Competition for self-peptide-MHC complexes and cytokines between naïve and memory CD8⁺ T cells expressing the same or different T cell receptors

Abstract

To study competition between naïve and memory T cells, we examined proliferation of adoptively transferred naïve CD8⁺ T cells in lymphopenic recipients or recipients containing a clonal population of CD8⁺ T cells. We find a hierarchy in the extent of T cell proliferation that appears to correlate with the strength of T cell receptor (TCR)-self-peptide-MHC (pepMHC) interactions. CD8⁺ T cells also proliferate in recipients containing a full complement of CD8⁺ cells with a different TCR if the transferred T cells experience stronger TCR-self-pepMHC interactions than the resident T cells. Furthermore, CD8⁺ T cells proliferate in recipients that contain memory CD8⁺ cells with a different TCR, but in this case the relative strengths of TCR-self-pepMHC interactions are not as critical. In contrast, CD8⁺ T cells do not proliferate significantly in recipients harboring naïve or memory CD8⁺ cells that bear the same TCR as the transferred cells. These results suggest that, among naïve T cells and between naïve and memory T cells, CD8⁺ cells having the same TCR compete for both self-pepMHC and cytokines, whereas TCR-different CD8⁺ cells compete for cytokines. These competitive relationships probably help maintain the size and TCR diversity of naïve and memory T cell populations required for optimal immune responses.

In a normal adult individual, peripheral T cells include both naïve cells that have not yet encountered antigen and antigen-experienced memory T cells. Both the naïve and memory cell compartments are composed of large numbers of T cells that express diverse antigen-specific T cell receptors (TCRs). Naïve T cells are critical for initiating immune responses to novel antigens, whereas memory T cells mount more rapid and vigorous responses on reencountering the same antigens. Because the total number of peripheral T cells is maintained at a relatively constant level (homeostasis) (1, 2), a proper size and sufficient diversity of naïve and memory T cell compartments are critical for the immune system's ability to respond to unpredictable encounters with an enormous number of different antigens (3). The molecular mechanisms that regulate the size and diversity of naïve and memory T cell compartments are under active investigation but are still not well understood.

The number of naïve T cells in the periphery is determined by their production in the thymus and by their survival, proliferation, and differentiation in peripheral lymphoid organs. Studies have shown that the number of T cells remains constant irrespective of whether the pool is oversupplied or undersupplied (4), indicating mechanisms of regulation in the periphery. Indeed, survival of naïve CD4⁺ and CD8⁺ T cells requires both soluble factors, such as IL-7 (5, 6), and interactions between T cells' TCR and self-peptide-MHC complexes (pepMHC) (MHC class I for CD8⁺ T cells and MHC class II for CD4⁺ T cells) (7-11). The numbers of memory T cells are similarly regulated by their generation, survival, proliferation, and differentiation. Unlike naïve T cells, survival of memory CD4⁺ and CD8⁺ T cells does not appear to require interactions between TCR and self-pepMHC complexes (12-14). Maintenance of a stable number of memory CD8⁺ T cells requires IL-15 (or IL-7 in the absence of IL-15), which stimulates memory CD8⁺ T cells to proliferate and replenish those lost by attrition (6, 15-17). However, neither IL-15 nor IL-7 (or other factors) is known to be required for survival or proliferation of memory CD4⁺ T cells (17).

Naïve T cells do not proliferate significantly in normal adults in the absence of cognate foreign antigen (pepMHC complexes, usually with self-MHC plus peptides derived from foreign proteins). However, after severe T cell depletion induced by ionizing radiation, chemotherapy, or virus infection, the residual T cells undergo expansion in the absence of foreign antigens (18, 19). Similarly, proliferation occurs when small numbers of naïve T cells are adoptively transferred into syngeneic lymphopenic hosts, such as mice deficient in recombination activating gene (RAG). The spontaneous T cell proliferation under lymphopenic conditions is often referred to as homeostatic proliferation because it increases cell number, and it also requires IL-7 and interactions between T cells' TCR with self-pepMHC complexes (5-9, 11, 12). After proliferation in lymphopenic mice, the persisting T cells acquire surface markers and functions characteristic of antigen-induced memory T cells (20-23). Yet, after proliferation, the total T cell numbers in lymphopenic recipients are not restored to the levels found in normal adult mice (24, 25). Hence, we prefer to call this process lymphopenia-induced proliferation and differentiation. Lymphopenia-induced T cell proliferation and differentiation also occurs under normal physiological conditions, as in neonates (26-28), and the process appears to play a role in generating and/or maintaining an appropriate size and diversity of the memory T cell compartment.

