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The Journal of Immunology, 2004, 172: 7399-7407.
Copyright © 2004 by The American Association of Immunologists

Activation of Naive CD4+ T Cells In Vivo by a Self-Peptide Mimic: Mechanism of Tolerance Maintenance and Preservation of Immunity1

Jin-Young Choi* and Joe Craft2,*,{dagger}

Sections of * Rheumatology and {dagger} Immunobiology, Yale School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intrathymic selection generates a peripheral repertoire of CD4+ T cells with receptors that retain low affinity for self-peptide MHC complexes. Despite self-recognition, T cells remain tolerant even in the setting of microbial challenge and resultant costimulatory signals. We demonstrate here a novel mechanism for tolerance maintenance under conditions of self-recognition and strong costimulation. TCR engagement in vivo with a low-avidity peptide, as a mimic of self, provided with poly(I:C) (dsRNA) led to division of naive T cells that was dependent upon costimulatory signals; however, the dividing cells rapidly underwent deletion. By contrast, the surviving cells that were activated as evidenced by up-regulation of CD69 did not become effectors upon restimulation with the same ligand and maintained an effective response against agonist peptide. We suggest TCR engagement with self-peptide MHC complexes promotes tolerance maintenance during pathogen challenge, while preserving efficient reactivity for subsequent encounter with foreign Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The maintenance of the naive CD4+ T cell repertoire is dependent upon TCR contact with self-peptide MHC (self-pMHC)3 complexes in secondary lymphoid tissues (1, 2, 3, 4, 5, 6), interactions that also enhance the sensitivity of mature CD4+ T cells to foreign stimuli (7). In naive CD4+ T cells, TCR engagement with self-pMHC complexes leads to phosphorylation of the TCR {zeta}-chain without phosphorylation of {zeta}-associated protein 70 (8, 9), with maintenance of the former dependent upon ongoing TCR contact with self-pMHC (7). Even though TCR-self-peptide interactions are required for normal T cell homeostasis, autoreactivity does not typically ensue.

Microbial infection changes the host cytokine environment and induces expression of accessory molecules on APC (10, 11). Upon pathogen challenge, naive T cells are presumably engaged with self-pMHC complexes on APC in the setting of costimulation, events that are theoretically capable of T cell activation. Nevertheless, such self-pMHC-TCR interactions do not typically lead to autoreactive T cell responses in the absence of genetic predisposition (12, 13). The lack of autoreactivity under these circumstances has been explained by several mechanisms (14), including tolerance induction by immature dendritic cells (15, 16, 17, 18); however, these latter studies have analyzed T cell tolerance induction upon contact with high-avidity peptide ligands, rather than T cells with low avidity for self-pMHC that populate the periphery (19).

We speculated that additional mechanisms of tolerance maintenance in the setting of enhanced costimulation might apply to the mature T cells with low avidity for self-pMHC. To address this question, we developed a system in which naive CD4+ TCR-transgenic cells were engaged with a low-affinity peptide ligand, as a mimic of a self-peptide, in the presence of poly(I)·poly(C) (poly(I:C); dsRNA), analogous to a viral infection (20). We used AND-transgenic TCR T cells, specific for residues 88–104 of pigeon cytochrome c (PCC88–104) (21), and as a model peptide, K99Q, a weak antagonist of the transgenic TCR (22, 23). K99Q is of such low affinity for the AND TCR that when presented in vitro by APC, it does not lead to CD69 up-regulation, T cell activation, or ZAP-70 phosphorylation, along with only minimal phosphorylation of CD3{zeta} (23, 24, 25). This pattern is consistent with a weak partial agonist or antagonist peptide (23) and is similar to the interaction of CD4+ T cells and self-pMHC ex vivo (7, 9).

We found that upon adoptive transfer into wild-type nonirradiated syngeneic hosts and priming with K99Q and poly(I:C), AND T cells divided, with division dependent upon costimulatory signals; however, the dividing cells rapidly died by apoptosis as demonstrated by in vitro experiments. By contrast, the surviving cells that did not divide, but were activated as evidenced by up-regulation of CD69, maintained an effective response against agonist peptide. We propose that this response of naive CD4+ T cells is operative in circumstances with pathogen infections in which T cells remain tolerant to self-peptides but preserve reactivity to foreign Ags.


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

B10.BR mice were purchased from The Jackson Laboratory (Bar Harbor, ME). AND mice expressing the V{alpha}11+V{beta}3+ TCR transgenes recognizing PCC88–104 were originally provided by S. Hedrick (University of California, San Diego) (21) and were maintained in the B10.BR background (B10.AND). We produced B10.BR recombination activating gene 1-deficient (RAG-1–/–) and B10.BR Thy1.1 (CD90.1) mice by serial backcross of B6;129S-Rag1tm1Mom and C57BL/6J-IghaThy1aGpi1a mice to the B10.BR background for >20 and 6 generations, respectively, followed by breeding to B10.AND animals to generate B10.AND RAG-1–/– and B10.AND Thy1.1 mice. AND TCR-transgenic mice were maintained as heterozygotes and screened as described previously (26). The animals were identically housed in specific pathogen-free facilities at the Yale Animal Resources Center and the Institutional Animal Care and Use Committee at the Yale School of Medicine approved all experiments.

