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We used clinically relevant murine allogeneic bone marrow transplantation (BMT) models to study the mechanisms by which IL-7 administration can improve posttransplant peripheral T cell reconstitution. After transplant we could distinguish two populations of mature donor T cells: (a) alloreactive T cells with decreased expression of CD127 (IL-7 receptor α chain) and (b) nonalloreactive T cells, which express CD127 and undergo homeostatic proliferation. IL-7 administration increased the homeostatic proliferation of nonalloreactive T cells, but had no effect on alloreactive T cells and the development of graft-versus-host disease. Allogeneic transplant of purified hematopoietic stem cells and adoptive transfer of thymocytes into lethally irradiated hosts suggested that recent thymic emigrants can undergo homeostatic proliferation and acquire a memory-like phenotype. We found by BrdU pulse-chase, cell cycle, and annexin V analyses that IL-7 administration has significant proliferative and antiapoptotic effects on posttransplant peripheral T cells. We conclude that homeostatic expansion is important for T cell reconstitution after allogeneic BMT and involves both transferred mature T cells and recent thymic emigrants. Apart from its thymopoietic effects, IL-7 promotes peripheral T cell reconstitution through its selective proliferative and antiapoptotic effects on nonalloreactive and de novo–generated T cells, but has no effect on alloreactive T cells.
Recipients of allogeneic hematopoietic stem cell transplantation (HSCT) suffer from a prolonged posttransplant immune deficiency that results in significant morbidity and mortality from infections (1–3). This immune deficiency is most pronounced in the T cell lineage due to a severe delay in T cell reconstitution. Reconstitution of CD4+ T cells in particular is severely impaired in adult recipients, and the rate of recovery of CD4+ T cells is inversely related to the age of the recipient and correlates with the risk of developing infections (3–5). Posttransplant T cell recovery is affected by a number of factors, including previous chemo/radiation therapy, a lack of sustained transfer of donor immunity, recapitulation of immunological ontology, age of the recipient, infections, thymic involution after puberty, donor/host histoincompatibility, graft-versus-host disease (GVHD), graft rejection, and drug-induced immunosuppression such as from antibiotics or GVHD prophylaxis (reviewed in ref. 6).
Prolonged T cell deficiency after transplant continues even after quantitative regeneration has taken place, suggesting that a T cell dysfunction occurs in the posttransplant period that is still poorly understood (7–10). This impaired T cell immunity is at least partially explained by the limited diversity of the posttransplant T cell receptor (TCR) repertoire (11).
Posttransplant T cell reconstitution involves thymus-dependent de novo T cell generation, extrathymic expansion of mature T cells that were transferred with the allograft, and possibly de novo T cell generation from extrathymic sites such as mucosa-associated lymphoid tissues in the gut (12). Previous studies in mice and humans have suggested that T cell reconstitution in older recipients is more dependent on extrathymic expansion of mature T cells than on thymopoiesis; this conclusion was based upon the delayed reappearance of naive T cells (especially CD4+ T cells) and a diminished T cell repertoire (13–18).
The size of the peripheral T cell pool can rapidly expand and contract during an antigen-specific immune response, but remains remarkably constant throughout life due to tight regulation by a number of cytokines (especially IL-7 and IL-15), and in the case of naive T cells, the interaction of the TCR with self-peptide–MHC ligands (reviewed in ref. 19). Homeostatic expansion/proliferation is an important regulatory mechanism of T cell homeostasis and is defined as the proliferative response of mature T cells in a lymphopenic environment. Naive T cells convert to a memory-like phenotype and require IL-7 and TCR recognition during homeostatic proliferation.
IL-7 is a γ-common chain cytokine, which is secreted by stromal cells in bone marrow (BM) and thymus and plays an important role in the development, survival, and homeostatic proliferation of T cells (19–22).
In this study, we examined the contribution of homeostatic proliferation, which is associated with the acquisition of a memory-like phenotype, to T cell reconstitution in recipients of allogeneic HSCT. Our data suggest that in addition to mature T cells (transferred in the allograft), de novo–generated T cells (derived from donor hematopoietic precursor cells) also undergo homeostatic proliferation in the lymphopenic posttransplant environment of the recipient. We found that posttransplant IL-7 administration promotes T cell reconstitution not only through its effects on thymopoiesis (as we have previously shown, as described in ref. 23), but also by stimulating homeostatic proliferation of nonalloreactive donor T cells (either de novo–generated or transferred with the allograft) and antiapoptotic effects on peripheral T cells.
