Homeostatic Proliferation of a Qa-1b-Restricted T Cell: A Distinction between the Ligands Required for Positive Selection and for Proliferation in Lymphopenic Hosts

Barbara A. Sullivan, Lisa M. Reed-Loisel, Gilbert J. Kersh and Peter E. Jensen
J Immunol November 15, 2004, 173 (10) 6065-6071; DOI: https://doi.org/10.4049/jimmunol.173.10.6065

Abstract

Naive T cells proliferate in response to self MHC molecules after transfer into lymphopenic hosts, a process that has been termed homeostatic proliferation (HP). Previous studies have demonstrated that HP is driven by low level signaling induced by interactions with the same MHC molecules responsible for positive selection in the thymus. Little is known about the homeostatic regulation of T cells specific for class Ib molecules, including Qa-1 and H2-M3, though it has been suggested that their capacity to undergo homeostatic expansion may be inherently limited. In this study, we demonstrate that naive 6C5 TCR transgenic T cells with specificity for Qa-1b have a capacity similar to conventional T cells to undergo HP after transfer into sublethally irradiated mice. Proliferation was largely dependent on the expression of β2-microglobulin, and experiments with congenic recipients expressing Qa-1a instead of Qa-1b demonstrated that HP is specifically driven by Qa-1b and not through cross-recognition of classical class I molecules. Thus, the same MHC molecule that mediates positive selection of 6C5 T cells is also required for HP. Homeostatic expansion, like positive selection, occurs in the absence of a Qa-1 determinant modifier, the dominant self-peptide bound to Qa-1 molecules. However, experiments with TAP−/− recipients demonstrate a clear distinction between the ligand requirements for thymic selection and HP. Positive selection of 6C5 T cells is dependent on TAP function, thus selection is presumably mediated by TAP-dependent peptides. By contrast, HP occurs in TAP−/− recipients, providing an example where the ligand requirements for HP are less stringent than for thymic selection.

The size of the lymphocyte pool is regulated through homeostatic mechanisms that appear to act independently on various lymphocyte subpopulations, including naive and memory T cells, B cells, and NK cells (1, 2, 3). Naive T cells have a long lifespan and a very low rate of proliferation in the absence of overt antigenic stimulation. However, naive T cells actively proliferate after transfer into T cell-deficient hosts, a phenomenon that has been termed homeostatic proliferation (HP)3 or lymphopenia-induced homeostatic expansion. Both long-term survival and HP of naive T cells require TCR interactions with appropriate self MHC-peptide ligands (1, 4, 5, 6, 7, 8, 9, 10). Low level signaling through interactions with self MHC-peptide complexes appears to occur constitutively in vivo, resulting in partial tyrosine phosphorylation of TCR ζ-chains (11). Removal of T cells from self-ligands results in rapid loss of the ζ-chain phosphorylation and reduced sensitivity to stimulation with foreign Ag (12). Thus, self-recognition by naive T cells modulates the activation threshold as well as promoting long-term survival and HP in lymphopenic hosts. The nature of the specific MHC-peptide ligands required for survival and HP remains an open question. It is appealing to consider the possibility that the same self-ligands that mediate positive selection in the thymus also stimulate the low-level signaling required for survival and HP in the periphery. A number of studies have provided evidence that the selecting MHC ligand is required for HP, and that there is a strong correlation between the self peptide repertoires that mediate thymic selection and HP (6, 8, 9, 10, 13). However, other evidence has been interpreted to indicate that the peptides that control proliferation of naive T cells in lymphopenic mice are distinct from those responsible for thymic selection (7, 14).