Adoptive transfer of naïve T cells into lymphopenic mice has been the model of choice to investigate factors that regulate the size and diversity of T cell compartments, because it requires the same factors (IL-7 and cognate self-pepMHC complexes) as those essential for naïve T cell survival. Using this model, two recent studies examined competition among naïve T cells that express different TCRs. In one study, a clonal population of naïve CD4⁺ T cells was adoptively transferred into either RAG^- recipients or transgenic mice expressing a different TCR on CD4⁺ T cells. The transferred CD4⁺ T cells were reported to proliferate to the same extent in the two recipients, although one had no T cells and the other had a relatively normal number of CD4⁺ T cells (29). The transferred CD4⁺ T cells presumably proliferated in the CD4⁺ TCR transgenic mice because the two clonal populations of T cells did not compete for the same self-pepMHC complexes. Because lymphopenia-induced T cell proliferation also requires IL-7, these results suggest that IL-7 was sufficient for proliferation of the introduced CD4⁺ T cells but not the resident CD4⁺ T cells. In the other study, when naïve CD8⁺ T cells expressing one TCR were transferred into CD8⁺ transgenic mice expressing another TCR, or vice versa, the transferred CD8⁺ T cells proliferated to the same extent (30). Although the level of IL-7 in the transgenic hosts does not support the spontaneous proliferation of the resident CD8⁺ T cells, it is apparently sufficient to support proliferation of the transferred CD8⁺ T cells. Based on the assumption that the amount of IL-7 in normal, RAG^-, and TCR transgenic mice is finite and that endogenous naïve T cells compete for IL-7, these results are puzzling and warrant further examination.

There is also little known about the factors that regulate the relative size of naïve and memory T cell compartments. Several studies have concluded that naïve and memory CD8⁺ T cell compartments are regulated independently (12, 31, 32). If this conclusion were correct, a mechanism would be required to keep naïve and memory T cells with the same TCR from competing for the same cognate self-pepMHC complexes. That there may be such a mechanism is suggested by the finding that survival of memory T cells does not require interactions between TCR and self-pepMHC (12-14). However, in the absence of TCR-self-pepMHC interactions, memory CD4⁺ T cells lose many of their functional properties (33), suggesting that these interactions normally occur and are essential. Nevertheless, whether naïve and memory T cells with the same TCR compete for self-pepMHC complexes has not been unequivocally demonstrated. Furthermore, memory CD8⁺ T cells express receptors for both IL-7 and IL-15 and can use IL-7 for their survival and proliferation in the absence of IL-15 (6, 17). The purported independent regulation of naïve and memory CD8⁺ T cell compartments would therefore require mechanisms to prevent competition for IL-7 between naïve and memory CD8⁺ T cells. To date, no such mechanism has been found. Thus, whether naïve and memory T cell compartments are regulated independently needs to be reevaluated.

In the present study, we investigated competition among naïve CD8⁺ T cells and between naïve and memory CD8⁺ T cells expressing the same or different TCRs. We show that naïve CD8⁺ T cells can undergo limited proliferation when transferred into hosts whose naïve CD8⁺ T cells express a different TCR; whether the transferred T cells proliferate or not depends on the relative strengths of the TCR-self-pepMHC interactions of the two T cell populations. We also show that naïve CD8⁺ T cells proliferate in hosts that harbor memory CD8⁺ T cells with a different TCR, but, in this case, the relative strengths of TCR-self-pepMHC interactions are not as critical. In contrast, naïve CD8⁺ T cells do not proliferate significantly when transferred into hosts with naïve or memory CD8⁺ T cells that bear the same TCR as the transferred cells. Thus, within the naïve T cell compartment and between naïve and memory T cell compartments, CD8⁺ T cells with the same TCR likely compete for cognate self-pepMHC complexes and cytokines, whereas CD8⁺ T cells having different TCR compete for cytokines.

