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The Journal of Immunology, 2007, 179, 4988-4995
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Surprising Kinetics of the T Cell Response to Live Antigenic Cells1

Aaron J. Tyznik and Michael J. Bevan2

Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cooperation between CD4+ and CD8+ T cells is required for the proper development of primary effector and memory CD8+ T cells following immunization with noninflammatory immunogens. In this study, we characterized murine CD4+ and CD8+ T cell responses to male-specific minor histocompatibility (HY) Ags following injection of live male cells into females of the same strain. Male cells are rejected 10–12 days after transfer, coinciding with the expansion and effector function of CD8+ CTLs to two H-2Db-restricted epitopes. Although anti-HY CD4+ T cell responses are readily detectable day 5 posttransfer, CD8+ responses are undetectable until day 10. The early CD4+ response is not dependent on direct presentation of Ag by donor male cells, but depends on presentation of the male cells by recipient APC. The CD4+ T cell response is required for the priming of CD8+ T cell effector responses and rejection of HY-incompatible cells. Unexpectedly, HY-specific CD4+ T cells are also capable of efficiently lysing target cells in vivo. The delay in the CD8+ T cell response can be largely abrogated by depleting T cells from the male inoculum, and donor male CD8+ T cells in particular suppress host anti-HY CD8+ responses. These data demonstrate dramatic differences in host T cell responses to noninflammatory Ags compared with responses to pathogens. We explain the delayed CD8+ response by proposing that there is a balance between cross-presentation of Ag by helper cell-licensed dendritic cells, on the one hand, and veto suppression by live male lymphocytes on the other.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Studies of endogenous T cell responses to foreign Ag have focused on the rapid and extensive response of Ag-specific CD8+ T cells during acute bacterial and viral infections. Following encounter with these pathogens, naive CD8+ T cells become activated and rapidly undergo 15 or more divisions to establish an effector CTL population 7–8 days postinfection. This extensive proliferation is followed by contraction over the next 7–14 days during which 95% of Ag-specific cells undergo apoptosis, leaving a long-lived memory population (1, 2). Similar to CD8+ T cells, Ag-specific CD4+ T cells undergo a coordinate response of activation, expansion, contraction, and establishment of Ag-specific memory. The main differences observed were that CD4+ T cells had a roughly 20-fold lower expansion, peaked a day later than CD8+ T cells, had a protracted contraction phase, and memory cell numbers declined over time (3, 4).

Following immunization with most pathogens, primary CD8+ T cell responses are independent of CD4+ T cell help (5, 6, 7). Despite CD4+ T cell help being dispensable for primary CD8+ T cell responses, it is necessary for the maintenance and functionality of memory T cells (8, 9, 10, 11). This is in contrast to the priming of most CD8+ T cell responses to cell-associated Ags where CD4+ T cells are necessary for the induction of optimal primary T cell responses (12, 13, 14). It has been demonstrated that the requirement for CD4+ T cell help is due to the requirement for APC conditioning and activation before priming CD8+ T cells. These differences appear to be related to the inflammatory nature of the immunogen such that antigenic tumor cells, minor H-disparate cells, and osmotically loaded cells are relatively noninflammatory and require help from activated CD4+ T cells. It is believed that activated CD4+ T cells reciprocally counter stimulate and license Ag-presenting dendritic cells (DC)3 through a CD40-CD40 ligand interaction, and the licensed APC can then properly stimulate naive CD8+ T cells (15, 16, 17). In contrast, inflammatory stimuli provided by viral and bacterial infections can overcome the CD4+ T cell requirement by directly licensing APC through TLR ligands or other intracellular innate receptors (18).

Although it is clear that the nature of the immunogen can dramatically affect the requirements for APC conditioning and CD4+ T cell help, the exact kinetics and functionality of CD4+ and CD8+ T cell populations following immunization with noninflammatory Ags have not been analyzed. To evaluate T cell responses to noninflammatory Ags, we used the well-characterized minor H Ag HY. This system allows us to track the survival of Ag-bearing cells and analyze both CD4+ and CD8+ Ag-specific T cell responses to three immunodominant epitopes with minimal introduction of inflammatory stimuli. Using this system, we describe the previously unappreciated differential kinetics of CD8+ and CD4+ T cell responses to HY Ags.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

B6.129-H2-Ab1tm1Gru N12 (MHC class II-deficient) and B6.SJL (129)-Ptprca/BoyAiTac B2mtm1Jae N9 + N1 (beta2m–/– SJL) mice were purchased from Taconic Farms. C57BL/6 (B6), B6(C)-H2-Ab1bm12/KhEgJ (H2bm12), B6.SJL-PtprcaPep3b/BoyJ (B6.SJL), and C57BL/6-Tg(UbC-GFP)30Scha/J (UbC-GFP) mice were purchased from The Jackson Laboratory and housed in specific pathogen-free conditions in the animal facilities at the University of Washington (Seattle, WA). Experiments were initiated when mice were 12–14 wk of age and were performed according to Institutional Animal Care and Use Committee guidelines.

In vivo immunizations

B6.SJL or UbC-GFP male mice were sacrificed and spleens were harvested in PBS supplemented with 0.5% mouse serum albumin to avoid coating the cells with foreign protein, such as those from FCS. Single-cell suspensions were prepared on ice and washed twice in PBS 0.5% mouse serum albumin and filtered twice. In brief, 2 x 107 male splenocytes were transferred i.v. into female B6 mice.