In addition to conventional TCRαβ T cells with specificity for MHC class II and class Ia molecules, there are subpopulations of T cells with specificity for nonclassical class I (class Ib) MHC molecules, such as Qa-1 and H2-M3. The physiological role of these T cells is actively being investigated and little is known about their homeostatic regulation. However, it is clear that class Ib-restricted T cells represent only a small fraction of the peripheral T cell pool. Very few CD8+CD4− single positive (SP) thymocytes and CD8+ peripheral T cells are present in KbDb−/− mice, which express class Ib but not class Ia molecules (15, 16). Low thymic output of class Ib-restricted T cells might reflect a low frequency of TCR with appropriate specificity for class Ib molecules in the preselection T cell repertoire. Alternatively, the relatively low levels of cell surface expression of class Ib molecules or the restricted diversity of self-peptides presented by these molecules may limit selection efficiency. It is also clear that these T cells do not proliferate extensively after leaving the thymus to fill the peripheral CD8 T cell compartment. It is possible that the abundant CD4+ T cells in these mice inhibit the expansion of the small population of class Ib-restricted T cells. Alternatively, ligand availability or intrinsic properties of class Ib-restricted T cells may limit peripheral expansion. Kurepa et al. (16) recently reported that CD8+ T cells from KbDb−/− donors proliferate poorly in lymphopenic sublethally irradiated syngeneic hosts. This suggests that the paucity of CD8+ T cells in KbDb−/− might reflect an inability of T cells selected on class Ib molecules to undergo homeostatic expansion. It may be relevant to note that CD1d-restricted NKT cells also have a markedly reduced capacity to proliferate in lymphopenic hosts and the proliferation that is observed is not dependent on expression of the selecting ligand CD1d (17). The pathways for thymic selection by CD1d, Qa-1, and H2-M3 appear to differ from that required for selection of conventional T cells by class Ia molecules. In each of these cases, positive selection can be mediated by interaction of thymocytes with the relevant ligand expressed selectively on hemopoietic lineage cells in the thymus (18, 19, 20, 21). By contrast, conventional T cells are selected by interaction with MHC molecules expressed on radioresistant epithelial cells in the thymus (22, 23). This alternative developmental pathway might imprint class Ib-restricted T cells with intrinsic properties that limit their capacity to undergo homeostatic expansion in the periphery (21).

In the present study, we used TCR transgenic T cells with specificity for Qa-1b to investigate the potential for Qa-1-restricted T cells to undergo HP after transfer to lymphopenic sublethally irradiated hosts. The H-2 T23-encoded class Ib molecule Qa-1 assembles with β2-microglobulin (β2m) and it is expressed in a wide range of tissues similar to class Ia molecules. However, Qa-1 is relatively nonpolymorphic, it is expressed at lower levels than class Ia molecules, and it predominantly binds a single nonameric self-peptide, “Qa-1 determinant modifier” (Qdm), derived from the leader sequence of H-2D/L class Ia molecules (24, 25, 26, 27). Presentation of Qdm is strictly dependent on TAP function (25). Despite a highly restricted diversity of presented self-peptides, Qa-1 has been demonstrated to have the capacity to present foreign and self peptides to CD8+ T cells (28, 29, 30, 31) through both TAP-dependent (28, 32) and TAP-independent (31) presentation pathways. We previously characterized the requirements for thymic development of TCR transgenic 6C5 CD8+ T cells with specificity for an insulin B chain epitope presented by Qa-1b (20). Positive selection of 6C5 transgenic T cells requires interaction with Qa-1b expressed on either hemopoietic or epithelial cells in the thymus. The dominant Qa-1-associated peptide, Qdm, is not required for selection, because 6C5 T cells develop efficiently in Db−/− hosts, which lack a source of Qdm. However, positive selection is strictly dependent on TAP function, despite the fact that the cognate Ag (insulin) is presented to these T cells through a TAP-independent pathway. These findings suggest that 6C5 T cells are selected in the thymus through interaction with Qa-1 molecules bearing TAP-dependent peptide(s) other than Qdm. In the current study, we report that 6C5 T cells undergo robust proliferation after transfer into sublethally irradiated hosts. HP is dependent on expression of the selecting MHC molecule Qa-1b. However, the observation that HP occurs in TAP-deficient hosts suggests that the peptide ligand(s) required for HP may differ from those required for positive selection in the thymus.

Materials and Methods

Mice

C57BL6/J, β2m −/− (B6.129P2-B2mtm1Unc), B6.PL (B6.PL-Thy1a/Cy), congenic B6.Tlaa (B6.A-H2-T18a/BoyEg), and TCR transgenic P14 mice (with specificity for lymphocytic choriomeningitis virus gp33/H-2Db) were purchased from The Jackson Laboratory (Bar Harbor, ME). TAP−/− mice (B6.129S2-Abcb2tm1Arp), Db−/− mice (33), OT-1 TCR transgenic (with specificity for OVA 357–364/Kb) (9), and 6C5, 6C5.β2m−/−, and 6C5.RAG−/− TCR transgenic mice (20) have been described previously. All mice were maintained following institutional guidelines.

