Antigen-Specific Modulation of an Immune Response by In Vivo Administration of Soluble MHC Class I Tetramers¹

Major histocompatibility complex/peptide tetramers that can directly bind the TCR allow the direct visualization and quantitation of Ag-specific T cells in vitro and in vivo. Staining with soluble MHC class I tetramer complexes has focused on re-evaluation of the frequencies of Ag-specific CD8⁺ T cells in animal models of bacterial (1) and viral infection (2, 3, 4), resulting in a new understanding of the frequencies of Ag-specific T cells. Tetramers have also been used to characterize and even isolate circulating T cells specific for tumor-associated Ags in patients (5, 6). Although much information has been gained from the use of soluble MHC class I tetramers to evaluate numbers of Ag-specific T cells in transgenic or manipulated mice (3, 7, 8), surprisingly little information is known about the frequencies of Ag-specific CD8⁺ T cells in naive mice.

Despite their widespread acceptance as tools to monitor CD8 responses, tetramers have not been used as triggers of cell activation. In principle, they should be far superior to peptides in engaging TCRs and should have a longer life span in the recipient. Thus, they could be used in many situations where peptides have been used to induce or block immune responses in intact animals. It is known that multiple exposures or high doses of Ag induce CD8⁺ T cell unresponsiveness in vivo.

Many approaches have been made to specifically “silence” alloreactive or autoreactive T cells, mainly through the induction of anergy (9) and deletion of T cells by overstimulation with high concentrations of specific Ag (10). These experiments have normally been performed in transgenic systems and few reports exist in which tolerance to alloantigen or autoantigen has been achieved in wild-type mice. The mechanisms responsible for such tolerance are poorly understood. In TCR-transgenic models, physical deletion of T cells (11, 12) has been observed as well as down-regulation of TCR surface levels (13) after tolerogenic exposure to exogenous Ag.

To test whether providing such a strong signal 1 to specific CD8⁺ T cells in vivo yields a form of “high zone tolerance,” we constructed soluble MHC class I tetramers loaded with a peptide described as a highly immunogenic epitope for an H-2D^b-restricted anti-H-Y (anti-male) response (14). In naive female B6 mice, we used this tetramer to detect male-specific T cells in the normal spleen. Furthermore, these naive cells were activated by injection of soluble MHC class I tetramer in both HY-TCR-transgenic and normal B6 females. Such injections primed female B6 mice for second-set skin graft rejection as effectively as injection of male spleen cells. Interestingly, multiple injections of relevant tetramer reversed the priming effect, suggesting elimination of the tetramer-reactive T cells by either anergy induction or activation-induced cell death, as demonstrated by surface annexin V staining (our unpublished observations).

HY TCRαβ-transgenic mice (15) that carry a transgene specific for male HY Ag were obtained from the National Institute of Arthritis and Infectious Diseases via Taconic Farms (C57BL6, TgN(TcrHY); Germantown, NY). These mice are referred to as HY-TCR mice. Normal B6 mice were purchased from (Charles River Breeding Laboratories, Wilmington, MA). 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 wk of age.

HY peptide (KCSRNRQYL) (14) and gp33 peptide (KAVYNFATM) were synthesized by the University of North Carolina Microchemical Facility, purified by HPLC, and tested for purity by mass spectroscopy.

Recombinant protein was prepared as previously described by Wang et al. (16). For in vivo tetramer injection experiments, HY-D^b was prepared in sterile PBS and 30 μg/mouse in 150 μl was injected directly into the peritoneal cavity.

The directly conjugated anti-mouse Abs used for cell surface staining in this study were anti-CD8 (53-6.7), anti-B220 (RA3-6B2), and anti-MHC class II (I-A^b, 25-9-17) purchased from BD PharMingen (San Diego, CA). Two- and three-color staining was performed using standard methods. List mode data were collected on a FACScan (BD Biosciences, Mountain View, CA) and analyzed using Summit software (Cytomation, Ft. Collins, CO).

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, Naperville, IL) to eliminate adherent cells before purification. CD8⁺ T cells were negatively selected by depletion of CD4⁺, MHC class II⁺, and CD11b⁺ cells (16) using the MACS magnetic separation system according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA).

Splenocytes were prepared from male B6 mice and resuspended at 2 × 10⁶ cells/ml in RPMI 1640 medium. Cells were then irradiated by exposure to 3000 rad emitted from a Gammacell 40 Cesium-137 source (Atomic Energy of Canada Limited, Ottawa, Canada).

