␥␦ T cell homeostasis is established in competition with ␣ T cells and NK cells Jena D. French, Christina L. Roark, Willi K. Born, and Rebecca L. O’Brien* Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado Health Sciences Center, 1400 Jackson Street, Denver, CO 80206 Communicated by John W. Kappler, National Jewish Medical and Research Center, Denver, CO, September 2, 2005 (received for review April 15, 2005)
CD4 T cells 兩 CD8 T cells 兩 homeostatic proliferation
he ␥␦ T cells are a diverse population of lymphocytes that have been shown to play an important role in immune regulation (1). Consequently, strict control of ␥␦ T cell function and population size is likely essential for the generation of an optimal immune response. Maintenance of lymphocyte populations, or lymphocyte homeostasis, is achieved by balancing the generation of new cells and clonal expansion with cell death. Uncovering the mechanisms responsible for this process has contributed to our understanding of the immune system and is essential for the future success of cellular immunotherapy. Lymphocyte populations have been shown to undergo spontaneous expansion when adoptively transferred into lymphopenic hosts (2–10). Characterization of this phenomenon, termed homeostatic proliferation, has provided insight into the mechanisms normally responsible for lymphocyte homeostasis and shaping the lymphocyte repertoire. The mechanisms responsible for regulating lymphocyte homeostasis vary between lymphocyte subsets. For example, whereas IL-7 is essential for naı¨ve ␣ T cell homeostasis (11–15), IL-15 plays a key role in regulating the CD8⫹ memory ␣ T cells (16–18), natural killer (NK) cells (7, 16, 18–21), and NK T cells (5, 8). Both IL-7 and IL-15 have been implicated in ␥␦ T cell biology and might play a role in ␥␦ T cell homeostasis. IL-7 is essential for rearrangement of the TCR␥ gene during ␥␦ T cell development (22, 23) and may also increase ␥␦ T cell life span in the periphery (24). Although IL-15 is not required for ␥␦ T cell development in general, IL-15⫺/⫺ mice lack V␥5⫹ epidermal ␥␦ T cells (25) and Thy1⫺ splenic and intestinal intraepithelial ␥␦ T cells (16, 26). To investigate the mechanisms responsible for regulating lymphoid ␥␦ T cell homeostasis, we developed a model of lymphopenia-induced ␥␦ T cell homeostatic expansion. Our studies reveal that ␥␦ T cell homeostasis is regulated not only by ␥␦ T cells themselves but also by ␣ T cells and NK cells. The effect of ␣ T cells appears to be mediated primarily by CD8⫹ ␣ T cells and results, at least in part, from the ability of ␣ T cells to monopolize IL-15 resources.
T
Materials and Methods Mice. Wild-type C57BL兾6, C57BL兾6 TCR  ⫺/⫺ , C57BL兾6 TCR␦⫺/⫺, C57BL兾6 CD8␣⫺/⫺, C57BL兾6 2m⫺/⫺, and C57BL兾6 TCR⫺/⫺兾␦⫺/⫺ mice were purchased from The Jackson Laboratory and were maintained and bred in our facility. Both male and female www.pnas.org兾cgi兾doi兾10.1073兾pnas.0507520102
mice were used in these experiments at 6–12 weeks of age. For any given experiment, mice were matched for both age and sex. In Vivo ␥␦ T Cell Proliferation. Donor ␥␦ T cells were isolated from either C57BL兾6 wild-type or TCR⫺/⫺ mice. Single-cell suspensions were prepared from donor spleens. After red blood cell lysis with Gey’s solution, cell suspensions were passed over nylon wool columns to enrich for T lymphocytes. ␣ T cells were depleted from C57BL兾6 samples by using the magnetic-activated cell sorting (MACS) system (biotinylated anti-TCR (27) plus streptavidincoated magnetic microbeads, Miltenyi Biotec, Auburn, CA). Cell purity was determined by flow cytometry (see below) before adoptive transfer, and ␥␦ T cell number was determined by multiplying the total cell number after nylon wool purification by the percentage of live cells and by the percentage of TCR␦⫹兾CD3⫹ cells. C57BL兾6-derived ␥␦ T cells comprised 20% of the final cell suspension. TCR⫺/⫺-derived ␥␦ T cells were enriched to 50% of the final cell suspension after nylon wool purification. The contaminating cells were composed of mainly B cells and NK cells. Enriched populations were resuspended in PBS at 2 ⫻ 106 cells per ml and labeled with 0.1 M 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) at 37°C for 15 min. Cells were washed twice with 50 ml of PBS and resuspended in injection saline. 