Abs and peptides

The following purified, fluorescently conjugate Abs were purchased from BD Biosciences: anti-IFN-{gamma}-allophycocyanin (clone XMG1.2), anti-CD44-FITC (clone IM7), anti-CD4-FITC (clone RM4-4), anti-CD4-PerCP (clone RM4-5), anti-CD4-allophycocyanin (clone RM4-5), anti-CD8-FITC (clone 53-6.7), and anti-CD8-PerCP (clone 53-6.7). Anti-MHC class II-allophycocyanin (clone M5/114.15.2), anti-CD45.1/Ly5.1-PE-Cy7 (clone A20), anti-CD62L-PE-Cy7 (clone MEL-14) anti-TNF-{alpha}-FITC (clone MP6-XT22), and anti-IL-2-PE (clone JES6-5H4) were purchased from eBioscience. Anti-CD8-allophycocyanin-Alexa Fluor 750 (clone 5H10) was purchased from Caltag Laboratories. Synthetic peptides representing the defined HY epitopes (I-Ab-restricted Dby608–622 (NAGFNSNRANSSRSS) and the H-2Db-restricted Smcy738–746 (KCSRNRQYL) and Uty246–254 (WMHHNMDLI)) were purchased from Chemicon International. Irrelevant synthetic H-2Db-restricted GP33–41 and I-Ab-restricted listeriolysin O (LLO)190–201 peptides were purchased from Invitrogen Life Technologies.

Intracellular IFN-{gamma} staining

HY-specific CD8+ and CD4+ T cells were detected by measuring IFN-{gamma} secretion in response to ex vivo stimulation by MHC class I or II peptides using the Cytofix/Cytoperm Kit Plus (with GolgiPlug; BD Biosciences) according to the manufacturer’s instructions. For T cell stimulation, 2 x 106 lymphocytes resuspended in complete DMEM supplemented with 10% FCS were incubated with or without synthetic peptides in a 96-well plate in a volume of 200 µl. Cells were stimulated with the MHC class I H-2Db-restricted peptides (1 µM) Smcy or Uty or the MHC class II I-Ab-restricted peptide (1 µM) Dby for 4–5 h in the presence of 1 µg/ml GolgiPlug (BD Biosciences) at 37°C in 7% CO2. Cells were washed, resuspended in FACS staining buffer (PBS containing 1% FCS and 0.1% sodium azide) and typically stained on ice for 20 min with anti-CD8-allophycocyanin-Alexa Fluor 750, anti-CD4-PerCP, and anti-CD45.1-PE-Cy7. Following surface staining, cells were washed, resuspended in permeabilization-fixation buffer, washed, and stained for intracellular cytokine expression with anti-IFN-{gamma}-allophycocyanin and anti-IL-2-PE for 30 min at 4°C. Cells were washed in PermWash solution, resuspended in PBS plus 1% paraformaldehyde, and performed on a FACSCanto using CellQuest software (BD Biosciences).

Tetramer staining

H-2Db tetramers bound to the Smcy-derived peptide KCSRNRQYL were generated as described elsewhere (19). Single-cell suspensions were prepared from spleen and lymph nodes by mechanical dissociation and lysis of RBCs in hypotonic buffer. Cells were washed and resuspended in FACS staining buffer and stained on ice for 1 h with H-2Db-PE tetramers and the following surface Abs: CD44-FITC, CD8-PerCP, CD4-allophycocyanin, and CD62L-PE-Cy7. Flow cytometry was performed on a FACSCanto and analyzed using FlowJo software (Tree Star).

In vivo cytotoxicity assay

The analysis of in vivo cytotoxicity was performed similarly to previously described protocols (20, 21). Naive male or female B6.SJL splenocytes were differentially labeled with either 5.0 or 0.5 µM CFSE (Molecular Probes). For peptide-pulsed target cell killing, female B6.SJL CFSEhigh cells were pulsed with 1 µM Uty or Smcy peptides, whereas female CFSElow cells were pulsed with an irrelevant peptide, GP33–41. Cells were washed and transferred i.v. (1 x 107 cells of each population) into naive or day 15 immune mice. For killing of target cells expressing endogenous levels of HY Ag, B6.SJL male CFSEhigh and female CFSElow cells were transferred i.v. into naive or day 15 immune mice. Lymph nodes were harvested from donor mice at the indicated time points and analyzed by FACS for target cell clearance. For MHC class II-restricted killing, splenocytes from B6.SJL female or beta2m–/– SJL male or female mice were differentially labeled with either 5.0 or 0.5 µM CFSE. CFSEhigh B6.SJL cells were pulsed with 1 µM Dby peptides, whereas CFSElow cells were pulsed with an irrelevant peptide, LLO190–201. Subsequently, naive and day 9 immune mice were coinjected i.v. with 1 x 107 cells of each population. For endogenous MHC class II-restricted killing, naive or day 9 immune mice received 1 x 107 male CFSEhigh beta2m–/– SJL and 1 x 107 female CFSElow beta2m–/– SJL cells. Host mice were depleted of NK cells with 100 µg of purified PK136-depleting Ab at day 5 postimmunization. At the indicated time points, lymph nodes were harvested and analyzed by flow cytometry for target cell clearance. For gating on CD45.1+ target cells for MHC class I-restricted killing or CD45.1+ I-Ab+ target cells for MHC class II-restricted killing, the percentage of killing was calculated as follows: 100% – [(percent peptide pulsed in immune/percent unpulsed in immune)/(percent peptide pulsed in naive/percent unpulsed in naive)] x 100.

In vivo depletion of CD4+ and CD8+ donor cells

Male mice were injected i.p. with 200 µg (day –3) and 50 µg (day –1) of purified anti-mouse CD4 (clone GK1.5) or CD8 (clone 2.43) mAb before harvest of splenocytes for use as immunogen. Using noncompeting FITC-conjugated Abs for CD4 (clone RM4-4) and CD8 (clone 53-6.7), we observed that >99% of CD4+ or CD8+ T cells were depleted in the donor inoculums.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In the B6 strain, female mice can recognize two known MHC class I-associated epitopes and one MHC class II-associated epitope presented by male cells. The peptides derive from proteins encoded by Y chromosome genes and are referred to as Uty and Smcy presented by H2-Db and Dby presented by I-Ab (22, 23, 24). The nature and kinetics of the CD8+ and CD4+ T cell response of females to immunization with live male cells that had not been exposed to foreign proteins was followed in this study. In some cases, we have used cells from mice expressing GFP in all tissues (UbC-GFP mice) to allow accurate quantitation of the rate of rejection of male cells by female mice (25). It is important to note that no rejection response is mounted against the GFP, i.e., female mice that have rejected male UbC-GFP cells do not subsequently reject female UbC-GFP cells although they rapidly reject male cells.

