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The Journal of Immunology, 2007, 179, 120-125
Copyright © 2007 by The American Association of Immunologists, Inc.
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IL-15 Is Required for Sustained Lymphopenia-Driven Proliferation and Accumulation of CD8 T Cells1

Michelle M. Sandau*,{dagger}, Colleen J. Winstead*,{ddagger} and Stephen C. Jameson2,*,{dagger},§

* Center for Immunology, {dagger} Department of Laboratory Medicine and Pathology, {ddagger} Department of Medicine, and § Cancer Center University of Minnesota, Minneapolis MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naive T cells undergo slow homeostatic proliferation in response to T cell lymphopenia, which is also called lymphopenia-induced proliferation (LIP). IL-7 is critically required for this process, but previous studies suggested IL-15 was expendable for LIP of naive CD8 T cells. In contrast, we show that IL-15 is important for sustained CD8 T cell proliferation and accumulation in a lymphopenic setting, as revealed by truncated LIP in IL-15–/– hosts. At the same time, we find that IL-12 enhances LIP by acting directly on the CD8 T cells and independently of IL-15, suggesting distinct pathways by which cytokines can regulate homeostatic proliferation. Interestingly, the memory-phenotype CD8 T cell generated by LIP in IL-15–/– hosts are phenotypically distinct from the rare endogenous memory-phenotype cells found in IL-15–/– animals, suggesting these cells are generated by different means. These findings demonstrate that cytokine requirements for LIP change during the process itself, illustrating the need to identify factors that regulate successive stages of lymphopenia-driven proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The total number of T cells remains relatively constant throughout an individual’s life. Such homeostasis requires a balance between T cell generation, survival, proliferation, and cell death, and there appear to be distinct mechanisms for the maintenance of naive vs memory T cell pools (1). Various factors regulate naive T cell survival, including the survival cytokine IL-7 and, in at least some models, self-peptide-MHC ligands (2).

In contrast, IL-15 plays a pivotal role in driving basal proliferation of CD8 memory cells (2, 3). IL-15- and IL-15R{alpha}-deficient (IL-15–/– and IL-15R{alpha}–/–) animals have a severely reduced CD8 memory pool (4, 5, 6, 7), and the few remaining CD44high CD8 cells in IL-15–/– mice are atypical in that they lack expression of CD122 (IL-2Rbeta, a key signaling component of the IL-15R) and do not respond to IL-15 (8). In addition, it has been proposed that other cytokines (IL-12, IL-18, type I IFN, and IFN-{gamma},) promote memory CD8 T cell proliferation indirectly by inducing elevated production of IL-15 (9, 10). In contrast to the dramatic effect on memory CD8 T cells, lack of IL-15 or IL-15R{alpha} causes a partial reduction in naive CD8 T cell numbers and little discernible effect on either naive or memory-phenotype CD4 T cells (4, 5).

T cell homeostasis is severely perturbed by factors that lead to large scale loss of mature T cells, such as chemotherapy or radiotherapy or certain infections, which results in transient or sustained lymphopenia (2). During recovery from T cell depletion, residual naive and memory T cells are driven into slow cell division, a process called homeostatic proliferation or lymphopenia-induced proliferation (LIP)3 (2, 11, 12). This response may arise from decreased competition for T cell survival factors, such as IL-7. For naive CD8 T cells, both IL-7 and self-peptide-MHC ligands are required for optimal LIP, whereas memory CD8 T cells respond to either IL-7 or IL-15, yet are independent of self-peptide-MHC ligands (2, 11, 12, 13, 14, 15, 16).