Female C57BL/6 (B6, H-2b), C3FeB6F1 (H-2b/k), B6D2F1/J (H-2b/d), B10.BR (H-2k), CBA/J (H-2k), and C57BL/6 (Ly5.1+) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). For some experiments, C57BL/6 (Ly5.1+) mice were obtained from the National Cancer Institute (Frederick, Maryland, USA). Mice used in BM transplantation (BMT) experiments were between 8 weeks and 10 weeks of age or were 9 months (CBA/J) of age. BMT protocols were approved by the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee. BM cells were removed aseptically from femurs and tibias. Donor BM was T cell depleted (TCD) by incubation with anti–Thy-1.2 for 30 minutes at 4°C, followed by incubation with Low-TOX-M rabbit complement (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) for 40 minutes at 37°C. Splenic T cells were obtained either by purification over a nylon wool column, by positive selection with anti-CD5 antibodies conjugated to magnetic beads (Miltenyi Biotec, Auburn, California, USA), or by negative selection using lineage-specific antibodies (NK1.1, Gr-1, Mac-1, B220) conjugated to FITC or phycoerythrin (PE) followed by anti-FITC or anti-PE antibodies coupled to magnetic beads (Miltenyi Biotec). Cells (5 × 106 to 10 × 106 BM cells with or without splenic T cells) were resuspended in DMEM (Life Technologies, Grand Island, New York, USA) and transplanted by tail vein infusion (0.25 ml total volume) into lethally irradiated recipients on day 0. On day 0, prior to transplantation, recipients received 1,300 cGy total body irradiation (137Cs source) as a split dose with a 3-hour interval between doses (to reduce gastrointestinal toxicity). Mice were housed in sterilized microisolator cages and received normal chow and autoclaved hyperchlorinated drinking water (pH 3.0).
Anti-murine CD16/CD32 FcR block (2.4G2) and all of the following fluorochrome-labeled antibodies against murine antigens were obtained from BD PharMingen (San Diego, California, USA): Ly9.1 (30C7), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD62L (MEL-14), CD122 (TM-B1), CD44 (IM7), CD45R/B220 (RA3-6B2), NK1.1 (PK136), CD11b (M1/70), CD25 (PC61), CD127 (B12-1), CD69 (H1.2F3), c-kit (2B8), SCA-1 (D7), Bcl-2 (3F11), annexin V, the BrdU detection kit, 7-amino-actinomycin D (7-AAD), isotypic controls, rat IgG2a,κ (R35-95), rat IgG2a,λ (B39-4), rat IgG2b,κ (A95-1), rat IgG1,κ (R3-34), hamster IgG1,κ (A19-3), and hamster IgG2,λ (Ha4/8). Streptavidin-FITC, -PE, and –peridinin chlorophyll protein were also obtained from BD PharMingen. Recombinant human IL-7 was kindly provided by Michel Morre (Cytheris, Vanves, France). CFSE was obtained from Molecular Probes Inc. (Eugene, Oregon, USA). BrdU (Sigma-Aldrich, St. Louis, Missouri, USA) and deoxyribonuclease I (DN-25; Sigma-Aldrich) were used in BrdU and cell cycling studies.
Tissue culture medium consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine.
Recipients of allogeneic BMT received BrdU (0.8 mg/ml) from day 15 to day 33 after transplant in their drinking water, which was prepared fresh daily and protected from light.
For immune reconstitution studies, IL-7 was given from day 21 to day 27 (10 μg/day intraperitoneally). For GVHD studies, IL-7 was administered on days 0–7 (10 μg/day intraperitoneally). In adoptive transfer experiments with CFSE-labeled T cells, IL-7 was administered at a dose of 10 μg/day intraperitoneally from day 0 to day 3.
BM cells from donor mice were obtained as described above. Lineage-negative cells were obtained by magnetic bead cell separation using lineage-specific antibodies against CD3, NK1.1, Gr-1, Mac-1, B220, CD4, and CD8. These lineage-negative cells were incubated with antibodies against SCA-1 and c-kit, and then lineage-negative SCA-1+ and c-kit+ cells were obtained by flow cytometric–assisted cell sorting (MoFlo; Cytomation Inc., Fort Collins, Colorado, USA) as previously described (24).
The severity of GVHD was assessed with a clinical GVHD scoring system as previously described (25). Briefly, ear-tagged animals in coded cages were individually scored every week for five clinical parameters on a scale from 0 to 2: weight loss, posture, activity, fur, and skin. A clinical GVHD index was generated by summation of the five criteria scores to give a total score of 0–10. Survival was monitored daily. Animals with scores of 5 or higher were considered moribund and were sacrificed.
As described before, splenic T cells were obtained by purification over a nylon wool column, which resulted in greater than 70% purity. Single-cell suspensions were prepared from inguinal and axillary lymph nodes and spleens according to standard protocols and used for flow cytometric analysis. Intraepithelial lymphocytes from gut were obtained as published previously (26). Briefly, mesenteric tissues were removed from intestines that were then cut longitudinally, washed with PBS, and put into 250-ml cell culture flasks. Segments of intestines were incubated with RPMI with 10% FCS for 1 hour at 37°C in a shaker. Finally, cells released from the intestine into the medium were obtained using either a nylon wool column or Percoll gradient separation. Single-cell suspensions of liver cells were obtained by Percoll gradient separation.
Cells were labeled with CFSE as described previously (27). Briefly, cells (thymocytes or splenocytes) were incubated with CFSE at a final concentration of 2.5 μM in HBSS at 37°C for 10 minutes. Cells were then washed three times with HBSS before intravenous injection.