Kinetics of the CD8 response

CD8+ T cell responses to Uty and Smcy were detectable 9–10 days after transfer of male cells into female mice and peaked at day 15 as measured by IFN-{gamma} production (Fig. 1, A and C). To ensure that effector responses measured by intracellular cytokine staining (ICS) identified all HY-specific cells, we enumerated the percentage of Smcy-specific CD8+ T cells by staining with H-2Db/Smcy tetramer. We did not find any significant difference in the percentage of Smcy-specific cells measured by ICS or tetramer staining (Fig. 1B). To follow the rejection of male cells by female mice, we injected naive females with male or female UbC-GFP cells. One day following cell transfer, male and female donor cells had a systemic distribution representing 0.8–1.0% of all lymphocytes in the spleen, lymph nodes, blood, lungs, and liver (Fig. 1D and our unpublished observations). Donor male cells were rejected between days 8 and 14 while female donor cells were easily detectable 24 days after transfer. Thus, rejection of the male graft correlates with the appearance of CTL.


Figure 1
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  		FIGURE 1. Primary CD8+ T cell responses to HY coincide with the clearance of male cells. Female mice were injected with 2 x 107 live male splenocytes and survival of donor cells and host CD8+ T cell responses were followed. At the indicated time points, splenocytes were stained directly ex vivo with H-2Db/Smcy-specific tetramers or stimulated in vitro with Uty, Smcy, or no peptide and stained for levels of intracellular IFN-{gamma}. Numbers indicate the percentage of IFN-{gamma}+ cells in the CD8+ population. A, Response of host CD8+ T cells to Uty or Smcy peptides in the spleen. B, HY-specific CD8+ T cells measured by intracellular IFN-{gamma} production as well as H-2Db/Smcy-specific tetramer staining. Results are representative of two independent experiments. C, Total HY-specific CD8+ T cell responses were followed for 4 wk postimmunization with male cells. The values represent the combined response to the dominant epitopes Uty and Smcy measured by ICS. Error bars, SEM. Results are representative of six independent experiments. D, Survival of donor male or female UbC-GFP-transgenic cells in the spleen of host mice. Error bars, SEM (n = 3 for each time point).

 
Lysis of male targets

The capacity of CD8+ T cells to lyse target cells in the periphery is a major effector mechanism by which CTLs clear infected cells (26). The importance of CTL responses has also been demonstrated in the clearance of tumors and rejection of foreign tissue grafts (27, 28). To determine the lytic capability of CTLs generated against HY, we performed in vivo cytotoxicity assays. In day 15 primed females, Uty- and Smcy-specific CTLs rapidly lysed HY peptide-pulsed female targets in an Ag-specific manner with Smcy-pulsed cells rejected more efficiently than Uty-pulsed cells (Fig. 2, A and B).


Figure 2
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  		FIGURE 2. In vivo cytolytic activity 15 days after primary immunization with male splenocytes. A, Equal numbers of Ly5.1 CFSEhigh HY peptide-pulsed female splenocytes and CFSElow control peptide-pulsed female splenocytes were injected i.v. into Ly5.2 C57BL/6 female mice (naive or day 15 postimmunization). In vivo cytotoxicity was assessed 8 h later by flow cytometry. Histograms are gated on Ly5.1+ splenocytes in recipient mice. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of the total Ly5.1+ donor cells recovered. Representative plots of Uty-pulsed (top panels) or Smcy-pulsed target cells (bottom panels). B, Cumulative data from three mice per group from two independent experiments representing Uty- or Smcy-pulsed target cells. C, Kinetics of cytolytic activity toward male splenocytes expressing endogenous levels of male Ag. Equal numbers of Ly5.1 CFSEhigh male and CFSElow female splenocytes were coinjected into Ly5.2 naive or day 15 immune female mice. In vivo cytotoxicity was assessed 24, 48, and 72 h after adoptive transfer by flow cytometry. Histograms are gated on Ly5.1+ donor cells recovered in the spleen of host mice. D, Cumulative data from two independent experiments of three mice per group at each indicated time point.

 
Efficient lysis of peptide-pulsed target splenocytes has been shown in many systems, but little is known about the lysis of target cells presenting endogenous levels of self- or pathogen-derived peptides. To address this, an in vivo cytotoxicity assay was performed comparing the survival of unpulsed male and female target cells. Male-specific target cell lysis was detectable 24 h after transfer, with 94% specific lysis within 72 h (Fig. 2, C and D).

The CD4 response

In the absence of inflammation caused by pathogens or innate signals, helper determinants and CD4+ T cells are necessary to activate and license professional APC to efficiently prime CD8+ T cell responses (12, 29). Our data would suggest that CD4+ T cells have already responded before day 12 after cell transfer. To test this, we performed a time course experiment to determine the kinetics of the primary CD4+ T cell response to the Dby peptide measured by ICS directly ex vivo. CD4+ T cell responses to Dby/I-Ab were detectable 5 days postimmunization and peaked at day 9, much earlier than the CD8+ T cell response (Fig. 3, A and C). At 5 days postimmunization, 50% of the IFN-{gamma}+ Dby-specific CD4+ T cells also produced IL-2. By day 9 after transfer, the percentage of CD4+ T cell producing IFN-{gamma} peaked at 1.7%; however, the percentage of IFN-{gamma}+ cells producing IL-2 decreased to 12% at this time point. By day 15 postimmunization, very few HY-specific IFN-{gamma}+ CD4+ T cells produced IL-2 (Fig. 3B).


