cells from female transgenic HY-TCR mice, the soluble MHC class I tetramer-peptide complex alone (signal 1) is sufficient for activation and differentiation into effector cells in the absence of costimulation (signal 2) (24). It is possible that CD8low T cells are a population of CD8+ T cells arising after incomplete stimulation by MHC class I tetramers. It has been proposed that during the contraction phase of a T cell response, surviving nonresponder T cells continue to form the Ag-specific memory T cell compartment, possibly via the process of homeostatic proliferation (38, 39). A recent study has provided evidence that homeostatic proliferation after T cell depletion can act to prevent allograft rejection (40). We hypothesize that the female CD8low T cells described here are a memory-like population of nonresponding T cells that survived and proliferated after overt stimulation by HY-Db tetramers which caused activation-induced cell death of some of the responding CD8high T cells (data not shown). However, it is apparent that this is a finely balanced outcome, dependent in a large part on the effective pMHC/TCR avidity. This study provides some weight to the theory that Ag stimulation can result in responder and nonresponder CD8+ T cell populations. This and other studies show that Ag stimulation can result in the induction of a graded continuum of nonresponsiveness to subsequent activation (41, 42, 43, 44, 45).
Provision of a pMHC with a higher avidity (in this case, C2A-Db) for the same TCR yielded a population of CD8low T cells with a much higher frequency of CD44 expression. These memory-like CD8 T cells possess corresponding enhanced recall responses to HY smcy Ag in vitro. This augmented T cell response confers accelerated rejection of male grafts in wild-type female B6 mice. Although the effect on grafting per se was similar to untreated control recipient mice, we noticed that an expansion of CD8low T cells occurred in wild-type female B6 mice even after a low avidity interaction such as with K1A-Db. This was possibly due to restimulation of polyclonal (possibly cross-reactive) HY-reactive T cells in the nontransgenic mice after grafting of wild-type Ag-bearing male skin. It is also possible that there is some enhanced ability of pMHC injected into a CD4/CD8 competent host to stimulate the CD8low T cells response, possibly via bystander or “self” help (46).
It is noteworthy that in wild-type mice the HY-Db tetramer stains a population of CD8 T cells which occupy the lower range of the total CD8 expression of a complete CD8+ T cell repertoire in wild-type untreated grafted (Fig. 9 A) and ungrafted mice (data not shown) in this and other studies (47), suggesting that any differences in CD8 expression might have significant effects on the low binding avidity of this particular pMHC/TCR interaction.
Interestingly, we have characterized a strong agonist APL (C2A) for the HY TCR that has an avidity (KD) less affected by reduced CD8 expression than wild-type smcy HY-Db. Strikingly, this peptide APL is able to fully stimulate CD8low T cells from male HY TCR mice which have been thought to be refractory to cognate autoantigen stimulation. The fact that C2A stimulated HY CD8low T cells can proliferate and kill to the same extent as female C2A-stimulated female HY CD8high T cells argues that the signaling pathways and cells are not defective, but rather that the threshold for effective signal transduction has been raised. Thus, an increased intrinsic avidity of pMHC for TCR is required to overcome the lowered avidity controlled by lowered CD8 expression. This finding strengthens the hypothesis that it is the low CD8 coreceptor expression level on male self-reactive T cells in vivo which is a means of escape from central deletion and/or a mechanism of peripheral tolerance to male Ag (48, 49).
From recent studies, it is apparent that physiological TCR affinities have arisen to be in the low range that requires CD8 to be present during syngeneic binding to MHC (4). It has been recently suggested that T cells bearing higher avidity TCRs are negatively selected in the thymus, which results in a population of more diversely reactive (possibly even cross-reactive) repertoire of T cells yet with lower T cell activity (50, 51, 52). Indeed, strength of pMHC/TCR avidity has also been postulated as crucial for a T cell clone to compete in vivo for homeostatic growth (53). It is possible that CD8 acts normally to amplify these T cell responses significantly. Data presented here support the theory that reduced expression of CD8 can indeed act to limit a T cell response by limiting the avidity window of effective signal transduction, and that peripheral CD8low T cells can arise dynamically after repeated antigenic stimulation in vivo. Use of well-defined pMHC tetrameric complexes allows us to correlate the contribution of initial Ag avidity “fine tuned” by CD8 to the activation of CD8+ T cells. The ability to dynamically modulate the formation (size and responsiveness) of an Ag-specific T cell pool by pMHC/TCR “activation threshold tuning” (54) during the early phases of an immune response has important implications for the balance of responsiveness, memory, and tolerance.
We thank Carie Barnes for technical assistance, Shannon Pop for critical reading of the manuscript, and members of the Frelinger, Collins, Cairns, and Tisch laboratories for helpful discussion.
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.
This work was supported by National Institutes of Health Grants R01 GM 67143 and AI 52435, American Heart Postdoctoral Fellowship No. 0225372U (to R.M.), and the Jaycee Burn Center, University of North Carolina Hospitals.
Abbreviations used in this paper: pMHC, peptide-MHC; APL, altered peptide ligand; MFI, median fluorescence intensity; MST, median survival time.