Cells were washed in FACS buffer (PBS with 0.5% BSA and 0.1% sodium azide), and 106 cells/ml were incubated for 30 minutes at 4°C with CD16/CD32 FcR block. Subsequently, cells were incubated for 30 minutes at 4°C with antibodies and washed twice with FACS buffer. The stained cells were resuspended in FACS buffer and analyzed on a FACScan flow cytometer (Becton, Dickinson and Co., San Jose, California, USA) with CellQuest software or with FlowJo software (Tree Star Inc., San Carlos, California, USA).
Cells were incubated for 5 hours with 10 ng/ml PMA and 2 μM ionomycin. Brefeldin A (10 μg/ml) was added after 2 hours of incubation. The cells were then harvested, washed, and stained with fluorochrome-conjugated antibodies against cell surface antigens. Subsequently, cells were fixed and permeabilized with Cytofix/Cytoperm Kit reagents (BD PharMingen) and stained with PE-conjugated antibodies.
Single-cell suspensions of spleens were obtained from allogeneic BMT recipients at day 28 or day 33. Cells were stained with fluorochrome-conjugated antibodies against cell surface antigens. The cells were then washed, fixed, permeabilized, and refixed according to manufacturer protocol using the BrdU Flow Kit (BD PharMingen). Fixed cells were incubated with DNase for 1 hour at 37°C and then washed and stained with anti-BrdU antibody or 7-AAD. Finally, flow cytometric analysis was conducted by gating for the designated populations and the FL3 fluorescence detector was used in linear mode for 7-AAD analysis.
Splenocytes (5 × 104 cells/well; prepared as described above) were incubated for 5 days with irradiated (2,000 cGy) C3FeB6F1 splenocytes as stimulators (2 × 105 cells/well) in 96-well plates. Cultures were pulsed during the final 18 hours with 1 microcurie/well 3H-thymidine and cells were harvested with a Topcount Harvester (Packard Bioscience Co., Meriden, Connecticut, USA).
All values are expressed as mean ± SEM. Statistical comparison of experimental data was performed with the nonparametric unpaired Mann-Whitney U test, whereas the Mantel-Cox log rank test was used for survival data. A P value below 0.05 was considered statistically significant.
To study peripheral T cell reconstitution after allogeneic HSCT and the effects of IL-7, we used well-described clinically relevant MHC-matched and -mismatched murine allogeneic BMT models. First, we analyzed the donor-derived immune reconstitution after a TCD allogeneic BMT (in which case no GVHD occurs) or an allogeneic BMT with GVHD in an MHC-matched model with a disparity in minor histocompatibility antigens: B10.BR → CBA/J (23, 28, 29). GVHD was induced by the addition of donor splenic T cells to the allograft. We chose this model because of its clinical relevance for and resemblance to patients receiving an allogeneic HSCT from an MHC-matched unrelated donor. These patients suffer from a profound and prolonged posttransplant immune deficiency associated with high mortality from infections, which is in most studies even higher than mortality from relapse (2, 30–34).
Donor-derived T cell reconstitution was severely delayed, and even at 3 months after BMT the numbers of CD3+, CD4+, and CD8+ T cells had not reached the levels found in normal host or donor mice (Figure (Figure1a).1a). Similar to T cell reconstitution in allogeneic HSCT patients (3), early CD4+ T cell reconstitution was slower than CD8+ T cell reconstitution, and the CD4/CD8 ratio was decreased during the first 6 weeks after transplant (data not shown). Donor-derived T cell reconstitution in recipients with GVHD remained severely suppressed until the mice died; this is reminiscent of the severe immunosuppression found in patients with GVHD (35).
In contrast to the severely suppressed B cell reconstitution in recipients with GVHD, the numbers of donor-derived B cells in allogeneic TCD-BMT recipients reached the levels of those in nontransplanted host animals by 6 weeks after transplant (Figure (Figure1b).1b). Numbers of CD11b+ monocytes and granulocytes in the spleen of allogeneic TCD-BMT recipients were three- to fourfold higher than in normal donor and host animals in the first 2 weeks after transplant, and normalized by 6 weeks after transplant (data not shown). This early increase in CD11b+ cells is probably be due to granulopoiesis in the spleen during the first weeks after transplant. Interestingly, recipients with GVHD also had high numbers of CD11b+ cells, which had not decreased by 6 weeks after transplant when all animals had died from GVHD. Finally, NK reconstitution was rapid, and numbers of donor-derived NK cells reached the levels of normal donor and host animals by 4 weeks (data not shown).
In addition to the profound delay in T cell reconstitution, we noted that most donor-derived CD4+ and CD8+ T cells had a memory-like CD44high phenotype at day 28 after an allogeneic TCD-BMT (Figure (Figure1c).1c). In our previously published experiments, we did not observe any difference in T cell recovery in the thymus or periphery between IL-7–treated and control groups when IL-7 was administered from day 0 to day 14 after BMT (23). However, we found increased immune reconstitution after IL-7 administration from day 14 to day 28 (continuous by subcutaneous pump) or from day 21 to day 27 (intraperitoneally). Therefore, we treated recipients from day 21 to day 28 after BMT with IL-7. Because IL-7 administration could stimulate early lymphoid precursors, which can express IL-7R (36) and need 15–18 days to differentiate into naive T cells (37), we measured donor-derived naive and memory T cell populations at varying times after transplant. This also allowed us to assess the duration of the IL-7–induced increase in T cell numbers after discontinuation of IL-7 administration. We detected no increase in naive CD4+ T cells and only a small (but significant) increase in naive CD8+ T cells at day 28 after transplant (Figure (Figure1d).1d). In contrast, memory-like CD4+ and CD8+ T cells in IL-7–treated recipients were significantly increased, and donor-derived memory-like CD4+ T cells on day 28 reached even higher levels than in normal animals.