Adoptive transfer

For analysis of lymphopenia-induced proliferation, cells were labeled with the fluorescent dye CFSE (Molecular Probes, Eugene, OR). Briefly, ∼107 cells (from a single cell suspension of splenic lymphocytes) were washed twice with room temperature PBS and incubated with 2.5 μM CFSE in a volume of 2 ml for 5 min. The reaction was quenched by the addition of 400 μl of calf serum for 30 s and the cells were immediately washed twice with PBS. Cells were then transferred i.v. into mice that had been sublethally irradiated (600–700 rad) 3–24 h earlier. Six days after transfer, spleen and inguinal lymph node cells were combined and analyzed for CFSE expression by flow cytometry.

Bead sorting

Single cell suspensions prepared from freshly isolated lymph nodes (inguinal) and spleens were incubated with bead-conjugated anti-CD8α (53-6.7) (1/10 dilution) in MACS buffer (PBS with 2 mM EDTA, 0.2% BSA) for 25 min at 4°C. Cells were washed twice with cold PBS and passed over a MACS column (LS or MS) according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Positively selected cells were 90–95% CD8α+.

Abs and flow cytometry

Single cell suspensions prepared from freshly isolated lymph nodes (inguinal) and spleens were incubated with mAbs (1/100 dilution) for 25 min at 4°C. Cells were washed twice, fixed with 1% paraformaldehyde, and analyzed by flow cytometry. FITC, PE, PerCP, or biotin-conjugated Abs specific for murine CD3 (17A2), CD4 (GK1.5), CD5 (53-7.3), CD8α (53-6.72), CD8β (53-5.8), CD11a (2D7), CD24 (M1/69), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD90.1 (OX-7), CD122 (TM-β1), CD127 (B12-1), Ly6C (AL-21), Vα2 (B20.1), Vα3.2 (RR3-16), Vβ5.1/5.2 (MR9-4), Vβ8.1/8.2 (MR5-2), (BD Pharmingen, Mountain View, CA) and biotinylated anti-CD4 (GK1.5) (American Type Culture Collection (ATCC), Manassas, VA) were used in various combinations for flow cytometric analysis. Biotinylated Abs were developed with allophycocyanin-conjugated streptavidin (Molecular Probes, Eugene, OR). Fluorescence was analyzed using a FACSCalibur (BD Biosciences, San Diego, CA). Live gates were set on lymphocytes by forward and side scatter profiles. Live lymphocytes (∼106) were collected and then analyzed using CellQuest software (BD Biosciences). For sorting, a single cell suspension of mature lymphocytes from 6C5.RAG−/− mice was washed and stained for expression of CD8α and CD44, or first bead sorted for CD8α (53-6.72) according to the manufacturer’s protocol (Miltenyi Biotec) and then stained for CD44 expression. Cells were sorted from the lymphocyte gate on the basis of CD8α and CD44 expression on a FACSVantage (BD Biosciences) to 98% purity.

Radiation bone marrow chimeric mice

CD8-depleted bone marrow cells (107) from various donors were injected i.v. into lethally irradiated (1060 rad) recipient mice. After an 8 wk recovery, spleens were removed and analyzed by flow cytometry.

Results

Only a small fraction of Qa-1-restricted 6C5 T cells have a memory phenotype

Unlike conventional CD8+ T cells, the large majority of MHC class Ib-restricted CD8+ T cells present in naive class Ia-deficient KbDb−/− mice show a CD44high activated/memory phenotype (16, 21, 34). Urdahl et al. (21) reported that activation/memory markers are already present on a significant subpopulation of mature CD8+ SP thymocytes in KbDb−/− mice, suggesting that the memory phenotype is acquired during development in the thymus. Analysis of bone marrow chimeric mice demonstrated that polyclonal class Ib (H2-M3) restricted T cells with specificity for Listeria monocytogenes differ from conventional T cells in that they are efficiently selected when class I molecules are expressed only on hemopoietic cells in the thymus (21). This finding, coupled with similar results from experiments with Qa-1-restricted TCR transgenic 6C5 T cells (20), supports the idea that class Ib-restricted T cells might in general differ from conventional T cells in their mechanism for thymic selection. This alternative developmental pathway might lead to differences in both the phenotype (memory markers) (21) and functional properties (for example, capacity for homeostatic expansion) between class Ib- and class Ia-restricted T cells.