Materials and Methods

Mice. F5, 2C, and OT-1 TCR transgenic mice were on the RAG^- background. The transgenic mice had been backcrossed with C57BL/6 (B6, H-2^b) mice for >10 generations. RAG^- mice were backcrossed with B6 mice for 13 generations. All mice were kept in a specific pathogen-free facility and used between 6 and 10 weeks of age.

Adoptive Transfer. CD44^-/lo cells were purified by depleting CD44⁺ cells from lymph node and spleen cells of TCR transgenic mice by using magnetic beads (Miltenyi Biotec, Auburn, CA; T cell purity > 98%). To track T cell proliferation in vivo, T cells were labeled with carboxyfluorescein diacetate-succinimidyl ester (CFSE) and injected retroorbitally into various nonirradiated recipients. Proliferation of the transferred T cells in lymph nodes and spleens of recipients was assayed at various times after transfer. 2C or OT-1 “memory” mice were made by transferring 2C or OT-1 T cells into RAG^- recipients 2 months before secondary adoptive transfer.

Antibodies and Flow Cytometry. Antibodies to TCR, Vα2, CD8, CD5, CD2, CD127, and CD122 were purchased as conjugates from Pharmingen. Clonotypic antibody 1B2, specific for the 2C TCR, was conjugated to biotin. Cells were stained in the presence of 2.5 μg/ml anti-FcR antibody in PBS containing 0.1% BSA and 0.1% NaN₃ and analyzed on a FACSCalibur, collecting 10⁴ to 10⁶ live cells (propidium iodide-negative) per sample.

Results

To investigate competition among naïve CD8⁺ T cells and between naïve and memory CD8⁺ T cells, we used an adoptive transfer model involving TCR transgenic mice that expressed the F5, 2C, or OT-1 TCR on CD8⁺ T cells. Because the TCR transgenes were all backcrossed on a RAG1-deficient background (RAG^-), the CD8⁺ T cells in each of the strains expressed a single TCR. 2C and OT-1 T cells recognize distinctly different peptide-K^b complexes (34, 35) and thus are likely to recognize different self-peptide-K^b complexes. F5 T cells recognize D^b-associated peptides (36) and thus likely recognize still other self-pepMHC complexes. All three TCR transgenic strains had been backcrossed to the C57BL/6 (B6) background for at least 10 generations, allowing syngeneic adoptive transfers among these mice and into RAG^- mice that were also on the B6 background. This greatly simplified model made it possible to probe for evidence of competition for self-pepMHC and/or other factors, such as cytokines, between CD8⁺ T cells with the same or different TCRs. For simplicity, the TCR transgenic mice on the RAG-deficient B6 background are referred to as F5/RAG^-, 2C/RAG^-, and OT-1/RAG^- mice; CD8⁺ T cells from these mice are called F5, 2C, and OT-1 T cells, respectively.

Analysis of T cells from the transgenic mice revealed that the TCR on 2C T cells were about twice as abundant as on OT-1 and F5 T cells (Fig. 1A). In contrast, the levels of CD5 were highest on OT-1 T cells, lowest on F5 T cells, and intermediate on 2C T cells. CD5 levels on T cells correlate with the strength of interactions between TCR and self-pepMHC complexes (37, 38). Thus, despite the much higher level of 2C TCR expression, OT-1 TCR appears to interact more strongly with cognate self-pepMHC complexes, because of either a greater abundance of these complexes or this TCR's higher intrinsic reactivity with them. Nevertheless, all three types of T cells had similar levels of CD8, CD2, CD122 (IL-2Rβ), and CD127 (IL-7Rα). The great majority of CD8⁺ T cells from the transgenic mice exhibited a naïve phenotype as indicated by low levels of CD44, CD122, and Ly6C expression (Fig. 1B and data not shown).

Fig. 1.