T cell purification and flow cytometry

Pooled axillary and inguinal lymph nodes, and/or spleens from B10.AND, B10.AND Thy1.1, or B10.AND RAG-1–/– mice were dissociated and single-cell suspensions were prepared, followed by hypotonic shock for RBC lysis. CD4+ TCR-transgenic T cells were purified by negative selection using mAb and magnetic beads. Briefly, the cells were labeled with biotinylated Abs to CD8 (53-6.7), CD16/CD32 (2.4G2), B220 (RA3-6B2), I-Ek (14-4-4S), CD11b (M1–70), and {gamma}{delta} (GL3) (all from BD PharMingen, San Diego, CA) followed by addition of streptavidin microbeads (Miltenyi Biotec, Auburn, CA) and passage through a magnetic column using the protocol of the manufacturer. Negatively selected CD4+ T cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA) for purity and activation status: cells were >98% CD4+ and >97% naive (CD62Lhigh and CD44low) (27). TCR-transgenic T cells in peripheral tissues, including the gut (intestinal epithelial lymphocytes), liver, and lung, were isolated as described previously (28). To assess costimulatory molecule expression, CD11c+ cells were enriched from spleens using magnetic beads (Miltenyi Biotec). Abs used for T cell and APC analysis included PE-conjugated anti-CD62L (MEL-14), anti-CD25 (PC61), anti-CD69 (H1.2F3), anti-B7-1 (CD80; 16-10A1), anti-B7-2 (CD86; GL1), anti-I-Ek (14-4-4S), PE-Cy5-conjugated anti-CD44, PerCP-conjugated anti-Thy1.1 (OX-7), and allophycocyanin-conjugated anti-CD4 (L3T4) (BD PharMingen).

Adoptive transfers and immunizations

A total of 5–8 x 106 negatively selected AND T cells were labeled with CFSE and injected i.v. into wild-type (T cell-replete) B10.BR recipients in 200 µl of sterile PBS (11). Recipients of naive AND T cells were simultaneously given i.v. 250 µg of the agonist peptide PCC88–104 or peptides altered by single amino acid substitution (altered peptide ligands (APL)) including K99R, K99Q, and K99A (Ref.12 ; synthesized by the Yale KECK Biotechnology Resource Center), with or without 150 µg of poly(I:C) (Amersham Pharmacia Biotech, Piscataway, NJ), or poly(I:C) alone. In certain experiments, LPS (LPS 055:B5, 25 µg; Sigma-Aldrich, St. Louis, MO) was substituted for poly(I:C). For Ag rechallenge studies, mice were administered the indicated peptides 11 days after initial treatment with poly(I:C) and/or peptide. For costimulatory blockade, mice were treated i.p. on days –1, 0, and 2 (relative to immunization) with 200 µg of CTLA-4 Ig or mouse IgG (Sigma-Aldrich). The CTLA-4 Ig was a kind gift from Drs. Charlotte Eiselson and David Rothstein at Yale.

Apoptosis studies

CFSE-labeled naive AND T cells (1 x 106/well) were activated in the presence of 3 x 106 APC (T cell-depleted spleen cells from poly(I:C)-treated B10.BR mice) for 96 h at 37°C. APC were preloaded with either 1 µM PCC88–104 or 25 µM K99Q. Annexin V staining was performed using the apoptosis detection kit (BD PharMingen), and the percentage of apoptotic cells was determined by assessment of annexin V+ cells, gating on the CFSE+ CD4+ T cell population.

Intracellular cytokine analysis

Lymph node cells obtained from immunized mice were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml; Sigma-Aldrich) for 6 h. After 2 h of culture, Golgi Plug (BD PharMingen) was added to promote intracellular cytokine accumulation (29). Cells were then washed, fixed with 2% paraformaldehyde for 20 min, and permeabilized with PBS containing 0.5% saponin (Sigma-Aldrich) for 10 min. Cells were stained with PE-labeled anti-IL-2 mAb or anti-IFN-{gamma} mAb, allophycocyanin-conjugated CD4, and PerCP-conjugated Thy1.1 (all from BD PharMingen), followed by flow cytometric analysis. The percentage of transgenic cells producing cytokines was determined by gating on the adoptively transferred Thy1.1+CD4+ AND T cell population.

