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The Journal of Immunology, 2008, 180, 2805-2814
Copyright © 2008 by The American Association of Immunologists, Inc.
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Polyclonal MHC Ib-Restricted CD8+ T Cells Undergo Homeostatic Expansion in the Absence of Conventional MHC-Restricted T Cells1

David C. Jay2, Lisa M. Reed-Loisel2 and Peter E. Jensen3

Department of Pathology, University of Utah, Salt Lake City, UT 84112


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naive T cells have the capacity to expand in a lymphopenic environment in a process called homeostatic expansion, where they gain a memory-like phenotype. Homeostatic expansion is dependent on competition for a number of factors, including growth factors and interactions with their selecting self-MHC molecules. In contrast to conventional T cells, it is unclear whether class Ib-restricted CD8+ T cells have a capacity to undergo homeostatic expansion. In this study, we demonstrate that polyclonal MHC Ib-restricted CD8+ T cells can undergo homeostatic expansion and that their peripheral expansion is suppressed by conventional MHC-restricted T cells. The acute depletion of CD4+ T cells in MHC class Ia-deficient Kb–/–Db–/– mice led to the substantial expansion of class Ib-restricted CD8+ T cells. Adoptive transfer of class Ib-restricted CD8+ T cells to congenic lymphopenic recipients revealed their ability to undergo homeostatic expansion in a MHC Ib-dependent manner. To further study the homeostatic expansion of MHC Ib-restricted T cells in the absence of all conventional MHC-restricted T cells, we generated mice that express only MHC Ib molecules by crossing H-2Kb–/–Db–/– with CIITA–/– mice. CD8+ T cells in these mice exhibit all of the hallmarks of naive T cells actively undergoing homeostatic expansion with constitutive memory-like surface and functional phenotype. These findings provide direct evidence that MHC Ib-restricted CD8+ T cells have the capacity to undergo homeostatic expansion. Their peripheral expansion is suppressed under normal conditions by a numerical excess of conventional MHC class Ia- and class II-restricted T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The size and repertoire of peripheral lymphocyte populations, including naive and memory CD4+ or CD8+ T cells, B cells, and NK cells are controlled independently by homeostatic mechanisms (1, 2, 3). Under steady-state conditions, the regulation of peripheral T cell homeostasis is likely to be maintained predominately by a balance between survival and proliferation in the periphery rather than the output of T cells from the thymus (4). When mature naive T cells are transferred into a lymphopenic environment, they actively proliferate in a process termed homeostatic expansion. The factors that control homeostatic expansion are thought to be similar to the requirements for the long-term survival of naive T cells under normal circumstances. The mechanisms guiding the homeostatic expansion of naive T cells are many, including competitive interactions for self-MHC-peptide complexes, TCR affinity and promiscuity for self-MHC-peptide complexes, strength of downstream TCR signaling, IL-7 availability, and regulatory T cell (Treg)4-mediated suppression (5, 6, 7, 8, 9, 10, 11, 12, 13, 14). The competition for these homeostatic factors between memory and naive CD8+ and CD4+ T cells in the periphery may limit each respective T cell subset’s ability to undergo homeostatic expansion (15, 16, 17, 18, 19). Interestingly, naive CD8+ T cells undergoing homeostatic expansion gain a memory-like phenotype, with the up-regulation of typical memory surface markers, such as CD44, Ly6c, and CD122, rapid production of IFN-{gamma} and acquisition of enhanced cytolytic capabilities following TCR stimulation, as well as a memory-like gene expression profile (20, 21, 22, 23).

Most CD8+ T cells in the periphery have been selected by classical MHC class Ia molecules (24). In addition to these conventional CD8+ T cells, there are subpopulations of T cells with specificity for MHC Ib molecules, such as H-2M3 and Qa-1. Little is known about the functional roles of these MHC Ib-restricted T cells and the mechanisms that control their homeostasis. Class Ib-restricted T cells represent only a small percentage of the peripheral T cell population in normal mice. In Kb–/–Db–/– mice, which lack MHC class Ia molecules but express class Ib molecules, CD4–CD8+ T cells exist at markedly reduced levels in thymus and they represent a very minor subpopulation in peripheral lymphoid organs (25, 26, 27, 28). These residual CD8+ T cells are thought to represent a low frequency of CD8+ T cells selected by class Ib molecules in the thymus. The low thymic output, as demonstrated by the relatively small fraction of CD4–CD8+ thymocytes in Kb–/–Db–/– mice, may result from a number of possible factors. In general, class Ib molecules are expressed at low cell surface levels, they are oligomorphic, and they bind a relatively limited repertoire of peptides. The efficiency of thymic selection may be reduced because of limited ligand availability and diversity (29, 30, 31). Also, the frequency of developing T cells with TCR that have potential specificity for class Ib molecules might be low, inherently restricting their thymic output.

Although thymic production of class Ib-restricted CD8+ T cells is limited, it is evident that these cells exist as a distinct population in the thymus and periphery. CD8+ T cells represent a very small fraction of T cells in the peripheral lymphoid organs of Kb–/–Db–/– mice, despite the absence of conventional class Ia-restricted T cells, indicating that CD8+ T cells selected by class Ib molecules have a restricted capacity to undergo homeostatic expansion to fill the CD8+ T cell niche in these animals. It is possible that polyclonal class Ib-restricted T cells are inherently limited in their capacity to undergo homeostatic proliferation compared with conventional T cells. In support of this, Kurepa et al. (27) reported that polyclonal CD8+ T cells from Kb–/–Db–/– mice expand poorly after transfer to irradiated hosts. In several model systems, class Ib-restricted T cells have been shown to participate in an alternative developmental pathway, involving selection by class Ib molecules expressed on hemopoietic lineage cells in the thymus, as opposed to thymic epithelial cells (32, 33, 34). Urdahl et al. (32) reported that CD4–CD8+ thymocytes in Kb–/–Db–/– mice begin to express activation markers before exit from the thymus, suggesting that an alternative developmental pathway may result in a distinct phenotype for class Ib-restricted T cells.