Extent of proliferation of naïve CD8⁺ T cells expressing F5, 2C, or OT-1 TCR in RAG^- recipients is correlated with their levels of CD5 expression. (A) Comparison of selected surface markers among CD8⁺ T cells expressing F5, 2C, or OT-1 TCR. Lymph node cells from F5/RAG^-, 2C/RAG^-, or OT-1/RAG^- mice were assayed for levels of TCR, CD5, CD127, CD8, CD2, and CD122. Shown are the levels of various markers on live TCR⁺CD8⁺ cells. Mean fluorescence intensity (MFI) values of TCR and CD5 on CD8⁺ T cells are indicated. (B) Comparison of proliferation and differentiation of CD8⁺ T cells expressing F5, 2C, or OT-1 TCR in RAG^- recipients. CD44^-/lo cells were purified from F5/RAG^-, 2C/RAG^-, or OT-1/RAG^- mice, labeled with CFSE, and transferred into nonirradiated RAG^- recipients (1 × 10⁶ per mouse). Five days after transfer, lymph node and spleen cells were recovered from some recipients and analyzed for TCR, CD8, and CFSE. CFSE profiles are shown for TCR⁺CD8⁺ cells. The numbers refer to the average number of divisions. Thirty days after transfer, lymph node and spleen cells were recovered from other recipients and analyzed for TCR and CD8 plus CD44, CD122, or Ly6C. Expression of CD44, CD122, and Ly6C on TCR⁺CD8⁺ T cells is shown. The top row shows CD44, CD122, and Ly6C on naïve CD8⁺ cells, using 2C cells to represent naïve CD8⁺ T cells from the other TCR transgenic mice. Shown are results from one representative experiment of three.

Competition Between Naïve CD8⁺ T Cells Having Different TCRs. To compare the ability of the three transgenic T cells to proliferate in lymphopenic hosts, CD44^-/lo naïve T cells were purified from each of the TCR transgenic mice and labeled with CFSE, and the same numbers of T cells were injected into nonirradiated RAG^- mice. Five days later, some F5 cells had divided, as indicated by loss of CFSE intensity (Fig. 1B): the average number of divisions was ≈0.6. In contrast, over the same period 2C and OT-1 T cells proliferated more extensively, with an average of 2.5 and 3.4 divisions, respectively. The extent of proliferation of F5, 2C, and OT-1 T cells in RAG^- recipients correlated with their levels of CD5, suggesting that the strength of TCR-self-pepMHC interactions determines the rate of proliferation of TCR-different T cells in the same cytokine environment.

Consistent with the extent of proliferation, almost all OT-1 T cells that persisted in RAG^- recipients had acquired memory T cell markers by day 30, including up-regulation of CD44, CD122, and Ly6C (Fig. 1B). Most 2C cells that persisted in RAG^- recipients also expressed high levels of these surface markers, except CD44, which was still low on some of these cells. In contrast, by day 30 only a small fraction of F5 T cells up-regulated CD44, CD122, and Ly6C expression.

To investigate competition between T cells with different TCRs, CFSE-labeled CD44^-/lo OT-1 T cells were transferred into RAG^-, F5/RAG^-, 2C/RAG^-, and OT-1/RAG^- mice, and proliferation was examined 5 and 30 days after transfer. As shown above, OT-1 T cells proliferated extensively in RAG^- recipients; they also proliferated significantly in F5/RAG^- mice (Fig. 2A), despite the presence of a full complement of F5 T cells in these recipients. After 5 days, there were 0.8 divisions on average, and by 30 days almost all persisting OT-1 T cells had divided. Similarly, a significant fraction of the persisting OT-1 T cells in 2C/RAG^- mice had proliferated 30 days after transfer, but there was less proliferation in these recipients than in F5/RAG^- mice. In contrast, the transferred OT-1 T cells had hardly proliferated at all 30 days after transfer into OT-1/RAG^- mice. In agreement with the extent of proliferation, OT-1 T cells that proliferated in F5/RAG^- or 2C/RAG^- mice also up-regulated CD44, indicating acquisition of the memory phenotype. The relatively slow proliferation and absence of CD69 up-regulation by proliferating OT-1 cells in F5/RAG^- or 2C/RAG^- recipients (data not shown) are characteristic of lymphopenia-induced (homeostatic) T cell proliferation. These results suggest that transferred OT-1 T cells had sufficient access to self-pepMHC and IL-7 in F5/RAG^- and 2C/RAG^- mice to undergo proliferation.

Fig. 2.