Purified CD8⁺ T cells (4 × 10⁵/well) were stimulated with Ags or tetramer at different concentrations in 200 μl of complete RPMI 1640 in 96-well flat-bottom plates. The cultures were incubated for 48 h, and 1 μCi [³H]thymidine was added to each well for the final 10 h of culture. Cells were harvested using a multiple sample harvester (Otto Hiller, Madison, WI), and incorporation of [³H]thymidine was measured by scintillation counting using a Beckman LS5000 counter (Palo Alto, CA). All data represent the average counts per minute of triplicate determinations. All proliferation experiments were repeated at least three times.

Tail grafting was performed as previously described (17). 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.

To investigate the ability of HY-D^b-reactive T cells to stain with PE-labeled HY-D^b tetramer, we used the HY-TCR-transgenic mouse strain (14). Fig. 1 illustrates peptide-specific staining of CD8^high (male-reactive) T cells with HY-D^b tetramer in female HY-TCR splenocytes. To demonstrate the ability of MHC class HY-D^b tetramers to identify the presence of HY-reactive T cells in naive, unmanipulated nontransgenic mice, splenocytes from 8-wk-old female B6 mice were stained with PE-conjugated labeled HY-D^b and counterstained according to the figure legend. Fig. 2, a and b, shows that HY-D^b tetramer staining revealed the presence of a surprisingly large population of HY-specific CD8⁺ T cells, which represent 1.5–2.0% of splenic CD8⁺ T cells. A control D^b tetramer, assembled with lymphocytic choriomeningitis virus (LCMV)³ gp33 peptide stained <0.6% of CD8⁺ T cells. Furthermore, after adoptive (i.v.) transfer of irradiated male splenocytes into female B6 mice, the HY-D^b tetramer staining population had expanded ∼2.5 fold at 5 days after transfer (Fig. 2,d). Control female B6 mice receiving irradiated female splenocytes did not expand the HY-reactive CD8⁺ T cells (Fig. 2,c). Fig. 2 e illustrates the summary of the data (three mice per group). There was significant (Student’s t test, p < 0.005) priming with male but not with female splenocytes. These observations demonstrate that naive female B6 have detectable male-specific T cells in the spleen.

In our previous studies (16), using soluble MHC class I tetramer alone to stimulate naive CD8⁺ T cells into effector cells, we showed that the response to LCMV in vitro was blocked by addition of Abs to the TCR coreceptor CD8. Since the LCMV-transgenic TCR has a relatively high affinity to the immunodominant gp33 peptide (18) we wanted to be certain that the same argument pertained to the relatively low-affinity HY-TCR (our unpublished observations). Here, we stimulated purified CD8⁺ HY-TCR T cells with HY-D^b tetramer in the presence of 20 μg/ml anti-CD8 Ab (53-6.7) or control Ig. This concentration of anti-CD8 Ab was found to completely block HY-TCR T cell proliferation in dose-response experiments (data not shown). As in our LCMV study, treatment with anti-CD8 Ab blocked the T cell proliferative response to HY-D^b tetramer (Fig. 3), suggesting an important role for the CD8 coreceptor in the activation of HY-TCR T cells by specific HY-D^b tetramer.

We have previously shown that soluble MHC class I tetramer alone is sufficient for in vitro activation and differentiation into effector cells of naive CD8⁺ T cells from transgenic HY-TCR mice (16). We wished to determine whether direct injection of HY-D^b into female B6 mice would result in functional priming of a specific immune response in vivo, measured here by accelerated rejection of male skin grafts. One group of mice was injected with 1 × 10⁷ male splenocytes as a positive control. Twenty days later groups of naive female mice were treated with either 30 μg/mouse HY-D^b tetramer or PBS (unprimed control). Three days after HY-D^b injection, we grafted tail skin grafts from unmanipulated male B6 donors onto all the female recipients. Fig. 4 a depicts the data from a representative (one of three) experiment. A single dose of HY-D^b tetramer to female B6 mice caused significant priming and earlier rejection of graft tissue (Wilcoxon rank order, p < 0.0001). Indeed, this response had identical kinetics to that of male splenocyte-primed female mice, demonstrating that tetramer injection had primed normal female B6 recipients. This also provides further evidence that the HY (KCSRNRQYL) peptide is an important epitope recognized in the anti-male response by female mice in vivo.