Statistics

Statistical significance was evaluated by two-tailed, unpaired Student’s t test. A p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation of naive CD4+ T cells following challenge with a low-affinity peptide ligand plus poly(I:C)

Recipient mice that received CFSE-labeled naive B10.AND Thy1.1+ T cells were first primed with PCC88–104 or a panel of APL with varying affinities for the AND TCR, alone or in conjunction with poly(I:C). For these experiments, we selected APL-bearing single amino acid substitutions at position 99 in the cognate peptide PCC88–104: K99R, K99Q, and K99A (22). Based on inhibition assays by Lyons and colleagues, these ligands showed a range of dissociation constants for the AND TCR: 330 µM for K99R and 2080 µM for K99Q, compared with 40 µM for the cognate peptide. K99R and K99Q have also been classified as a antagonist and a weak antagonist, respectively, as demonstrated by strong or partial inhibition of IL-2 production of 2B4 hybridoma cells stimulated by the cognate PCC peptide (22, 30). K99A is defined as a null peptide based upon the observation that K99A had no effect on 2B4 T cell development in thymic organ culture (30). All four peptides have similar affinities for I-Ek (31).

Challenge with PCC or K99R, the cognate Ag or a antagonist, respectively, led to cell division within 3 days of priming (Fig. 1, A and B). For both peptides, poly(I:C) treatment promoted proliferation of AND CD4+ T cells and a 4- to 5-fold increase in cell number compared with T cells primed with ligand alone. By contrast, adoptively transferred naive AND T cells did not undergo proliferation 3 days after i.v. administration of K99Q or K99A, a weak antagonist and a null peptide, respectively, an expected result given previous in vitro studies (22, 23) (Fig. 1, A and B). In contrast, when recipient mice were primed with either K99Q or K99A and simultaneously administered poly(I:C), adoptively transferred AND T cells divided several times, whereas naive AND T cells in hosts treated with poly(I:C) alone did not divide (Fig. 1, A and B). Since priming with K99Q induced a similar response in terms of cell division to that observed with K99A and since as noted above the former is of such low affinity for the AND TCR (23, 24, 25) that it mimics interaction of CD4+ T cells and self-pMHC ex vivo (7, 9), we decided to use it as a model self-ligand for the remainder of our studies.



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  		FIGURE 1. Naive CD4+ T cells proliferate after TCR engagement with low-affinity ligand and poly(I:C) in vivo. A and B, AND CD4+ T cells purified from lymph nodes of B10.AND mice were labeled with CFSE and adoptively transferred into T cell-replete B10.BR mice. Recipient mice were immunized i.v. with the indicated peptide with or without poly(I:C), poly(I:C) alone, or PBS (control) alone. Divisions of transferred CFSE+ AND T cells were assessed by flow cytometry 3 days after priming (A; cells in boxes). Panels in A are gated on live lymph node cells and the data are representative of seven experiments with similar results obtained using splenic T cells. Panels in B are gated on CD4+ CFSE+ T cells and are representative of two experiments. C, Recipient mice were immunized i.v. with K99Q plus LPS or given LPS alone i.v. Three days after priming, the transferred CFSE+ AND T cells were assessed by flow cytometry. D, Expression of V{alpha}11 on dividing cells from a host immunized with K99Q plus poly(I:C).

 
To determine whether Toll-like receptor ligands other than poly(I:C) can also potentiate K99Q-dependent proliferation, recipients of naive CFSE-labeled AND T cells were primed with K99Q and 25 µg of LPS or LPS alone. Three days after priming, adoptively transferred AND T cells divided several times when recipient mice were primed with K99Q plus LPS, whereas cells in host treated with LPS alone did not divide (Fig. 1C).

These experiments were done with AND T cells isolated from RAG-intact hosts. Despite the purity of the transferred population, heterogeneity in TCR {alpha}-chain usage could influence the proliferative potential of T cells. Given the low avidity interaction of the AND TCR with K99Q, we suspected that the dividing cells all expressed the V{alpha}11 and V{beta}3 receptors of the transgenic receptor. To confirm this, we stained the dividing cells and found that all were V{alpha}11bright (Fig. 1D). Moreover, we repeated the above experiments, using B10.AND RAG-1–/– mice as T cell donors with the same results as for RAG-intact B10.AND mice (data not shown), demonstrating that T cell proliferation following priming with K99Q plus poly(I:C) was not due to activation of T cells bearing endogenous TCR{alpha} chains.

CD4+ T cell proliferation induced by lymphopenia requires TCR interaction with self-pMHC complexes and a CD28-mediated signal and is inhibited by the presence of memory or naive CD4+ T cells (4, 32, 33, 34). To determine whether proliferation of naive AND T cells upon priming with K99Q in the presence of poly(I:C) was likewise dependent upon cell number, different numbers of CFSE-labeled naive AND T cells were adoptively transferred and primed with either PCC88–104 plus poly(I:C) or K99Q plus poly(I:C). With transfer of 2 x 106 cells, at least six divisions were evident within 3 days of transfer with both priming regimens (Fig. 2). By contrast, when 25 x 106 cells were transferred, proliferation was dramatically reduced (Fig. 2). The reduction of proliferation engendered by K99Q plus poly(I:C) administration was also observed in AND T cells primed with PCC88–104 plus poly(I:C). Thus, the extent of proliferation was greater when fewer cells were transferred, suggesting that cell division after priming with K99Q or with PCC88–104 follows the same rule of space constraints as homeostatic proliferation in lymphopenic hosts (35).