An important aspect of LIP is that naive T cells going through this process convert into memory-like cells, based on changes in phenotype and gene expression profiles (2, 11, 17). Such changes are accompanied by appearance of memory-like functional properties, including the ability to rapidly produce cytokines and mediate protection against pathogens in vivo (2, 11, 18). An unresolved question is whether these memory-like cells also share the same requirements for maintenance and proliferation with true memory cells. If so, we might expect to see a differential requirement of factors, such as cytokines, during initiation vs maintenance of T cell LIP. In apparent contradiction with this idea, however, initial studies suggested that IL-15 was nonessential for LIP of naive CD8 T cells because LIP of such cells was similar in wild-type (WT) and IL-15–/– hosts (14). In contrast, exogenous recombinant IL-15 (14) and IL-12 (19) have both been shown to enhance LIP of CD8 T cells. Together, these reports suggest that endogenous IL-15 is dispensable for LIP of naive CD8 T cells, but that elevated levels of IL-15 (as might be induced by IL-12) could enhance LIP of this subset.

In this study, we re-evaluate the role of IL-15 in LIP of naive CD8 T cells, and find that endogenous IL-15 is not required for initial proliferation of naive CD8 T cells, but is needed for sustained proliferation and accumulation of these cells, as they differentiate into memory-phenotype cells. These findings help define the role of IL-15 in generating and maintaining CD8 memory pools, as well as demonstrating that cytokine requirements are not static during LIP, but change with differentiation of the cells.


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

C57BL/6 (B6) mice were purchased from The Jackson Laboratory and C57BL/6 IL-15–/– were purchased from Taconic Farms. OT-I TCR transgenic (20) or P14 TCR transgenic (TgN(TCRP14 LCMV) Rag2tm1; Taconic Farms) were bred to B6.PL (Thy1.1) mice (The Jackson Laboratory) to generate the OT-I.PL and P14.PL strains. IL-12Rbeta1–/– mice (B6.129S1-Il12rb1tm1Jm) (21) were crossed to OT-I transgenic mice to produce mice referred to in this study as IL-12R–/– OT-I, generously provided by M. Mescher (University of Minnesota, Minneapolis, MN). All mice were maintained under specific pathogen-free conditions at the University of Minnesota. All animal handling and experiments were conducted with approval from the University of Minnesota Institutional Animal Care and Use Committee.

Adoptive transfer

CD44low OT-I.PL, IL-12R–/– OT-I, or P14 cells were purified by negative selection and CFSE-labeled as previously described (19). For cotransfer experiments, equal numbers of OT-I and IL-12R–/– OT-I cells were CFSE-labeled and injected into irradiated hosts. B6 and IL-15–/– mice were sublethally irradiated (440 cGy) and 24 h later 0.5–1 x 106 CFSE-labeled OT-I cells were injected via the tail vein.

IL-12 injections

Recombinant IL-12 was a gift of Genetics Institute (Cambridge, MA) and M. Mescher (University of Minnesota, Minneapolis, MN). Mice were i.p. injected with IL-12 (1 µg, equivalent to 2700 ± 1200 U) in a carrier of 100 µl of PBS containing 1% mouse serum, or with carrier alone (for control animals) on days 1, 2, and 3 posttransfer of T cells, as described (19).

Flow cytometry

Single-cell suspensions from spleen and pooled lymph nodes were analyzed by flow cytometry. Donor OT-I.PL cells were identified by co-staining with CD8{alpha} and CD90.1 (Thy1.1) and cell phenotype determined using the indicated Abs from BD Pharmingen and eBioscience. For cotransfer experiments, OT-I T cells were first identified by staining with anti-CD8{alpha} and OVA/Kb tetramer, as described (22), then WT OT-I cells were identified as Thy1.1+, whereas the IL-12R–/– OT-I cells were Thy1.1–. Flow cytometry was performed on a FACSCalibur (BD Biosciences) and data analyzed using FlowJo software (Tree Star).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-15 is required for sustained LIP of CD8 T cells

Although exogenous IL-15 can enhance naive CD8 T cell LIP, previous work suggested the endogenous cytokine was dispensable because naive CD8 T cell LIP appeared normal in lymphopenic IL-15–/– animals (14). However, the ability to respond to IL-15 requires expression of CD122 (8, 23) and this receptor chain is up-regulated after a few rounds of cell division once a naive CD8 T cell has undergone LIP (24). Hence, it was possible that reactivity to IL-15 would also gradually appear as LIP proceeded.