To further examine the effects of tetramer injection into female mice, 30 μg HY-D^b or irrelevant gp33-D^b tetramer was injected i.p. into female HY-TCR mice. CD8⁺ T cells were purified from the spleens of HY-TCR female mice harvested 72 h after tetramer injection and restimulated in vitro with HY-D^b tetramer or irrelevant gp33-D^b tetramer. Fig. 4 b shows that CD8⁺ T cells purified from tetramer injected female mice responded in an Ag-specific manner, with an increased proliferative response to HY-D^b and not to gp33-D^b. Additionally, splenocytes from transgenic mice treated with HY-D^b exhibited an increased proliferative response to HY-D^b tetramer than gp33-D^b-treated littermates. It was apparent that in mice 48 h after HY-D^b tetramer injection, very few cells stained with either anti-CD8 or anti-Vβ8.2 but had a phenotype consistent with T cell activation (data not shown). We believe that this represents down-regulation of TCR and CD8, as observed in other studies with peptide stimulation (19, 20). These data demonstrate that administration of HY-D^b tetramer in vivo has the ability to prime transgenic mice to HY peptide.

To examine the ability of the in vivo-primed T cells to respond to natural HY Ag, we stimulated them with B6 male spleen cells. Fig. 4 c illustrates the restimulation response of tetramer-treated CD8⁺ T cells to irradiated male B6 splenocytes. Again, CD8⁺ T cells from tetramer-treated female mice responded much more vigorously than cells from gp33-D^b-treated controls. The lack of an apparent response of CD8⁺ T cells from gp33-D^b-treated female mice to male splenocytes is due to the short assay time (48 h), typical of primed T cells. Unprimed HY-TCR shows a peak response at 72 h at this cell density. These data demonstrate that soluble HY-D^b tetramer can prime the female HY-TCR mouse to respond to normally processed male Ag as well as HY-D^b tetramer.

In studies of peptide-induced T cell nonresponsiveness, multiple doses of Ag often had to be administered to observe Ag-specific tolerance (21, 22, 23). We wanted to examine whether the priming observed with a single tetramer dose could be changed to exhaustion using multiple tetramer doses.

We injected one, two, or three doses of tetramer or PBS (i.p.) into female HY-TCR mice, each dose separated by 2-day intervals. CD8⁺ T cells were purified from the spleens and stimulated in vitro with HY-D^b tetramer, HY peptide, or irrelevant gp33. Fig. 5 summarizes these data. Mice injected with a single dose of relevant HY-D^b tetramer (all PBS-treated groups exhibited high responses to HY-D^b tetramer and HY peptide, but no response to gp33 peptide, as in untreated mice; data not shown) exhibited a primed response to HY peptide and tetramer (Fig. 5,a). In contrast, if two injections were administered, then the proliferative response to peptide and tetramer is greatly diminished (Fig. 5,b) and after three injections becomes undetectable (Fig. 5 c).

Flow cytometric examination of the splenocytes in the tetramer-injected mice showed a marked dose-dependent difference in their levels of CD8 expression compared with other cell surface markers (CD69, CD62L, CD44, CD25, Vβ8.2; data not shown) 3 wk after the final tetramer injection. Fig. 6,a shows that in HY-TCR mice treated with three doses of PBS, the majority of HY-D^b tetramer staining cells reside in the CD8^high compartment. However, with increasing tetramer doses, the majority of tetramer-positive T cells are CD8^low. This is a result of a loss of tetramer-reactive CD8^high T cells and an increase in CD8^low numbers. Importantly, if the same experiment was repeated in a group of naive B6 female mice, a very similar pattern of tetramer and CD8 staining emerged (Fig. 6 b), an increase in CD8^lowtetramer⁺ cells and a decrease in CD8^hightetramer⁺ cells. This suggests that sustained T cell unresponsiveness associated with multiple tetramer doses is due to sustained decreased expression levels of CD8. Treatment with anti-CD8 Ab blocked an in vitro T cell proliferative response to HY-D^b tetramer (data not shown), further confirming an important role for CD8 coreceptor in the activation of HY-TCR T cells by specific HY-D^b tetramer.

Experiments with B6 females (Fig. 4,c) suggested that a single dose of HY-D^b tetramer induced priming to HY peptide and earlier rejection of male skin grafts. However, the above data demonstrated that multiple doses of specific HY-D^b tetramer ablated an in vitro proliferative response to HY Ag. We therefore asked whether multiple tetramer exposures would change rejection of male skin grafts. One group of B6 female mice was injected with 1 × 10⁷ male splenocytes as a positive control. Twenty days after priming this group, three groups (six mice) of naive female mice were injected with one, two, or three doses of 30 μg/mouse HY-D^b tetramer or PBS. Three days after tetramer administration, tail skin grafts from naive male B6 donors were grafted onto all the B6 female recipients. Fig. 7 illustrates the data from a representative experiment; a single dose of HY-D^b tetramer to female B6 mice caused priming and rapid rejection of skin grafts. However, two or three doses of tetramer reversed this effect; grafts on mice receiving three doses of tetramer surviving significantly (Wilcoxon rank order, p < 0.001) longer than the control unprimed mice. Indeed, 25% of treated mice showed long-term graft survival. Thus, although there is at least one other defined HY epitope (15, 24, 25), induction of unresponsiveness to the KCSRNRQYL epitope resulted in prolonged graft survival.