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  		FIGURE 2. Cell division after priming with K99Q or with PCC88–104 follows the same rule of space constraints as homeostatic proliferation in lymphopenic hosts. The indicated number of CFSE-labeled AND T cells were transferred into B10.BR mice, followed by priming with PCC plus poly(I:C) or K99Q plus poly(I:C). Divisions of transferred CFSE+ AND T cells were assessed by flow cytometry 3 days after priming. Histograms represent gates on CFSE+ AND T cells and the data are representative of two experiments.

 
Cell surface markers on CD4+ T cells after engagement with low-affinity peptide ligand plus poly(I:C)

One day after priming with PCC88–104, TCR-transgenic T cells had not yet divided; nevertheless, CD69, an early activation marker (36), was up-regulated, with enhanced expression in animals also given poly(I:C) (Fig. 3A, left panel). Although administration of K99Q alone minimally up-regulated expression of CD69 on transferred T cells, treatment of mice with K99Q plus poly(I:C), or only poly(I:C), led to its up-regulation in a pattern analogous to that seen after administration of cognate peptide; however, CD69 expression was significantly higher on AND T cells from mice treated with K99Q plus poly(I:C) (mean channel fluorescence (MCF) = 978) than those from mice treated with poly(I:C) alone (MCF = 418; Fig. 3A, left panels). Poly(I:C) treatment also led to up-regulation of CD69 on host CD4+ T cells (data not shown), suggesting either that its expression in the presence of this adjuvant is induced by contact with endogenous self-pMHC complexes or does not require TCR engagement.



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  		FIGURE 3. AND T cells differentially express activation markers and costimulation blockade abolishes naive CD4+ T cell proliferation following low-affinity TCR engagement. AND T cells purified from B10.AND mice were labeled with CFSE and adoptively transferred into B10.BR mice. Recipient mice were immunized as described in Materials and Methods. A and B, Lymph node cells were prepared from recipient mice 1or 3 days after priming, and the expression of CD69, CD25, CD44, and CD62L by individual AND T cells was assessed by flow cytometry. At day 1, histograms in gray represent the expression of each molecule on AND T cells from unprimed recipients, whereas open lines represent expression on AND T cells from primed animals. Boxes in dot plots for cells stained with CD62L at day 3 indicate the population of AND T cells that express a high density of this molecule. The data shown are representative of five separate experiments. C, One day after priming, spleen cells were prepared from poly(I:C)-treated mice and CD11c+ cells were enriched as described in Materials and Methods, and the expression of B7-1, B7-2, and I-Ek was assessed by flow cytometry. Shaded histograms represent the expression of each molecule on cells from PBS-primed (control) recipients. Dotted lines represent isotype controls. The data shown are from one representative of three independent experiments with two mice per group. D, Recipients of adoptive transfers of CFSE+ AND T cells were treated i.p. with CTLA-4 Ig or control IgG on days –1, 0, and 2 relative to immunization with PCC or PCC or K99Q plus poly(I:C). Three days later, cell divisions were assessed. The data shown are from lymph node cells and are identical to that for splenocytes and are representative of two experiments.

 
By contrast to CD69 up-regulation, challenge with K99Q and poly(I:C) did not promote CD25 expression within 4 h or within 1 day after priming (data not shown and Fig. 3A, middle panel). Such challenge also did not lead to CD44 up-regulation or CD62L (L-selectin) down-regulation 1 day or 3 days after priming (Fig. 3, A, right panels, and B). For example, the MCF for CD44 of cells 3 days after exposure to K99Q plus poly(I:C) at division 4 was only 156, roughly equivalent to the MCF of 100 for undivided cells (Fig. 3B, left panels). Similarly, dividing AND T cells primed with K99Q plus poly(I:C) did not have a reduction in their expression of CD62L 3 days after challenge, with a MCF after 4 divisions of 545 compared with 621 at baseline (Fig. 3B, right panels).

In contrast, CD25 was up-regulated on AND T cells primed with PCC88–104 plus poly(I:C) (Fig. 3A) in concert with CD62L shedding (Fig. 3B). CD44 was also up-regulated, albeit to a lesser extent than that observed with PCC priming alone (Fig. 3, A, right panels, and B). Thus, costimulation plus strong TCR engagement are required for CD25 expression (37). Despite division and CD69 up-regulation following low-affinity ligand and poly(I:C) challenge, TCR-transgenic T cells did not otherwise exhibit an activation phenotype.