1 ⫻ 106 CFSE-labeled ␥␦ T cells were injected intravenously into recipient mice. Spleens were harvested at various time points after adoptive transfer, T cells were isolated as described above, and cell proliferation was determined by flow cytometry. For cotransfer experiments, splenic ␣ T cells were isolated from TCR␦⫺/⫺ donors, enriched by nylon wool purification, and injected i.v. with ␥␦ T cells at the designated ratios into TCR⫺/⫺兾␦⫺/⫺ recipients. C57BL兾6 2m⫺/⫺ and CD8⫺/⫺ recipients were exposed to 600 cGy whole-body irradiation 1 day before cell transfer. Listeria Infection. Mice were injected intravenously with Listeria monocytogenes (strain EGD, 2.5 ⫻ 103 colony-forming units in 200 l) or saline alone 24 h after ␥␦ T cell adoptive transfer and harvested on day 4 of the infection. Isolation of Purified ␣ T Cell Subsets and Long-Term Reconstitution Studies. After treatment with Gey’s solution to lyse red blood
cells, C57BL兾6 spleen cell suspensions were passed over nylon wool columns. The enriched T cell population was divided into three samples to generate total ␣ T cells, CD4⫹ ␣ T cells, or CD8⫹ ␣ T cells. Cells were resuspended in PBS兾0.5% BSA (200 l兾1 ⫻ 107 cells) and preincubated with unlabeled 2.4G2 mAb (28) to reduce nonspecific mAb binding by Fc receptors. All populations were incubated with a biotinylated anti-TCR␦ antibody (GL3). CD8⫹ and CD4⫹ T cell preparations were also incubated with biotinylated anti-CD4 (GK1.5) and anti-CD8 Freely available online through the PNAS open access option. Abbreviations: CFSE, carboxyfluorescein diacetate succinimidyl ester; NK, natural killer. *To whom correspondence should be addressed. E-mail:
[email protected]. © 2005 by The National Academy of Sciences of the USA
PNAS 兩 October 11, 2005 兩 vol. 102 兩 no. 41 兩 14741–14746
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␥␦ T cells are a diverse population of lymphocytes that play an important role in immune regulation. The size of the ␥␦ T cell pool is tightly regulated, comprising only 1–10% of total lymphoid T cells in mice and humans. We examined the homeostatic regulation of ␥␦ T cells using a model of lymphopenia-induced homeostatic expansion. We found that IL-15 and, to a lesser extent, IL-7 play an important role in lymphoid ␥␦ T cell homeostasis. Moreover, ␥␦ T cell homeostatic expansion was limited not only by ␥␦ T cells themselves but also by natural killer cells and ␣ T cells. Our results suggest that CD8ⴙ ␣ T cells are the most potent inhibitors of ␥␦ T cell homeostasis and exert their effect by competing for IL-15.
(53.6.7) antibodies, respectively. After antibody binding, cells were incubated with streptavidin-coated magnetic microbeads, passed over a miniMACS separation column (Miltenyi Biotec), and the nonadherent cells were collected. Purity was determined by flow cytometry before adoptive transfer (⬍1% contaminating CD4⫹ or CD8⫹ ␣ T cells). TCR⫺/⫺-derived ␥␦ T cells (5 ⫻ 105) (see above) were injected i.v., either alone or in combination with 5 ⫻ 105 MACS-purified ␣ T cells, into TCR⫺/⫺兾␦⫺/⫺ mice. Antibody-Mediated Depletions. ␣ T cells and NK cells were depleted from recipient TCR␦⫺/⫺ and TCR⫺/⫺兾␦⫺/⫺ mice by using antibodies targeted against the TCR chain (H57–597) or NK1.1 (PK136), respectively. Mice received 200 g of control antibody (hamster IgG or mouse IgG, Jackson ImmunoResearch) or cellspecific antibody i.v. 3 days before adoptive transfer. The extent of cell depletion was measured by flow cytometry. To ensure that ␣ T cells and NK cells were truly depleted, we stained for alternative surface markers. ␣ T cell depletion was verified by staining with anti-CD3, anti-CD4, and anti-CD8 antibodies. Depletion of NK cells was verified by the loss of the IL-2Rhi兾CD3⫺ population. Cytokine Injections. Recombinant IL-7 and IL-15 were purchased from PeproTech (Rocky Hill, NJ). Cytokines were resuspended in sterile PBS and stored at ⫺70°C. TCR⫺/⫺兾␦⫺/⫺ mice received PBS or 1 g of cytokine per i.v. injection. Cytokine-Blocking Experiments. Anti-IL-7 (M25) (29), anti-IL-7R␣