To better understand the preferential effect of IL-7 on the posttransplant reconstitution of memory/activated T cells, we first analyzed the phenotype and proliferation of the various populations of donor T cells after allogeneic BMT, including (a) alloreactive T cells, mature T cells contained in the allograft that recognize host allo-antigens and cause GVHD; (b) nonalloreactive T cells, mature T cells in the allograft that do not recognize alloantigens but have been suggested to contribute to T cell reconstitution, especially in older recipients with diminished thymic function (17); and (c) donor hematopoietic stem cell–derived T cells, newly derived T cells that originate from the transplanted donor hematopoietic stem cells and have been generated through thymic (and possibly also through extrathymic) differentiation.
To assess the proliferative activity and cell surface antigen expression patterns of alloreactive and nonalloreactive donor T cells after transplantation, we transferred CFSE-labeled purified splenic T cells from B6 (Ly5.1) donors into lethally irradiated syngeneic (B6) and allogeneic (C3FeB6F1) recipients and analyzed the number of cell divisions and cell surface antigen expression daily for 7 days (Figure (Figure2a,2a, 64 hours, and data not shown). When transferred into a lethally irradiated syngeneic recipient, B6 (Ly5.1) T cells undergo not more than three cell cycles in 64 hours, which is in agreement with the slow proliferation described previously for homeostatic proliferation of mature T cells in lymphopenic hosts (38–42).
These T cells acquire a partially activated memory-like cell surface expression pattern during their homeostatic proliferation, consisting of increased expression of CD44 and CD122. However, CD62L was downregulated on less than 40% of the cells and CD25 expression was not increased.
CFSE-labeled T cells transferred into lethally irradiated allogeneic recipients could be separated into two populations of cells based upon their proliferation and cell surface expression. The first population consisted of cells that had undergone more than seven cell cycles in 64 hours and acquired a fully activated cell surface expression pattern with increased expression of CD44, CD122, and CD25, and decreased expression of CD62L. Based upon their rapid proliferation with a cell cycle duration of 6–9 hours and their acquisition of an activated cell surface antigen pattern, which is identical to the antigen-driven rapid proliferative response of alloreactive T cells described previously (43, 44), we conclude that these cells represent alloreactive T cells that respond to host antigens.
The second population of T cells has slower proliferative kinetics (not more than three cell cycles in 64 hours) similar to those of T cells transferred into syngeneic hosts. These slowly proliferating T cells in allogeneic recipients acquire a cell surface expression pattern similar to that of T cells transferred into syngeneic recipients: increased CD44 and CD122 expression, but only limited downregulation of CD62L and no increase of CD25 expression. Based upon their similarity in proliferation kinetics and cell surface expression pattern, we conclude that these cells represent nonalloreactive T cells undergoing homeostatic proliferation.
We also assessed the expression of the early activation antigen CD69, and found increased expression on CD4+ and CD8+ T cells transferred to syngeneic or allogeneic recipients at 16 hours; this increase disappeared by the 40-hour timepoint (Figure (Figure22b).
The differences in activation state between nonproliferating, nonalloreactive T cells and alloreactive T cells are further illustrated by differences in cell size as determined by flow cytometry (Figure (Figure2c).2c). Alloreactive T cells have a larger, blast-like size than nonalloreactive proliferating T cells, which are only slightly larger than nonproliferating T cells.
To further define the functional differences between the fast-dividing alloreactive and slow-dividing nonalloreactive T cells, we purified fast-proliferative and slow-proliferative T cells from recipients of allogeneic CFSE-labeled T cells by flow cytometry and incubated these populations with irradiated host cells for 5 days. We found that alloreactive fast-dividing T cells had a significantly greater proliferative response to host antigen than did the slower-dividing nonalloreactive T cells (Figure (Figure22d).
To assess the sensitivity of these nonalloreactive and alloreactive T cells to IL-7 administration, we analyzed the expression of the IL-7 receptor α chain (IL-7Rα, CD127). We found that most naive CD44low and memory/activated CD44high CD4+ and CD8+ T cells in normal mice expressed IL-7R (Figure (Figure3a).3a). We then analyzed the IL-7Rα expression on these same populations of naive and memory/activated T cells in recipients of allogeneic TCD-BMT (which incur no GVHD) and found IL-7Rα expression on the majority of cells even in recipients treated with IL-7.