Based on these findings, we were interested in knowing whether Qa-1-restricted 6C5 T cells display a memory phenotype similar to the polyclonal class Ib-restricted CD8+ T cells in KbDb−/− mice. As a positive control, we used B6→β2m−/− bone marrow chimeric mice (21), containing peripheral CD8+ T cells that are exclusively selected by interaction with class I molecules expressed on hemopoietic cells. Confirming previous results (21), the CD8+ T cells in these chimeric mice almost uniformly have a CD44highLy6ChighCD122highCD11ahigh activated/memory phenotype (Fig. 1⇓). By contrast, a relatively small subpopulation of CD8+ T cells with memory phenotype is present in unmanipulated B6 mice, and even fewer cells with this phenotype are present in 6C5 mice. The fraction of CD8+ cells with activated/memory phenotype in 6C5 mice was quite similar to the fraction present in class Ia-restricted OT-1 (Kb) and P14 (Db) TCR transgenic animals. A somewhat larger fraction of CD8+ 6C5 T cells displayed a CD62Llow memory phenotype, but this was also true for OT-1 T cells. We conclude that 6C5 T cells largely retain a naive phenotype similar to class Ia-restricted T cells. Thus, the acquisition of an activated/memory phenotype is not an obligatory consequence of positive selection by the class Ib molecule Qa-1.

FIGURE 1.
FIGURE 1.

Mature 6C5 transgenic T cells do not have an activated phenotype. Analysis of lymphocytes from C57BL/6, 6C5, OT1, and P14 TCR Tg+/− mice and B6→β2m chimeric mice. Lymphocytes from the spleen were analyzed by flow cytometry for cell surface expression of CD11a, CD44, CD62L, Ly6C, and CD122 on CD8+ TCR transgenic T cells (CD8+Vα3.2+ lymphocytes for 6C5 and CD8+Vα2+ lymphocytes for P14 and OT1 mice) or polyclonal CD8+ T cells (for B6 and B6→β2m bone marrow chimeric mice). Filled histograms are shown overlaid with open histograms for the isotype control. Numbers indicate the percentage of indicated subpopulations. Data are representative of at least three mice in each group.

6C5 T cells proliferate after transfer into sublethally irradiated mice

As noted above, there is evidence that class Ib-restricted T cells may have a restricted capacity for homeostatic expansion (16). To further investigate this issue, we measured the expression of the IL-7R (CD127) on 6C5 transgenic T cells compared with CD8+ T cells from C57BL/6 and P14 mice. IL-7 signaling is required for naive T cell HP (1). 6C5 T cells expressed high levels of the IL-7R, equivalent to the level on P14 CD8+ T cells and polyclonal CD8+ T cells (Fig. 2⇓ and data not shown). Next, we determined the ability of the 6C5 T cells to proliferate in lymphopenic mice. We transferred CFSE-labeled CD8+ T cells (1–2 × 106) from P14 and 6C5 mice into sublethally irradiated C57BL/6 recipients. Six days after transfer, the combined inguinal lymph nodes and the spleen were analyzed for the dilution of the CFSE dye indicative of proliferation. The 6C5 and P14 CD8+ T cells each proliferated in the lymphopenic C57BL/6 host after 6 days (Fig. 2⇓). The extent of proliferation was only slightly reduced for 6C5 cells as compared with P14 T cells. As expected, no proliferation was observed after transfer of CFSE-labeled 6C5 T cells into unirradiated B6 recipients (data not shown).

FIGURE 2.
FIGURE 2.

6C5 transgenic T cells undergo efficient lymphopenia-induced proliferation. Left panels, CD8+ T cells from 6C5 and P14 TCR transgenic mice were labeled in vitro with CFSE and transferred into sublethally irradiated (600 rad) B6 mice. Six days after transfer, lymphocytes from the spleens of recipient mice were analyzed for cell surface expression of CD8α and either Vα3.2 (6C5) or Vα2 (P14). Proliferation of CD8+ T cells is shown by the dilution of CFSE. Right panels, Lymphocytes from the spleens of 6C5 and P14 TCR transgenic mice were analyzed for cell surface expression of the IL-7R, CD127. Filled histograms of CD127 expression are shown overlaid with open histograms for the isotype control.