(A7R34) (30), anti-IL-2R (TM1) (31), anti-IL-2 (S4B6) (32), and anti-IL-2R␣ (PC-61, American Type Culture Collection) antibodies were isolated from in vitro-cultured hybridomas. Rat IgG was used as a nonspecific control antibody. TCR⫺/⫺兾␦⫺/⫺ mice received 300 g of total antibody (150 g of each antibody) per i.v. injection. Anti-IL-2R was injected with 150 g of rat IgG to control for total protein levels. Flow Cytometry. Nylon-wool-enriched T cells were resuspended in staining buffer (Hanks’ BSS兾2% FBS兾0.1% sodium azide, pH 7.1) and aliquoted into round-bottom 96-well polystyrene tissue culture plates (105–106 cells per well). Cells were preincubated with 2.4G2 to avoid nonspecific antibody binding by Fc receptors. ␥␦ T cells were identified by staining with biotinylated or phycoerythrin (PE)-conjugated anti-TCR␦ antibodies (GL3, BD Pharmingen) in combination with anti-CD3-PE-Cy5 (145–2C11, BD Pharmingen). ␣ T cells were identified by staining with anti-CD3-PE-Cy5, anti-CD3-FITC (KT3) (33), anti-TCR-PE (H57–597, BD Pharmingen), anti-CD8␣-PE (53.6.7, BD Pharmingen), and anti-CD4-PE (GK1.5, BD Pharmingen) antibodies. Anti-NK1.1-PE (PK136, eBioscience, San Diego, CA) was used to identify NK cells. Anti-IL-2R (5H4, eBioscience), anti-IL-7R␣ (A7R34, eBioscience), and anti-IL15R␣-biotin (R & D Systems) antibodies were used to assess cytokine receptor expression levels on gated T cell populations. Biotinylated antibodies were detected with streptavidin-PE (BioSource International, Camarillo, CA) or streptavidinPE-Cy5 (BD Pharmingen). Cells were fixed with 1% paraformaldehyde in PBS and analyzed for immunofluorescence on a FACScan analyzer (Becton Dickinson). Collected data were analyzed by using FLOWJO 4.4.3 software. Briefly, live ␥␦ T cells or ␣ T cells were gated and assessed for proliferation or surface-marker expression. Percent proliferation was determined as the percentage of total T cells that had undergone at least one cell division at the time of harvest. With each cell division, CFSE fluorescence is reduced by 50%, allowing the determination of proliferation by flow cytometry (34). Total T cell number was determined by multiplying the total number of cells recovered after nylon wool enrichment by the percentage of live cells and by the percentage of each cell type as determined by flow cytometry. 14742 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0507520102
Fig. 1. ␥␦ T cells are capable of homeostatic expansion in the absence of endogenous ␥␦ T cells and ␣ T cells. Splenic ␥␦ T lymphocytes obtained from TCR⫺/⫺ donors were labeled with CFSE and injected into uninfected (shaded) and Listeria-infected (black line) TCR␦⫺/⫺ mice (A), TCR␦⫺/⫺ and TCR⫺/⫺兾␦⫺/⫺ mice (B), or TCR⫺/⫺兾␦⫺/⫺ (black line) and TCR⫺/⫺ (shaded) mice (C). (D) Homeostatic expansion of TCR⫺/⫺-derived ␥␦ T cells (shaded) was compared with that of C57BL兾6-derived ␥␦ T cells (black line) in TCR⫺/⫺兾␦⫺/⫺ recipients. Recipient splenocytes were harvested at the designated time points (B) or day 5 (A, C, and D) after adoptive transfer, enriched for T cells by nylon wool purification, and analyzed by flow cytometry.
Statistical Analysis. Each experiment in this study was performed at least three times. In general, three mice were used per condition in each experiment. To control for potential variability in donor cells, sham or untreated mice that received the same donor cells were included in relevant experiments and were analyzed at the same time point. Data shown are either representative of numerous experiments or display the combined data for multiple experiments. For combined data sets, the mean value is shown ⫾SEM. The statistical relevance of our studies was determined by using the Mann–Whitney t test for nonparametric data, with a 95% confidence interval.
Results ␥␦ T Lymphocytes Undergo Homeostatic Expansion in T Cell-Deficient Mice. To assess the homeostatic potential of ␥␦ T cells, splenic ␥␦
T cells were obtained from TCR⫺/⫺ (35) donors, labeled with CFSE, and adoptively transferred into either TCR␦⫺/⫺ (36) recipients, which lack endogenous ␥␦ T cells, or TCR⫺/⫺兾␦⫺/⫺ (36, 37) mice, which lack both ␣ and ␥␦ T cells. Despite their ability to undergo proliferation in TCR␦⫺/⫺ mice after infection with L. monocytogenes, donor ␥␦ T cells did not proliferate in uninfected TCR␦⫺/⫺ recipients (Fig. 1A). In contrast, ␥␦ T cells underwent extensive homeostatic proliferation after transfer into TCR⫺/⫺兾 ␦⫺/⫺ recipients (Fig. 1B). Cell division was apparent 3 days after cell transfer, and ⬇80% of the donor cells had undergone at least one division by day 5. The transferred ␥␦ T cells population continued to expand over time in TCR⫺/⫺兾␦⫺/⫺ mice, such that, 2 months after cell transfer, total ␥␦ T cell numbers were similar to that observed in TCR⫺/⫺ mice (1–2 ⫻ 106 ␥␦ T cells per spleen, data not shown). Of note, ␥␦ T cells did not proliferate when adoptively French et al.