We also analyzed IL-7Rα expression on CFSE-labeled donor T cells transferred to syngeneic or allogeneic recipients and found intact IL-7Rα expression on homeostatically proliferating T cells in syngeneic or allogeneic recipients even after more than five cell cycles (Figure (Figure3,3, b and c). When we analyzed CFSE-labeled purified T cells 16 hours after injection into lethally irradiated recipients, we observed a decrease in IL-7Rα expression on donor T cells transplanted into allogeneic recipients compared with syngeneic recipients: 51% versus 83% for CD4+ T cells and 58% versus 86% for CD8+ T cells (data not shown and Figure Figure3b).3b). Moreover, alloreactive T cells that had gone through more than eight cell divisions in 7 days, expressed low levels of IL-7Rα (Figure (Figure3c).3c). When we compared the proliferation and IL-7Rα expression on CFSE-labeled T cells in the lymph nodes and the spleen 3 days after infusion, we noted only a small fraction of fast-proliferating alloreactive T cells in the lymph nodes compared with the spleen (Figure (Figure3d).3d). In recipients with clinical GVHD, IL-7Rα expression on T cells in the target organs of GVHD (such as gut and liver) was evaluated, and donor-derived TCR-αβ+ intraepithelial lymphocytes in the gut and T cells in the liver were found to have downregulated their IL-7Rα expression on day 7 (gut) or day 14 (spleen, lymph node, and liver) (Figure (Figure33e).
These data suggest that alloreactive T cells are not sensitive to IL-7 and therefore that IL-7 administration should have no (or little) effect on the development of GVHD. We have previously published that IL-7 administration had no effect on GVHD mortality in two different GVHD models. However, a recent study showed increased GVHD mortality in IL-7–treated recipients (45). The authors suggested that the difference could be due to the fact that in our previous study all experimental groups had high mortality (75–100%) from GVHD, which could impair the sensitivity to find smaller differences in GVHD mortality between groups. Therefore, we studied GVHD mortality in recipients of varying doses of T cells, but could still not detect a significant increase in GVHD mortality between IL-7–treated and control groups, even when GVHD mortality was only 50% after more than 100 days (Figure (Figure33f).
Donor-derived peripheral T cells in recipients of purified allogeneic hematopoietic stem cells have predominantly a memory-like phenotype. As stated before, previous studies have suggested that the predominance of T cells with a memory/activated phenotype after allogeneic BMT is due to the homeostatic expansion of nonalloreactive mature T cells, which are transferred with the allograft. However, in our experiments with TCD allografts (Figure (Figure1c),1c), we noted that in the first weeks after transplant, donor CD4+ and CD8+ T cells are mostly of a memory/activated phenotype. To rule out that this early reconstitution of memory/activated T cells was due to homeostatic expansion of a few contaminating mature nonalloreactive donor T cells that had escaped the T cell depletion, we repeated our T cell reconstitution experiments with highly purified allogeneic lineage-negative SCA-1+c-kit+ hematopoietic stem cells (Figure (Figure4).4). At day 28 after transplant we again found a predominance of memory/activated CD4+ and CD8+ T cells. IL-7 administration to recipients of allogeneic hematopoietic stem cells resulted not only in enhanced donor T cell reconstitution, but also significantly increased naive donor CD4+ and CD8+ T cells. We conclude from these reconstitution experiments with purified allogeneic hematopoietic stem cells that early T cell reconstitution with mostly memory/activated T cells can also occur in the absence of mature nonalloreactive donor T cells.
To explain the predominance of memory-like T cells in recipients of allogeneic hematopoietic stem cells, we hypothesized that recent thymic emigrants (RTEs) with a naive phenotype in the lymphopenic environment of an allogeneic recipient will undergo homeostatic expansion and acquire a memory-like phenotype (CD44highCD122+; see Figure Figure2a)2a) similar to that of mature T cells, which undergo homeostatic proliferation.
To test this hypothesis, we transferred thymocytes from CBA/J donors or from recipients of allogeneic (B10.BR → CBA/J) TCD-BMT into lethally irradiated CBA/J recipients, and assessed their proliferation 5 days later. When we compared the transfer of CFSE-labeled thymocytes (Figure (Figure5a)5a) and splenocytes (Figure (Figure5b)5b) from CBA/J donors into a lethally irradiated CBA/J host, we observed an identical slow homeostatic proliferation (with a cell cycle longer than 24 hours) in both cell populations. This was true for both CD4+ and CD8+ T cells, and we did not detect any CD4+CD8+ double-positive cells in recipients of thymocytes. All slowly proliferating CD4+ and CD8+ thymocytes had upregulated CD44+ expression, which is consistent with homeostatic proliferation (data not shown). These results demonstrated that thymocytes can undergo homeostatic proliferation in a lymphopenic environment.
We then performed similar experiments with CFSE-labeled thymocytes and purified splenic T cells from recipients of allogeneic (B10.BR → CBA/J) TCD-BMT transferred into a lethally irradiated CBA/J host (Figure (Figure5,5, c and d). We found that both populations could undergo homeostatic proliferation, although we noted a second rapidly proliferating population among transferred thymocytes. As expected for homeostatically proliferating cells, we observed increased expression of CD44 and CD122 in the slow-proliferating populations of transferred thymocytes (data not shown). When we compared the two rapid- and slow-proliferating populations of thymocytes with the proliferation kinetics of syngeneic or allogeneic T cells transferred into lethally irradiated recipients, we noted that the rapidly proliferating thymocytes had the same proliferation pattern as allogeneic T cells (Figure (Figure5e),5e), whereas the slowly proliferating thymocytes had proliferation kinetics similar to those of syngeneic T cells. These rapidly proliferating thymocytes could represent de novo–generated alloreactive thymocytes or mature alloreactive T cells that had been transferred with the TCD allograft and had invaded the thymus.
