Survival of peripheral CD8+ T cells requires TCR interactions with peptide-MHC complexes (p-MHC). In the adult mouse, in the presence of homeostatic mechanisms that strictly control T cell numbers, it is likely that diverse T cell clones may compete for shared patterns of p-MHC. In the present study, we investigate whether the recognition of p-MHC overlaps between different T cell populations and what role does this process plays in the establishment of the peripheral T cell pools. Using an experimental strategy that follows the fate of adoptively transferred polyclonal T cells into RAG0/0 or different TCR transgenic RAG0/0 hosts, we demonstrate that T cells bearing different TCR specificities share identical TCR-specific requirements for survival and lymphopenia driven proliferation (LDP). This interclonal competition applies to both naive and activated/memory T cells and is partially determined by the clone size of the established/resident T cells. However, clonal competition with activated/memory resident T cells impacts differently on the fate of newly produced bone-marrow-derived T cells or adoptively transferred peripheral T cells. Overall, our findings indicate that p-MHC define multiple diverse resource niches that can be shared by T cells from different compartments.

A major criterion of successful immune reconstitution following infection (HIV) or therapeutic-induced lymphopenia is the recovery of a diverse T cell repertoire, capable to face any new microbial invasion. In this context, it is important to understand how the different processes involved in peripheral T cell pool reconstruction may interplay with the final composition of the T cell repertoire.

In the adult mouse, the number of peripheral T cells is maintained within narrow ranges and is largely independent of the numbers of precursor and thymus cells (1). The T cell repertoire size is supposed to be mainly controlled by the number of T cell clones contained in the naive pool (2). Using competitive bone marrow (BM)3 reconstitution strategies to study the establishment of the peripheral CD8+ T cell compartments, it was shown that, in the presence of continuous new cell production, each lymphocyte had to compete with other newly produced or resident cells for survival (3). Several different lines of evidence suggest the important role of TCR/self-peptide-MHC complexes (sp-MHC) interactions in the process of T cell competition. First, TCR/sp-MHC interactions are required for peripheral T cell survival, as demonstrated by the findings that the absence of either the TCR (4, 5) or specific MHC-molecules (6, 7, 8, 9, 10) leads to the disappearance of the peripheral MHC-restricted mature T cells. Secondly, in monoclonal RAG-deficient TCR transgenic (Tg) mice, the number of T cells differs according to the TCR expressed (11), suggesting that TCR specificity for sp-MHC plays an important role to define the final clone size (11, 12, 13). Finally, TCR recognition of sp-MHC has been shown to be required for the expansion of mature T cells during the peripheral T cell repopulation of immune-deficient hosts (1, 14, 15, 16). The lower naive T cell proliferation in germfree T cell-deficient hosts (17) suggests that intestinal flora drives T cell expansion either by presentation of p-MHC from commensal bacteria or by inducing the hosts APCs to increase the levels of sp-MHC expression and the release of homeostatic cytokines.

Because of the involvement of p-MHC complexes in many homeostatic T cell processes, competition among T cells in limiting conditions for defined TCR specific ligands are likely to occur. Different studies have shown that a defined TCR Tg T cell population transferred into hosts containing T cells bearing the same TCR Tg could not proliferate, despite the relative host lymphopenia (18, 19, 20, 21). These observations were consistent with the existence of competition between resident naive T cells and transferred T cells. Nevertheless, because most of these experiments involved T cells bearing identical TCRs, they did not allow any conclusion whether different T cell clones could compete for identical p-MHC. Lymphopenia driven proliferation (LDP) of transferred polyclonal CD4+ T cells has been shown to be inversely correlated to the repertoire diversity of resident LDP-derived T cells (22). However, this study only addressed the role of the number and diversity of the resident T cell clones, and provided no information concerning the potential role of the TCR specificity of each of the competing T cell clones. A more recent study suggests that interclonal competition extends largely beyond TCR specificity (21). Moreover, the occurrence of clonal competition among T cells from different compartments, which, despite sharing some cytokine resources (23), are supposed to be independently regulated (24), have never been addressed.

We have now studied the role of TCR specificity in T cell competition in different homeostatic situations. Using different TCR transgenic RAG-deficient hosts, we show that diverse polyclonal CD8+ T cell clones compete for identical p-MHC and that the clone size of resident T cells influences the expansion and accumulation of the transferred T cells in a TCR-dependent manner. We found that this interclonal competition applied to both naive and activated/memory T cells. In addition, we show that resident LDP-derived activated T cells can interfere with the peripheral establishment of newly introduced populations of monoclonal T cells. However, while newly produced BM-derived T cells were out-competed according to their TCR specificity, the accumulation of a second population of transferred mature T cells occurred in a nonspecific manner. These results indicate that p-MHC may define TCR-specific niches shared by T cells belonging to different compartments and controlled by different homeostatic processes.