Ligand requirements for homeostatic proliferation of Qa-1-restricted 6C5 T cells

We next evaluated the role of MHC class I expression and class I-bound peptides in HP of the 6C5 T cells by transferring MACS bead-sorted CD8+ 6C5 T cells into sublethally irradiated β2m−/−, TAP−/−, Db−/−, and T region congenic B6.Tlaa mice (Fig. 3⇓A). The 6C5 T cells consistently proliferated to a greater extent in B6 hosts as compared with irradiated class I-deficient β2m−/− hosts, demonstrating that MHC ligand interactions promote the HP of these T cells. The small degree of proliferation observed in β2m−/− recipients might reflect low level expression of Qa-1 H chains and/or the previously described capacity of naive T cells to undergo one or two rounds of cytokine-driven proliferation after transfer to irradiated recipients (7, 35). The specific role of the selecting MHC molecule Qa-1b was evaluated by transferring 6C5 T cells into B6.Tlaa, which express the nonselecting Qa-1a allele. The degree of proliferation observed in these recipients was less than or equal to that observed in β2m−/− hosts. Thus, the selecting MHC molecule rather than class Ia molecules is specifically required to promote HP. Several previous studies have provided support for the idea that the same peptides that mediate positive selection in the thymus may also mediate peripheral HP of naive T cells (6, 8, 9, 10). Consistent with this idea, the degree of HP of 6C5 T cells observed after transfer to Db−/− recipients was equal to that observed in B6 mice. Lacking the Db leader sequence, Db−/− mice do not have a source of Qdm, the dominant self-peptide constitutively presented by Qa-1 molecules (33, 36). Thus, peptides other than Qdm mediate HP of 6C5 T cells. Previous experiments demonstrated that Qdm is not required for positive selection of 6C5 T cells (20). However, TAP function is required for positive selection, suggesting that undefined TAP-dependent peptides other than Qdm mediate selection (20). This is confirmed in the experiment shown in Fig. 3⇓B, demonstrating that very few CD8+Vβ5+ 6C5 T cells are detected in the periphery of lethally irradiated TAP−/− recipients after reconstitution with bone marrow from 6C5.β2m−/− mice. Unexpectedly, 6C5 T cells were observed to proliferate in irradiated TAP−/− recipients to an extent similar to that observed in control B6 mice (Fig. 3⇓A). This result suggests that the Qa-1b-associated self-peptides that promote HP of 6C5 T cells may differ from those that mediate positive selection.

FIGURE 3.
FIGURE 3.

Homeostatic proliferation of 6C5 transgenic T cells requires Qa-1b but does not require TAP. A, CD8+ T cells from 6C5.B6 TCR Tg+/− transgenic mice were labeled in vitro with CFSE and transferred into sublethally irradiated (600 rad) B6, B6.Tlaa, β2m−/−, Db−/−, and TAP−/− mice. Six days after transfer, lymphocytes from the spleens of recipient mice were analyzed for cell surface expression of CD8α and Vα3.2. Proliferation of TCR transgenic CD8+ T cells is shown by the dilution of CFSE. B, Requirement for TAP in positive selection of 6C5 T cells. CD8-depleted bone marrow cells from β2m−/− 6C5 TCR+ donors were injected i.v. into lethally irradiated (1060 rad) B6, β2m−/−, or TAP−/− mice. After an 8 wk recovery, spleens were removed and analyzed by flow cytometry for the presence of mature CD8+Vβ5+ 6C5 T cells. C, CD8+ T cells from 6C5 TCR Tg+/− transgenic mice were analyzed for cell surface expression of CD8α and CD44. CD8+CD44low T cells were FACS sorted to 99% purity, labeled in vitro with CFSE, and transferred into sublethally irradiated (600 rad) B6 mice, β2m−/−, and TAP−/− mice. Six days after transfer, lymphocytes from the spleens of recipient mice were analyzed for cell surface expression of CD8β and Vα3.2. Proliferation of TCR transgenic CD8+ T cells is shown by the dilution of CFSE. Numbers indicate the percentage of indicated subpopulations. Data are representative of at least three mice in each group.