Therefore we tested whether sustained homeostatic expansion of CD8 T cells was influenced by the presence or absence of IL-15. To address this effect, CFSE-labeled (CD44low) OT-I CD8 T cells were adoptively transferred into lymphopenic B6 or IL-15–/– hosts, and LIP assessed at different time points after transfer.

Consistent with an earlier report (14), we found initial OT-I T cell proliferation was similar in either host strain (Fig. 1A). However, as early as 14 days posttransfer, we observed reduced OT-I proliferation in the IL-15–/– hosts (Fig. 1A). This trend becomes even more extreme at later time points (days 21 and 27) (Fig. 1A), suggesting that OT-I cells continued to divide in the B6 hosts (eventually becoming CFSE–), whereas proliferation in the IL-15–/– hosts had "stalled," with CFSE levels remaining similar to those seen at day 14. This stalling effect is most clearly seen when CFSE levels at the various time points are overlaid (Fig. 1B).


Figure 1
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  		FIGURE 1. IL-15 is required for the sustained LIP and accumulation of CD8 T cells. CFSE-labeled CD44low OT-I were injected into irradiated hosts and analyzed on days indicated. CFSE levels were assessed on OT-I T cells as a method to compare cell division history on various time points. A, OT-I CFSE levels from B6 and IL-15–/– hosts are compared in each histogram on the day indicated. B, CFSE levels are compared as a function of time for each host. C, The number of OT-I T cells in lymph nodes in host mice at the indicated time point was calculated by multiplying the percentage of CD8{alpha}+ or Thy1.1+ cells by the total number of cells. Data are representative of at least three experiments.

 
Differences in proliferation do not necessarily predict differences in cell accumulation, and it was important to determine whether the abrupt termination of naive CD8 T cell LIP in the IL-15–/– animals correlated with an impairment in OT-I expansion. At early time points (day 5) during LIP, the number of OT-I present in IL-15–/– and B6 hosts was similar in both lymph node (Fig. 1C) and spleen (data not shown). However, by 14 days posttransfer, more OT-I cells had accumulated in the B6 host than in the IL-15–/– host (Fig. 1C), corresponding to differences in proliferation in these hosts (Fig. 1, A and B). Furthermore, the number of OT-I cells in the B6 continued to increase from days 14 to 21, whereas the number of OT-I in the IL-15–/– remained low from day 14 through day 27 (Fig. 1C) and even later time points (through to day 34, data not shown). We obtained similar results when using CD44low Rag–/– OT-I CD8 T cells as donors, indicating that the observed impact of IL-15 deficiency was not due to usage of endogenous TCR in the OT-I T cell pool. Specifically, at 21 days following transfer into irradiated mice, Rag–/– OT-I T cells showed reduced proliferation and accumulation in IL-15–/– compared with B6 hosts (data not shown).

Together, these experiments indicate that sensitivity to IL-15 is acquired during the course of CD8 T cell LIP. This response correlates with the coordinate up-regulation of CD122 during LIP, which permits responsiveness of CD8 T cells to IL-15 (2, 3, 25). It was possible that other differences in the IL-15–/– lymphopenic hosts might influence LIP, but we found that LIP of CD4 T cells (which appear to be insensitive to IL-15 (2, 3, 25)) was similar in both irradiated WT and IL-15–/– hosts (data not shown), arguing against indirect effects of IL-15 deficiency on LIP. These findings help define changes in requirements for induction vs maintenance of LIP, and highlight the fact that naive CD8 T cells undergoing LIP are moving targets, and their reliance on specific cytokines changes during the process.