Costimulation blockade abolishes naive CD4+ T cell proliferation following low-affinity TCR engagement

One day after administration of poly(I:C), with or withoutPCC88–104 or K99Q priming, the levels of B7-2 and, to a lesser extent, levels of B7-1 and class II MHC were up-regulated on splenic CD11c+ cells of the recipients of adoptive transfers (Fig. 3C). To prove that costimulation led to the proliferation of AND T cells primed with the low-affinity ligand K99Q, we blocked the interaction between B7-1/B7-2 and CD28 on T cells using soluble CTLA-4 Ig (38) before and after peptide priming. Treatment with CTLA-4 Ig led to a reduction in proliferation of AND T cells from recipient mice primed with PCC88–104 plus poly(I:C) and, to a somewhat lesser extent, in proliferation of T cells from mice primed with PCC88–104 alone (Fig. 3D, middle to left panels). Proliferation in either case was not abolished, suggesting that at least in part proliferation after challenge with a high-affinity peptide was costimulation independent. By contrast, CTLA-4 Ig treatment nearly abolished the proliferation of AND T cells primed with K99Q plus poly(I:C) (Fig. 3D, right panel). These results indicate that poly(I:C) can initiate proliferation of naive CD4+ T cells despite a weak TCR signal and that such proliferation is strongly dependent upon B7 expression induced by poly(I:C) with subsequent interaction with CD28 on T cells.

T cells proliferating in response to a low-affinity ligand undergo apoptosis

To determine the fate of naive CD4+ T cells induced to proliferate with weak TCR engagement, we sacrificed recipient mice 5 and 15 days after peptide plus poly(I:C) priming and determined the number of adoptively transferred AND Thy1.1+ T cells remaining. Five days after priming with PCC88–104, the vast majority (>97%) of transferred AND T cells resident in lymph nodes had divided more than once (Fig. 4A). At 15 days following transfer, we detected a similar percentage (98.2%) of cells that had divided, although we recovered a lower number of cells than at day 5: recovered Thy1.1+ AND T cells constituted 4% of total lymph node cells at day 5 compared with 1.2% of total lymph node cells at day 15 (Fig. 4B, left panel). Thus, even with cell division and expected migration to nonlymphoid tissues, lymph nodes still contained a significant number of dividing cells 15 days following AND T cell transfer and PCC plus poly(I:C) priming. By contrast, 5 days following priming with K99Q plus poly(I:C), only ~45% of the transferred cells in the lymph nodes had divided (Fig. 4A). By day 15 following priming, the total recovered cells was further reduced: recovered Thy1.1+ AND T cells constituted 0.83% of total lymph node cells at day 5 compared with <0.1% of total lymph node cells at day 15 (Fig. 4B, right panel). In addition, only 15.2% of the remaining cells were dividing (Fig. 4A). Thus, compared with AND TCR-transgenic cells primed in vivo with PCC plus poly(I:C), cells primed with K99Q plus poly(I:C) divided, but dividing cells were no longer present in the recipient lymph nodes 15 days after priming.



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  		FIGURE 4. T cells proliferating in response to a low-affinity ligand undergo apoptosis. A and B, CFSE+ B10.AND Thy1.1+ cells were transferred into B10.BR Thy1.2+ mice and recipients were immunized with PCC88–104 plus poly(I:C) or K99Q plus poly(I:C). Lymph node cells were prepared from the recipient mice 5 or 15 days after priming. A, Cell divisions were assessed by flow cytometry by gating on CD4+ Thy1.1+ cells. B, The number of recovered Thy1.1+ AND T cells was assessed as a percentage of total lymph node cells. C, CFSE-labeled naive AND T cells (1 x 106/well) were activated for 96 h in the presence of 3 x 106 APC (T cell-depleted splenocytes) obtained from poly(I:C)-treated B10.BR mice. APC were preloaded with either 1 µM PCC88–104 or 25 µM K99Q, and annexin V staining was used to assess apoptosis. Dots in each box represent annexin V+ cells in each division as measured by gating on the CFSE+CD4+ T cell population. The data shown are representative of two independent experiments.

 
To determine why we could no longer detect AND T cells triggered to divide by K99Q plus poly(I:C), we asked whether they had migrated out of the lymph nodes, seeking them in the liver, lung, and gut (intestinal epithelial lymphocytes). As expected (28, 39), AND T cells were found in peripheral tissues after priming with PCC88–104 plus poly(I:C), and such cells had divided, as determined by CFSE dilution. By contrast, we found few, if any, AND-transgenic cells in peripheral tissues of mice primed with K99Q plus poly(I:C) (data not shown). This finding was not surprising given the lack of CD44 up-regulation and CD62L shedding of these cells compared with those primed with PCC or PCC plus poly(I:C) (Fig. 3B). We surmised that the cells dividing after K99Q and poly(I:C) stimulation had died, a notion suggested by their lack of CD25 expression (Fig. 3A).