It is also possible that peripheral homeostatic expansion is limited by the availability of appropriate class Ib ligands in the periphery. Homeostatic expansion of naive conventional CD4+ and CD8+ T cells requires expression of the selecting MHC molecule in the periphery, as well as the availability of appropriate MHC-bound self-peptides. In a given peripheral location, the selecting class Ib molecules might not be expressed at sufficient levels with the appropriate ligands to support homeostatic proliferation.

The evidence described above supports the possibility that class Ib-restricted T cells may not be able to undergo substantial peripheral expansion because of intrinsic properties acquired in the thymus or the limited availability of appropriate class Ib ligands in the periphery. Opposing this idea are results from experiments with TCR-transgenic 6C5 T cells. These CD8+ TCR-transgenic T cells are specific for beef insulin in the context of Qa-1b and have been shown to require Qa-1b for selection in the thymus. Furthermore, their selection can be mediated by thymic hemopoietic cells, and their specificity for Qa-1b is maintained after maturation and emigration to the periphery (33, 35, 36). Despite these properties, 6C5 T cells have a capacity to undergo homeostatic expansion similar to class Ia-restricted T cells and their expansion is dependent upon peripheral expression of Qa-1b. Thus, there is no inherent limitation in the ability of class Ib-specific T cells to expand, nor is the availability of peripheral Qa-1b limiting, at least for this clonal population of T cells.

The current study was performed to address the hypothesis that the peripheral expansion of CD8+ T cells observed in Kb–/–Db–/– mice may be limited as a result of the large peripheral population of CD4+ T cells that remain present in these animals. CD4+ T cells in the periphery of the Kb–/–Db–/– mouse might inhibit the expansion of the class Ib-restricted CD8+ T cells through competition for shared factors dictating their homeostasis. To reduce the peripheral CD4+ T cell population and examine the impact on class Ib-restricted CD8+ T cells, we crossed Kb–/–Db–/– mice with mice that lack expression of the master MHC class II (MHC II) transcriptional regulator CIITA. CIITA–/– mice lack expression of MHC II molecules and exhibit a very small population of CD4+CD8– T cells in the periphery and thymus (37, 38). The resulting Kb–/–Db–/–CIITA–/– mice lack expression of both MHC Ia and MHC II molecules, expressing only class Ib molecules. Using Kb–/–Db–/–CIITA–/– mice, we observed that in the absence of MHC II-restricted CD4+ T cells, class Ib-restricted CD8+ T cells expand in the periphery. Furthermore, the expanded CD8+ T cells in the Kb–/–Db–/–CIITA–/– mice show a memory-like phenotype by surface marker expression, rapid IFN-{gamma} production and robust proliferation following T cell stimulation, as well as extensive peripheral proliferation in vivo. These observations are characteristic of T cells actively undergoing homeostatic proliferation, suggesting that polyclonal class Ib-restricted CD8+ T cells are not inherently defective in their capacity to proliferate in the periphery, but are inhibited from doing so by excessive numbers of conventional MHC-restricted T cells.


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

C57BL/6J, B6.PL-Thy1a/Cy (B6.Thy1.1), CIITA–/–, and β2m–/– mice were purchased from The Jackson Laboratory. Kb–/–Db–/– mice were obtained from Taconic Farms. Kb–/–Db–/– mice were previously described (8). Kb–/–Db–/–.CIITA–/– mice we generated by breeding Kb–/–Db–/– mice with CIITA–/– mice. Kb–/–Db–/–.Thy1.1 mice were generated by breeding Kb–/–Db–/– mice with B6.Thy1.1 mice. Mice, 10–15 wk old, of either sex were used. Mice were housed in the specific pathogen-free colonies at the vivarium of the Woodruff Memorial Research Building (Emory University, Atlanta, GA) and the Emma Eccles Jones Medical Research Building (University of Utah, Salt Lake City, UT). All experiments were done according to institutional guidelines.

Flow cytometry and analysis

Single-cell suspensions from spleens, lymph nodes (pooled inguinal), or thymi were obtained and RBC were lysed in a hypotonic buffer for 10 min at room temperature. Cells were resuspended in staining buffer (pH 7.4, PBS and 2 mM EDTA) at 1–2 x 107 cells/ml. One hundred-microliter cell suspensions were incubated for 30 min with conjugated Abs at 4°C. Cells were then washed and fixed in 1% paraformaldehyde. The following mAbs were used for flow cytometry: anti-CD4 (GK1.5), anti-CD8{alpha} (53-6.7), anti-CD44 (IM7), anti-CD62L (MEL-14), CD122 (TM-β1), anti-β7 integrin (M293), anti-Ly6c (AL-21), anti-H-2KbDb (28-8-6), anti-I-A/I-E (M5), anti-Qa-1b (6A8.6F10.1A6), anti-Qa-2 (1-1-2), anti-H2-M3 (130), anti-B220 (RA3-6B2), anti-CD11c (HL3), anti-Thy1.1 (OX-7), anti-Thy1.2 (53-2.1), anti-IFN-{gamma} (XMG1.2), and anti-BrdU were purchased from BD Biosciences. Anti-CD11c (N418), anti-CD1d (1B1), and anti-TCRβ (H57-597) were purchased from eBioscience. Staining with biotinylated mAbs was revealed with streptavidin-Alexa Fluor 647 (Invitrogen Life Technologies/Molecular Probes). Flow cytometry was collected on a FACSCalibur and FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Magnetic bead sorting

Single-cell suspensions were prepared from spleen and inguinal lymph nodes and then incubated with magnetic bead-conjugated anti-CD8{alpha} in MACS buffer (PBS (pH 7.4) with 3/500 dilution of citrate-dextrose solution (Sigma-Aldrich) and 0.2% BSA) for 15 min at 4°C. Cells were washed twice with cold MACS buffer and passed over a LS MACS column according to the manufacturer’s protocol (Miltenyi Biotec). Positively selected cells were 92–98% CD8{alpha}+.