The 6C5 T cells are predominantly phenotypically naive, however, there is a small population in unimmunized animals that express a memory phenotype (Fig. 1⇑). In contrast to naive T cells, memory T cells do not require interaction with MHC ligands for survival or HP (1, 37, 38, 39). To ensure that the proliferation observed after transfer into TAP −/− mice reflects the expansion of naive 6C5 T cells, we FACS-sorted CD44low 6C5 T cells before CFSE labeling and transferred them into sublethally irradiated B6, β2m−/−, and TAP−/− mice (Fig. 3⇑C). We observed a similar pattern of proliferation compared with the unsorted 6C5 T cell population. Little or no proliferation was observed after transfer into β2m−/− hosts, whereas the degree of proliferation observed in TAP−/− recipients was similar to that observed in wild-type B6 hosts. We conclude that the 6C5 T cells require TAP for positive selection but not for HP, indicating that the peptides required for homeostatic expansion may differ from those that mediate positive selection.

HP of polyclonal CD8+ T cells in TAP−/− hosts

Based on the results with 6C5 T cells, we were interested in reexamining the role of TAP in HP of polyclonal CD8+ T cells, compared with its role in positive selection. Most CD8+ T cells require β2m and TAP expression for positive selection, as there is an overall reduction of CD8 SP thymocytes (2% in C57BL/6 mice, 1% in β2m−/−, or TAP−/− mice) and of those CD8 SP, few of them have a mature CD24low phenotype (29% in β2m−/− and 17% in TAP−/− mice) compared with C57BL/6 CD8 SP cells (78%) (Fig. 4⇓A). Similarly, in the spleen of β2m−/− and TAP−/− mice, there is a marked reduction in the number of CD3+CD8+ lymphocytes, 1 and 2%, respectively, compared with B6 mice (38%). This presumably reflects a limited capacity for homeostatic expansion in addition to a reduced thymic output. A minor fraction of polyclonal CD8+ T cells from B6 donors undergo one or more rounds of proliferation after transfer to sublethally irradiated β2m−/− recipients, presumably representing memory cells or naive cells responding to cytokines and/or low-level MHC H chain expression (Fig. 4⇓B). The degree of proliferation observed in TAP−/− recipients was only slightly and variably enhanced as compared with that seen on β2m−/− hosts, indicating that the fraction of polyclonal CD8+ T cells that can respond to TAP-independent peptide complexes is very small. The fraction of T cells that undergo multiple rounds of proliferation after transfer into TAP−/− hosts was noticeably increased when the CD8+ T cells were isolated from TAP−/− donors (Fig. 4⇓C). The proliferating cells may represent T cells that are positively selected in a TAP−/− environment and retain a capacity to respond to TAP-independent peptide complexes after transfer to lymphopenic recipients. However, CD8+ T cells with properties similar to 6C5 T cells, retaining the capacity to undergo MHC-dependent homeostatic expansion in TAP-deficient recipients, are clearly very rare.

FIGURE 4.
FIGURE 4.

Polyclonal CD8+ T cells require MHC class I and TAP for positive selection and homeostatic proliferation. A, Left panel, Cells from thymus lobes of B6, β2m−/−, and TAP−/− mice were analyzed for cell surface expression of CD4, CD8, and CD24 (heat stable Ag). Right panel, Lymphocytes from the spleens of B6, β2m−/−, and TAP−/− mice were analyzed for cell surface expression of CD3, CD4, and CD8. B, CD8+ T cells from B6.PL (Thy1.1) mice were labeled in vitro with CFSE and transferred into sublethally irradiated (600 rad) B6 mice, β2m−/−, and TAP−/− mice. Six days after transfer, lymphocytes from the spleens of recipient mice were analyzed for cell surface expression of CD8β and Thy1.1. Proliferation of polyclonal CD8+Thy1.1+ T cells is shown by the dilution of CFSE. C, CD8+ T cells from B6 and TAP−/− mice were purified using magnetic beads, labeled in vitro with CFSE, and transferred into sublethally irradiated (600 rad) β2m−/−, and TAP−/− mice. Six days after transfer, CD3+CD8β+ lymphocytes from the spleens of recipient mice were analyzed for dilution of CFSE. Data are representative of at least three mice in each group.