Figure 3
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  		FIGURE 3. Primary CD4+ T cell responses to HY are rapid compared with the CD8+ T cell response. Female mice were injected with 2 x 107 male splenocytes and host CD4+ T cell responses were followed. At the indicated time points, splenocytes were harvested and stimulated in vitro for 4 h with Dby or no peptide and stained for intracellular IFN-{gamma} and IL-2 accumulation. A, Intracellular IFN-{gamma} staining in the CD4+ T cell population following in vitro peptide stimulation at the indicated times postimmunization. Numbers indicate the percentage of CD4+ T cells producing IFN-{gamma}. Results are representative of six separate experiments of two to three mice per time point. B, Percentage of Dby-specific CD4+ T cells producing IFN-{gamma} and IL-2 at the indicated time points. Numbers in parentheses indicate the percentage of IFN-{gamma}+, CD4+ T cells producing IL-2. C, Kinetics of the Dby-specific CD4+ T cell response. Values represent percentage of IFN-{gamma}+ cell in the CD4+ T cell population. Results are representative of six separate experiments of two to three mice per time point. Error bars, SEM.

 
Recent studies have demonstrated that CD8+ T cell responses to HY in the B6 strain are primed by uptake of donor male cells by host APC and cross-presented to host CD8+ T cells (30). It is possible that the disparate CD8+ and CD4+ T cell kinetics we observed could be the result of a delay in cross-presentation of MHC class I-restricted epitopes by host APC while B cells or DC in the donor male inoculum directly present MHC class II-restricted Ag to host CD4+ T cells. To rule out direct presentation to CD4+ T cells, female mice were immunized with MHC class II-deficient male cells. The kinetics of the host CD4+ and CD8+ T cell responses to HY were unaffected when MHC class II-restricted Ag was cross-presented exclusively by host APC (Fig. 4A).


Figure 4
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  		FIGURE 4. Direct Ag presentation by donor male cells is not necessary for the rapid Dby-specific T cell response, but host CD4+ T cell responses are necessary for rejection of male cells and development of CD8+ effector T cell responses. A, Female mice received 2 x 107 live male MHC class II-deficient splenocytes and CD4+ and CD8+ T cell responses were monitored. Values represent the combined percentage of IFN-{gamma}+ cells in the CD8+ T cell population responding to Uty and Smcy or the percentage of Dby-specific cells in the CD4+ T cell population. Results are representative of three separate experiments with two mice per time point. B, MHC class II-deficient female mice received 2 x 107 B6 male splenocytes. Response of host CD8+ T cells to Uty or Smcy peptides in the spleen were analyzed at various time points. Numbers indicate the percentage of IFN-{gamma}+ cells in the CD8+ population.

 
Requirement for CD4+ T cells

To confirm the requirement of CD4+ T cell help for primary CD8+ T cell responses in our model, we transferred male cells into female mice deficient in CD4+ T cells. HY-specific CD8+ T cell responses were not detectable in these mice and donor male cells were not rejected up to 45 days after transfer (Fig. 4B and our unpublished observations). However, it was possible that Ag-nonspecific CD4+ T cells could provide the necessary help for the primary CD8+ T cell response. To address this, we analyzed HY responses in H2bm12 mice. These mice have an H2 I-Ab mutation, but possess an intact CD4+ T cell compartment. It has been suggested that these mice are unable to present antigenic peptides derived from male cells to CD4+ helper T cells (31). Similar to the result in the CD4+ T cell-depleted mice, female H2bm12 mice were unable to mount an effector CTL response or reject male cells despite the presence of CD4+ T cells (our unpublished observations).

MHC class II-restricted killing has been observed following immunization with pathogens (32, 33). To determine whether CD4+ T cell lytic activity played any role in the HY cell transfer model, we examined the killing of Dby peptide-pulsed and control LLO peptide-pulsed female target cells in vivo. By 48 h after transfer, 65% specific lysis was seen in the lymph nodes (Fig. 5). We also observed efficient MHC class II-restricted lysis of unpulsed male target cells presenting endogenous levels of HY peptide. To demonstrate this, beta2m-deficient male and female spleen cells were used as target cells to avoid class I killing. They were injected into naive or day 9 immunized, NK-depleted mice and showed a specific loss of male cells only in the HY-primed recipients (Fig. 5).


Figure 5
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  		FIGURE 5. MHC class II-restricted cytolytic activity following immunization with male cells is detectable 9 days postimmunization. A, Top panels, equal numbers of Ly5.1 CFSEhigh Dby peptide-pulsed and CFSElow control peptide-pulsed female splenocytes were injected i.v. into Ly5.2 naive or day 9 immune female mice. In vivo cytotoxicity was assessed in the lymph nodes 48 h after transfer. Representative histograms are gated on Ly5.1+MHC class IIhigh donor cells. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of the total Ly5.1+MHC class IIhigh donor cells recovered. A, Lower panels, equal numbers of Ly5.1 beta2m–/– CFSEhigh male and CFSElow female splenocytes were coinjected into Ly5.2 naive or day 9 immune NK-depleted female mice. In vivo cytotoxicity was assessed in the lymph nodes 48 h after transfer. Representative histograms are gated on Ly5.1+MHC class IIhigh donor cells. Numbers at the top of each plot represent the percentage of CFSElow or CFSEhigh cells of the total Ly5.1+MHC class IIhigh donor cells recovered. B, Cumulative data from three independent experiments of three mice per group. Error bars, SEM.