We next attempted to measure cell death in vivo; however, we were unable to reproducibly quantify apoptotic cells due to the small numbers of cells available for study after adoptive transfer and K99Q plus poly(I:C) priming (Fig. 4B) and presumably due to their rapid clearance. Even after priming with different doses of K99Q along with poly(I:C), we were unable to quantify dying cells in vivo. Thus, we established an in vitro system to measure AND T cell death following activation with peptides presented by T cell-depleted APC taken from poly(I:C)-treated mice. In these experiments, we tested multiple doses of PCC and K99Q (1–100 µM). We found that 1 µM PCC and 25 µM K99Q induced proliferation of naive AND T cells to a degree similar to that shown in vivo (Fig. 4C, left panels, cf with Fig. 1A). Lower doses of K99Q did not lead to proliferation in vitro and higher doses of PCC or K99Q did not substantively change the proliferation patterns from those observed with 1 and 25 µM, respectively (data not shown). Using these peptide concentrations, following in vitro stimulation with PCC88–104 plus poly(I:C), cycling AND T cells in each division were 4–5% annexin V+ compared with proliferating cells after K99Q plus poly(I:C) stimulation that were 20–50% annexin V+ in each division (overall 5.8-fold increase; Fig. 4C, right panel). Undivided AND T cells in both groups were annexin V– (data not shown). These in vitro results suggest that T cells that divided in response to low-affinity ligand plus poly(I:C) in vivo underwent apoptotic death.

TCR engagement with low-affinity ligand and poly(I:C) does not promote effector function upon restimulation with low-affinity Ag

Although naive T cell activation upon low-affinity TCR engagement in the presence of costimulation promoted cell death, at least as determined by in vitro analysis, ~0.1% of lymph node cells (6–8 x 103 undivided Thy1.1+ cells) remained alive even 15 days following initial priming in vivo (Fig. 4B, right panel). These cells had been activated, as evidenced by up-regulation of CD69 to a significantly greater extent than that induced by poly(I:C) alone (Fig. 3A). Thus, we asked whether low-affinity Ag encounter had altered their Ag responsiveness, determining whether they were capable of effector function if rechallenged with K99Q alone or K99Q plus poly(I:C).

First, we determined that recovery of AND T cells before rechallenge (day 11 after priming) was equivalent between animals given K99Q plus poly(I:C) and as control, poly(I:C) alone (Fig. 5A; ~0.21–0.22% of total lymph node cells in both cases compared with ~0.05–0.06% of total lymph node cells from mice given PBS alone). When rechallenged with K99Q plus poly(I:C), AND T cells from mice primed with K99Q plus poly(I:C) underwent minimal division, with only 20.8% total AND T cells dividing, in comparison to 13.8% cells in animals that had been initially administered poly(I:C) alone (Fig. 5B, right panels). Patterns of cell division in both cases were similar to those observed in animals initially given K99Q plus poly(I:C) or poly(I:C) alone and that were not rechallenged (Fig. 5B, left panels). Upon ex vivo stimulation with PMA/ionomycin, AND T cells from rechallenged mice did not produce IFN-{gamma}, although 22–23% were IL-2+ in both cases, approximately equivalent to cells from mice given K99Q plus poly(I:C) or poly(I:C) alone and that were not rechallenged (Fig. 5B). Thus, T cells exposed to a weak TCR ligand in the setting of strong costimulatory signals neither expand nor become effector cells upon rechallenge with the same peptide ligand, even if the latter is provided with exogenous costimulation via poly(I:C).



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  		FIGURE 5. T cells activated after TCR engagement with low-affinity ligand do not produce IFN-{gamma} after rechallenge with the same ligand. A, Naive CFSE+ B10.AND Thy1.1+CD4+ T cells were transferred to Thy1.2+ recipients, followed by administration of PBS (control), poly(I:C), or K99Q plus poly(I:C). At day 11, a group of mice were sacrificed for assessment of AND T cell recovery (day 11). Other animals were rechallenged with K99Q plus poly(I:C) and sacrificed 3 days later along with animals that were not rechallenged (day 14). Data represent mean ± SD of lymph nodes from three mice and are representative of three experiments. B, Animals were primed as in A and 11 days later rechallenged with K99Q plus poly(I:C). Three days after rechallenge, lymph node cells were incubated with PMA and ionomycin for 6 h and stained for IL-2 and IFN-{gamma} as determined by gating on adoptively transferred Thy1.1+CD4+ cells. The data are representative of seven independent experiments.