transferred into TCR⫺/⫺ mice, which contain ␥␦ T cells but lack ␣ T cells (Fig. 1C). Because ␥␦ T cell development might be altered in the TCR⫺/⫺ mice that we used as donors (38), we also tested the ability of donor ␥␦ T cells isolated from wild-type C57BL兾6 mice to expand in TCR⫺/⫺兾␦⫺/⫺ recipients and obtained similar results (Fig. 1D). Thus, ␥␦ T cells undergo homeostatic expansion only in the absence of both ␥␦ T cells and ␣ T cells, suggesting that both ␥␦ T cells and ␣ T cells regulate ␥␦ T cell homeostasis. Antibody-Mediated Depletion of ␣ T Cells from TCR␦ⴚ/ⴚ Mice Allows ␥␦ T Cell Homeostatic Expansion. To test whether ␣ T cell deficiency
is sufficient to allow for ␥␦ T cell homeostatic expansion in the absence of endogenous ␥␦ T cells, we depleted TCR␦⫺/⫺ mice of ␣ T cells by using a pan-specific anti-TCR antibody before ␥␦ T cell adoptive transfer. This treatment resulted in a 4-fold reduction in ␣ T cell number at the time of harvest (data not shown). Although ␥␦ T cells did not undergo expansion in TCR␦⫺/⫺ mice treated with a control nondepleting antibody, mice treated with the anti-TCR antibody became permissive for homeostatic expansion of donor ␥␦ T cells (Fig. 2 A and B). On average, ⬇30% of the donor ␥␦ T cells had undergone at least one division in anti-TCR-treated TCR␦⫺/⫺ mice 5 days after cell transfer. ␣ T Cells Inhibit ␥␦ T Cell Homeostatic Expansion When Cotransferred into TCRⴚ/ⴚ兾␦ⴚ/ⴚ Mice. To assess whether ␣ T cells could inhibit
␥␦ T cell homeostasis when cotransferred into TCR⫺/⫺兾␦⫺/⫺ recipients, splenic ␣ T cells were injected with ␥␦ T cells at a 1:1 ratio into TCR⫺/⫺兾␦⫺/⫺ recipients. One month after cell transfer, the number of ␥␦ T cells was reduced approximately 2-fold in the presence of ␣ T cells, compared with the number recovered from mice that received only ␥␦ T cells (Fig. 2C). Surprisingly, ␣ T cells displayed an obvious homeostatic advantage over ␥␦ T cells. Although TCR⫺/⫺兾␦⫺/⫺ recipients received an equal number of ␥␦ T cells and ␣ T cells, ␣ T cells were at least 17-fold more abundant than ␥␦ T cells in TCR⫺/⫺兾␦⫺/⫺ recipient spleens after 1 month (Fig. 2C). As shown in Fig. 2D, the negative regulatory effect of ␣ T cells is due, at least in part, to early inhibition of ␥␦ T cell homeostatic expansion, because cotransfer of either excess ␥␦ T cells or ␣ T cells reduced ␥␦ T cell proliferation to a similar extent 5 days after cell transfer. French et al.