C57BL/6(B6).Ly5.1, B6.PL.Thy1.1, and B6.RAG20/0 mice were from the Centre de Distribution, Typage and Archivage animal (CDTA, Orleans, France). The TCR Tg strains on a B6 background used were: the aHY RAG20/0 mice Tg for a H-2Db-restricted TCR specific for the HY-male Ag; the P14 RAG20/0 mice Tg for a H-2Db-restricted TCR specific for the gp33–41 epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV); the OT-1 RAG10/0 mice Tg for a H-2Kb-restricted TCR specific for the 257–264 peptide of OVA. All Tg mice were crossed to be Ly5.1+ or Ly5.2+ and were maintained in our specific pathogen free animal facilities according to the Experimental Ethics Committee guidelines.

Donor lymph nodes (LN) CD8+ T cells (2 × 106), enriched after depletion of CD19+ and CD4+ cells by MACS (Miltenyi Biotec), were labeled with CFSE (Molecular Probes) as described (25) and injected i.v. into RAG0/0 hosts, transgenic or not for different TCRs. For secondary transfers, donor CFSEhigh and CFSE CD8+ T cells recovered from the first hosts were sorted by flow cytometry (MoFlow, DakoCytomation). The CFSE T cells were relabeled with CFSE and ∼3000–6000 cells were transferred into secondary RAG20/0, P14 RAG20/0, or OT-1 RAG10/0 hosts. In competition experiments, sorted CFSEhigh and CFSE (relabeled with CFSE) donor CD8+ T cells were transferred alone or together with the same number of CFSE-labeled LN OT-1 T cells into RAG20/0 hosts. Hosts were killed at different times after transfer. Spleen, inguinal, and mesenteric LN cells were pooled and counted and analyzed by flow cytometry. For the sequential cell transfers, 2–3 × 106 P14 or OT-1 LN T cells were injected i.v. into RAG20/0 hosts and 4 wk later, a second subset of CFSE-labeled P14 or OT-1 LN T cells (2–3 × 106) was injected into the same hosts. The mice were killed 4 wk after the second transfer. In all experiments, we used mice expressing different Ly5 or Thy1 allotypes to discriminate the T cells from different hosts and donors.

Spleen, inguinal, and mesenteric LN cells were stained with appropriate combinations of different FITC, PE, PercP or PercPCy5.5, allophycocyanin, PE-Cy7 and allophycocyanin-Cy7 or allophycocyanin Alexa 750-conjugated anti-CD3, CD4, CD8, CD44, Ly5.1, Ly5.2, Thy1.1, Thy1.2, Vα2, Vβ8, and Vβ5 mAbs (BD Pharmingen). Acquisitions were done with LSR or Canto (BD Biosciences) flow cytometers interfaced to the CellQuest or FlowJo software.

RAG20/0 mice were injected i.v. with 2 × 106 LN T cells from P14 or OT-1. At the same time or 4 wk later, 2–7 × 106 T cell-depleted (by MACS; Miltenyi Biotec) Ly5.1+ BM precursors from P14 or OT-1 donors were injected i.v. into the same hosts. Please note that the mice were not irradiated to preserve the existing first T cell population and to avoid irradiation-induced inflammation. In these conditions, some of the transferred precursor cells colonize the empty RAG0/0 thymus and reestablish T cell development. Chimeras were analyzed 8 wk after BM cell transfer.

To evaluate the influence of resident naive T cells and their TCR specificity on the fate of newly transferred T cells, we injected CFSE-labeled polyclonal B6 CD8+ LN T cells into different lymphopenic RAG0/0 hosts, non-Tg or Tg for different class I-restricted TCRs (aHY, P14 and OT-1Tg mice), i.e., empty hosts or hosts containing monoclonal CD8+ T cell populations. As expected, after transfer into RAG20/0 hosts, the transferred T cells expanded and the vast majority of cells recovered 7 wk later had divided and lost their CFSE labeling (Fig. 1,A). When the same number of T cells was transferred into TCR Tg RAG20/0 hosts, an increased fraction of the transferred T cells did not divide and remained CFSEhigh, following the hierarchy OT-1>P14>aHY≥RAG20/0. Symmetrically, the number of CFSE cells recovered in these hosts followed the opposite hierarchy RAG20/0>aHY>P14>OT-1 (Fig. 1,B). Because the proliferation of some donor polyclonal CD8+ T cells could be just delayed by the presence of the resident T cells, we evaluated the number of nondividing (CFSEhigh) CD8+ T cells after transfer into RAG20/0 or OT-1 TCR Tg RAG10/0 hosts at different time intervals. As shown in Fig. 1,C, the total number of CD8+ T cells recovered from both RAG20/0 and OT-1 hosts reached a plateau ∼1 mo after transfer. At equilibrium the number of T cells recovered in the OT-1 hosts was 2.5 × lower than in the RAG20/0 hosts (1.5 × 106 vs 3.5 × 106). The accumulation of CFSE T cells followed the same kinetics in both types of hosts (data not shown). In contrast, while in RAG20/0 hosts virtually all nondividing (CFSEhigh) CD8+ T cells end up disappearing after transfer, in OT-1 hosts some CD8+ T cells still not divided even 2 mo after transfer (Fig. 1 D). These findings indicate that the proliferation of a fraction of the polyclonal CD8+ T cells was stably prevented in OT-1 hosts.

FIGURE 1.

