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
). 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
). 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+
) 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
). 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
). However, some studies have argued that survival and/or homeostatic proliferation of CD4+
T cells can occur without matching MHC class II ligands (62
). 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
), 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 a, 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
). 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
The important role of homeostatic proliferation in posttransplant T cell reconstitution could also partially explain the differences in CD4+
T cell reconstitution. CD8+
T cells survive better and engage faster in homeostatic proliferation than CD4+
T cells (40
). 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.