Intracellular IFN-{gamma} staining

CD8+ T cells were purified via magnetic sorting (Miltenyi Biotec) and activated with PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich) for 5 h in an incubator at 37°C/5% CO2 in the presence of brefeldin A (Sigma-Aldrich) in complete MEM-{alpha} (Invitrogen Life Technologies) (supplemented with 10% FCS (HyClone), 2 mM L-glutamine, 0.5 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µM nonessential amino acids (Invitrogen Life Technologies)). Cells were washed and stained for surface CD8{alpha} and TCRβ, washed, and then stained intracellularly for IFN-{gamma} using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s protocol.

T cell adoptive transfer

For analysis of lymphopenia-induced proliferation, purified CD8+ T cells were labeled with CFSE (Sigma-Aldrich). Briefly, ~107 cells/ml were washed twice with room temperature PBS (pH 7.4) and then incubated with 2.5 µM CFSE for 5 min. The labeling reaction was quenched by adding one-fifth total volume of FCS for 30 s. The cells were then immediately washed twice with PBS. Cells were then transferred into anesthetized recipient mice that had been sublethally irradiated (600 rad) 24 h earlier and acutely depleted of NK cells 48 h earlier via an i.p. injection of 250 µg of anti-NK1.1 (PK136) in sterile PBS. Recipient mice were warmed until conscious. Five days following adoptive transfer, spleen and inguinal lymph nodes were pooled, surface stained for CD8{alpha} and Thy1.1, then analyzed by flow cytometry for CFSE dilution.

In vitro T cell activation

CD8+ T cells were purified via magnetic sorting and labeled with CFSE, as described above. Cells were then cultured in complete MEM-{alpha} in the presence or absence of 20 U/ml IL-2 (PeproTech) and a 1:1 ratio of anti-CD3- and anti-CD28-coated beads to cells (Invitrogen Life Technologies) for 72 h. Cells were surface stained for CD8{alpha} and TCRβ and CFSE dilution was analyzed via flow cytometry.

In vivo BrdU incorporation

Mice were treated with 0.8 mg/ml BrdU (Sigma-Aldrich) and 1% glucose (Sigma-Aldrich) in their drinking water for 9 days. Spleens were harvested and homogenized to a single-cell suspension and RBC were lysed in a hypotonic solution. Cells were surface stained for CD8{alpha}, CD4, and TCRβ expression. Cells were then stained intracellularly for BrdU using the FITC BrdU Flow Kit per the manufacturer’s protocol (BD Biosciences).

Peripheral CD4+ T cell depletion

Twelve-week age-matched and sex-matched mice were injected i.p. with 250 µg of anti-CD4 mAb GK1.5 in sterile PBS on days 1, 4, 7, and 10 over a 2-wk time course. Control mice were similarly injected with equal volumes of PBS on the days indicated. On day 14, single-cell suspensions of spleen and inguinal lymph nodes from each mouse were counted and stained for CD4, CD8{alpha}, and TCRβ and analyzed by flow cytometry. Total numbers of CD8+ T cells were calculated based on the total percentage of CD8{alpha}+TCRβ+ per organ (total no. of splenocytes x percentage of total collected CD8{alpha}+TCRβ+ events = total no. of CD8{alpha}+TCRβ+ T cells). Depletion was >98% in the spleen and inguinal lymph nodes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Kb–/–Db–/– CD8+ T cells exhibit homeostatic expansion following the acute depletion of CD4+ T cells

Conventional naive CD4+ and CD8+ T cells share a number of requirements for their homeostasis in the periphery, such as IL-7 and interactions with autologous MHC molecules. Little is known about the homeostatic requirements of unconventional class Ib-restricted T cells as a polyclonal population. For instance, the homeostasis of invariant NK T (iNKT) cells requires IL-15 and, to a lesser degree, IL-7, but not interactions with the selecting class Ib molecule CD1d (39). The homeostatic requirements for iNKT point to an overlapping niche with NK cells and memory T cells, instead of naive CD4+ and CD8+ T cells. However, Qa-1b-restricted CD8+ TCR-transgenic 6C5 T cells have the capacity to undergo homeostatic expansion in a Qa-1b-dependent manner, similar to the MHC interaction requirements for the homeostasis of conventional MHC-restricted naive T cells (36). In the peripheral lymphoid organs of Kb–/–Db–/– mice, there is a large population of CD4+ T cells and a very small population of polyclonal class Ib-restricted CD8+ T cells. This situation suggests that naive conventional CD4+ T cells and class Ib-restricted CD8+ T cells compete for similar homeostatic factors, which may suppress the peripheral expansion of the class Ib-restricted CD8+ T cell population in Kb–/–Db–/– mice.

Based on this hypothesis, we were interested in whether the small population of polyclonal class Ib-restricted CD8+ T cells in the Kb–/–Db–/– mouse has the capacity to expand following the acute peripheral depletion of CD4+ T cells. Following a 14-day period of acute peripheral CD4 depletion mediated by i.p. injections of purified anti-CD4 mAb, the CD8+ T cell population in B6 and Kb–/–Db–/– mice was enumerated and compared with the PBS-injected control mice. In the spleen and inguinal lymph nodes of the CD4-depleted Kb–/–Db–/– mice, we observed an expansion of CD8+ T cells compared with the PBS-injected control (Fig. 1). Thus, in the absence of naive CD4+ T cells, polyclonal class Ib-restricted CD8+ T cells appear to have the capacity to expand in the periphery, suggesting that these two populations may share an overlapping niche.


Figure 1
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  		FIGURE 1. Effect of CD4+ T cell depletion on total numbers of peripheral CD8+ T cells. Age- and sex-matched B6 or Kb–/–Db–/– mice were treated with acute CD4+ T cell-depleting anti-CD4 mAb (GK1.5) or PBS. On day 14, the total number of CD8+ T cells in the spleen and the inguinal lymph nodes was assessed as described in Materials and Methods. Depletion was >98% per organ.