Lack of IL-15 does not impact the phenotype of LIP memory CD8 T cells

Acquired responsiveness to IL-15 during naive CD8 T cell LIP correlates with up-regulation of CD122 as shown below and by others (24, 26, 27). Interestingly, the few memory phenotype CD44high CD8 T cells in IL-15–/– mice are CD122low (5, 8) and, unlike their CD122high counterparts, show robust proliferation in the absence of IL-15 (8). Hence, we considered that LIP of naive CD8 T cells in IL-15–/– hosts might generate similar CD44highCD122low memory-like T cells.

To test this model, we analyzed the surface expression of various molecules on the cells at multiple time points after adoptive transfer. First, we analyzed the expression of CD44, a marker of memory/activated cells. At early time points (<7 days) the expression of CD44 was similar on OT-I T cells expanding in the B6 and IL-15–/– host (data not shown); in contrast by 14 days after adoptive transfer, CD44 levels were higher on donor cells in the B6 host compared with the IL-15–/– hosts (Fig. 2A). The difference in CD44 expression on OT-I from B6 vs IL-15–/– host was likely due to the higher proliferative capacity of OT-I cells in the B6 hosts (Fig. 1) because CD44 levels increase with cell division (Fig. 2B), as previously noted. However, CD44 is clearly increased in expression (compared with naive cells) following LIP in either host (Fig. 2A), suggesting that these cells have begun differentiation to the memory-like state.


Figure 2
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  		FIGURE 2. LIP gradually induces a memory-phenotype that confers sensitivity to IL-15. Flow cytometry of adoptively transferred OT-I T cells (A and B) or P14 T cells (C) from indicated hosts at day 14 (A and B) or day 21 (C). A, CD44 expression of OT-I cells depicts B6 irradiated host (gray-shaded histogram), IL-15–/– irradiated host (solid line histogram), and CD8{alpha}+ T cells from an unirradiated B6 control (dotted line histogram). B, The percentage for OT-I T cell expression of CD44 correlated with CFSE levels is shown. C, The percentage for P14 T cell expression of CD44 correlated with CFSE levels. Mean fluorescence intensity (in parentheses) of CD44 expression of gated population is shown. Data are representative from at least four (A and B) or three (C) similar experiments.

 
We also investigated the role of IL-15 in LIP of P14 TCR transgenic T cells. Similar to the OT-I studies, CD44low P14 CD8+ T cells were allowed to proliferate in irradiated B6 or IL-15–/– mice for 21 days. In our hands, P14 CD8 T cells undergo fewer rounds of cell division in irradiated hosts, as determined by CFSE loss, when compared with OT-I cells (Fig. 2, B compared with C and data not shown), yet P14 T cells also up-regulate CD44 as they proliferate in the lymphopenic B6 hosts (Fig. 2C) (24). However, as for OT-I T cells, we observed a reduction in CFSE dye dilution and CD44 up-regulation (reflected by CD44 mean fluorescence intensity) in P14 T cells following their LIP in IL-15–/– hosts (Fig. 2C). These data reinforce the model that CD8 T cells undergoing LIP acquire a memory phenotype and develop sensitivity to IL-15 for their maintenance.

Next, we assessed surface expression of various markers, starting with up-regulation of CD122 (IL-2Rbeta). As expected from previous studies (24, 26, 27), OT-I T cells undergoing LIP in irradiated WT hosts up-regulated CD122, compared with OT-I cells transferred into a unirradiated host (Fig. 3). However, surprisingly, similar up-regulation of CD122 was observed during OT-I LIP in IL-15–/– hosts (Fig. 3), a pattern that was observed at all time points analyzed (data not shown). Hence, CD8 T cell LIP does not appear to generate the population of CD122lowCD44high CD8 T cells observed in intact IL-15–/– animals (8).