We conclude from these studies that thymocytes have a capacity to undergo homeostatic expansion similar to that of splenic T cells in lymphopenic lethally irradiated syngeneic or allogeneic recipients. These data suggest that RTEs in a lymphopenic recipient of allogeneic BMT could acquire a memory-like phenotype and undergo homeostatic proliferation, which could explain the predominance of donor-derived memory-like T cells even in recipients of purified donor hematopoietic stem cells.
We also assessed the effects of IL-7 administration on the homeostatic proliferation of CFSE-labeled thymocytes transferred into lethally irradiated syngeneic recipients. Four days after transfer, we observed an increased fraction of slowly proliferating CD4+ and CD8+ thymocytes in the IL-7–treated recipients (data not shown). These results provide further evidence that IL-7 administration can stimulate homeostatic proliferation of RTEs.
To further analyze the effects of IL-7 administration on peripheral T cell reconstitution in recipients of allogeneic BMT, we assessed the fraction of peripheral donor T cells in S/G2/M phase and found a significant increase in IL-7–treated recipients (Figure (Figure6a).6a). These data suggest that IL-7 has a proliferative effect on peripheral T cells after allogeneic BMT. This proliferative effect of posttransplant IL-7 administration affected mostly memory-like T cells, as demonstrated with pulse-chase BrdU labeling studies (Figure (Figure6b).6b). We further assessed the specific effects of IL-7 administration on homeostatic proliferation of donor T cells and found an increase in the number of cell divisions of CD4+ and CD8+ T cells in lethally irradiated syngeneic recipients (Figure (Figure6c).6c). In IL-7–treated allogeneic recipients, we observed a remarkable increase in nonalloreactive slowly proliferating CD8+ T cells, whereas IL-7 administration (again) did not increase the proliferation of the alloreactive cells (Figure (Figure6d).6d). When we determined the potential to secrete IFN-γ, we found, as expected, that only fast-dividing alloreactive T cells demonstrated increased IFN-γ secretion upon a short stimulation with PMA-ionomycin (Figure (Figure6e).6e). IL-7 administration to the recipients of CFSE-labeled T cells did not affect IFN-γ secretion by alloreactive T cells.
To test a possible antiapoptotic effect of IL-7 on peripheral T cell reconstitution after allogeneic BMT, we assessed the number of apoptotic splenic T cells in young (3-month-old) and middle-aged (9-month-old) allogeneic recipients (Figure (Figure7,7, a and b). We found an increased number of apoptotic CD4+ and CD8+ T cells in middle-aged (but not young) recipients of allogeneic BMT that was significantly decreased by IL-7 administration. We determined Bcl-2 expression levels by flow cytometry and found a decrease in Bcl-2+ cells in peripheral T cells of allogeneic BMT recipients compared with normal animals, especially in middle-aged recipients. However, we could not detect a significant increase in Bcl-2+ T cells in IL-7–treated middle-aged recipients of allogeneic BMT.
The majority of donor-derived T cells in the first weeks (mouse) or months (man) after allogeneic HSCT have a memory-like phenotype, and this has been interpreted previously as evidence for a predominance of antigen-driven peripheral expansion of mature T cells from the allograft and a deficit in thymic output in the posttransplant period (14, 15). A number of studies measuring RTEs by TCR rearrangement excision circles (TRECs) have questioned the lack of posttransplant thymic function and were able to demonstrate significant thymic output even in older recipients of allogeneic HSCT (46–49). Murine studies have demonstrated that aged recipients of T cell–depleted BMT display normal thymic subsets, appropriate negative selection, and significant thymic T cell output (50% of the thymic output of young recipients) (17).
Our results regarding the predominance of memory-like donor T cells after transplantation of purified hematopoietic stem cells challenge the hypothesis that memory-like donor T cells during posttransplant reconstitution are being generated exclusively by the expansion of mature T cells in the allograft. Moreover, our data are in agreement with human studies regarding T cell reconstitution following an allogeneic peripheral blood stem cell transplant with CD34+-selected cells, which demonstrated that most CD4+ T cells in the posttransplant period expressed CD45RO (50).
Our data suggest that the homeostatic expansion of peripheral T cells in a lymphopenic recipient involves both thymic emigrants and nonalloreactive T cells in the allograft and has the characteristics of homeostatic expansion (slow proliferation kinetics and partially activated cell surface antigen pattern) instead of classic antigen-driven T cell proliferation (with rapid proliferation and full activated cell surface antigen pattern).
In agreement with our findings that RTEs can undergo homeostatic proliferation, Le Campion et al. (51) have demonstrated recently that RTEs in neonatal mice undergo homeostatic proliferation. Interestingly, neonatal RTEs entering into the periphery of a neonatal mouse maintained their naive phenotype during proliferation, whereas RTEs that exited from a neonatal thymic graft in an adult mouse acquired an activated phenotype during homeostatic proliferation in the adult lymphoid environment.