 
Polyclonal class Ib-restricted CD8+ T cells can undergo homeostatic proliferation

It has been suggested that polyclonal class Ib-restricted CD8+ T cells do not undergo homeostatic expansion or at least have a very limited capacity to do so (27). Further investigation into the class Ib-restricted CD8+ T cells in the Kb–/–Db–/– mouse by analyzing recent thymic emigrants into the spleen following their direct labeling with an intrathymic injection of FITC shows an activated phenotype gained in the periphery, which is indicative of homeostatic expansion (25). Additionally, the Qa-1b-restricted CD8+ TCR-transgenic T cell 6C5 undergoes homeostatic expansion in a Qa-1b-dependent manner (36). Based on these observations and the peripheral expansion of class Ib-restricted CD8+ T cells seen in the Kb–/–Db–/–CIITA–/– mice, we explored whether polyclonal class Ib-restricted CD8+ T cells were capable of undergoing homeostatic expansion after transfer into lymphopenic hosts and whether they do so in a class Ib-dependent manner.

We adoptively transferred purified CFSE-labeled Thy1.1 CD8+ T cells from B6 or Kb–/–Db–/– mice into NK cell-depleted, sublethally irradiated Thy1.2, B6, Kb–/–Db–/–, and β2m–/– recipients. In support of previous studies, B6 CD8+ T cells actively proliferated in B6 recipients, but they proliferated very little in β2m–/– recipients, demonstrating a requirement for MHC class I molecules. Kb–/–Db–/– CD8+ T cells also proliferated very little when transferred into lymphopenic β2m–/– recipients, but we consistently observed a small number of proliferating donor cells; the degree to which these proliferating CD8+ T cells may represent MHC-independent homeostatic expansion or cross-reactivity is unknown. However, Kb–/–Db–/– CD8+ T cells preferentially proliferated in Kb–/–Db–/– recipients, although to a slightly lesser extent than when B6 CD8+ T cells were transferred into B6 recipients (Fig. 2). These data suggest that the homeostatic proliferation of class Ib-restricted CD8+ T cells requires interactions with class Ib molecules and that this proliferation is not dependent on cross-reaction with class Ia molecules. When B6 CD8+ T cells were transferred into Kb–/–Db–/– recipients, little to no proliferation occurred (Fig. 2). However, this is not to say that the class Ib-restricted CD8+ T cells represented in the B6 CD8+ T cell population do not undergo homeostatic expansion. It is likely that class Ib-restricted CD8+ T cells represent such a small fraction of transferred B6 CD8+ T cells that their proliferation is not detected. They may also be outcompeted by transferred MHC Ia-restricted CD8+ T cells for shared factors required for homeostatic expansion. When Kb–/–Db–/– CD8+ T cells were transferred into B6 recipients, homeostatic expansion occurred, but with an additional population of highly proliferative class Ib-restricted CD8+ T cells. This observation supports previous work showing that the TCRs of class Ib-restricted CD8+ T cells can cross-react with MHC Ia molecules (40, 41, 42, 43).


Figure 2
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  		FIGURE 2. Homeostatic proliferation of Kb–/–Db–/– CD8+ T cells. CFSE-labeled CD8+ T cells were purified from B6.Thy1.1 or Kb–/– Db–/–.Thy1.1 donor mice and transferred i.v. into the indicated sublethally irradiated and NK cell-depleted recipient mice (donor -> recipient). Five days following transfer, splenocytes were analyzed for expression of CD8{alpha} and Thy1.1 by flow cytometry. CD8{alpha}+ Thy1.1+ T cells were gated and proliferation of polyclonal CD8+Thy1.1+ is shown by CFSE dilution analyzed by flow cytometry.

 
Kb–/–Db–/–CIITA–/– mice express class Ib molecules, but not MHC class Ia or class II molecules

To further investigate the overlapping niche between conventional naive MHC II-restricted CD4+ T cells and naive class Ib-restricted CD8+ T cells in the Kb–/–Db–/– mouse, we used a genetic approach by generating Kb–/–Db–/– mice deficient in MHC II expression. Because the MHC Ia genes for Kb and Db and the MHC II gene for I-Ab colocalize in the H-2 locus, crossing the Kb–/–Db–/– genotype onto a MHC II genetic knockout is not feasible. However, MHC II expression can be abolished through gene targeting of the MHC CIITA, which serves as the master transcriptional regulator of MHC II expression. CIITA–/– mice are unable to effectively select CD4+ T cells due to the abrogation of MHC II expression (37, 38, 44). Therefore, we crossed Kb–/–Db–/– mice with CIITA–/– mice to produce Kb–/–Db–/–CIITA–/– mice, with the goal of generating animals deficient in MHC class Ia and class II molecules.

Staining of splenic B cells and dendritic cells for expression of both MHC Ia and II molecules revealed that Kb–/–Db–/–CIITA–/– mice lack surface expression of MHC Ia molecules, similar to Kb–/–Db–/– mice, and MHC II molecules, similar to CIITA–/– mice (Fig. 3A). Expression of H-2 locus-linked class Ib molecules Qa-1b, Qa-2, and H2-M3, and the MHC-unlinked class Ib molecule CD1d, was detected on Kb–/–Db–/–CIITA–/– B cells and dendritic cells. Expression of these class Ib molecules was similar to that found in B6, Kb–/–Db–/–, and CIITA–/– mice, although Qa-2 expression was consistently slightly higher on the splenic B cells of Kb–/–Db–/–CIITA–/– mice (Fig. 3B). Therefore, Kb–/–Db–/–CIITA–/– mice do not express conventional MHC Ia or MHC II molecules, leaving class Ib molecules as the sole MHC ligands available to mediate T cell selection.


Figure 3
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  		FIGURE 3. MHC expression on splenocyte subsets. A, Splenocytes from Kb–/–Db–/–.CIITA–/– (gray tinted with black outline), B6 (solid black line), Kb–/–Db–/– (black-filled histogram), and CIITA–/– (gray tinted) mice were surface stained for H-2KbDb (MHC Ia) and I-A/I-E (MHC II) expression on gated B220+ B lymphocytes or CD11c+ dendritic cells as indicated. B, Splenocytes from B6 (solid line), CIITA–/– (dashed line), Kb–/–Db–/– (dotted line), and Kb–/–Db–/–.CIITA–/– (gray shaded) mice were stained for expression of the MHC Ib molecules Qa-1b, Qa-2, H2-M3, and CD1d or isotype (black filled) on gated B220+ B lymphocytes or CD11c+ dendritic cells as indicated.