Figure 3
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  		FIGURE 3. Similar phenotype of LIP cells in the presence and absence of IL-15. Flow cytometry of adoptively transferred OT-I T cells from the spleen of indicated hosts at day 21. B6 irradiated host (gray-shaded histogram), IL-15–/– irradiated host (solid line histogram), and CD8{alpha}+ T cells from an unirradiated B6 control (dotted line histogram) are depicted. CD122 (IL-2Rbeta), IL-7R{alpha}, IL-15R{alpha}, L-selectin, and CD27 expression levels on OT-I are similar regardless of host. Representative data of four experiments are shown.

 
Although high affinity binding of IL-15 depends on the IL-15R{alpha}, recent data from our group and others have questioned whether expression of this chain on T cells has functional relevance because IL-15R{alpha} may be more critical on bystander cells that "present" IL-15 through transpresentation (23, 28, 29). In any case, examination of the levels of IL-15R{alpha} on cells undergoing LIP was increased on cells in both hosts (Fig. 3), thus suggesting the receptor is up-regulated regardless of the presence of IL-15 in the lymphopenic hosts. Likewise, up-regulation of the receptor for a distinct homeostatic cytokine, IL-7, was similar on OT-I cells undergoing LIP in either host strain (Fig. 3). Finally, we examined expression of markers (CD62L and CD27), which have been used to discriminate "central" from "effector" memory CD8 T cells. As reported previously (18, 24, 26, 27), LIP of CD8 T cells typically yields cells of central memory phenotype, and this response was observed regardless of the host animals ability to produce IL-15 (Fig. 3).

Together, these data argue that LIP of naive CD8 T cells induces a set of maturation changes that produce cells resembling conventional central memory T cells. These changes include up-regulation of IL-15R{alpha} and, probably more important, of CD122, which allows the cells to respond to available IL-15. However, the presence or absence of IL-15 does not seem to influence the phenotype of these cells.

IL-12 augments LIP of CD8 T cells in the absence of IL-15

Previous studies have indicated that IL-12 can enhance LIP of CD8 T cells (19). Because activated CD8 T cells can express IL-12R, this could potentially be a direct effect. In addition, the enhanced proliferation caused by IL-12 may result in rapid acquisition of the "memory-phenotype" earlier thus revealing IL-15 dependency. Conversely, Sprent and coworkers (10, 30) have suggested that the ability of IL-12 to induce proliferation of memory-phenotype CD8 T cells is indirect, acting through induction of IFN-{gamma}, which in turn promotes elevated IL-15 levels. In this model then, IL-15 would be the critical effector cytokine in IL-12-mediated induction of memory CD8 T cell turnover and enhanced proliferation would not be observed in the absence of IL-15.

To test whether the enhancement of LIP by IL-12 depends on host cell production of IL-15, we injected recombinant IL-12 into sublethally irradiated B6 or IL-15–/– hosts that had received CFSE-labeled OT-I the day before. We studied proliferation of the donor OT-I cells in lymph nodes and spleen 7 days after adoptive transfer, as previously described (19).

As expected, exogenous IL-12 enhanced OT-I T cell LIP in B6 hosts (Fig. 4A). Interestingly, the proliferation of the OT-I in the IL-15–/– hosts was also augmented by IL-12 (Fig. 4B). In addition, the administration of IL-12 enhanced CD44 expression on the donor cells to a similar level in the IL-15–/– and B6 hosts, both of which were higher levels than seen in the untreated B6 host (Fig. 4D). These data further reinforce the correlation between expression of this marker and the extent of proliferation (Fig. 2). However, we did observe a reproducible difference in the extent of proliferation induced by IL-12 in the two hosts, with the extent of CFSE dilution being slightly less in the IL-15–/– hosts (Fig. 4C). This difference is noteworthy because at this time point (day 7 posttransfer), there is very little difference in donor cell proliferation in untreated B6 and IL-15–/– hosts (Fig. 4, A compared with B, grayed histograms), in keeping with our earlier observations (Fig. 1). Hence, we found that IL-12 was still clearly capable of inducing more robust donor cell LIP in either host, but the optimal effect was observed when hosts were capable of making IL-15.