 
Delayed kinetics due to direct presentation

Previous studies have suggested that live antigenic donor T cells may alter the ability of the recipient to mount an effective immune response against the donor Ag (34). To initially test this, we immunized female mice with male splenocytes depleted of CD4+ and CD8+ cells and followed the kinetics of host T cell responses by ICS. Unexpectedly, HY-specific CD8+ T responses were detectable and peaked 3 days earlier following immunization with T-depleted vs intact splenocytes (Fig. 6A). In the same animals, host CD4+ T cell responses remained unaffected regardless of the treatment of the male inoculum (our unpublished observations). We further defined the subset of cells responsible for delaying the host CD8+ T cell response. Donor male cells were depleted of either CD8+ or CD4+ T cells and used as immunogen. Donor CD8+ cells appeared to be more efficient at delaying host CD8+ T cell responses than donor CD4+ cells (Fig. 6, A and B).


Figure 6
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  		FIGURE 6. CD8+ T cell responses to HY are accelerated when the immunogen is depleted of T cells. Female mice were injected with 2 x 107 male intact or T subset-depleted splenocytes and host CD8+ T cell responses were followed. Host mice were sacrificed at days 9, 12, and 15 postimmunization and splenocytes were assessed for intracellular IFN-{gamma} production. A, Representative flow plots displaying CD8+ T cell response to Smcy peptide in the spleen following immunization with total, T-depleted, CD8-depleted, or CD4 depleted male splenocytes. Numbers indicate the percentage of IFN-{gamma}+ cells in the CD8+ population. B, Total HY-specific CD8+ T cell responses to intact and T subset-depleted splenocytes as indicated. Values represent the combined response to Uty and Smcy peptides in each mouse at the indicated time points. Values plotted are the mean of two to three mice per time point from a minimum of four independent experiments. Statistically significant differences using the equal variable Student t test are indicated with * (p ≤ 0.0005) or ** (p ≤ 0.03). C, CD8+ T cell response to HY Ags was suppressed with increasing numbers of male donor cells. Female mice received 1 x 107 ({blacksquare}), 2 x 107 (Figure 6), or 4 x 107 ({square}) male UbC-GFP splenocytes and host T cell responses were measured at days 9, 12, and 15. Numbers represent the combined CD8+ T cell responses to Uty246–254 and Smcy738–746 measured by intracellular IFN-{gamma} production. Error bars, SEM (n = 4 mice for each group) and are representative of two independent experiments. Statistically significant differences using Student’s t test are indicated with * (p ≤ 0.02).

 
Despite evidence that cross-presentation primes CD8+ effector T cell responses to live HY-bearing cells in the B6 strain, there was a possibility that some direct presentation of HY Ag was occurring by donor DC, and our immunization strategy with T-depleted splenocytes introduced a higher proportion of donor APC to more efficiently directly prime the CTL response. It is also possible that the male-specific genes are differentially expressed in different cell populations within the spleen, altering the amount of Ag introduced in our system. To address this, we immunized female mice with varying doses of unfractionated male splenocytes. This allowed us to immunize mice with the same proportion of each cell population, but allowed us to add varying numbers of donor male T cells. Female mice were immunized with increasing doses of male cells and the kinetics of the CD8+ T cell response was measured by ICS. When female mice were immunized with a low dose of male cells, effector host CD8+ T cell responses were rapid and easily detected 9 days after cell transfer. At this time, ~1.3% of all host CD8+ T cells were Uty or Smcy specific compared with 0.2 and 0.0% in mice immunized with 2 x 107 and 4 x 107 splenocytes, respectively. By 12 days postimmunization, HY-specific CD8+ T cell responses were comparable in mice immunized with 1 x 107 and 2 x 107 splenocytes with 2.8 and 2.2% of CD8+ T cells producing IFN-{gamma}, respectively, but these responses were significantly greater than the HY-specific response in mice immunized with 4 x 107 splenocytes. By 15 days after cell transfers, responses to all three doses of male cells were equivalent (Fig. 6C). Therefore, higher doses of antigenic male cells delayed the host CTL response.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we provide a detailed analysis of the surprising in vivo kinetics of CD8+ and CD4+ T cell responses of female B6 mice immunized with live male splenocytes. Although the primary effector CD8+ T cell response expands and contracts in a manner observed in other immunization strategies, we find that this response is considerably delayed following immunization with HY. The lack of detectable HY-specific CD8+ T cells capable of IFN-{gamma} production before day 10 postimmunization was due to the absence of Ag-specific effector T cells as confirmed by tetramer staining. This is in contrast to primary effector responses peaking 7–8 days postimmunization with irradiated cells, peptide-pulsed DC, or acute infections (14, 35, 36, 37). Despite numerous studies analyzing responses to HY, the delay in the CD8+ T cell response has not been appreciated. In addition to the slow CD8+ effector response, the time required to clear the immunogen from the host was also delayed and correlates with the expansion of effector CD8 cells. Despite this delay, HY-specific CD8+ T cells were similar to pathogen-induced CTLs as measured by effector cell surface molecule expression, cytokine production, and CTL activity in vivo (Fig. 1 and data not shown).

Recently, many studies have analyzed the role of CD4+ T cells in the generation of functional primary, memory, and recall CD8+ T cell responses (18). It is generally accepted that CD8+ T cell responses to noninflammatory, cell-associated Ag require CD4+ T cell help. In ours and similar models of immunization, generation of a primary CD8+ T cell response requires an active CD4+ T cell response to HY (Fig. 4B and our unpublished observations) (38). Because a CD4+ helper response is required, we examined whether the delay in the CD8+ T cell response could be attributed to a slow CD4+ T cell response. On the contrary, we found that the kinetics of the CD4+ T cell response to HY was similar to that seen postimmunization with pathogen and peaks many days before the detection of a primary CD8 response (Fig. 3). The CD4+ T cell response is characterized by a high percentage of IL-2 and IFN-{gamma} double-producer HY-specific cells detectable as early as 5 days postimmunization. The fraction of cells making IL-2 declines rapidly however.