Goldrath et al. showed that the memory phenotype (CD44+CD122+Ly6C+) acquired by naive CD8+ T cells during homeostatic-driven proliferation in sublethally irradiated syngeneic hosts was transient, and naive T cells started to accumulate around 3 weeks after transfer of spleen and lymph node cells (52). This was explained as the reversion of memory to naive phenotype by homeostatic CD8+ T cells. However, several studies have suggested that this reversal does not occur, and that the increase of naive T cells over time is due to de novo T cell development in the thymus (53–55). This would suggest that homeostasis-driven proliferation will replete only the memory T cell pool, and thymopoiesis is required for reconstitution of the naive T cell pool. We hypothesize that one reason for the delay in reconstitution of the naive T cell pool is related to the conversion of RTEs to memory-like T cells, which will then undergo homeostatic proliferation until the total T cell pool has been repleted.
Our data demonstrate that the expansion of alloreactive and nonalloreactive T cells in allogeneic recipients can be distinguished based upon the number of divisions in the first days after transplant, cell size, and the acquisition of a partially or fully activated cell surface pattern. Interestingly, the early activation marker CD69 is temporarily upregulated on both alloreactive and nonalloreactive T cells before the first cell division has been completed. In our studies, CD25 expression was the most reliable activation marker for differentiation between alloreactive and nonalloreactive donor T cell populations. We found a greater proliferative and cytokine response in vitro to host antigens by alloreactive (fast-dividing) cells compared with nonalloreactive (slow-dividing) cells, and these results confirm our hypothesis that the fast-dividing population represents alloreactive T cells.
Most studies have indicated that naive T cells require expression of their TCR and contact with the correct restricting MHC class I or II allele for their survival and/or homeostatic proliferation (40, 56–61). However, some studies have argued that survival and/or homeostatic proliferation of CD4+ T cells can occur without matching MHC class II ligands (62–64). These studies have been questioned because of the possibility of contamination with MHC-expressing antigen-presenting cells among the transferred CD4+ T cells (19). Seddon and Zamoyska (65) have suggested that TCR and IL-7R signals can drive homeostatic proliferation independently of each other, and we are currently analyzing the requirement for IL-7 and IL-15 in this situation.
We found that posttransplant IL-7 administration in allogeneic HSCT recipients will predominantly result in an expansion of memory-like donor T cells. Apart from its thymopoietic effects (23, 66, 67), IL-7 has been proposed as an important regulator of lymphoid homeostasis (reviewed in ref. 19). In normal mice, most IL-7 is bound to its receptor on T cells, levels of free IL-7 are low, and T cells have to compete for the limited IL-7 supply. However, in lymphopenic mice, levels of unbound IL-7 increase and naive T cells have increased access to IL-7 and can undergo homeostatic expansion. It is thought that IL-7 potentiates the reactivity of the residual naive T cells against self-ligands, which results in their differentiation into effector T cells and proliferative response. In support of this theory, IL-7 transgenic mice have an expanded memory T cell pool (68). Our data suggest that IL-7 administration to allogeneic BMT recipients, in addition to its thymopoietic effects as previously reported (23), has important extrathymic effects and can stimulate survival and homeostatic proliferation of RTEs and mature T cells in the periphery.
Our findings regarding the importance of homeostatic proliferation for T cell reconstitution after an allogeneic BMT would suggest that administration of IL-15, which is another important γc cytokine that can regulate CD8+ T cell homeostatic expansion (69), can enhance posttransplant T cell reconstitution. Indeed, we have found that IL-15 administration increased CD8+ T cell numbers and function after allogeneic BMT and specifically enhanced homeostatic CD8+ T cell proliferation (Ö. Alpdogan, unpublished observations).
When we analyzed CFSE-labeled purified T cells at different timepoints after injection into lethally irradiated recipients, we observed a decrease in IL-7Rα expression on donor T cells transplanted into allogeneic recipients compared with syngeneic recipients. These data suggest that alloreactive T cells undergo rapid downmodulation of IL-7R upon alloactivation. In addition, in a series of mixed lymphocyte reaction (MLR) experiments with splenic T cells from naive animals, or from recipients with GVHD as effectors and irradiated host splenocytes as stimulators, we saw no increase in proliferation with the addition of IL-7 (0.5–500 ng/ml).