 
Peripheral expansion of CD8+ T cells in Kb–/–Db–/–CIITA–/– mice

We next wanted to see whether we observed an expansion of CD8+ T cells in the peripheral lymphoid organs of the Kb–/–Db–/–CIITA–/– mice. We found that Kb–/–Db–/–CIITA–/– mice exhibited a substantial expansion in CD8+ T cells in the spleen and inguinal lymph nodes compared with their Kb–/–Db–/– counterparts, in both percentage and total numbers. The percentage and total numbers of CD8+ T cells in spleen and inguinal lymph nodes of the Kb–/–Db–/–CIITA–/– mice were generally less than one-half those found in B6 mice (Fig. 4, A and B). The striking increase in the percentage and total numbers of CD8+ T cells in the periphery of Kb–/–Db–/–CIITA–/– mice occurred despite a limited thymic output as illustrated by a reduced percentage of CD8+CD4– thymocytes, similar to that observed in Kb–/–Db–/– mice (Fig. 4A). Despite a slightly higher average number of CD8+CD4– thymocytes in the thymus of Kb–/–Db–/–CIITA–/– mice compared with the Kb–/–Db–/– mice, this difference was not significant (p < 0.05; data not shown). Thymi were also similar in size and in total number of thymocytes among the B6, Kb–/–Db–/–, and Kb–/–Db–/–CIITA–/– mice (data not shown). This result suggests that the increased numbers of CD8+ T cells in the peripheral lymphoid organs of the Kb–/–Db–/–CIITA–/– mice is not necessarily due to increased thymic output. Splenomegaly and lymphoproliferative disease were ruled out as potential explanations for the peripheral expansion of CD8+ T cells in the Kb–/–Db–/–CIITA–/– mice since total splenocyte numbers were comparable to those of B6, CIITA–/–, and Kb–/–Db–/– mice (Fig. 4C). Although the means of the total splenocyte numbers tend to be slightly lower for the CIITA–/– and the Kb–/–Db–/–CIITA–/– mice compared with the B6 and Kb–/–Db–/– mice, this difference was statistically insignificant (p > 0.05). Additionally, the Vβ repertoire of the CD8+ T cells from the Kb–/–Db–/–CIITA–/– mice was polyclonal with a wide variety of detectable Vβ chain usage and no clonal expansion or overly overt biases compared with CD8+ T cells from B6 or Kb–/–Db–/– mice, similar to previously published data with Kb–/–Db–/– mice (data not shown and Ref. 28). These observations strongly suggest that class Ib-restricted CD8+ T cells undergo extensive homeostatic expansion in the periphery of the Kb–/–Db–/–CIITA–/– mice to fill the niche left partially empty by the ablated thymic output of MHC II-restricted CD4+ T cells. Additionally, a substantial number of CD4+ T cells exist in the periphery of Kb–/–Db–/–CIITA–/– mice (Fig. 4A). The characterization of these cells is currently in progress.


Figure 4
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  		FIGURE 4. Peripheral expansion of CD8+ T cells in the Kb–/–Db–/–.CIITA–/– mice. A, Specified organs from the indicated mouse strains were stained with anti-CD4 and anti-CD8{alpha}. Total percentages of CD4+ vs CD8+ T cells are indicated in the upper left or lower right corner, respectively. B, The total number of CD8+TCRβ+ T cells in the spleen (left panel) and inguinal lymph nodes (right panel) was calculated in the indicated mouse strains. C, Total number of splenocytes was calculated for the indicated mouse strains.

 
CD8+ T cells in Kb–/–Db–/–CIITA–/– mice have a memory-like phenotype

One of the hallmarks of CD8+ T cells undergoing homeostatic expansion is a memory-like phenotype, including memory surface marker expression and function. We wanted to determine whether the peripherally expanded population of class Ib-restricted CD8+ T cells in Kb–/–Db–/–CIITA–/– mice exhibited this memory-like phenotype indicative of ongoing homeostatic expansion. We stained splenocytes from Kb–/–Db–/–CIITA–/– mice with a panel of memory surface marker mAbs to CD44, CD122, Ly6c, and β7 integrin. We found that most of the CD8+ T cells in the Kb–/–Db–/–CIITA–/– and Kb–/–Db–/– mice had a memory-like surface phenotype, CD44high, Ly6chigh, β7 integrinlow, and CD122high, with most all Kb–/–Db–/–CIITA–/– CD8+ T cells being CD44high (Fig. 5A). This memory-like surface phenotype was more prominent among Kb–/–Db–/–CIITA–/– and Kb–/–Db–/– CD8+ T cells compared with their B6 and CIITA–/– counterparts.


Figure 5
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  		FIGURE 5. Memory-like phenotype of Kb–/–Db–/–, CIITA–/– CD8+ T cells. A, Splenocytes from B6 (black line), CIITA–/– (dashed), Kb–/–Db–/– (dotted), and Kb–/– Db–/–.CIITA–/– (gray shaded) mice were stained for CD8+ T cells and analyzed by flow cytometry for the expression of the memory surface markers CD44, CD122, Ly6c, and β7 integrin. Histograms are gated on CD8{alpha}+TCRβ+ T cells. B, Purified CD8+ T cells from the specified mouse strains were stimulated with PMA/ionomycin in vitro in the presence of brefeldin A for 5 h, then stained for expression of surface CD8{alpha} and TCRβ and intracellular IFN-{gamma} and analyzed by flow cytometry. The percentage of IFN-{gamma} expressing gated CD8{alpha}+TCRβ+ T cells is shown. C, CFSE-labeled purified CD8+ T cells from the indicated mouse strains were cultured in vitro in the presence or absence of 20 U/ml IL-2 and a 1:1 ratio of anti-CD3:anti-CD28-coated beads for 72 h. Proliferation of CD8{alpha}+TCRβ+ gated T cells is shown by CFSE dilution analyzed by flow cytometry.