We investigated the possibility that HY-specific CD4+ T cells had effector lytic activity in vivo. Similar to previous reports following immunization with pathogen, HY-specific CD4+ T cells efficiently killed peptide-pulsed target cells in an Ag- and MHC class II-dependent manner in vivo (Fig. 5). Additionally, to determine the ability of CD4+ T cells to lyse targets with endogenous levels of HY Ag, we performed an in vivo killing assay with beta2m–/– male and female target cells in mice acutely depleted of NK cells. MHC class II-restricted CD4+ T cells lysed male target cells in a HY-specific manner, suggesting that rejection of the male cells between days 8 and 12 after transfer may be partially attributed to MHC class II-restricted killing of donor B cells. However, CD4+ T cells alone are unable to mediate complete rejection of male splenocytes (38). It remains possible that in vivo killing of male cells by lytic CD4+ T cells contributes to the available Ag load necessary to cross-prime effector CD8+ T cells. In support of this idea, CD4+ and CD8+ T cell responses to irradiated male cells were weak relative to immunization with live male cells. This may be due to the rapid clearance of the male cells and the absence of prolonged Ag exposure necessary to prime efficient CD4+ and CD8+ T cell responses.

A concern with our immunization strategy is that the injected, live male cells are capable of directly presenting HY Ag to host T cells. A recent study has determined that the primary CD8+ T cell response to immunization with live male cells is differentially presented directly by donor cells or cross-presented by host APC depending on the epitope and strain of mouse analyzed (30). A possible explanation for the rapid CD4+ and delayed CD8+ response was that host CD4+ T cells are directly primed by donor male B cells or DC, while the CD8+ T cell responses are slowly cross-presented by host APC. Following immunization with class II-deficient male cells incapable of directly priming host CD4 cells, the kinetics of the primary CD4+ and CD8+ T cell responses remained unchanged (Fig. 4A). This would suggest that donor male cells are taken up by host APC and presumably both MHC class I- and MHC class II-restricted peptides are presented by host DC as early as 5 days postimmunization. Despite cross-presentation of HY by female APC, CD8+ T cell responses were not detectable for another 5 days.

Another explanation for the delayed CD8 response is the action of suppressor cells in the immunizing donor cell population. FoxP3+CD4+ T regulatory cells are thought to have a self-reactive TCR repertoire that is necessary to suppress autoreactive T cells that escaped negative selection (39). Our immunization strategy with live male cells introduces a significant number of FoxP3+CD4+ T regulatory cells that may be capable of suppressing host cells that recognize HY as foreign. To investigate this, female mice were immunized with donor male cells depleted of total T cells. Following T cell depletion of the male inoculum, the CD8+ T cell response was accelerated by 3 days while the CD4+ T cell response remained unaffected. We further defined the T cell subset responsible for delaying the host CD8+ T cell response by depleting either CD4+ or CD8+ subsets from the donor inoculum. Although depletion of CD4+ cells from the male cell immunogen accelerated the CD8+ T cell response to HY, the effect was not as great as depleting donor splenocytes of CD8+ cells. This indicates that the CD8+ donor cell population was predominantly responsible for delaying the host CD8+ T cell response to HY (Fig. 6). To determine whether the delay in the CD8+ T cell response was dependent on the number of CD8+ cells transferred during the immunization, female mice were immunized with varying doses of male cells. Despite receiving a lower dose of Ag, female mice mounted a more rapid CD8+ T cell response compared with mice receiving a higher dose of male cells (Fig. 6C). These data demonstrate that despite an increased Ag load, the host CD8+ T cell response to male cells is delayed in a dose-dependent manner. This observation has been described previously as a "veto" cell phenomenon observed following immunization with live allografts (34, 40, 41). The veto cell is characterized by the capacity of certain subsets of cells to specifically suppress CTL precursors directed against Ag presented by the veto cells themselves (40, 41, 42, 43). It has been shown that veto cells are ubiquitously distributed and include cells of T cell origin (34, 44, 45, 46, 47, 48, 49). The TCR specificity of the veto cell is inconsequential, but instead the suppression is likely dependent on the specificity of the precursor CTL recognizing Ag on the veto cell itself (50, 51, 52). As a result, there is recognition of the veto cell by the precursor CTL but not vice versa. According to this phenomenon, when naive responder CD8+ T cells meet Ag on other T cells, their response is delayed.

It has been suggested that veto cell activity is mediated by apoptosis (53). We investigated the possibility that CD8+ veto cells kill naive HY-specific CD8+ T cells and the delay in the host CD8+ response requires de novo generation of HY-specific precursor cells. However, the host T cell kinetics was unaffected when wild-type mice were immunized with perforin- or granzyme B-deficient male splenocytes or when thymectomized female mice were immunized with wild-type male cells (our unpublished observations). Instead, our data would suggest that the veto phenomenon is due to sequestration of naive HY-specific CD8+ T cells around male donor T cells that have migrated into the T cell zones of secondary lymph nodes presenting self-HY Ag on MHC class I molecules. This is supported by the absence of a veto phenomenon when male CD8 cells lacking beta2m isolated from mixed radiation bone marrow chimeras are used as the immunogen (our unpublished observations). This interaction does not appear to be permanently tolerizing because the host ultimately generates an HY-specific CD8+ T cell response presumably primed from cross-presented Ag on host APC. This explanation accounts for the fact that donor T cells veto a primary CD8+ T cell response more effectively than B cells (because of the different homing to T or B areas). However, it does not explain why CD8+ T cells are more effective than CD4+ T cells, since both subsets commingle in the T areas.