IL-15 Plays a Dominant Role in ␥␦ T Cell Homeostatic Expansion. To determine whether the inhibitory effect of ␣ T cells on ␥␦ T cell homeostasis results from their superior ability to compete for key growth or survival factors, we next investigated the role of IL-7 and IL-15. Here, we used cytokine and cytokine-receptor-specific antibodies to block the effects of endogenous cytokines (Fig. 3 A and B). TCR⫺/⫺兾␦⫺/⫺ mice received a combination of anti-IL-7 and anti-IL-7R␣ antibodies to inhibit endogenous IL-7, or anti-IL-2R antibody to block IL-15. Because IL-2R is also a component of the IL-2 receptor complex, we used antibodies against both IL-2 and IL-2R␣ to assess the role of IL-2 in our model. ␥␦ T cell homeostatic proliferation was somewhat reduced in mice that received anti-IL-7 plus anti-IL-7R␣ and markedly diminished in TCR⫺/⫺兾␦⫺/⫺ mice that received anti-IL-2R treatment. Importantly, blocking IL-2 alone by anti-IL-2兾anti-IL-2R␣ treatment did not inhibit ␥␦ T cell expansion, indicating that the blocking effect of anti-IL-2R is due to inhibition of IL-15 rather than IL-2. The number of ␥␦ T cells recovered from antibody-treated mice was also reduced in anti-IL7兾anti-IL-7R␣-treated mice and greatly decreased in mice that received anti-IL-2R treatment (Fig. 3C). Consistent with these findings, treating mice with recombinant IL-7 enhanced ␥␦ T cell homeostatic proliferation in TCR⫺/⫺兾␦⫺/⫺ mice, and recombinant IL-15 had an even stronger effect (Fig. 3D). These data suggest that both IL-15 and IL-7 contribute to ␥␦ T cell homeostasis, and that IL-15 may play a dominant role. In support of this hypothesis, increased expression of both IL-15R␣ and IL-2R, but not IL-7R␣, correlate with ␥␦ T cell homeostatic proliferation after transfer into TCR⫺/⫺兾␦⫺/⫺ mice (Fig. 3E). CD8ⴙ兾IL-2Rⴙ ␣ T Cells Are Potent Inhibitors of ␥␦ T Cell Reconstitution. In our model, ⬎90% of CD8⫹ ␣ T cells but only 50% of
CD4⫹ ␣ T cells have undergone cell division in TCR⫺/⫺兾␦⫺/⫺ recipients by day 5 (data not shown), and similar results have been reported in other models (12, 39, 40). Thus, the robustly proliferating CD8⫹ subset of ␣ T cells might play a predominant inhibitory role. To test this possibility, we assessed the effects of purified CD4⫹ and CD8⫹ ␣ T cells on ␥␦ T cell reconstitution in TCR⫺/⫺兾␦⫺/⫺ mice (Fig. 4A). Compared with mice that received only ␥␦ T cells, cotransfer of purified CD4⫹ ␣ T cells resulted in only a slight reduction in ␥␦ T cell number, which was not statistically significant. In contrast, ␥␦ T cell recovery was considPNAS 兩 October 11, 2005 兩 vol. 102 兩 no. 41 兩 14743
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Fig. 2. ␣ T cells inhibit ␥␦ T cell homeostasis. (A) TCR␦⫺/⫺ mice were treated with hamster IgG control antibody or an antibody against TCR 3 days before adoptive transfer. Splenic ␥␦ T cells obtained from TCR⫺/⫺ donor mice were labeled with CFSE and injected into antibodytreated TCR␦⫺/⫺ mice. Splenocytes were recovered from recipient mice 5 days after adoptive transfer, T cells were enriched by nylon wool, and ␥␦ T cell proliferation was assessed by flow cytometry (***, P ⬍ 0.001). (B) Representative histogram of ␥␦ T cell proliferation in antibody-treated mice. (C) Splenic ␥␦ T cells obtained from TCR⫺/⫺ donor mice were injected alone or in the presence of ␣ T cells (wild-type, C57BL兾6) at a 1:1 ratio into TCR⫺/⫺兾␦⫺/⫺ mice. Splenocytes were recovered from recipient mice 1 month after adoptive transfer, T cells were enriched by nylon wool, and ␥␦ T cell (anti-TCR␦⫹兾CD3⫹) or ␣ T cell (antiTCR⫹兾CD3⫹) reconstitution was determined by flow cytometry. (D) CFSE-labeled donor ␥␦ T cells from TCR⫺/⫺ mice were transferred alone (filled histogram) or in the presence of unlabeled ␥␦ T cells (gray line) or unlabeled ␣ T cells (black line) from TCR␦⫺/⫺ mice into TCR⫺/⫺兾␦⫺/⫺ recipients. ␥␦ T cell homeostatic expansion was assessed 5 days after adoptive transfer (percent proliferation: ␥␦ alone, 86.9%; ␥␦ plus unlabeled ␥␦, 62.4%; ␥␦ plus ␣, 67.4%).
Fig. 3. IL-15 plays a dominant role in ␥␦ T cell homeostasis. (A) TCR⫺/⫺兾␦⫺/⫺ mice were treated with the designated antibodies on days ⫺2, 0, and 2, relative to transfer of CFSE-labeled ␥␦ T cells. Recipient splenocytes were harvested 5 days after adoptive transfer. (B) Representative histograms of ␥␦ T cell proliferation in mice treated with control IgG (shaded), anti-IL-7兾antiIL-7R␣ (gray line), or anti-IL-2R (black line). (C) ␥␦ T cell number was determined by flow cytometry 5 days after cell transfer. (D) TCR⫺/⫺兾␦⫺/⫺ recipients received PBS (shaded), IL-7 (gray line), or IL-15 (black line) on days 0, 1, and 2, relative to ␥␦ T cell transfer, and splenocytes were harvested on day 3. (E) ␥␦ T cell cytokine receptor expression was determined by flow cytometry 5 days after cell transfer. ns, not statistically significant; *, P ⬍ 0.1; **, P ⬍ 0.01; ***, P ⬍ 0.001.