Proliferation of donor polyclonal CD8+ T cells in different lymphopenic hosts. A and B, Two × 106 CFSE-labeled CD8+Thy1.1+ LN T cells were transferred into Thy1.2+RAG0/0 hosts transgenic or not for the different class I-restricted TCRs: aHY, P14 and OT-1. Mice were sacrificed 7 wk after adoptive transfer. A, CFSE profile of CD3+CD8+Thy1.1+ is shown in each case. Each FACS profile is representative of one of five mice for each type of hosts. B, Numbers of donor CFSE (left) and CFSEhigh (right) CD8+Thy1.1+ LN T cells recovered in RAG20/0 (white bars), aHY (pale gray bars), P14 (dark gray bars), and OT-1 (black bars) TCR Tg hosts. Each bar represents the mean ± SD of five mice. C and D, Total number (C) and the number of CFSEhigh (D) donor CD8+ T cells recovered at different times after the transfer of 2 × 106 polyclonal Ly5.1+CD8+ LN T cells into RAG0/0 (open symbols) or OT-1 RAG0/0 (closed symbols) hosts. E, Numbers of T cells in the different intact TCR Tg RAG0/0 hosts.

FIGURE 1.

Proliferation of donor polyclonal CD8+ T cells in different lymphopenic hosts. A and B, Two × 106 CFSE-labeled CD8+Thy1.1+ LN T cells were transferred into Thy1.2+RAG0/0 hosts transgenic or not for the different class I-restricted TCRs: aHY, P14 and OT-1. Mice were sacrificed 7 wk after adoptive transfer. A, CFSE profile of CD3+CD8+Thy1.1+ is shown in each case. Each FACS profile is representative of one of five mice for each type of hosts. B, Numbers of donor CFSE (left) and CFSEhigh (right) CD8+Thy1.1+ LN T cells recovered in RAG20/0 (white bars), aHY (pale gray bars), P14 (dark gray bars), and OT-1 (black bars) TCR Tg hosts. Each bar represents the mean ± SD of five mice. C and D, Total number (C) and the number of CFSEhigh (D) donor CD8+ T cells recovered at different times after the transfer of 2 × 106 polyclonal Ly5.1+CD8+ LN T cells into RAG0/0 (open symbols) or OT-1 RAG0/0 (closed symbols) hosts. E, Numbers of T cells in the different intact TCR Tg RAG0/0 hosts.

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We then asked about the mechanism that prevented division of a fraction of the transferred CD8+ T cells in the TCR Tg hosts. The decreased T cell proliferation could be due to the reduced availability of non-TCR related (IL-7, IL-15) and/or TCR-specific (p-MHC ligands) resources, sequestered by the resident TCR Tg T cells. If this inhibition was due to the consumption of non-TCR specific resources, it would likely be proportional to the number of peripheral T cells (Fig. 1,E) and not related to their TCR specificity. In contrast, if it was due to reduced TCR-specific resources, it should be related to the TCR specificity of the hosts T cells, i.e., the proliferation of the undivided CFSEhigh T cells recovered from a defined TCR Tg host would be only specifically blocked by the presence of T cells from the same host. To test this, we sorted polyclonal CFSE and CFSEhigh Ly5.1+CD8+ T cells from primary injected OT-1 hosts and transferred each subset (<10.000 cells) into different non-Tg and Tg Ly5.2+ secondary RAG0/0 hosts. The retransferred CFSE (relabeled with CFSE) CD8+ T cells divided extensively, losing their CFSE and becoming relatively easily detectable, whatever the type of secondary hosts they were transferred into (Fig. 2, A and C). In contrast, the CFSEhighLy5.1+CD8+ T cells also divided extensively, becoming CFSE, after transfer into non-Tg or P14 Tg RAG20/0 hosts (Fig. 2, B and C), but were not detectable in secondary OT-1 RAG10/0 hosts. This indicates that the transferred cells either died, did not proliferate, or if so, they proliferated far less than in the RAG20/0 or P14Tg RAG20/0 hosts (Fig. 2 B). Thus, while these observations exclude any intrinsic proliferation defect of these cells, they indicate that their inability to expand and accumulate was dictated by the TCR specificity of OT-1 T cells rather than due to competition for other nonspecific factors like IL-2, IL-7, or IL-15 cytokines previously involved in CD8+ T cell LDP (23, 26). Similarly, OT-1 cells proliferate in P14Tg RAG20/0 hosts, but not in OT-1 Tg RAG10/0 hosts, while P14 cells proliferate in OT-1 Tg RAG10/0 hosts, but not in P14 Tg RAG20/0 hosts (supplemental Fig. S1)4 (18). Therefore, our findings confirmed that TCR-specific resources, likely recognition of p-MHC (most probably self-ligands), control CD8+ T cell LDP. These resources seem identical with those required by resident naive T cells to survive. They may define TCR-specific niches, which both control the number of CD8+ T cells that survive and proliferate.

FIGURE 2.