 
Lack of significant cell division after K99Q plus poly(I:C) rechallenge was in marked contrast to that observed after initial priming of naive T cells 3 days after adoptive transfer (Fig. 1A). We also asked whether T cells without initial priming (PBS control) divided upon initial challenge with K99Q plus poly(I:C) 11 days after adoptive transfer. Although division of T cells was observed 3 days later (day 14), the number of dividing cells was quite small, presumably a consequence of the poor cell recovery in transfer recipients only given PBS at the time of AND T cell transfer (Fig. 5A, see bar graph labeled control and data not shown).

TCR engagement with low-affinity ligand and poly(I:C) does not alter effector function upon restimulation with high-affinity peptide ligand

Next, we asked whether AND T cells primed with low-affinity ligand and poly(I:C) and that survived in vivo responded to rechallenge with agonist peptide. Eleven days after priming and 3 days after rechallenge with PCC88–104 or PCC88–104 plus poly(I:C) or as control PBS alone, lymph node T cells were removed from recipients and analyzed for the frequency of cytokine-secreting cells. As expected, in the absence of in vivo PCC88–104 rechallenge, AND T cells initially primed with K99Q plus poly(I:C) did not produce IFN-{gamma} and only 35% produced IL-2 ex vivo (Fig. 6, left four panels). By contrast, in response to rechallenge with PCC88–104 alone, cells initially primed with K99Q plus poly(I:C) showed rapid proliferation and IL-2 production, although without IFN-{gamma} production (Fig. 6, middle four panels). Rechallenge with PCC88–104 plus poly(I:C), on the other hand, induced proliferation and cytokine production, including IL-2 and IFN-{gamma} (Fig. 6, right four panels). T cells initially primed with poly(I:C) alone behaved similarly after rechallenge with PCC, or PCC plus poly(I:C), compared with T cells primed with K99Q plus poly(I:C) (data not shown); the number of cells producing IFN-{gamma} was only minimally, if at all, greater in mice initially primed with K99Q plus poly(I:C) than in mice treated with poly(I:C) alone (p = 0.064; data not shown). Thus, TCR engagement with low-affinity ligand in the setting of poly(I:C) does not significantly affect the sensitivity of subsequent responses to agonist peptide.



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  		FIGURE 6. T cells activated after TCR engagement with low-affinity ligand have unaltered responsiveness after rechallenge with a high-affinity ligand. Adoptively transferred, CFSE+ naive Thy1.1+ AND CD4+ T cells were primed with K99Q plus poly(I:C). Eleven days later, recipients were rechallenged with PBS (control), PCC88–104, and PCC88–104 plus poly(I:C), and 3 days later proliferation and cytokine production were assessed by gating on adoptively transferred Thy1.1+CD4+ T cells. The data shown are representative of four independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present work demonstrates that naive CD4+ T lymphocytes undergo dynamic responses in vivo following interaction with a low-affinity peptide for the TCR in the setting of strong costimulatory signals. After adoptive transfer into wild-type nonirradiated syngeneic hosts and priming with K99Q and poly(I:C) or with LPS, naive CD4+ T cells divide dependent upon B7-CD28 costimulation. The dividing cells have a distinctive pattern of surface molecule expression characterized by up-regulation of CD69 without change in expression of CD25, CD44, and CD62L and appear to rapidly undergo deletion. Cells that do not divide, yet have been activated as measured by CD69 up-regulation, do not become effector cells if restimulated with the same low-affinity ligand, even if given with poly(I:C); nevertheless, such cells maintain an effective response against agonist peptide.

T cell interaction with self-pMHC in the setting of pathogen challenge is potentially deleterious to the host due to the possible initiation of self-reactive responses. Our experiments suggest that activation and proliferation of naive T cells can occur upon initial TCR engagement with low-affinity interactions with self-ligands when costimulation is provided at the time of priming, but such activation is short-lived and leads to cell death via apoptosis. Likewise, studies in an in vitro system have demonstrated that a short or weak TCR stimulation can induce naive CD4+ T cell proliferation in the presence of costimulation, but does not promote T cell survival, eventually leading to cell death by neglect (40, 41). Although we do not know why the cells primed with K99Q and poly(I:C) underwent apoptosis, we suspect that lack of expression of the high-affinity IL-2 receptor (CD25) is a critical factor. Support for this notion comes from the observation that brief TCR stimulation of C8+ T cells in vitro is sufficient to initiate proliferation, but these cells fail to sustain CD25 up-regulation and undergo apoptosis (37). Lack of survival following such stimulation is likely a consequence of the lack of both IL-2 production and responsiveness to this cytokine. In contrast, although only a minority of cells primed by the low-affinity ligand and poly(I:C) produced IL-2 upon ex vivo stimulation with PMA and ionomycin (Fig. 5B), such low levels of endogenous IL-2 binding to the low-affinity IL-2 receptor (CD122) may be sufficient for the initial cell division that we observed (Fig. 1A).