erably reduced in the presence of purified CD8⫹ ␣ T cells. Importantly, the total number of ␣ T cells recovered 1 month after adoptive transfer was similar whether mice received unfractionated ␣ T cells, purified CD4⫹ ␣ T cells, or purified CD8⫹ ␣ T cells (Fig. 4B), even though their initial proliferation rates differ. Of interest, at the time of harvest, CD4⫹ and CD8⫹ ␣ T cell populations expressed similar levels of IL-7R␣. In contrast, ⬎40% of the CD8⫹ population expressed IL-2R, whereas ⬍10% of the CD4⫹ population was positive for IL-2R (Fig. 4C). These data suggest that IL-15-responsive (IL-2R⫹) CD8⫹ ␣ T cells are largely responsible for the inhibitory effect of ␣ T cells on ␥␦ T cell homeostasis. ␥␦ T Cell Homeostatic Expansion Is Enhanced After NK Cell Depletion.
If competition for IL-15 is at least partly responsible for ␣ T cell-mediated inhibition of ␥␦ T cell homeostasis, it follows that 14744 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0507520102
Fig. 4. Increased levels of IL-2Rhi CD8⫹ ␣ T cells coincide with poor ␥␦ T cell reconstitution. Splenic ␥␦ T cells obtained from TCR⫺/⫺ donor mice were injected alone or in the presence of total ␣ T cells, purified CD4⫹ ␣ T cells, or purified CD8⫹ ␣ T cells at a 1:1 ratio into TCR⫺/⫺兾␦⫺/⫺ mice. Splenocytes were recovered from recipient mice 1 month after adoptive transfer, T cells were enriched by nylon wool, and ␥␦ T cell (A) and ␣ T cell (B) reconstitution was determined by flow cytometry. (C) Expression of IL-7R␣ (Left) and IL-2R (Right) was assessed on CD4⫹兾TCR⫹ and CD8⫹兾TCR⫹ cells recovered from TCR⫺/⫺兾␦⫺/⫺ mice that were originally reconstituted with either ␥␦ T cells plus purified CD4⫹ ␣ T cells or ␥␦ T cells plus purified CD8⫹ ␣ T cells, respectively. Shaded histograms represent the background fluorescence of each population. The percent of T cells that express each cell surface protein is shown. *, P ⫽ 0.0071; ns, not statistically significant; ***, P ⫽ 0.0001.
NK cells, which also rely on IL-15, might inhibit ␥␦ T cell homeostasis. To test this hypothesis, TCR⫺/⫺兾␦⫺/⫺ mice were treated with an anti-NK1.1 antibody 3 days before ␥␦ T cell adoptive transfer, resulting in a 30-fold reduction of endogenous NK cells, compared with mice that received mouse IgG control antibody (data not shown). As predicted, ␥␦ T cell homeostatic expansion was substantially increased in mice that received anti-NK1.1 (Fig. 5A). On average, 70% of the donor ␥␦ T cells had undergone at least one division 3 days after adoptive transfer into anti-NK1.1-treated TCR⫺/⫺兾␦⫺/⫺ mice, whereas only 20% had divided in control mice. Similarly, ␥␦ T cell homeostatic expansion was enhanced in TCR␦⫺/⫺ mice that received both anti-TCR and anti-NK1.1, compared with those treated with anti-TCR alone (Fig. 5B). In contrast, ␥␦ T cells did not undergo significant homeostatic expansion in TCR␦⫺/⫺ mice treated with anti-NK1.1 (Fig. 5B). These data suggest that, in the absence of ␣ T cells, NK cells are capable of inhibiting ␥␦ T cell homeostatic proliferation. French et al.
␥␦ T Cells Do Not Require MHCI Molecules During Homeostatic Expansion. Although the role of MHC in ␥␦ T cell biology remains
Exogenous IL-15 Restores ␥␦ T Cell Homeostatic Expansion in the Presence of ␣ T Cells. The inhibitory effects of CD8⫹ ␣ T cells and
NK cells may stem from their ability to outcompete ␥␦ T cells for endogenous IL-15. To assess this possibility, we tested whether exogenous IL-15 could alleviate the inhibitory effect of ␣ T cells on ␥␦ T cell homeostatic expansion in TCR⫺/⫺兾␦⫺/⫺ recipients (Fig. 6A). As expected, ␥␦ T cell proliferation was reduced in mice that also received ␣ T cells (54 ⫾ 1.8%), compared with mice that received only ␥␦ T cells (73 ⫾ 0.9%). However, ␥␦ T cell proliferation was restored to near or above the levels achieved in the absence of ␣ T cells when recipient mice also received injections of recombinant IL-15 (83 ⫾ 0.7%). Thus, ␣ T cells did not inhibit short-term ␥␦ T cell proliferation in the presence of excess IL-15.