Clonal inhibition of CFSEhigh donor CD8 T+ cells. CFSE-labeled Ly5.1+CD8+ polyclonal T cells were injected into Ly5.2+OT-1 RAG0/0 hosts. Five to 6 wk later, CFSEhigh and CFSELy5.1+CD8+ T cells were sorted and each population (after relabeling the CFSE T cells with CFSE) was retransferred into new RAG0/0, OT-1 RAG0/0, or P14 RAG0/0 hosts. Six weeks later, the secondary hosts were sacrificed and the presence of donor Ly5.1+CD3+CD8+ T cells derived from the CFSE (A) and CFSEhigh (B) cell subsets was assessed by flow cytometry. C, Shows the absolute number of CFSEhigh (○) or CFSE (•) derived Ly5.1+CD3+CD8+ donor T cells recovered in the OT-1 RAG0/0, P14 RAG0/0, or RAG0/0 secondary hosts. This experiment is representative of four different experiments. D, Two million CFSE-labeled polyclonal Ly5.1+CD8+ LN T cells were transferred into Ly5.2+OT-1 RAG0/0 hosts. The CD44 expression by the donor Ly5.1+CD3+CD8+ T cells was assessed by flow cytometry before transfer (histogram) and 6 wk after transfer as a function of CFSE profile (dot plot). E, CFSE-labeled Ly5.1+CD8+ polyclonal T cells were injected into Ly5.2+OT-1 RAG0/0 hosts. Six weeks later, CFSEhighLy5.1+CD8+ T cells were sorted and re-injected into secondary RAG0/0 hosts. Six weeks later, the transferred Ly5.1+CD8+ T cells, which have expanded extensively (see Fig. 2 B), were sorted again, relabeled with CFSE, and transferred into tertiary RAG0/0 or OT-1 RAG0/0 hosts. Six weeks later, the expansion of the donor T cells in RAG0/0 vs OT-1 RAG0/0 hosts was assessed by flow cytometry. Dot plots show Ly5.1+CD8+ donor T cells after gating on CD3+ T cells. Please note that, while donor T cell were present in the tertiary RAG0/0 hosts, we were unable to detect any in the tertiary OT-1 RAG0/0 hosts. This experiment is representative of two experiments.

FIGURE 2.

Clonal inhibition of CFSEhigh donor CD8 T+ cells. CFSE-labeled Ly5.1+CD8+ polyclonal T cells were injected into Ly5.2+OT-1 RAG0/0 hosts. Five to 6 wk later, CFSEhigh and CFSELy5.1+CD8+ T cells were sorted and each population (after relabeling the CFSE T cells with CFSE) was retransferred into new RAG0/0, OT-1 RAG0/0, or P14 RAG0/0 hosts. Six weeks later, the secondary hosts were sacrificed and the presence of donor Ly5.1+CD3+CD8+ T cells derived from the CFSE (A) and CFSEhigh (B) cell subsets was assessed by flow cytometry. C, Shows the absolute number of CFSEhigh (○) or CFSE (•) derived Ly5.1+CD3+CD8+ donor T cells recovered in the OT-1 RAG0/0, P14 RAG0/0, or RAG0/0 secondary hosts. This experiment is representative of four different experiments. D, Two million CFSE-labeled polyclonal Ly5.1+CD8+ LN T cells were transferred into Ly5.2+OT-1 RAG0/0 hosts. The CD44 expression by the donor Ly5.1+CD3+CD8+ T cells was assessed by flow cytometry before transfer (histogram) and 6 wk after transfer as a function of CFSE profile (dot plot). E, CFSE-labeled Ly5.1+CD8+ polyclonal T cells were injected into Ly5.2+OT-1 RAG0/0 hosts. Six weeks later, CFSEhighLy5.1+CD8+ T cells were sorted and re-injected into secondary RAG0/0 hosts. Six weeks later, the transferred Ly5.1+CD8+ T cells, which have expanded extensively (see Fig. 2 B), were sorted again, relabeled with CFSE, and transferred into tertiary RAG0/0 or OT-1 RAG0/0 hosts. Six weeks later, the expansion of the donor T cells in RAG0/0 vs OT-1 RAG0/0 hosts was assessed by flow cytometry. Dot plots show Ly5.1+CD8+ donor T cells after gating on CD3+ T cells. Please note that, while donor T cell were present in the tertiary RAG0/0 hosts, we were unable to detect any in the tertiary OT-1 RAG0/0 hosts. This experiment is representative of two experiments.

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To determine whether naive and activated/memory CD8+ T cells are equally affected by interclonal competition for TCR-specific ligands during LDP, we first characterized the phenotype of the CFSEhighCD8+ T cells, which did not proliferate after transfer into TCR Tg hosts. We observed that the frequency of CD44low (90% naive) and CD44high (10% activated/memory) in the nondividing CFSEhighCD8+ T cell fraction after transfer was similar to the frequency found in the initial CD8+ polyclonal T cells before transfer (Fig. 2 D). This observation suggests that the LDP of both naive and activated/memory T cells was inhibited through TCR-specific interactions. More precisely, we studied whether the LDP of polyclonal activated/memory CD8+ T cells was inhibited by the presence of resident T cells. For this, we first transferred sorted CFSEhigh polyclonal CD8+ T cells, recovered from previous transfer into OT-1 hosts, into RAG-deficient hosts. We tested whether these cells, once expanded in these secondary RAG-deficient hosts, would be then able to expand in tertiary OT-1 hosts. We found that, even after extensive proliferation in secondary hosts, the CD8+ memory-like T cells derived from previous undivided CFSEhigh T cells were still able to proliferate and repopulate tertiary RAG0/0 hosts, but unable to proliferate remaining undetectable in new OT-1 hosts (Fig. 2 E). Altogether, we concluded that resident host naive T cells inhibit, in a clonal specific manner, the LDP of not only naive but also activated/memory transferred CD8+ T cells.