Figure 4
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  		FIGURE 4. IL-12 enhances LIP of OT-I independently of IL-15. LIP of OT-I was assessed in irradiated hosts with or without exogenous IL-12 on day 7. OT-I CFSE levels from B6 hosts with or without IL-12 (A), IL-15–/– host with or without IL-12 (B), and B6 and IL-15–/– hosts with IL-12 (C) are shown. D, OT-I CD44 expression from IL-15–/– hosts with IL-12 (thin line histogram), B6 hosts with IL-12 (thick line histogram), and B6 hosts without IL-12 (gray-shaded histogram). E, The number of OT-I in the indicated hosts was calculated as in Fig. 2. Data are representative of three experiments.

 
In addition to enhanced proliferation, we previously showed that IL-12 can augment expansion of CD8 T cells undergoing LIP (19). We therefore also analyzed accumulation of donor T cell numbers with or without IL-12 administration. Again, OT-I T cell accumulation at day 7 was similar in untreated B6 and IL-15–/– hosts (Fig. 4E), as shown earlier (Fig. 2). IL-12 was capable of inducing a substantial increase in the accumulation of donor OT-I cells in B6 hosts, but this effect was somewhat attenuated in IL-15–/– hosts (Fig. 4E).

IL-12 acts directly on CD8 T cells to enhance LIP

Although exogenous IL-12 could enhance OT-I LIP in IL-15–/– hosts, the fact that the OT-I proliferation and expansion was reduced compared with that in B6 hosts made it difficult to determine whether the effect of IL-12 was chiefly direct (acting on the OT-I T cells themselves) or indirect (acting on other cells to induce production of homeostatic cytokines including IL-15). To clarify this effect, we adoptively cotransferred IL-12R–/– OT-I T cells and WT OT-I.PL T cells into either B6 or IL-15–/– irradiated hosts. Half of the recipients were also injected with IL-12, and all animals were analyzed a week after transfer. In this approach, we expected that both populations of OT-I cells would be able to respond to indirect effects induced by exogenous IL-12, but only the WT OT-I cells would be able to directly respond to IL-12. We detected OT-I cells using OVA/Kb tetramers and distinguished WT vs IL-12R–/– OT-I cells by using Thy1.1 congenic WT donor cells.

First, we established the proliferative capacity of WT and IL-12R–/– cells in B6 hosts in the presence or absence of exogenous IL-12. Both OT-I groups underwent similar proliferation in irradiated B6 hosts, but exogenous IL-12 selectively enhanced the proliferation of the WT OT-I donor cells (Fig. 5A). These data argue that exogenous IL-12 acts, for the most part, directly on the CD8 T cell to enhance its LIP. We also found similar responses in IL-15–/– hosts (Fig. 5A), which further reinforce the argument that the enhanced proliferation induced by IL-12 is not primarily acting through IL-15 induction. These conclusions were supported by examination of the proliferative response of the IL-12R–/– OT-I cells in the various hosts (Fig. 5, B and C), which indicated little effect of either exogenous IL-12 or endogenous IL-15 on their proliferation. There was a modest (but reproducible) increase in proliferation of IL-12R–/– OT-I cells in IL-12-treated B6 compared with IL-15–/– hosts, which is consistent with some induction of host IL-15 following IL-12 treatment (Fig. 5C); however, this effect pales in comparison with the effect of IL-12 acting directly on WT OT-I T cells (Fig. 5A).