Fig. 6. ␥␦ T cells and ␣ T cells compete for IL-15 but not MHCI during homeostatic expansion. (A) Donor ␥␦ T cells from TCR⫺/⫺ mice were labeled with CFSE and transferred alone (Left, black line) or in the presence of ␣ T cells (Left, shaded) from TCR␦⫺/⫺ mice into TCR⫺/⫺兾␦⫺/⫺ recipients. Cotransfer recipients received PBS (Right, shaded) or IL-15 (black line, Right) on days 0, 1, 2, 3, and 4, relative to ␥␦ T cell transfer, and splenocytes were harvested on day 5. ␥␦ T cell homeostatic expansion was assessed by flow cytometry. (B) CFSElabeled ␥␦ T cells from TCR⫺/⫺ mice were transferred into sublethally irradiated CD8⫺/⫺ or 2m⫺/⫺ recipients. Splenocytes were harvested on day 6.
French et al.
Discussion In the last decade, lymphocyte homeostasis has been the subject of extensive study. Here, we report the ability of ␥␦ T cells to undergo homeostatic expansion in a T cell-deficient environment (TCR⫺/⫺兾␦⫺/⫺). In support of our findings, recent work by Baccala et al. (2) revealed that ␥␦ T cells also undergo expansion in irradiated and Rag⫺/⫺ recipients. Our studies provide direct evidence that ␣ T cells, in addition to ␥␦ T cells, regulate ␥␦ T cell homeostasis. This finding is consistent with earlier observations that ␥␦ T cell number is increased in C57BL兾6 TCR⫺/⫺ mice, compared with wild-type C57BL兾6 mice (37, 43) and may also explain why mice expressing a mutant form of linker for activation of T cells (LAT) (LATY7/8/9F) develop a ␥␦ T cell lymphoproliferative disorder (44). Although the authors suggest that LAT plays a negative regulatory role in ␥␦ T cell homeostasis, the ␣ T cell population is dramatically reduced in LATY7/8/9F mice. Many studies have focused on defining the relationship between distinct subsets within a major lymphocyte population in maintaining homeostasis. ␣ T cell subsets appear to overlap, to some extent, in their homeostatic requirements, such that the CD8⫹ population is expanded in CD4⫺/⫺ mice (45), and memory CD8⫹ ␣ T cells are capable of inhibiting the homeostatic expansion of both CD4⫹ and CD8⫹ naı¨ve ␣ T cells (46). Similarly, follicular B cells may inhibit the homeostatic expansion of both mature and immature B cells (4). In contrast, T cells and B cells are thought to constitute independent homeostatic pools (47, 48). Despite the apparent autonomy of B cell and T cell populations, recent data suggest that competition between heterologous lymphocytes may also play a role in lymphocyte homeostasis. Specifically, NK T cell homeostatic expansion is enhanced in the absence of NK cells (5, 8). Here, we show that both NK cells and CD8⫹ ␣ T cells influence ␥␦ T cell homeostasis. Because ␥␦ T cells have been shown to regulate both ␣ T cell and NK cell function (49, 50), it is intriguing that these cells inversely play a role in regulating ␥␦ T cell homeostasis. The ability of ␣ T cells to inhibit ␥␦ T cell homeostasis may be due simply to their greater cell number. Such reasoning could explain why depletion of NK cells, a smaller lymphocyte population, does not allow ␥␦ T cell homeostatic expansion in TCR␦⫺/⫺ mice. Additionally, although ␥␦ T cells are capable of inhibiting their own homeostatic expansion, this effect was detectable only when the competing ␥␦ T cells greatly outnumbered the population of interest. Nevertheless, greater cell number alone is not sufficient to explain the inhibitory effect of ␣ T cells on ␥␦ T cell homeostasis. Despite similar numbers at the time of harvest, purified CD8⫹ ␣ T cells dramatically reduced ␥␦ T cell reconstitution, whereas CD4⫹ ␣ T cells had no significant effect. Our studies suggest that IL-15 and, to a lesser extent, IL-7 contribute to ␥␦ T cell homeostasis. Consistent with our results, a recent study using a different system also suggested that both IL-7 and IL-15 support ␥␦ T cell homeostatic proliferation (2). The dual contribution of these cytokines to lymphoid ␥␦ T cell homeostasis likely explains the observed persistence of Thy-1⫹ lymphoid ␥␦ T cells in IL-15⫺/⫺ mice (26). Competition for cytokine resources, PNAS 兩 October 11, 2005 兩 vol. 102 兩 no. 41 兩 14745
IMMUNOLOGY
Fig. 5. Antibody-mediated depletion of NK cells results in enhanced ␥␦ T cell homeostatic expansion in the absence of ␣ T cells. (A) TCR⫺/⫺兾␦⫺/⫺ mice were treated with control mouse IgG or anti-NK1.1 antibody 3 days before adoptive transfer of CFSE-labeled ␥␦ T cells. (B) TCR␦⫺/⫺ mice were treated with control IgG, anti-TCR, anti-NK1.1, or anti-TCR plus anti-NK1.1 3 days before adoptive transfer of CFSE-labeled ␥␦ T cells. Recipient splenocytes were harvested 3 (A) or 5 (B) days after adoptive transfer. ␥␦ T cell proliferation was determined by flow cytometry. *, P ⬍ 0.1.