Because resident naive OT-1 T cells could specifically prevent the proliferation of a fraction of polyclonal CD8+ T cells, we asked whether these nonproliferating CD8+ T cells could out-compete the expansion of naive OT-1 T cells during LDP. In other words, we wanted to study whether the TCR-specific requirements of OT-1 naive T cells and polyclonal CD8+ T cells for survival and LDP would somehow overlap. We cotransferred OT-1 T cells together with polyclonal CFSEhighCD8+ T cells, recovered from a primary transfer into OT-1 hosts, into new RAG20/0 hosts. We found that, 6 wk after transfer, the number of OT-1 T cells recovered in the new hosts was significantly lower when cotransferred with CFSEhighCD8+ T cells than when transferred alone, indicating that CFSEhighCD8+ T cells could out-compete OT-1 T cells (Fig. 3,A). In contrast, the cotransfer of OT-1 cells with the CFSECD8+ T cells recovered from primary OT-1 hosts did not modify the number of OT-1 T cells recovered (Fig. 3 B). It should be noted that although when transferred alone the number of CFSECD8+ T cells recovered is lower than their CFSEhigh counterpart, they proliferate as much as CFSEhighCD8+ T cells when in presence of OT-1 T cells. Thus, the CFSEhighCD8+ T cells, which did not divide in the first OT-1 host mice, specifically out-competed the OT-1 T cells during LDP, suggesting that the OT-1 and the polyclonal CFSEhighCD8+ T cells recovered from OT-1 hosts share similar TCR-specific resources to survive and proliferate.

FIGURE 3.

Clonal competition between polyclonal CFSEhighCD8+ T cells and OT-1 T cells. CFSE-labeled Ly5.1+CD8+ polyclonal T cells were injected into Ly5.2+OT-1 RAG0/0 hosts. Six weeks later, CFSEhigh (•, A) and CFSE (○, B) cells were sorted and injected alone or together with identical numbers of Ly5.2+OT-1 T cells (gray circles in both A and B into secondary RAG0/0 hosts). Six weeks after transfer, the secondary RAG20/0 hosts were sacrificed and the absolute number of each population was calculated. p values (Student’s t test) are indicated (NS = not significant). This experiment is representative of three experiments.

FIGURE 3.

Clonal competition between polyclonal CFSEhighCD8+ T cells and OT-1 T cells. CFSE-labeled Ly5.1+CD8+ polyclonal T cells were injected into Ly5.2+OT-1 RAG0/0 hosts. Six weeks later, CFSEhigh (•, A) and CFSE (○, B) cells were sorted and injected alone or together with identical numbers of Ly5.2+OT-1 T cells (gray circles in both A and B into secondary RAG0/0 hosts). Six weeks after transfer, the secondary RAG20/0 hosts were sacrificed and the absolute number of each population was calculated. p values (Student’s t test) are indicated (NS = not significant). This experiment is representative of three experiments.

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We next asked whether an established population of LDP-derived CD8+ T cells could also affect the fate of newly introduced CD8+ T cells, and whether these effects would be related to the TCR specificity of the populations involved. For this purpose, a first population of Ly5.1+ TCR Tg T cells was transferred into RAG0/0 hosts, followed 4 wk later by a second population of Ly5.2+ CFSE-labeled TCR Tg T cells. As control, each cell population was injected alone in RAG0/0 mice. Four weeks later, we studied both the CFSE dilution pattern and the number of cells recovered. The CFSE dilution profiles obtained showed that the presence of a first LDP-derived T cell subset reduced the proliferation of the second T cell subset and that this effect was more marked when resident T cells bore the same TCR as transferred T cells (Fig. 4, A and B). However, the accumulation of cells from the second injected population was significantly reduced in a nonspecific manner, i.e., independently of the TCR expressed by the first population (Fig. 4, C and D) following the rule “first come, first served”. It should be noted that the number of resident T cells was not modified by the introduction of a new cell subset (supplemental Fig. S2).

FIGURE 4.

Clonal inhibition of CD8+ T cell LDP in hosts containing LDP-derived T cells. A first subset of Ly5.1+ TCR Tg P14 or TCR Tg OT-1 LN CD8+ T cells was injected into RAG20/0 hosts. Four weeks later, a second subset of CFSE-labeled Ly5.2+ TCR Tg P14 (A and C) or OT-1 (B and D) LN CD8+ T cells was injected into RAG20/0 hosts previously injected or not with a first subset of Ly5.1+ TCR Tg P14 or OT-1 LN CD8+ T cells. Another 4 wk later, the hosts were sacrificed and the CFSE profiles of the second subset of Ly5.2+CD3+CD8+ P14 (A) or OT-1 (B) cells were analyzed after transfer into new RAG20/0 hosts empty (pale gray histogram), or containing LDP-derived P14 (black histogram) or OT-1 (dark gray histogram) T cells. Each histogram represents the data obtained from one mouse and is representative of seven to ten mice. C and D, Absolute number of cells of the second subset of Ly5.2+ TCR Tg P14 (C) or OT-1 (D) LN CD8+ T cells recovered after transfer into RAG20/0 hosts empty (pale gray circles), or containing LDP-derived P14 (•) or OT-1 (dark gray circles) T cells. p values are indicated. The data shown are a pool of two different experiments.