   Acknowledgment
 
We acknowledge B. Dere for technical assistance in the generation of MHC tetramers.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 the Howard Hughes Medical Institute and National Institute of Health Grant AI19335 (to M.J.B.) and by Ruth L. Kirschstein National Research Service Award CA009537 (to A.J.T.). Back

2 Address correspondence and reprint requests to Dr. Michael J. Bevan, Howard Hughes Medical Institute, Department of Immunology, Box 357370, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195. E-mail address: mbevan{at}u.washington.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; LLO, listeriolysin O; ICS, intracellular cytokine staining. Back

Received for publication May 3, 2007. Accepted for publication August 3, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Williams, M. A., M. J. Bevan. 2007. Effector and memory CTL differentiation. Annu. Rev. Immunol. 25: 171-192. [Medline]
  2. Kaech, S. M., E. J. Wherry, R. Ahmed. 2002. Effector and memory T-cell differentiation: implications for vaccine development. Nat. Rev. Immunol. 2: 251-262. [Medline]
  3. Homann, D., L. Teyton, M. B. Oldstone. 2001. Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory. Nat. Med. 7: 913-919. [Medline]
  4. Seder, R. A., R. Ahmed. 2003. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat. Immunol. 4: 835-842. [Medline]
  5. Buller, R. M., K. L. Holmes, A. Hugin, T. N. Frederickson, H. C. Morse, III. 1987. Induction of cytotoxic T-cell responses in vivo in the absence of CD4 helper cells. Nature 328: 77-79. [Medline]
  6. Rahemtulla, A., W. P. Fung-Leung, M. W. Schilham, T. M. Kundig, S. R. Sambhara, A. Narendran, A. Arabian, A. Wakeham, C. J. Paige, R. M. Zinkernagel, et al 1991. Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353: 180-184. [Medline]
  7. Wu, Y., Y. Liu. 1994. Viral induction of co-stimulatory activity on antigen-presenting cells bypasses the need for CD4+ T-cell help in CD8+ T-cell responses. Curr. Biol. 4: 499-505. [Medline]
  8. Shedlock, D. J., H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300: 337-339. [Abstract/Free Full Text]
  9. Sun, J. C., M. J. Bevan. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300: 339-342. [Abstract/Free Full Text]
  10. Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, S. P. Schoenberger. 2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421: 852-856. [Medline]
  11. Khanolkar, A., M. J. Fuller, A. J. Zajac. 2004. CD4 T cell-dependent CD8 T cell maturation. J. Immunol. 172: 2834-2844. [Abstract/Free Full Text]
  12. Bennett, S. R., F. R. Carbone, F. Karamalis, J. F. Miller, W. R. Heath. 1997. Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help. J. Exp. Med. 186: 65-70. [Abstract/Free Full Text]
  13. Guerder, S., P. Matzinger. 1992. A fail-safe mechanism for maintaining self-tolerance. J. Exp. Med. 176: 553-564. [Abstract/Free Full Text]
  14. Wang, J. C., A. M. Livingstone. 2003. Cutting edge: CD4+ T cell help can be essential for primary CD8+ T cell responses in vivo. J. Immunol. 171: 6339-6343. [Abstract/Free Full Text]
  15. Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393: 478-480. [Medline]
  16. Ridge, J. P., F. Di Rosa, P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393: 474-478. [Medline]
  17. Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393: 480-483. [Medline]
  18. Bevan, M. J.. 2004. Helping the CD8+ T-cell response. Nat. Rev. Immunol. 4: 595-602. [Medline]
  19. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8: 177-187. [Medline]
  20. Coles, R. M., S. N. Mueller, W. R. Heath, F. R. Carbone, A. G. Brooks. 2002. Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1. J. Immunol. 168: 834-838. [Abstract/Free Full Text]
  21. Mueller, S. N., C. M. Jones, C. M. Smith, W. R. Heath, F. R. Carbone. 2002. Rapid cytotoxic T lymphocyte activation occurs in the draining lymph nodes after cutaneous herpes simplex virus infection as a result of early antigen presentation and not the presence of virus. J. Exp. Med. 195: 651-656. [Abstract/Free Full Text]
  22. Greenfield, A., D. Scott, D. Pennisi, I. Ehrmann, P. Ellis, L. Cooper, E. Simpson, P. Koopman. 1996. An H-YDb epitope is encoded by a novel mouse Y chromosome gene. Nat. Genet. 14: 474-478. [Medline]
  23. Markiewicz, M. A., C. Girao, J. T. Opferman, J. Sun, Q. Hu, A. A. Agulnik, C. E. Bishop, C. B. Thompson, P. G. Ashton-Rickardt. 1998. Long-term T cell memory requires the surface expression of self-peptide/major histocompatibility complex molecules. Proc. Natl. Acad. Sci. USA 95: 3065-3070. [Abstract/Free Full Text]
  24. Scott, D., C. Addey, P. Ellis, E. James, M. J. Mitchell, N. Saut, S. Jurcevic, E. Simpson. 2000. Dendritic cells permit identification of genes encoding MHC class II-restricted epitopes of transplantation antigens. Immunity 12: 711-720. [Medline]
  25. Schaefer, B. C., M. L. Schaefer, J. W. Kappler, P. Marrack, R. M. Kedl. 2001. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell. Immunol. 214: 110-122. [Medline]
  26. Walsh, C. M., M. Matloubian, C. C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. Huang, J. D. Young, R. Ahmed, W. R. Clark. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91: 10854-10858. [Abstract/Free Full Text]
  27. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369: 31-37. [Medline]