 
To explore the function of peripheral CD8+ T cells from Kb–/–Db–/–CIITA–/– mice, we treated purified CD8+ T cells from B6, Kb–/–Db–/–, and Kb–/–Db–/–CIITA–/– mice in vitro briefly with PMA and ionomycin and measured IFN-{gamma} production via intracellular cytokine staining. The majority of CD8+ T cells from Kb–/–Db–/–CIITA–/– mice rapidly produced IFN-{gamma} following in vitro stimulation (Fig. 5B). Although the percentage of CD8+ T cells producing IFN-{gamma} from the Kb–/–Db–/– mice was slightly higher than that seen among CD8+ T cells from B6 mice, it was substantially lower than that observed from Kb–/–Db–/–CIITA–/– CD8+ T cells. This result may be a functional consequence of a more extensive history of homeostatic expansion among the Kb–/–Db–/–CIITA–/– CD8+ T cells compared with the Kb–/–Db–/– CD8+ T cells. Similar results were also obtained following in vitro stimulation of purified CD8+ T cells for 24 h with soluble or plate-bound anti-CD3{epsilon} and anti-CD28 mAb (data not shown).

In correlation with IFN-{gamma} production, memory and homeostatically expanding CD8+ T cells proliferate rapidly following TCR-induced T cell stimulation (20). To investigate whether CD8+ T cells from Kb–/–Db–/–CIITA–/– mice could similarly expand rapidly following T cell stimulation, we cultured CFSE-labeled CD8+ T cells from B6, Kb–/–Db–/–, and Kb–/–Db–/–CIITA–/– mice with anti-CD3{epsilon} and anti-CD28 for 3 days and observed their proliferation via CFSE dilution. Very few CD8+ T cells from Kb–/–Db–/–CIITA–/– mice underwent proliferation in the presence of medium and IL-2 alone, although there was a consistent background of proliferation in a small fraction of CD8+ T cells from Kb–/–Db–/– and Kb–/–Db–/–CIITA–/– mice compared with B6 and CIITA–/– controls. In response to anti-CD3{epsilon}- and anti-CD28-mediated T cell stimulation, CD8+ T cells from Kb–/–Db–/–CIITA–/– mice underwent robust proliferation compared with those from B6 and CIITA–/– mice (Fig. 5C). These findings provide strong evidence that naive class Ib-restricted CD8+ T cells, in the absence of MHC II- and MHC Ia-restricted T cells, are capable of undergoing homeostatic expansion and acquiring a characteristic memory-like phenotype.

Class Ib-restricted CD8+ T cells rapidly proliferate in the periphery in vivo

To confirm that the CD8+ T cells in the periphery of Kb–/–Db–/–.CIITA–/– mice were homeostatically expanding in vivo, we performed BrdU incorporation assays. A much larger fraction of CD8+ T cells in the spleens of Kb–/–Db–/–CIITA–/– mice incorporated BrdU compared with CD8+ T cells from B6, CIITA–/–, and Kb–/–Db–/– mice (Fig. 6). Thus, class Ib-restricted CD8+ T cells in Kb–/–Db–/–.CIITA–/– mice are actively undergoing homeostatic expansion in the periphery.


Figure 6
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  		FIGURE 6. In vivo proliferation of Kb–/–Db–/–.CIITA–/– CD8+ T cells in the periphery. Mice of the specified strains were given BrdU in their drinking water for a 9-day period. After 9 days, splenocytes were stained for the surface expression of CD8{alpha} and TCRβ and intracellularly for BrdU labeling, then analyzed by flow cytometry. Numbers in the upper right corner indicate the percentage of BrdU+ among CD8{alpha}+ CRβ+ gated T cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the current study, we demonstrated that naive polyclonal class Ib-restricted CD8+ T cells have the ability to homeostatically expand in the periphery in a largely class Ib-dependent manner in the relative absence of MHC II-restricted CD4+ T cells. However, this expansion is suppressed in the presence of abundant class II-restricted CD4+ T cells or class Ia-restricted CD8+ T cells. In the absence of MHC II-restricted CD4+ T cells, the peripheral class Ib-restricted CD8+ T cells of Kb–/–Db–/–CIITA–/– mice were able to expand markedly compared with that observed in Kb–/–Db–/– mice, despite the limited thymic production of CD8+CD4– T cells. Naive class Ib-restricted CD8+ T cells in the periphery of Kb–/–Db–/–CIITA–/– mice have the hallmark memory-like phenotype of homeostatically expanding naive class Ia-restricted CD8+ T cells. This phenotype included memory CD8+ T cell surface marker expression, as well as the rapid production of IFN-{gamma} and robust proliferation following T cell stimulation. The class Ib-restricted CD8+ T cells in the Kb–/–Db–/–CIITA–/– mouse were also found to be undergoing vigorous proliferation in the periphery in vivo, a strong sign of their ongoing homeostatic expansion.

Using 6C5 Qa-1b-restricted, beef insulin-specific, CD8+ TCR-transgenic T cells, our previous work determined that a monoclonal population of class Ib-restricted CD8+ T cells can undergo homeostatic proliferation when transferred into lymphopenic hosts (36). However, as a polyclonal population, it has been suggested that class Ib-restricted CD8+ T cells are unable to undergo homeostatic expansion in the periphery, based on the observations that there are so few CD8+ T cells in the periphery of the MHC Ia-deficient Kb–/–Db–/– mouse and adoptive transfer models. Our data indicate that in the Kb–/–Db–/– mouse, class II-restricted CD4+ T cells appear to be able to competitively suppress the ability of class Ib-restricted CD8+ T cells to expand in the periphery. Additionally, earlier studies in which lymphocytes from Kb–/–Db–/– mice where transferred into lymphopenic syngeneic hosts indicated that class Ib-restricted CD8+ T cells proliferated little or not at all (27). However, our data clearly show that a polyclonal population of naive class Ib-restricted CD8+ T cells is capable of undergoing extensive homeostatic expansion, similar to that that observed with naive MHC Ia-restricted CD8+ T cells.