FIGURE 4.

Clonal inhibition of CD8+ T cell LDP in hosts containing LDP-derived T cells. A first subset of Ly5.1+ TCR Tg P14 or TCR Tg OT-1 LN CD8+ T cells was injected into RAG20/0 hosts. Four weeks later, a second subset of CFSE-labeled Ly5.2+ TCR Tg P14 (A and C) or OT-1 (B and D) LN CD8+ T cells was injected into RAG20/0 hosts previously injected or not with a first subset of Ly5.1+ TCR Tg P14 or OT-1 LN CD8+ T cells. Another 4 wk later, the hosts were sacrificed and the CFSE profiles of the second subset of Ly5.2+CD3+CD8+ P14 (A) or OT-1 (B) cells were analyzed after transfer into new RAG20/0 hosts empty (pale gray histogram), or containing LDP-derived P14 (black histogram) or OT-1 (dark gray histogram) T cells. Each histogram represents the data obtained from one mouse and is representative of seven to ten mice. C and D, Absolute number of cells of the second subset of Ly5.2+ TCR Tg P14 (C) or OT-1 (D) LN CD8+ T cells recovered after transfer into RAG20/0 hosts empty (pale gray circles), or containing LDP-derived P14 (•) or OT-1 (dark gray circles) T cells. p values are indicated. The data shown are a pool of two different experiments.

Close modal

We also studied whether a previously established population of LDP-derived CD8+ T cells could affect the fate of a newly produced population of CD8+ T cells. To test this, we performed a set of experiments in which T cell-depleted BM TCR Tg precursors were injected into nonirradiated RAG20/0 hosts either simultaneously or 4 wk after TCR Tg LN T cells transfer. We choose to not irradiate the hosts to avoid any inflammatory response, which, through the release of cytokines, may disturb homeostatic process, and to preserve the existing first T cell population during sequential transfer. The mice were sacrificed 8 wk after BM engraftment and the numbers of each of the T cell subsets evaluated. In these settings, some of the transferred precursor cells colonize the empty RAG0/0 thymus and reestablish T cell development. The BM-derived T cells migrated from the thymus to the periphery at a time when “LN subset” already reached a plateau 3–4 wk after transfer. We first assessed the fate of BM-derived OT-1 T cells in the periphery of hosts containing LDP-derived OT-1 T cells. As shown in Fig. 5,A, the number of BM-derived OT-1 T cells is decreased more than twice (11.3 vs 4.8 × 106 cells) in the presence of LDP-derived OT-1 T cells, while the number of LDP-derived OT-1 T cells was unaffected by the generation of BM-derived T cells. To determine whether this inhibition of new T cell establishment depends on the TCR specificity of the preinstalled LDP-derived T cell population, we performed another experiment in which the T cell-depleted BM cells exhibited either the same or different TCR specificity as the LDP-derived T cells. As exemplified for the transfer of OT-1 BM precursors into RAG0/0 containing or not LDP-derived OT-1 T cells, the presence of the LDP-derived resident T cells did not alter either the DN, DP, and SP thymus subsets or the subsequent CD44 expression level of the peripheral BM-derived T cells (Fig. 5,B). Thus, resident T cells did not affect either the thymic T cell development from the subsequently injected BM precursors, or their phenotype at the periphery. However, as shown in Fig. 5,C, the number of BM-derived peripheral P14 and OT-1 T cells was significantly decreased in the presence of LDP-derived T cells bearing the same TCR specificity, i.e., from the same TCR Tg donors. In contrast, the phenotype and the number of LDP-derived T cells remained unaffected by the new incoming BM-derived T cells (Fig. 5, B and D). These results indicate that the peripheral accumulation of newly developing T cells is impaired by the presence of resident T cells bearing the same TCR specificity, i.e., in a TCR-dependent manner.

FIGURE 5.