Figure 5
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  		FIGURE 5. CD8 T that cannot directly respond to IL-12 proliferate minimally in response to IL-15. OT-I.PL (Thy1.1+) and IL-12R–/– OT-I (Thy1.1–) T cells were cotransferred into irradiated B6 and IL-15–/– hosts. IL-12 was administered as indicated, and donor cells were assessed for proliferation and accumulation on day 7. A, Cell division as measured by CFSE loss of WT cells or IL-12R–/– cells in either B6 (top row) or IL-15–/– (bottom row) hosts, in the absence of IL-12 (left) or presence of IL-12 (right). B, Histograms of CFSE content of IL-12R–/– OT-I T cells in B6 (left) or IL-15–/– (right) hosts in the absence (gray-shaded histogram) or presence (black line histogram) of IL-12. C, Histogram of CFSE content of IL-12R–/– OT-I T cells after LIP in recipients receiving exogenous IL-12. Data from B6 hosts (gray-shaded histogram) and IL-15–/– (black line histogram) hosts are shown. D, Number of either OT-I or IL-12R–/– OT-I T cells recovered from indicated hosts when IL-12 was administered.

 
Finally we enumerated both donor OT-I populations in the different hosts after IL-12 administration, to determine how accumulation of the cells was influenced by direct responsiveness to IL-12 and to host IL-15 expression. As expected from Fig. 4, there was enhanced accumulation of WT OT-I in B6 compared with IL-15–/– hosts. However the IL-12R–/– OT-I T cells exhibited minimal accumulation in either host (Fig. 5D), reflecting their relatively poor proliferation.

Together, these data suggest IL-12 can act independently of IL-15 to support CD8 T cell LIP, but that the presence of IL-15 allows for their continued proliferation and accumulation. A potential model for this effect is that IL-12 may accelerate the differentiation of naive CD8 T cells into LIP memory cells, and by doing so, allow them to acquire sensitivity to IL-15 earlier than cells not exposed to supplementary IL-12.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These findings are important for understanding the essential requirements for induction and persistence of LIP. Our data indicate that, at least in regard to cytokine requirements, CD8 T cells undergoing LIP are a moving target, and their reliance on specific cytokines changes during the process. This difference is analogous to that found in cytokine requirements for maintenance of naive and conventional memory CD8 T cells, but while those differentiation stages are interrupted by the massive expansion of activated/effector T cells in normal immune responses, the transition between naive and memory-phenotype cells is seamless and gradual in LIP.

The data from these experiments reveal that in the absence of IL-15, naive CD8 T cells can undergo a few rounds of division in a lymphopenic environment (Fig. 1). However, IL-15 is necessary to maintain their proliferation to enable maximal expansion (Fig. 1). We have not ruled out the possibility that IL-15 also enhances survival of CD8 T cells, and previous studies clearly show IL-15 can promote expression of anti-apoptotic Bcl-2 family proteins (31). It is unlikely, however, that survival alone explains the large difference in cell numbers we observe at late time points because the donor OT-I cells appear to be maintained long term in IL-15–/– hosts following the halt in cell division (Fig. 1).

Our data on LIP memory CD8 T cells corresponds with previous studies on conventional (Ag-driven) CD8 memory T cells, which also indicated that up-regulation of CD122 is not dependent on the availability of IL-15 (6). Recent data indicate that the endogenous CD44highCD122low pool responds to self-peptide-MHC class I ligands for survival and proliferation in normal and lymphopenic settings (32). That study went on to propose that at least some cells in this CD44highCD122low subset can eventually up-regulate CD122, and more closely resemble conventional memory-phenotype CD8 T cells (32). LIP of naive CD8 T cells also involves a response to specific self-peptide-MHC class I ligands (2, 11, 12), and hence we might have expected to see at least a transient appearance of CD44highCD122low OT-I cells during LIP, especially in IL-15–/– hosts. Our data however, indicate that CD44highCD122low is not an intermediate in the LIP process for OT-I CD8 T cells. Whether the CD44highCD122low CD8 cells found at steady state in IL-15–/– and IL-15R{alpha}–/– animals and the LIP memory cells described in this study derive from different precursors is not clear. It is certainly possible that, despite their different characteristics, these populations both can arise from naive CD8 T cells that find distinct homeostatic niches. It is possible that up-regulation of CD122 during LIP in IL-15–/– hosts equips the cells with the ability to respond to IL-2. However, CD25 (the IL-2R{alpha}-chain) remains low on CD8 T cells undergoing LIP (24, 26, 27), so they would be relatively insensitive to IL-2. At least in WT hosts, IL-2 appears to play a minimal role in supporting naive CD8 T cell LIP (19, 27), although these studies focused on initiation of LIP and, as in the current study, interactions with this cytokine may also change as the process continues.