Discussion

In the current study, we demonstrate that naive TCR transgenic T cells with specificity for Qa-1b have a capacity similar to that observed with class Ia-restricted T cells to undergo homeostatic expansion after transfer into sublethally irradiated hosts. The observed proliferation was largely dependent on the expression of β2m (class I molecules) in the recipient animals. Experiments with congenic recipients expressing Qa-1a instead of Qa-1b demonstrated that MHC class I-dependent HP is specifically driven by interaction of the T cells with Qa-1b and not through cross-recognition of classical class I molecules. Thus, the same MHC molecule that mediates positive selection of 6C5 T cells is also required for HP. Homeostatic expansion, like positive selection, occurs in the absence of Qdm, the dominant self-peptide bound to Qa-1 molecules under normal conditions. However, experiments with TAP−/− recipients demonstrate a clear distinction between the ligand requirements for thymic selection and HP. Positive selection of 6C5 T cells is dependent on TAP function, thus selection is presumably mediated by TAP-dependent peptides. By contrast, HP is not dependent on TAP function, supporting the conclusion that different peptides mediate positive selection and HP.

The possibility that class Ib-restricted T cells might have a reduced capacity for homeostatic expansion is suggested by the observation that very few CD8+ T cells are present in KbDb−/− mice, which express class Ib but not class Ia molecules (15, 16). In addition, recently published data indicate that CD8+ T cells from KbDb−/− donors proliferate poorly in lymphopenic sublethally irradiated syngeneic hosts (16). It is clear from our results with a homogenous population of transgenic T cells with specificity for Qa-1b that selection by class Ib molecules during development in the thymus does not necessarily lead to a phenotype characterized by a reduced capacity to undergo HP. It is possible that 6C5 T cells are not representative of the majority of class Ib-restricted T cells. One might envision that class Ib-restricted T cells acquire different functional properties as a consequence of an alternative developmental pathway involving selection by interaction with hemopoietic lineage cells in the thymus, and thus they could have intrinsic limitations in their capacity for HP. In addition, reduced expression of class Ib molecules or reduced diversity of self-peptide ligands might limit homeostatic expansion of class Ib-restricted T cells. Alternatively, T cells selected by nonclassical MHC molecules may have a capacity for homeostatic expansion similar to conventional CD8+ T cells. The large population of CD4+ T cells present in KbDb−/− animals might limit the expansion of the relatively small output of CD8+ T cells (10, 40). It is also possible that the CD8+ T cells present in the periphery of KbDb−/− mice already have a history of substantial homeostatic expansion, but that this is insufficient to compensate for the small thymic output. This could in part explain the activated memory phenotype of the CD8+ T cells in KbDb−/− mice. Naive T cells undergoing HP acquire a stable memory like phenotype (1, 41, 42, 43, 44). Previous homeostatic expansion might limit that capacity of CD8+ KbDb−/− T cells to undergo further proliferation after transfer to lymphopenic adoptive recipients. Clearly, more work needs to be done to characterize the polyclonal repertoire of class Ib-restricted T cells and the homeostatic mechanisms that regulate the size of this population. Nevertheless, our results with transgenic 6C5 T cells indicate that at least some Qa-1-restricted T cells have a capacity for HP, and that the relatively low level of expression of Qa-1 does not inherently limit the capacity of these T cells to productively interact with Qa-1 in the periphery.

A striking finding in the current study is the observation that although 6C5 T cells require TAP function for thymic selection, they do not require TAP for homeostatic proliferation. This suggests that one or more TAP-dependent self-peptides are required for positive selection of these Qa-1b-restricted T cells, but these TAP-dependent peptides are not required for HP. Other TAP-independent Qa-1-bound peptides appear to be sufficient to drive proliferation in the periphery in lymphopenic hosts, although they are not sufficient to support thymic selection. A number of previous studies provided evidence that the same peptides required for positive selection may also be required for HP (6, 8, 9, 10, 13). For example, Ernst et al. (10) demonstrated that polyclonal CD4+ T cells selected on highly diverse IAb-peptide complexes in B6 mice undergo HP in irradiated B6 recipients, but not in H2-M−/− recipients, which express a highly restricted population of IAb-bound peptides dominated by CLIP. However, T cells that were selected by this restricted population of peptides in H2-M−/− mice proliferated after transfer into irradiated H2-M−/− recipients. H2-Kb-restricted TCR transgenic OT-1 CD8+ T cells proliferate in lymphopenic B6 recipients, but not in TAP-deficient recipients, which have a restricted repertoire of Kb-bound peptides as well as a marked reduction in total Kb expression (8, 9). However, proliferation could be rescued in TAP-deficient recipients expressing a transgene encoding an antagonist peptide that is capable of driving positive selection of OT-1 T cells in fetal thymic organ culture (9). Expression of Kb was not up-regulated in the transgenic recipients, indicating that recognition of the selecting peptide was directly responsible for rescue of HP.