Relative strength of TCR-self-pepMHC interactions (CD5 levels) correlates with the competitiveness of different naïve CD8⁺ T cells. (A) Naïve OT-1 T cells proliferate in F5/RAG^- and 2C/RAG^- recipients but not in OT-1/RAG^- recipients. CFSE-labeled CD44^-/lo OT-1 T cells were adoptively transferred into nonirradiated RAG1^-, F5/RAG^-, 2C/RAG^-, and OT-1/RAG^- recipients (1 × 10⁶ per mouse). Five and 30 days after transfer, lymph node and spleen cells were recovered from recipients and analyzed for Vα2 (specific for OT-1 TCR), CD8, and CFSE. CFSE profiles are shown for CD8⁺Vα2⁺ cells. The numbers indicate the average number of divisions. (Right) CD44 versus CFSE profiles are shown for CD8⁺Vα2⁺ cells from F5/RAG^- and 2C/RAG^- recipients 30 days after transfer. The hatched area at the left in some histograms (e.g., OT-1 cells transferred into OT-1/RAG^- recipients) is unlabeled host T cells, not CFSE-labeled donor cells that have divided many times. Shown are representative data from one of three experiments. (B) Naïve 2C T cells proliferate in F5/RAG^- recipients, but not in 2C/RAG^- and OT-1/RAG^- recipients. The experiments were performed in the same way as in A, except that 2C T cells were transferred and that clonal antibody 1B2 was used for their identification. CFSE profiles are shown for CD8⁺1B2⁺ cells. CD44 versus CFSE profiles are shown for CD8⁺1B2⁺ cells from F5/RAG^- recipients 30 days after transfer.

We also transferred CFSE-labeled CD44^-/lo 2C T cells into RAG^-, F5/RAG^-, 2C/RAG^-, and OT-1/RAG^- recipients. As expected, 2C T cells proliferated extensively in RAG^- recipients; they also proliferated in F5/RAG^- recipients 30 days after transfer (Fig. 2B). Consistent with the observed proliferation, the 2C T cells in F5/RAG^- mice up-regulated CD44, CD122, and Ly6C (Fig. 2B and data not shown). In contrast, transferred 2C T cells did not proliferate appreciably in OT-1/RAG^- or 2C/RAG^- recipients. Thus, a clonal population of naïve CD8⁺ T cells adoptively transferred into recipients harboring a full complement of a TCR-different clonal population of CD8⁺ T cells may proliferate if the strength of their TCR-self-pepMHC interactions exceeds those of resident T cells.

Competition Between Naïve and Memory CD8⁺ T Cells Having the Same or Different TCRs. To investigate competition between naïve and memory CD8⁺ T cells expressing the same TCR, we assayed proliferation of adoptively transferred naïve 2C T cells in mice that harbored a population of memory 2C T cells. Memory 2C mice were generated by adoptive transfer of naïve 2C T cells into RAG^- recipients for at least 2 months. By then, all persisting 2C T cells in the recipients expressed the memory T cell phenotype, and the number of memory 2C T cells was stable (≈2 × 10⁶; Table 1). When CFSE-labeled naïve 2C T cells were transferred into these memory recipients, only a small fraction of the transferred 2C cells proliferated 30 days after transfer (Fig. 3A), indicating that memory 2C cells effectively inhibited proliferation of the transferred naïve 2C cells in the virtually lymphopenic hosts. Because lymphopenia-induced proliferation requires TCR-self-pepMHC interaction and the presence of IL-7 (39), these results suggest that naïve and memory CD8⁺ T cells having the same TCR compete for self-pepMHC or IL-7 or both.

Table 1. Number of CD8⁺ T cells in various hosts.

	F5	2C	OT-1
TCR/RAG− donor (×107)	3.5 ± 1.1 (n = 5)	1.93 ± 0.78 (n = 10)	3.8 ± 0.9 (n = 5)
Recipients (3 months after transfer, ×106)
RAG−	0.46 ± 0.04 (n = 3)	2.4 ± 1.2 (n = 5)	2.9 ± 0.9 (n = 3)
F5/RAG−		1.78 ± 0.8 (n = 3)	1.92 ± 1.1 (n = 3)
2C/RAG−			1.1 ± 0.6 (n = 3)

Fig. 3.