Interestingly, the vast majority of the CD8+ T cells transferred from the Kb–/–Db–/– mice into irradiated hosts preferentially required interactions with class Ib molecules to undergo homeostatic expansion. This was demonstrated by the favored requirement of Kb–/–Db–/– CD8+ T cells for the presence of β2m, but not class Ia expression to undergo homeostatic proliferation. Previously, it had been assumed that the small population of CD8+ T cells that exist in Kb–/–Db–/– mice had been selected by class Ib molecules. However, Kb–/–Db–/– CD8+ T cells may also exist due to hitherto unknown MHC-independent developmental pathways, or be restricted by MHC II molecules, as observed in CD4-deficient mice (45). Interestingly, we do consistently observe a small number of proliferating Kb–/–Db–/– CD8+ T cells when transferred into sublethally irradiated β2m–/– recipients, which may represent a small portion of MHC-independent homeostatic expansion. However, our data clearly demonstrate that a major fraction of Kb–/–Db–/– CD8+ T cells is specific for class Ib molecules.

It is interesting to speculate as to the fate of the Qa-1b-restricted T cells among the adoptively transferred Kb–/–Db–/– CD8+ T cells into B6-irradiated recipients. It is plausible that the highly proliferative Kb–/–Db–/– donor T cells transferred into the B6-irradiated recipients are not solely cross-reactive with the host’s class Ia molecules, but include Qa-1b-restricted T cells reactive to the dominant Qa-1-binding self-peptide, Qa-1 determinant modifier, or Qdm. Qdm is a nonameric peptide derived from the leader sequence of H-2D and L molecules and predominantly occupies the Qa-1b binding site in the wild-type mouse (46, 47). However, because Kb–/–Db–/– mice do not express H-2D or L molecules, there is no leader sequence available, instead a heat shock protein 60 (hsp60)-derived peptide is likely to dominantly bind to Qa-1 (48). Therefore, Qa-1b-restricted CD8+ T cells from Kb–/–Db–/– will have never seen Qdm and will likely recognize Qdm in the context of Qa-1b in the B6 host as foreign and robustly proliferate in response.

In the Kb–/–Db–/–CIITA–/– mouse, these Qa-1b-restricted T cells may represent a potentially pathogenic population. In the absence of Qdm, these T cells may be cross-reactive with the hsp60-derived peptide being predominantly presented. This would be especially relevant in a stressed environment, such as during an infection, where hsp60 processing and presentation may be up-regulated (49, 50, 51). Important work from the Soloski laboratory (52) has illustrated that Qa-1-restricted CD8+ T cells that recognize a Salmonella typhimurium GroEL-derived epitope, and cross-react with a hsp60-derived peptide, lyse stressed, but not unstressed, murine target cells. This presents a hypothetical scenario in a stressed Kb–/–Db–/–CIITA–/– mouse where hsp60 cross-reactive Qa-1-restricted T cells are sensitive to the up-regulated presentation of hsp60-derived peptides by Qa-1 and mediate possible autoimmune reactions. As the T cells in the Kb–/–Db–/–CIITA–/– mouse have been undergoing extensive homeostatic expansion, these hsp60 cross-reactive Qa-1-restricted CD8+ T cells may represent a much more significant precursor population compared with wild-type and Kb–/–Db–/– mice. The degree to which these hsp60 cross-reactive Qa-1-restricted CD8+ T cells are represented among the repertoire of T cells in the Kb–/–Db–/–CIITA–/– mouse is unknown.

In contrast to previous reports concerning the memory-like surface phenotype of CD8+ T cells in Kb–/–Db–/– mice, their memory surface phenotype appears to be obtained in the periphery and not in the thymus as a result of their unique selection pathway (25, 32). This observation suggests that CD8+ T cells in the periphery of adult Kb–/–Db–/– mice already have a history of homeostatic expansion. The previous history of homeostatic proliferation among Kb–/–Db–/– CD8+ T cells might affect their ability to proliferate rapidly after transfer to a secondary lymphopenic environment. This could explain the somewhat reduced degree of proliferation seen when Kb–/–Db–/– CD8+ T cells were transferred into lymphopenic Kb–/–Db–/– recipients compared with B6 CD8+ T cells transferred into lymphopenic B6 recipients. However, the extent to which polyclonal populations of class Ib-restricted CD8+ T cells can undergo homeostatic expansion is likely to be influenced by the individual properties of the different subpopulations, their class Ib ligand specificity, and intercompetition among subpopulations for shared homeostatic factors.

More, the robust in vivo proliferation of the Kb–/–Db–/–CIITA–/– CD8+ T cells, as shown by BrdU incorporation, indicates their persistent expansion (Fig. 6). Interestingly, the robust peripheral proliferation of Kb–/–Db–/–CIITA–/– CD8+ T cells correlates with the majority of the cells staining for annexin V, indicating abundant cell death as well as constant expansion; this has also been observed for CD4+ T cells in the Kb–/–Db–/–CIITA–/– mouse (data not shown). Because of their persistent homeostatic expansion, it is possible that competition for growth factors and nutrients is fierce among T cells in the periphery of Kb–/–Db–/–CIITA–/– mice due to the need for homeostatic factors for survival and increased metabolic requirements. This competition among T cells could result in them not obtaining their required homeostatic factors and nutrients, leading to a proapoptotic state and a rapid rate of cell turnover (53, 54, 55).

It is also interesting that the limited thymic output of CD8+ T cells observed in Kb–/–Db–/– mice persists in Kb–/–Db–/–CIITA–/– mice. This appears to indicate that the intrathymic niche of most class Ib-restricted CD8+ and CD4+ T cell precursors is highly specialized, as inferred by their unconventional thymic selection (32, 56, 57). It is possible that the low cell surface expression level of class Ib molecules inherently limits their ability to efficiently select class Ib-restricted CD8+ T cells. Furthermore, because class Ib molecules are oligomorphic, they tend to bind a limited repertoire of peptides, although Qa-2 can bind a wider range of peptides than other class Ib molecules (58, 59, 60, 61). Also, although little is known about the stability of class Ib-peptide complexes, studies with Qa-1b indicate that Qa-1b-peptide complexes are highly unstable compared with optimally binding H-2Kb-SIINFEKL complexes, which may be characteristic of other class Ib molecules (31). The limited diversity of endogenous self-peptides, unstable class Ib-peptide complexes, and low class Ib surface expression taken together could lead to very restrictive competition for intrathymic ligands to mediate the selection of class Ib-restricted T cells. Competition for intrathymic ligands that mediate positive selection of T cells has been observed using CD4+ TCR-transgenic T cells specific for a limiting selecting peptide (62). Furthermore, alternative selection pathways for class Ib-restricted CD8+ T cells may be relatively inefficient, leading to limited thymic output. Continued study of the properties of class Ib molecules, the specificities of polyclonal T cells selected by class Ib molecules, and thymic developmental pathways will be critical in delineating the pressures defining their limited thymic output.