A recent human DNA microarray analysis of T cells incubated with IL-2 demonstrated that IL-7Rα was the most potently repressed (four- to fivefold) cytokine receptor (70). This IL-2–induced decrease of IL-7R expression was dependent on PI3K/Akt signaling. Therefore, we hypothesize that the downregulation of IL-7Rα on alloreactive T cells may be, at least partially, the result of the autocrine or paracrine effects of IL-2 secretion by these activated T cells. Interestingly, this downregulation of IL-7Rα on a population of T cells transferred to an allogeneic recipient occurred before the completion of the first cell cycle, as measured by CFSE expression. In this very early point of T cell infusion, local IL-2 concentrations are probably very low and difficult to determine. On the other hand, since IL-2 is a potent stimulator of IL-2Rα, and IL-2Rα induction and IL-7Rα repression by IL-2 occur concomitantly as demonstrated by Xue et al. (70), the level of IL-2Rα expression could be used as an indirect indicator of the presence of IL-2. Only 5.4% of CFSE-labeled alloreactive CD8+ T cells expressed IL-2Rα when analyzed 16 hours after infusion, and the expression of IL-2Rα on alloreactive T cells gradually increased over the course of several days. As shown in Figure Figure2a,2a, 3 days after transfer of CFSE-labeled donor T cells, all fast-proliferative T cells expressed IL-2Rα on their surface, whereas homeostatically, slow-proliferating CD8+ T cells did not express IL-2Rα on their surface at any timepoint (16 hours, 40 hours, 64 hours, 88 hours, and day 7). Taken together, these data suggest that other factors in addition to IL-2 can contribute to the downregulation of IL-7Rα on alloreactive T cells, especially in the early phase after activation against alloantigens.
In addition, a study using the P14 transgenic mouse to analyze the changes in gene expression (by DNA microarray) during the differentiation of naive antigen-specific CD8+ T cells into effector and memory T cells demonstrated that IL-7Rα expression was diminished (2.4-fold) during the transition from naive to effector T cells (71). Finally, in a recent review by Schluns and Lefrancois (72) regarding CD8+ memory T cells, it is suggested that IL-7R expression is downregulated on antigen-specific effector cells, but memory T cells will re-express IL-7Rα after antigen clearance and contraction of effector cells.
Important differences between our GVHD experiments and those by Sinha et al. (45), which demonstrated increased GVHD at some donor T cell doses, are the use of a different IL-7 preparation at a higher dose and for a longer period of treatment. We had previously observed that even BMT recipients generate neutralizing antibodies against recombinant human IL-7, which has only 81% homology with murine IL-7 (23). Therefore, in our experiments, recipients were never treated for more than 14 days (either from day 0 to day 14 or from day 14 to day 28), whereas Sinha et al. treated recipients continuously for 4 weeks. We are currently generating recombinant murine IL-7 to analyze the effects of prolonged administration of IL-7 after transplant in our mouse models.
Older recipients of allogeneic HSCT have been shown to have a five- to tenfold higher number of apoptotic T cells in the periphery than do healthy controls, and this was associated with GVHD, human leukocyte antigen disparity, time after transplant, and the expression of Fas and Bcl-2 (73, 74). In our experiments, IL-7 administration was capable of decreasing the number of apoptotic peripheral T cells by 50%. Although Bcl-2 levels were significantly decreased in T cells of older recipients compared with younger recipients (data not shown), we observed only a small increase in Bcl-2 levels in T cells of older IL-7–treated recipients. Therefore the protective effect of IL-7 administration on peripheral T cells cannot be explained by upregulation of Bcl-2, but could involve other IL-7–induced effects on survival, such as PI3K-dependent increased metabolic activity (75) or induction of the transcription factor lung Kruppel-like factor, which is required for naive T cell survival (76, 77).
The important role of homeostatic proliferation in posttransplant T cell reconstitution could also partially explain the differences in CD4+ versus CD8+ T cell reconstitution. CD8+ T cells survive better and engage faster in homeostatic proliferation than CD4+ T cells (40, 62, 78). In addition, homeostatic expansion of CD8+ T cells is not limited to secondary lymphoid organs and their antigen-presenting cells (79), and does not require ligands for CCR7 (80), whereas homeostatic expansion of CD4+ T cells is limited to lymphoid organs. This increased potential for CD8+ T cells to undergo homeostatic proliferation could result in a better reconstitution of CD8+ T cells than of CD4+ T cells after transplant.
In conclusion, this study demonstrates the importance of homeostatic proliferation by both mature T cells in the allograft and de novo–generated donor T cells for posttransplant T cell reconstitution in recipients of an allogeneic HSCT. IL-7 administration has clinical potential to improve posttransplant immune deficiency, because it can promote posttransplant T cell reconstitution through its antiapoptotic and proliferative effects on thymocytes and nonalloreactive mature T cells without stimulating alloreactive T cells.
The authors would like to thank the staff of the Research Animal Resource Center for excellent animal care, and Richard O’Reilly and Eric Pamer for helpful discussions and support throughout the project. We would like to thank Samuel Rice, Kartono Tjoe, Marsinay Smith, and Thomas van Huystee for their technical assistance. This work was supported by NIH grants HL-69929 and HL-72412 (M.R.M. van den Brink) and by a translational research grant from the Leukemia and Lymphoma Society (M.R.M. van den Brink). M.R.M. van den Brink is the recipient of a Damon Runyan Scholar Award of the Cancer Research Fund and a research award from the V scholar program of the V Foundation.
Conflict of interest: The authors have declared that no conflict of interest exists.
Nonstandard abbreviations used: hematopoietic stem cell transplantation (HSCT); graft-versus-host disease (GVHD); T cell receptor (TCR); bone marrow (BM); BM transplantation (BMT); phycoerythrin (PE); T cell depleted (TCD); 7-amino-actinomycin D (7-AAD); recent thymic emigrant (RTE); mixed lymphocyte reaction (MLR).