Competition between naïve and memory CD8⁺ T cells expressing the same or different TCRs. (A) Naïve and memory 2C T cells compete for self-pepMHC and/or cytokines in the same hosts. Naïve 2C T cells were transferred into RAG^- recipients for 2 months. The resulting mice were referred to as memory 2C mice because they contained stable numbers of memory 2C T cells. CFSE-labeled CD44^-/lo 2C T cells were transferred into RAG^- or memory 2C recipients (1 × 10⁶ per mouse). Five and 30 days after transfer, lymph node and spleen cells were recovered from the recipients and analyzed for 1B2, CD8, and CFSE. CFSE profiles are shown for CD8⁺1B2⁺ cells. Hatched areas at the left in some histograms represent unlabeled host 2C T cells. Shown are representative data from one of two experiments. (B) Naïve OT-1 T cells and memory 2C T cells compete for cytokines in the same hosts. CFSE-labeled CD44^-/lo OT-1 T cells were transferred into RAG^- or memory 2C recipients (1 × 10⁶ per mouse). Five days after transfer, lymph node and spleen cells were recovered from the recipients and analyzed for Vα2, CD8, and CFSE. CFSE profiles are shown for CD8⁺Vα2⁺ cells. The numbers indicate the average number of divisions. Shown are representative data from one of two experiments. (C) Naïve 2C T cells and memory OT-1 T cells compete for cytokines in the same hosts. CFSE-labeled CD44^-/lo 2C T cells were transferred into RAG^- or memory OT-1 recipients (1 × 10⁶ per mouse). Five and 20 days after transfer, lymph node and spleen cells were recovered from the recipients and analyzed for 1B2, CD8, and CFSE. CFSE profiles are shown for CD8⁺1B2⁺ cells. Shown are representative data from one of two experiments.

To distinguish between these possibilities, we investigated competition between naïve and memory CD8⁺ T cells with different TCRs by adoptive transfer of naïve OT-1 T cells into memory 2C mice. In contrast to the transferred naïve 2C T cells, the transferred naïve OT-1 T cells proliferated with an average of 1.0 division by day 5 (Fig. 3B). This exceeded the extent of proliferation of OT-1 T cells in 2C/RAG^- mice (Fig. 2A), but it was still considerably less than in RAG^- recipients. Because 2C and OT-1 TCRs likely recognize different self-pepMHC complexes, the lesser extent of proliferation of OT-1 T cells in memory 2C mice, which had only ≈2 × 10⁶ memory 2C T cells, than that seen in RAG^- mice suggests that naïve and memory T cells having different TCRs compete for the same cytokines and further implies that naïve and memory T cells with the same TCR also compete for the same cytokines. The limited proliferation of OT-1 T cells in 2C/RAG^- mice (Fig. 2A) could reflect more intense competition for the same cytokines between naïve 2C and naïve OT-1 T cells because there are many more naïve 2C T cells in 2C/RAG^- mice (≈1.9 × 10⁷) than there are memory 2C T cells in memory 2C mice (2 × 10⁶).

To further examine competition among naïve and memory CD8⁺ T cells with different TCRs, we prepared memory OT-1 mice by adoptive transfer of naïve OT-1 T cells into RAG^- mice for 2 months. Then, CFSE-labeled naïve 2C T cells were transferred into memory OT-1 mice and T cell proliferation was assayed 5 and 20 days after transfer. The naïve 2C cells proliferated significantly 20 days after transfer (Fig. 3C). 2C T cell proliferation in this situation was in stark contrast to the lack of proliferation of transferred 2C cells in OT-1/RAG^- mice (Fig. 2B) but was still less than in RAG^- mice. Again, these results suggest that naïve and memory T cells having different TCRs compete for the same cytokines, but the competition appears to be less intense than between TCR-different naïve T cells, either because naïve and memory CD8⁺ T cells differ in their preference for IL-7 and IL-15 (6, 17) or because the number of memory T cells in the memory recipients is relatively small.

Size of Naïve and Memory T Cell Compartments in Various Recipient Mice. To examine factors that regulate the size of T cell compartments, we enumerated the numbers of T cells in various transgenic mice before and after T cell transfer. The average number of CD8⁺ T cells in spleen plus lymph nodes of an adult F5/RAG^-, 2C/RAG^-, or OT-1/RAG^- mouse was 2-4 × 10⁷ (Table 1). Thus, in each of these hosts, there are sufficient self-pepMHC and cytokines to support ≈2-4 × 10⁷ clonal naïve CD8⁺ T cells in the steady state.