Clonal inhibition of BM-derived T cell accumulation in hosts containing LDP-derived T cells. A, LN CD8+ Ly5.2+ T cells (•) and BM Ly5.1+ (gray circles) cells from OT-1 RAG0/0 hosts were injected into empty RAG20/0 hosts either alone or together. The number of peripheral OT-1 T cells originated from each subset was determined 8 wk later. B–D, LN CD8+ Ly5.2+ T cells and BM Ly5.1+ precursor cells from either TCR Tg P14 or OT-1 RAG0/0 donor mice were sequentially injected into empty RAG20/0 hosts. Eight weeks after BM transfer, the hosts were sacrificed. B, The thymic (first column) and peripheral OT-1 BM-derived populations (second column) were compared after transfer into either empty RAG20/0 hosts (first row) or those containing LDP-derived OT-1 T cells (second row), for the representation of DN, DP, and SP subsets and CD44 expression levels, respectively. Third column, CD44 expression of LDP-derived resident OT-1 T cells upon adoptive transfer into RAG0/0 (first row) or into RAG0/0 in the presence of BM-derived T cells as well (second row). C, The absolute numbers of BM-derived P14 (diamonds) and OT-1 (circles) Ly5.1+CD3+CD8+ were calculated after transfer alone (pale gray symbols) or in the presence of LN P14 (black symbols) or OT-1 (dark gray symbols) CD8+ T cells. p values are indicated. D, The absolute numbers of LN-derived P14 (left) and OT-1 (right) Ly5.1+CD3+CD8+ were calculated after transfer alone (diamonds) or in the presence of BM-derived P14 (○) or OT-1 (•) T cells. p values are indicated.

FIGURE 5.

Clonal inhibition of BM-derived T cell accumulation in hosts containing LDP-derived T cells. A, LN CD8+ Ly5.2+ T cells (•) and BM Ly5.1+ (gray circles) cells from OT-1 RAG0/0 hosts were injected into empty RAG20/0 hosts either alone or together. The number of peripheral OT-1 T cells originated from each subset was determined 8 wk later. B–D, LN CD8+ Ly5.2+ T cells and BM Ly5.1+ precursor cells from either TCR Tg P14 or OT-1 RAG0/0 donor mice were sequentially injected into empty RAG20/0 hosts. Eight weeks after BM transfer, the hosts were sacrificed. B, The thymic (first column) and peripheral OT-1 BM-derived populations (second column) were compared after transfer into either empty RAG20/0 hosts (first row) or those containing LDP-derived OT-1 T cells (second row), for the representation of DN, DP, and SP subsets and CD44 expression levels, respectively. Third column, CD44 expression of LDP-derived resident OT-1 T cells upon adoptive transfer into RAG0/0 (first row) or into RAG0/0 in the presence of BM-derived T cells as well (second row). C, The absolute numbers of BM-derived P14 (diamonds) and OT-1 (circles) Ly5.1+CD3+CD8+ were calculated after transfer alone (pale gray symbols) or in the presence of LN P14 (black symbols) or OT-1 (dark gray symbols) CD8+ T cells. p values are indicated. D, The absolute numbers of LN-derived P14 (left) and OT-1 (right) Ly5.1+CD3+CD8+ were calculated after transfer alone (diamonds) or in the presence of BM-derived P14 (○) or OT-1 (•) T cells. p values are indicated.

Close modal

In the present study, we evaluated to which extent specific TCR/p-MHC interactions may overlap between different CD8+ T cell clones. We found that clonal competition, determined by specific TCR/p-MHC interactions (most probably self-ligands), affects both naive and activated CD8+ T cells and occurs during either peripheral cell accumulation after thymic output or peripheral T cell recovery after LDP.

LDP of monoclonal TCR Tg T cells is blocked after transfer into hosts containing monoclonal T cells bearing the same TCR (18, 19, 20) and our own results (supplemental Fig. 1). We now extend this concept of “intraclonal competition” during LDP, to that of “interclonal competition,” by showing that the proliferation of some adoptively transferred polyclonal CD8+ T cells is strictly and stably blocked by the presence of specific T cell clones in the lymphopenic hosts. This interpretation is strongly supported by the observation that the polyclonal CD8+ T cells that were unable to proliferate in OT-1 hosts specifically out-competed the OT-1 T cells when both T cell populations were cotransferred into RAG20/0 hosts, while other T cell clones could not. This indicates that these cells disclose the same TCR-specific ligands or requirements to survive and proliferate during LDP. In addition, we found that these requirements were shared by both naive and activated LDP-derived CD8+ T cells. It should be noted that after transfer into RAG20/0 hosts, we detected (day 9) a small fraction of CFSElow CD8+ polyclonal T cells. These cells were not observed after transfer into OT-1 hosts and likely correspond to a “slow proliferating” population (27). The progressive dilution of CFSE labeling over time (day 28; all cells become CFSE by day 60) led to their complete disappearance, suggesting that some of these cells proliferated in the hosts, probably in response to unlimited access to IL-7 and MHC-ligand resources (27).

The fraction of polyclonal CD8+ T cells whose proliferation was inhibited after adoptive transfer into TCR Tg hosts varied according to the TCR specificity of monoclonal resident T cells and followed the hierarchy OT-1>P14>aHY hosts. It was previously shown that the LDP of naive CD4+ T cells is not controlled by the number of resident T cells but by their repertoire diversity (22). In their study, Min et al., (22) generated RAG-deficient hosts containing identical numbers of memory CD4+ T cells, but with different T cell repertoires complexity, by transferring different numbers of naive CD4+ T cells. They showed that the expansion of a second polyclonal naive CD4+ T cell population transferred into these different hosts was inversely proportional to the TCR diversity of the resident memory T cells. Because in the monoclonal RAG-deficient TCR Tg mice, the number of T cells varies according to the TCR expressed and was higher in OT-1 than in P14 or aHY hosts, the differential expansion of polyclonal CD8+ T cells that we observed could be related to the clone size of the resident T cells rather than to their diversity. Recent findings from our laboratory, showing that T cell clone size is determined according to the TCR promiscuity, i.e., cells with a broader pattern of specificities occupying a larger space (11), reconcile our present results with those of Min et al. (22). Indeed, one can easily postulate that promiscuous TCR-bearing resident T cells, like OT-1 T cells, which interact with more p-MHC ligands (11) or the P14 T cells that also interact with different self-peptides (28), will be more numerous than the less promiscuous aHY T cells. In the case of the transferred polyclonal T cells, the number of different T cells that will not proliferate would be determined by the availability of p-MHC ligands that are not sequestered by the resident T cells.