In contrast to the above studies, Bender et al. (7) demonstrated that TCR interaction with the selecting peptide-MHC ligand is not always sufficient to cause proliferation in irradiated hosts under conditions where there is a highly restricted repertoire of MHC-bound peptides in transgenic mice expressing covalent peptide-IAb complexes. Correia-Neves et al. (14), using mice expressing a limited TCR repertoire, demonstrated that homeostatic expansion and survival can skew the postselection T cell repertoire. Variation in the potential of mature T cells to undergo HP in response to self peptide-MHC ligands is illustrated by the observation that only 30–50% of polyclonal T cells proliferate within 1–2 wk after transfer into lymphopenic recipients (7, 10, 42, 45). Heterogeneity in the capacity to respond to self MHC ligands in lymphopenic hosts appears to be controlled to a major extent by TCR specificity. Naive T cells from transgenic mice expressing the 2C, P14, OT-1, DO11, or 1H3.1 TCR proliferate, whereas T cells from H-Y, OT-2, and AND mice do not (5, 6, 7, 10, 40, 46). Thus, self peptide-MHC complexes capable of supporting positive selection are not always sufficient to stimulate T cell expansion after transfer to lymphopenic hosts. This might result from an increase in the signal threshold required for HP as compared with positive selection (47). In addition, differences in the level of expression and cellular distribution of self peptide-MHC ligands in the thymus as compared with periphery could lead to differences in signal strength.

Our results with T cells expressing the 6C5 TCR differ from previous findings in studies with transgenic and polyclonal T cells because the signal requirements for HP appear to be less stringent than for thymic selection, rather than more stringent. Our results illustrate that very few polyclonal CD8+ T cells from B6 mice proliferate in response to self MHC class I complexes after transfer into irradiated TAP-deficient hosts. Thus, T cells with the capacity to proliferate in TAP−/− recipients are rare in B6 mice. The vast majority of these T cells have specificity for Kb and Db class Ia molecules. In addition to a severe reduction in the diversity of peptides bound to Kb and Db, there is also a marked decrease in the total expression of these MHC molecules in TAP−/− mice. Thus, the reduced expression of class Ia molecules could be a limiting factor for HP, irrespective of the diversity of bound peptides. Qa-1 differs from class Ia molecules in that its expression is only slightly reduced in TAP-deficient cells (48, 49, 50). Thus, the major impact of TAP function is on the nature of Qa-1 bound peptides. It is unclear what peptides are bound to Qa-1 on the surface of TAP−/− cells and there is a significant likelihood that these Qa-1 molecules are “empty” (48). Given the limited diversity of natural peptide ligands bound to Qa-1 under physiological conditions, it is possible that T cells selected through interaction with Qa-1 in the thymus may have an inherent bias toward recognition of structural elements of Qa-1 with less contribution from TCR interactions with bound peptide. Indeed, recognition of empty Qa-1b molecules may be sufficient to drive the proliferation of T cells bearing the 6C5 receptor in lymphopenic hosts. Further investigation will be required to determine whether this is a general feature of T cells with dedicated specificity for Qa-1.

Acknowledgments

We thank Taku Kambayashi, Dominique Weber, Piotr Kraj, and Leszek Ignatowicz for helpful discussions.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by National Institutes of Health Grants AI33614 (to P.E.J.) and AI30554 (to P.E.J.).

  • ↵2 Address correspondence and reprint requests to Dr. Peter E. Jensen, Department of Pathology, Emory University School of Medicine, Room 7313 Woodruff Memorial Building, 101 Woodruff Circle, Atlanta, GA 30322. E-mail address: pjensen{at}emory.edu

  • ↵3 Abbreviations used in this paper: HP, homeostatic proliferation; SP, single positive; B2m, B2-microglobulin; Qdm, Qa-1 determinant modifier.

  • Received June 8, 2004.
  • Accepted September 9, 2004.

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