When 1 × 10⁶ OT-1 or 2C T cells were adoptively transferred into RAG^- recipients, the number of persistent OT-1 or 2C T cells was ≈2-3 × 10⁶ even 3 months after transfer (Table 1). By 1 month after transfer, the majority of the persistent OT-1 and 2C T cells in RAG^- recipients had lost the CFSE signal (Fig. 2) and had therefore divided more than six to eight times. Thus, >90% of the transferred OT-1 and 2C T cells did not survive after proliferation in RAG^- recipients. Consistent with a lesser extent of proliferation, only 4.6 × 10⁵ F5 T cells were recovered 3 months after transfer of 1-2 × 10⁶ naïve F5 T cells into RAG^- recipients. Because the survival of memory T cells does not require TCR-self-pepMHC interaction, the low level of memory T cell survival in RAG^- recipients (only 2-3 × 10⁶ or fewer clonal memory CD8⁺ T cells per RAG^- recipient) is most likely due to a lack of survival factors, such as IL-15.

When 1 × 10⁶ naïve OT-1 or 2C T cells were transferred into F5/RAG^- mice, ≈2 × 10⁶ memory T cells were recovered after 3 months. When the same number of OT-1 T cells was transferred into 2C/RAG^- mice, the number of memory OT-1 T cells recovered after 3 months was only slightly smaller (≈1 × 10⁶). Thus, the number of memory T cells expressing a particular TCR is quite similar in different hosts.

Discussion

By comparing the extent of proliferation of different clonal naïve CD8⁺ T cell populations in the same lymphopenic recipients, we found a hierarchy in the extent of proliferation among different T cells. OT-1 T cells proliferated most, F5 T cells proliferated least, and 2C T cells were intermediate. Because the RAG^- recipients should provide the same cytokine environment, our findings suggest that the extent of lymphopenia-induced T cell proliferation is likely determined by the strength of interactions between TCR and self-pepMHC complexes. Consistent with this view, the level of CD5, which indicates the strength of TCR-self-pepMHC interactions (37, 38), was highest on OT-1 T cells, intermediate on 2C T cells, and lowest on F5 T cells. This interpretation is consistent with the notion that the strength of TCR-self-pepMHC interactions determines T cell proliferation under lymphopenic conditions (29, 40) and that 2C T cells lacking CD8 or T cells lacking CD4 do not proliferate in RAG^- recipients (41, 42).

The same hierarchy was maintained when a clonal population of naïve CD8⁺ T cells was transferred into mice that contain a full complement of CD8⁺ T cells with a different TCR. Thus, the transferred OT-1 T cells proliferated in both F5/RAG^- and 2C/RAG^- mice, with more extensive proliferation in F5/RAG^- mice, whereas the transferred 2C T cells proliferated in F5/RAG^- mice but not in OT-1/RAG^- or 2C/RAG^- mice. F5/RAG^- mice have a full complement of CD8⁺ F5 T cells and do not proliferate significantly in the absence of stimulation by foreign antigen. The levels of cytokines and self-pepMHC with which F5 TCR reacts must therefore be sufficient to support F5 T cell survival but not proliferation. Nevertheless, F5/RAG mice can support limited proliferation of OT-1 and 2C T cells. OT-1, 2C, and F5 TCRs recognize different foreign peptides (with self MHC) and likely different self-pepMHC complexes as well. Although MHC class I molecules are widely expressed on almost all cells, lymphopenia-induced T cell proliferation occurs predominantly in the secondary lymphoid organs and requires interaction with professional antigen-presenting cells (APCs) (43, 44). Thus, those T cells that can interact more effectively with self-pepMHC on APCs also compete more effectively for IL-7, possibly because IL-7 is available only in the local environment where T cells and APCs interact.

Our results differ from those reported in a recent study that investigated competition between naïve CD8⁺ T cells. Troy and Shen (30) showed that transferred naïve OT-1 T cells proliferated in transgenic recipients expressing the P14 TCR but not in OT-1 TCR transgenic recipients. However, transferred P14 T cells proliferated to the same extent in OT-1 TCR transgenic recipients but not in P14 TCR transgenic mice, suggesting that OT-1 and P14 T cells in the same hosts do not compete. One explanation for the difference between the two studies could be that CD8⁺ T cells with different TCRs were used. However, this cannot be the entire explanation, because naïve T cells also compete for IL-7. In the Troy and Shen study (30), the OT-1 and P14 TCR transgenes were not on the RAG^- background. Because the TCRα gene does not undergo allelic exclusion, it is possible that some of the T cells might have expressed endogenous TCRα chains, yielding more than one type of TCR per cell. Consistent with this notion, a large fraction of the transferred OT-1 and P14 T cells failed to proliferate in recipient mice, whereas a small fraction of the transferred T cells that did not express the transgenic TCR proliferated many rounds (30). In contrast, we observed more uniform proliferation of transferred CD8⁺ T cells. In addition, in RAG⁺ TCR transgenic animals other types of T cells are also produced, including CD4⁺CD25⁺ T cells. The presence of these and other T cells may have affected the proliferation of transferred T cells.