Of further interest is the nature and specificity of the CD4+ T cells in the Kb–/–Db–/–CIITA–/– mouse. CD4+ T cells make up a large fraction of peripheral T cells in these animals, despite the absent expression of detectable MHC II molecules. The thymic output of CD4+ T cells is also markedly reduced. Like the CD8+ T cells, the CD4+ T cells are likely to be undergoing extensive homeostatic expansion, consistent with a memory/activation phenotype (data not shown). The peripheral T cell repertoire is clearly polyclonal in the Kb–/–Db–/–CIITA–/– mouse with diverse Vβ chain usage in both the CD8+ and CD4+ populations. Among the peripheral CD4+ T cells in the Kb–/–Db–/–.CIITA–/– mouse, CD4+CD25+/–FoxP3+ T regulatory cells persist to a large percentage, as do iNKT cells. However, the majority of these peripheral CD4+ T cells have unknown MHC specificity, possibly representing novel subpopulations of class Ib-restricted CD4+ T cells or T cells that develop through a MHC-independent pathway. Studies are in progress to further characterize the origins, specificity, and function of the CD4+ T cells in Kb–/–Db–/–CIITA–/– mice.

It has been documented that homeostatic expansion markedly shapes the peripheral T cell repertoire because of the differential ability of T cells to respond to homeostatic cues (63). Our current studies suggest that class Ib-restricted CD8+ T cells require interactions with class Ib molecules to undergo homeostatic proliferation. Previous reports have found that there is clonal competition among naive T cells in the periphery for self-MHC-peptide ligands and that the relative avidity of the TCR for these ligands enhances their ability to homeostatically expand (8, 12, 63). Given the unique characteristics of class Ib molecules and class Ib-restricted T cells, including the relative ligand promiscuity of H2-M3 and Qa-2-restricted TCRs, rapid off rate of Qa-1 peptides, and low class Ib surface expression, it would be interesting to see to what degree these homeostatic factors influence the peripheral class Ib-restricted T cell repertoire (31, 59, 60, 61, 64, 65). Also, the extent to which naive class Ib-restricted T cells require growth factors, such as IL-7 and IL-15, for their homeostasis and survival needs to be studied.

A recent publication in which Kb–/–Db–/–CIITA–/– mice were similarly generated reported that class Ib-restricted CD8+ T cells mediate systemic autoimmunity, including insulitis and inflammatory bowel disease by 12 wk of age (66). Our results are not consistent with these findings. Animals in our Kb–/–Db–/–CIITA–/– colony do not develop lymphoproliferative disease, splenomegaly, or intestinal swelling. We have observed few animals with rectal prolapse. It should be noted that the Kb–/–Db–/–CIITA–/– mice are particularly susceptible to common specific-pathogen free mouse facility infections, particularly Helicobacter, which is very prevalent and is not routinely tested for at most institutions (67). In our Kb–/–Db–/–CIITA–/– mouse colony, all mice that developed a rectal prolapse were also Helicobacter positive by PCR of feces, similar to findings in IL-10–/– mice (68, 69). It should be noted that all data shown represent Helicobacter-free mice because it has been eradicated from the colony. We have also grossly observed aberrant gut architecture, including enlarged mesenteric lymph nodes in infected animals. The degree to which this observation represents a break in tolerance by class Ib-restricted T cells in the gut is under investigation.

Interestingly, tolerance mechanisms persist in Kb–/–Db–/–CIITA–/– mice, with functional CD4+CD25+/–FoxP3+ Tregs existing despite the absence of MHC II expression (data not shown). Recently published studies show that functional Tregs, both CD4+ and CD8+, endure in MHC II-deficient mice (70, 71, 72). Other tolerance mechanisms are also likely to be functioning properly in Kb–/–Db–/–CIITA–/– mice. Previous work has implicated competitive homeostasis in the regulation of pathogenic autoreactive T cells (15). Furthermore, regulatory activity mediated by class Ib-restricted T cells has been implicated from several different subsets; however, much remains to be learned about the biology of these T cells and the mechanisms of their suppressive activity (73, 74, 75, 76, 77, 78). The extent to which class Ib-restricted T cells can mediate regulatory activity and are themselves subject to immunoregulation are currently being studied.


   Acknowledgments
 
We thank Dr. Xinjian Chen and Dr. Xiao He for helpful advice. We also thank James Johnson, Jacob Bailey, Carolyn Dehner, and Rita Jen for technical assistance.


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


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

1 This work was supported by National Institutes of Health Grants AI33614, AI20554 (to P.E.J.), and AI055434 (to D.C.J.). Back

2 D.C.J. and L.M.R.-L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Peter E. Jensen, Department of Pathology, University of Utah, 30 North 1900 East, Room 5C124, Salt Lake City, UT 84132. E-mail address: peter.jensen{at}path.utah.edu Back

4 Abbreviations used in this paper: Treg, regulatory T cell; MHC II, MHC class II; iNKT, invariant NKT; hsp60, heat shock protein 60. Back

Received for publication August 29, 2007. Accepted for publication December 13, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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P. A. Swanson II, A. R. Hofstetter, J. J. Wilson, and A. E. Lukacher
Cutting Edge: Shift in Antigen Dependence by an Antiviral MHC Class Ib-Restricted CD8 T Cell Response during Persistent Viral Infection
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