The results presented in this study demonstrate the dual ability of soluble MHC class I tetramers to directly visualize HY Ag-specific T cells in vivo without any immune manipulation and to directly stimulate HY Ag-specific T cells in vitro and in vivo. HY-D^b tetramer was used to measure the numbers of HY-reactive CD8⁺ T cells in naive, wild-type female B6 mice. Male-reactive CD8⁺ T cells were apparent after specific tetramer staining and expressed high levels of CD8 coreceptor. In female wild-type B6 mice, these cells represent ∼1.5–2.0% of the splenic CD8⁺ T cell population, the first time such an estimate has been made. This is consistent with the strong in vivo response to HY Ag.

We have previously demonstrated that for naive CD8⁺ T cells from female HY-TCR mice, soluble MHC class I tetramer alone (signal 1) is sufficient for activation and differentiation into effector cells (16). Two other studies have used tetramers to stimulate T cell clones or hybrids (26, 27). Soluble MHC class I tetramers have also been used to stimulate calcium flux in Ag-specific T cell clones (27), and some evidence exists that MHC class I monomers were able to activate CD8⁺ T cells in vivo (28). Here, we describe the use of soluble MHC class I tetramers to induce and modulate an Ag-specific immune response in vivo. Injection of the HY-D^b tetramer (but not the irrelevant D^b tetramer) into female HY-TCR mice induced a very rapid but transient activation, resulting in expansion of HY-specific CD8⁺ T cells. This response primed female HY-TCR mice to more rapid responses to both the HY peptide and irradiated male splenocytes. Reinforcing the notion that HY tetramers could influence a T cell response in normal mice, HY-D^b-treated B6 mice rejected male tail grafts more rapidly than untreated mice. In addition, our data show that after multiple in vivo doses of the HY-D^b tetramer, recovered CD8^low T cells proliferate poorly in vitro to HY Ag stimulation. In treated B6 mice, this results in longer survival times of male skin grafts.

Several studies investigating peripheral tolerance in the HY-transgenic mouse have involved 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 (29). Transfer of female B6 CD8⁺ T cells did not mediate this effect, suggesting an Ag-specific effect. Remaining recipient transgenic CD8^high T cells were responsive, but there was a large increase in the number of recipient transgenic CD8^low T cells that were nonresponsive to both male Ag and anti-CD3 Abs (29). 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 (13, 30, 31). Earlier studies have postulated that immune recognition of HLA molecules specifically down-modulates CD8 expression on CD8⁺ T cells (30).

Results from our study allow us to investigate this mechanism further. Injection with soluble MHC class I tetramer complexed with specific HY peptide into female HY-TCR mice showed a very similar effect as seen in other studies, but without the confounding signals provided by injected activated male T cells or other APC. It argues that a strong “signal 1” delivered to reactive T cell populations does indeed cause a similar initial expansion of female HY-reactive CD8⁺ T cells followed by a decline in numbers, likely due to activation-induced cell death as demonstrated by surface annexin V staining (our unpublished observations). Although we have shown that the CD8⁺ T cell response to HY peptide is not dependent on known costimulatory molecules (16), the response is CD8 dependent. This is possibly a reflection of a TCR with a low affinity to the HY epitope (our unpublished observations). Surviving CD8 HY-reactive T cells after multiple tetramer doses possessed a sustained lower expression of CD8, which may act as a mechanism of their nonresponsiveness. It will be interesting to determine whether this model of nonresponsiveness applies to T cells specific for a higher affinity epitope such as LCMV gp33. In such cases, the threshold for T cell activation might not be so dependent on levels of coreceptor molecules.

Although most other previous studies have been performed in transgenic mouse models, the tetramer-based system described here is applicable to other models, as demonstrated by the ability to stain scarce CD8⁺ T cells of known specificity with MHC class I tetramer and show that tetramer injection can influence the antimale rejection response in naive wild-type mice.

We thank Katherine Midkiff and Carrie Barnes for technical assistance.