  28. Simpson, E., D. Scott, E. James, G. Lombardi, K. Cwynarski, F. Dazzi, J. M. Millrain, P. J. Dyson. 2001. Minor H antigens: genes and peptides. Eur. J. Immunogenet. 28: 505-513. [Medline]
  29. Smith, C. M., N. S. Wilson, J. Waithman, J. A. Villadangos, F. R. Carbone, W. R. Heath, G. T. Belz. 2004. Cognate CD4+ T cell licensing of dendritic cells in CD8+ T cell immunity. Nat. Immunol. 5: 1143-1148. [Medline]
  30. Millrain, M., D. Scott, C. Addey, H. Dewchand, P. Ellis, I. Ehrmann, M. Mitchell, P. Burgoyne, E. Simpson, J. Dyson. 2005. Identification of the immunodominant HY H2-Dk epitope and evaluation of the role of direct and indirect antigen presentation in HY responses. J. Immunol. 175: 7209-7217. [Abstract/Free Full Text]
  31. de Waal, L. P., J. de Hoop, M. J. Stukart, H. Gleichmann, R. W. Melvold, C. J. Melief. 1983. Nonresponsiveness to the male antigen H-Y in H-2 I-A-mutant B6.C-H-2bm12 is not caused by defective antigen presentation. J. Immunol. 130: 655-660. [Abstract]
  32. Adhikary, D., U. Behrends, A. Moosmann, K. Witter, G. W. Bornkamm, J. Mautner. 2006. Control of Epstein-Barr virus infection in vitro by T helper cells specific for virion glycoproteins. J. Exp. Med. 203: 995-1006. [Abstract/Free Full Text]
  33. Jellison, E. R., S. K. Kim, R. M. Welsh. 2005. Cutting edge: MHC class II-restricted killing in vivo during viral infection. J. Immunol. 174: 614-618. [Abstract/Free Full Text]
  34. Fink, P. J., I. L. Weissman, M. J. Bevan. 1983. Haplotype-specific suppression of cytotoxic T cell induction by antigen inappropriately presented on T cells. J. Exp. Med. 157: 141-154. [Abstract/Free Full Text]
  35. Badovinac, V. P., K. A. Messingham, S. E. Hamilton, J. T. Harty. 2003. Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host. J. Immunol. 170: 4933-4942. [Abstract/Free Full Text]
  36. Busch, D. H., I. M. Pilip, S. Vijh, E. G. Pamer. 1998. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8: 353-362. [Medline]
  37. Janssen, E. M., N. M. Droin, E. E. Lemmens, M. J. Pinkoski, S. J. Bensinger, B. D. Ehst, T. S. Griffith, D. R. Green, S. P. Schoenberger. 2005. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death. Nature 434: 88-93. [Medline]
  38. Robertson, N. J., J. G. Chai, M. Millrain, D. Scott, F. Hashim, E. Manktelow, F. Lemonnier, E. Simpson, J. Dyson. 2007. Natural regulation of immunity to minor histocompatibility antigens. J. Immunol. 178: 3558-3565. [Abstract/Free Full Text]
  39. Hsieh, C. S., Y. Liang, A. J. Tyznik, S. G. Self, D. Liggitt, A. Y. Rudensky. 2004. Recognition of the peripheral self by naturally arising CD25+CD4+ T cell receptors. Immunity 21: 267-277. [Medline]
  40. Miller, R. G.. 1980. An immunological suppressor cell inactivating cytotoxic T-lymphocyte precursor cells recognizing it. Nature 287: 544-546. [Medline]
  41. Miller, R. G., H. Derry. 1979. A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen. J. Immunol. 122: 1502-1509. [Abstract/Free Full Text]
  42. Muraoka, S., R. G. Miller. 1980. Cells in bone marrow and in T cell colonies grown from bone marrow can suppress generation of cytotoxic T lymphocytes directed against their self antigens. J. Exp. Med. 152: 54-71. [Abstract/Free Full Text]
  43. Thomas, J. M., F. M. Carver, P. R. Cunningham, L. C. Olson, F. T. Thomas. 1991. Kidney allograft tolerance in primates without chronic immunosuppression: the role of veto cells. Transplantation 51: 198-207. [Medline]
  44. Cassell, D. J., J. Forman. 1990. Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens. J. Immunol. 144: 4075-4081. [Abstract]
  45. Claesson, M. H., R. G. Miller. 1984. Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. I. CTL clones express strong anti-self suppressive activity. J. Exp. Med. 160: 1702-1716. [Abstract/Free Full Text]
  46. Claesson, M. H., C. Ropke. 1986. Antiself suppressive (veto) activity of responder cells in mixed lymphocyte cultures. Curr. Top. Microbiol. Immunol. 126: 213-223. [Medline]
  47. Fink, P. J., R. P. Shimonkevitz, M. J. Bevan. 1988. Veto cells. Annu. Rev. Immunol. 6: 115-137. [Medline]
  48. Nakamura, H., R. E. Gress. 1990. Interleukin 2 enhancement of veto suppressor cell function in T-cell-depleted bone marrow in vitro and in vivo. Transplantation 49: 931-937. [Medline]
  49. Zhang, L. I., D. R. Martin, W. P. Fung-Leung, H. S. Teh, R. G. Miller. 1992. Peripheral deletion of mature CD8+ antigen-specific T cells after in vivo exposure to male antigen. J. Immunol. 148: 3740-3745. [Abstract]
  50. Claesson, M. H.. 1987. Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions. Cell. Immunol. 109: 360-370. [Medline]
  51. Sambhara, S. R., R. G. Miller. 1991. Programmed cell death of T cells signaled by the T cell receptor and the {alpha} 3 domain of class I MHC. Science 252: 1424-1427. [Abstract/Free Full Text]
  52. Sambhara, S. R., R. G. Miller. 1994. Reduction of CTL antipeptide response mediated by CD8+ cells whose class I MHC can bind the peptide. J. Immunol. 152: 1103-1109. [Abstract]
  53. Hiruma, K., H. Nakamura, P. A. Henkart, R. E. Gress. 1992. Clonal deletion of postthymic T cells: veto cells kill precursor cytotoxic T lymphocytes. J. Exp. Med. 175: 863-868. [Abstract/Free Full Text]

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