IL-12, as well as other cytokines (type I IFN, IFN-{gamma}, and IL-18) may stimulate memory CD8 T cell turnover indirectly, by promoting increased production of IL-15 (10). In contrast, we show in this study that augmentation of naive CD8 T cell LIP by IL-12 occurs in both WT and IL-15–/– hosts, arguing that host production of IL-15 is not essential for their response to IL-12 (Fig. 4). These studies could not exclude the possibility that IL-12 might augment LIP both directly (acting on the CD8 T cells themselves) and indirectly (through induction of IL-15 or other terminal homeostatic cytokines). However, our data showing that exogenous IL-12 has little impact on the LIP of IL-12R–/– OT-I cells, while promoting vigorous proliferation of WT OT-I cells (Fig. 5), suggests IL-12 is chiefly acting directly (in keeping with our earlier study (19)) and that there is minimal contribution of an indirect response. This conclusion is at odds with the reported lack of IL-12 reactivity by endogenous memory-phenotype CD8 T cells in vitro (10). Whether these different conclusions indicate that direct IL-12 sensitivity is transiently acquired during LIP (but not maintained into the memory-phenotype phase), or that there are fundamental differences between the IL-12 reactivity of endogenous memory-phenotype cells and the memory cells produced by LIP is unclear. In any case, these data argue that distinct pathways (involving IL-15 or IL-12) may operate independently on regulating proliferation of CD8 T cells in response to lymphopenia.

At the same time, our data indicate that direct IL-12 reactivity is not a prerequisite for LIP of naive CD8 T cells: We observed little difference between proliferation of IL-12R–/– and WT OT-I cells in the absence of exogenous IL-12 (Fig. 5A and data not shown), arguing that endogenous IL-12 has a minimal impact on their LIP, at least at the time points studied.

LIP is thought to occur during episodes of lymphopenia (which may be induced by therapeutic treatments or during various physiological states, including neonatal life) (2, 33, 34). Cells produced during LIP may acquire enhanced reactivity to pathogens, but may also cause autoimmune damage (2, 33, 34). Understanding the role of homeostatic cytokines in regulating LIP is critical in determining how this process is normally regulated and could be therapeutically controlled. Our data indicate that cytokine requirements for CD8 T cell LIP change during the process itself. Specifically, although LIP of naive CD8 T cells is initially insensitive to IL-15, the presence of endogenous IL-15 begins to have a critical effect at later stages of LIP and expansion. Hence, our data underscore the importance of studying factors that regulate not only initiation but also maintenance of the LIP process.


   Acknowledgments
 
We thank Kris Hogquist and members of the Jamequist lab for input, Martin Prlic for helpful suggestions, and Alex Khoruts for comments on the manuscript.


   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 Grant R37 AI38903 by the National Institutes of Health (to S.C.J.) and by an Immunology Predoctoral Training Grant T32 AI07313 (to M.M.S.). Back

2 Address correspondence and reprint requests to Dr. Stephen C. Jameson, Center for Immunology, University of Minnesota Medical School, Mayo Mail Code 334, 420 Delaware Street SE, Minneapolis MN 55455. E-mail address: james024{at}umn.edu Back

3 Abbreviations used in this paper: LIP, lymphopenia-induced proliferation; WT, wild type. Back

Received for publication October 6, 2006. Accepted for publication April 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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