Our results also differ from another recent study that investigated the competition between naïve CD4⁺ T cells with different TCRs. Moses et al. (29) showed that transferred HA-specific CD4⁺ T cells proliferated at a similar rate in both DO11.10 TCR transgenic mice and RAG^- mice, whereas they did not proliferate in normal BALB/c mice or HA TCR transgenic mice. It is likely that HA and DO11.10 TCRs recognize different self-pepMHC complexes. Nevertheless, the observation that HA T cells proliferated at the same rate in both DO11.10 TCR transgenic recipients and RAG^- recipients suggests that DO11.10 T cells did not compete with the introduced HA T cells for IL-7. Yet the level of IL-7 was unlikely to be in excess, because it was not sufficient to promote DO11.10 T cell proliferation in the same hosts. Because both the DO11.10 and HA TCR transgenes were on the RAG^- background, expression of endogenous TCRα chains cannot be a contributing factor. Whether disparities between Moses et al. (29) and the present study are due to differences between CD4⁺ and CD8⁺ T cells requires further investigation.

Our findings clearly demonstrate competition between naïve and memory CD8⁺ T cells with the same TCR. The presence of only ≈2 × 10⁶ memory 2C T cells in a RAG^- recipient almost completely inhibited proliferation of transferred naïve 2C T cells, indicating that naïve and memory CD8⁺ T cell compartments are not regulated independently, in contrast to a previous conclusion (32). These findings are consistent with the expression by naïve and memory T cells of the same levels of TCR and IL-7 receptor. Although TCR-self-pepMHC interactions are not required for survival and homeostatic proliferation of memory T cells (12-14), these interactions are required for maintenance of memory CD4⁺ T cell functions (33). Because memory 2C mice express sufficient amounts of cytokines to promote naïve OT-1 T cell proliferation, our findings unequivocally demonstrate that naïve and memory T cells with the same TCR compete for the same self-pepMHC complexes.

The present study also reveals competition between naïve and memory CD8⁺ T cells with different TCRs. Transferred naïve OT-1 T cells proliferated much more extensively in RAG^- recipients containing ≈2 × 10⁶ memory 2C cells than in 2C/RAG^- recipients, containing 10 times more naïve 2C cells, but not as much as in RAG^- recipients, containing no mature T cells. Because expression of self-pepMHC is probably similar in the three types of recipient mice (29), these results suggest competition for cytokines between naïve and memory T cells with different TCRs. They likewise imply that naïve and memory T cells having the same TCR also compete for available cytokines. Indeed, both IL-15 and IL-7 can support proliferation of memory CD8⁺ T cells in lymphopenic hosts (6, 17).

Both 2C/RAG^- and OT-1/RAG^- mice contain ≈2-4 × 10⁷ CD8⁺ T cells. The levels of self-pepMHC and IL-7 are thus sufficient to support the survival of this many naïve T cells. However, even 3 months after transfer and after extensive proliferation of transferred 2C or OT-1 T cells, the numbers of memory 2C or OT-1 T cells in RAG^- recipients were 10 times lower (≈2-3 × 10⁶ per mouse). Because RAG^- mice express sufficient amounts of self-pepMHC for survival of 2-4 × 10⁷ naïve 2C or OT-1 T cells, the dramatically reduced accumulation of memory 2C or OT-1 T cells in RAG^- recipients is likely due to insufficient factors required for memory CD8⁺ T cell survival, such as IL-15. Thus, there appears to be an upper limit on the size of a clonal memory T cell population. Because the total size of the memory T cell compartment is limited, a limit on the size of any particular clonal population of memory T cells likely plays a role in maintaining TCR diversity of the memory T cell pool.