controversial, MHCI molecules could provide an additional source of competition between CD8⫹ ␣ T cells and ␥␦ T cells during the establishment of homeostasis. To address the role of MHCI in ␥␦ T cell homeostasis, we compared ␥␦ T cell proliferation after adoptive transfer into sublethally irradiated CD8⫺/⫺ (41) and 2m⫺/⫺ (42) recipients, both of which lack a normal CD8⫹ ␣ T cell compartment. Consistent with previous studies (2), ␥␦ T cell proliferation was comparable in 2m⫺/⫺ and CD8⫺/⫺ mice (Fig. 6B), suggesting that, overall, MHCI does not influence ␥␦ T cell homeostatic proliferation. Consequently, MHCI molecules do not likely provide a source of competition between ␥␦ T cells and CD8⫹ ␣ T cells.
primarily IL-15, is at least partly responsible for the interplay between ␣ T cells and ␥␦ T cells during homeostatic expansion. Similar to ␥␦ T cells, both IL-7 and IL-15 have been implicated in maintenance of the CD8⫹ ␣ T cell population (13, 46). More than 50% of CD8⫹ ␣ T cells express high levels of both IL-7R␣ and IL-2R 5 days after adoptive transfer into TCR⫺/⫺兾␦⫺/⫺ hosts (data not shown). Furthermore, the inhibitory effect of ␣ T cells on ␥␦ T cell homeostatic expansion was eliminated in the presence of excess exogenous IL-15. Although we did not directly test the ability of IL-7 to rescue ␥␦ T cell homeostatic expansion, it is likely that competition for IL-7 plays only a secondary role in our model. If ␣ T cells are capable of outcompeting ␥␦ T cells for IL-15 stores, one might expect ␣ T cells to express higher levels of the required cytokine receptors, compared with ␥␦ T cells. However, the donor ␥␦ T cells used in our studies express IL-2R and IL-15R␣ at a level that is comparable with or exceeds that of ␣ T cells, both before and after adoptive transfer (data not shown). Thus, the relative disadvantage of ␥␦ T cells, compared with ␣ T cells, during homeostatic expansion (Fig. 3B) is not likely due to poor cytokine binding. Instead, they may have a decreased propensity for survival and兾or a more limited inherent proliferative potential, compared with ␣ T cells. In support of this hypothesis, the majority of lymphoid ␥␦ T cells are thought to be short-lived, compared with ␣ T cells (10, 51). Of note, whereas cytokines play an important role in ␣ T cell homeostasis, several studies have revealed a role for T cell antigen receptor (TCR)–MHC interactions in maintaining the ␣ T cell pool (52, 53). Such interactions
might prolong ␣ T cell survival, providing an early advantage over most ␥␦ T cells. Additionally, ␣ T cells may actively inhibit ␥␦ T cell homeostasis by inducing ␥␦ T cell apoptosis through apoptosismediating surface antigen Fas (CD95)–Fas ligand (CD95L) interactions, as has been suggested for memory T cell homeostasis (54). Given an initial advantage, cotransferred ␣ T cells could quickly exceed the ␥␦ T cell population and would then provide substantial competition for essential cytokine stores. ␥␦ T cells are known to have potent antitumor effects both in vitro and in murine tumor models (55–58). In human studies, in vivo ␥␦ T cell expansions have been shown to correlate with temporary tumor regression and increased survival of leukemia and myeloma patients (59–61). In light of these findings, the possibility of using ␥␦ T cells in cancer immunotherapy is now being explored, and such studies have already shown some promise. However, animal studies suggest that prolonged maintenance of donor ␥␦ T cells may be essential to achieve complete tumor regression (62, 63). Defining the mechanisms responsible for regulating ␥␦ T cell homeostasis may be critical to the success of such therapies.
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14746 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0507520102
We thank Dr. Philippa Marrack (National Jewish Medical and Research Center) for generously providing antibody-producing hybridomas used in our studies and Dr. Marrack and Dr. Laurent Gapin for critical reading of the manuscript. This work was supported by National Institutes of Health Grants 2R01AI44920 (to R.L.O.), 2T32AI000048 (to J.D.F.), and 1R01HL65410 and 2R01AI40611 (to W.K.B.). J.D.F. was also supported by a fellowship from the American Heart Association (05200005Z) and C.L.R. by an Arthritis Foundation Investigator Award.
French et al.