Previous studies have demonstrated that Ag-experienced CD8+ and CD4+ cells, as measured by CD44 up-regulation, divide following poly(I:C) challenge (42, 43, 44); however, to our knowledge, this adjuvant, or LPS, is not known to have a similar effect upon naive cells. Our studies demonstrate that division of naive cells can occur upon priming of poly(I:C) or LPS, in the setting of exogenously supplied self-peptide mimic. We would anticipate that some T cells seeing physiologically expressed self-pMHC in vivo in the setting of adjuvant would also divide and subsequently undergo apoptotic death; however, these events may be difficult to detect during assessment of polyclonal T cells in vivo. We would nevertheless speculate that such abortive responses would occur in naive T cells having higher affinity for self-Ags in vivo, thereby maintaining peripheral tolerance, although this idea remains to tested experimentally.

Although naive T cell activation by a low-affinity ligand and costimulation does not produce a productive immune response, it is as important to note that such interactions also do not adversely affect sensitivity for high-affinity ligands. Transferred AND T cells that up-regulated CD69 as a result of K99Q plus poly(I:C) administration and that did not die responded well to rechallenge with the cognate PCC peptide, including with generation of effector cells, when poly(I:C) was jointly administered. Similarly, initial challenge with poly(I:C) alone led to the same outcome, presumably as a consequence of engagement of physiologic self-ligands in vivo. These phenomena highlight an essential element in T cell physiology that lymphocytes recognize and respond actively to self-Ags under certain circumstances without autoreactivity, and, indeed, such responses may help to maintain sensitivity to foreign Ags (7). In our experiments, we have assumed that the surviving transgenic T cells following K99Q plus poly(I:C) challenge have undergone TCR engagement with the low-affinity ligand, based upon their CD69 up-regulation, in comparison to T cells exposed to poly(I:C) alone. However, we acknowledge the possibility that the former cells have not been so exposed. This question is currently under investigation in our laboratory.

We also noticed that T cells that up-regulated CD69 upon poly(I:C) administration were less responsive following restimulation with K99Q 10–14 days later, suggesting that exposure to poly(I:C) alone or in concert with K99Q may induce tolerance to Ags with low affinity for TCR. This phenomenon may be analogous to TCR tuning in response to self-peptide display in vivo, the "tunable activation threshold hypothesis" (45, 46, 47). This hypothesis predicts that the threshold for cell activation is adjustable to the stimulatory experience. By extension to our work, cells experienced with low-affinity ligand may be less responsive and/or ignore restimulation with the same ligand. However, we would add the following cautionary note: we were unable to convincingly demonstrate that AND T cells adoptively transferred and left unprimed for 11 days divided upon K99Q plus poly(I:C) challenge, in contrast to the effects observed 3 days after adoptive transfer upon challenge with an identical regimen. The reason for this finding is unclear, although we suspect it was due to the poor AND T cell recovery in unchallenged animals at day 11 (Fig. 5A), since it seems unlikely that duration of transfer should affect responsiveness of naive cells.

Although naive T cell activation by low-affinity self-peptides with costimulatory signals does not promote autoreactivity in nonautoimmune mice, it is possible that such encounters may have a different outcome in mice genetically predisposed to autoimmunity. Although the mechanism of activation of self-reactive T cells in lupus is unknown, self-reactive T cells in lupus-prone mice have heightened TCR-mediated activation in response to low-avidity peptides compared with cells from control mice (12), as well as anergy avoidance in vivo (48). In other systems, a lower threshold for activation through the TCR is also associated with autoreactivity (49).

In summary, we have shown here that low-affinity TCR engagement can induce division of naive T cells in the presence of strong costimulation. Importantly, such cell division is short-lived and does not induce memory responses. Moreover, T cells that encounter low-affinity TCR ligands in the setting of costimulation maintain an effective response to high-affinity Ags. These phenomena may represent a mechanism for avoiding autoimmunity while maintaining T cell responses to pathogens.


   Acknowledgments
 
We thank Ping Zhu and Christie Hawley for animal care and gratefully acknowledge the comments of members of the Craft laboratory.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (AR 40072 and 44076), Rheuminations, Inc., the Arthritis Foundation, and a Kirkland Scholar’s Award. J.-Y.C. is a Postdoctoral Fellow of the Arthritis Foundation. Back

2 Address correspondence and reprint requests to Dr. Joe Craft, P.O. Box 208031, Room S541D, Anlyan Center, New Haven, CT 06520-8031. E-mail address: joseph.craft{at}yale.edu Back

3 Abbreviations used in this paper: self-pMHC, self-peptide MHC; PCC, pigeon cytochrome c; RAG-1, recombination-activating gene 1; AP:, altered peptide ligand; MCF, mean channel fluorescence. Back

Received for publication February 19, 2004. Accepted for publication April 16, 2004.


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 Materials and Methods
 Results
 Discussion
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