Using sequential transfers of mature T cells, we found that, while a population of resident naive cells selectively inhibited the proliferation and accumulation of newly transferred CD8+ T cells bearing the same TCR specificity, LDP-derived resident T cells inhibited the expansion of all newly transferred T cells independently of their TCR specificity. They suggest that TCR interactions may also increase the first arrived LDP-derived T cells ability to compete for and consume non-TCR specific trophic resources required for the survival and accumulation of newly transferred T cells (20, 21). IL-7 or IL-15 cytokines, shown to be involved in memory CD8+ T cell survival, are the most likely candidates. Alternatively, LDP-derived resident cells could actively inhibit or eliminate the newly proliferating T cells through TNF-α or IFNα secretion, by a mechanism that was previously shown to be involved in memory CD8+ T cell attrition (29). Altogether, these observations strongly suggest that the survival requirements of naive and LDP-derived CD8+ T cells do not completely overlap. Although they both require recognition of p-MHC, the activated LDP-derived T cells seemed to be more capable of using other “non-TCR specific” resources like cytokines.

By transferring BM precursors instead of mature T cells into hosts containing LDP-derived T cells, we found that the resident population selectively impaired the accumulation of newly produced thymus emigrants bearing the same TCR, thus in a TCR-specific manner. Most likely, the LDP-derived long-term resident T cells monopolize TCR-specific resources required by recent T cells to accumulate in the peripheral pools. Despite the well-established differences of requirements between naive and memory T cell survival (24, 30, 31), our findings suggest that they may share enough resources to establish some form of pre-emptive competition as previously observed using parabiosis experiments (3). This pre-emptive competition would favor peripheral T cell diversity, since the new developed T cells, expressing redundant existing specificities, do not survive. Interestingly, LDP-derived resident T cells affect differently recent T cells arising from BM precursors and adoptively transferred mature T cells, suggesting that these two latter populations have different survival requirements and/or competitive fitness. Alternatively, compared with the limited number of cells introduced upon adoptive transfer, the higher number of continuously produced recent thymic emigrants, may justify the different final outcomes.

In contrast, resident activated monoclonal T cells were unaffected by the entry of newly thymus-produced monoclonal T cells into the peripheral pools (Fig. 5, B and D). Other studies have shown that polyclonal BM-derived T cells were able to replace CD4+ TCR Tg LDP-derived T cells, but not memory A1 T cells (32). These differences may suggest different behaviors for T cell populations involved and/or in the model systems used. Replacement of peripheral T cells should vary with the rates of production and the diversity of the T cells produced (33). Diverse polyclonal T cells by their increased ability to exploit different niches should in long-term replace, at least partially, established monoclonal T cells (3).

Overall, our results strongly support that in the absence of intentional immunization, competition for overlapping p-MHC ligands controls peripheral CD8+ T cell survival, proliferation, and accumulation. This implies the existence of a complex network of TCR/p-MHC interactions defining specific niches, which regulate several homeostatic events and dictate the final composition of the T cell pools (34). The fact that resident naive T cells prevent the establishment of new T cells expressing identical TCR specificities favors peripheral T cell repertoire diversity. In contrast, the fact that LDP-derived activated T cells prevents, in a non-TCR specific manner, the accumulation of other activated T cell subsets might enhance the efficiency of an immune response by avoiding bystander expansion of non-Ag specific cells and favoring the expansion of the appropriate set of responding cells. These findings may help understanding the homeostatic events leading to the establishment of a new T cell repertoire and occurring during the reconstitution of the peripheral T pools in individuals suffering from infection or therapeutic-induced lymphopenia and potentially subjected to LDP processes.

We thank Anne Louise from the Cytometry Platform at the Pasteur Institute for the FACS sorting experiments.

The authors have no financial conflict of interest.

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

C.L. is supported by a grant from the Portuguese Foundation for Science and Technology (FCT) and by a Pasteur-Weizmann grant. This work was supported by the Pasteur Institute, the Centre National de la Recherche Scientifique, and grants from Agence Nationale de Recherches sur le SIDA and Association pour la Recherche sur le Cancer.

3

Abbreviations used in this paper: BM, bone marrow; sp-MHC, self-peptide-MHC complex; Tg, transgenic; LDP, lymphopenia driven proliferation; LCMV, lymphocytic choriomeningitis virus; LN, lymph node.

4

The online version of this article contains supplemental material.

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