© Rockefeller University Press, 0022-1007 $8.00
JEM, Volume 200, Number 7, 825-834
Coordinate Expression and Trans Presentation of Interleukin (IL)-15R
and IL-15 Supports Natural Killer Cell and Memory CD8+ T Cell Homeostasis
Patrick R. Burkett,
Rima Koka,
Marcia Chien,
Sophia Chai,
David L. Boone, and
Averil Ma
Department of Medicine, University of California, San Francisco, San Francisco, CA 94143
Address correspondence to Averil Ma, Dept. of Medicine, University of California, San Francisco, 513 Parnassus Ave., S-1057, Box 451, San Francisco, CA 94143. Phone: (415) 502-9405; Fax: (415) 502-9404; email: ama1{at}itsa.ucsf.edu
 |
Abstract
|
---|
The high affinity interleukin (IL)-15 receptor, IL-15R
, is essential for supporting lymphoid homeostasis. To assess whether IL-15R
's role in vivo is to trans present IL-15, we generated mixed bone marrow chimera from IL-15R
and IL-2/15Rßdeficient mice. We find that IL-15R
competent, IL-2/15Rßdeficient cells are able to support IL-15R
deficient natural killer (NK) and memory CD8+ T cells, thus ruling out secondary signals on these cells and demonstrating that IL-15R
mediated presentation of IL-15 in trans is the primary mechanism by which IL-15R
functions in vivo. Surprisingly, using IL-15 and IL-15R
deficient mixed chimera, we also find that IL-15 and IL-15R
must be expressed by the same cells to present IL-15 in trans, indicating that IL-15R
is required on a cellular level for the elaboration of IL-15. These studies indicate that IL-15R
defines homeostatic niches for NK and memory CD8+ T cells by controlling both the production and the presentation of IL-15 in trans to NK and CD8+ memory T cells.
Key Words: intracellular cytokine receptor IL-15/IL-15R
preassociation mixed chimera IL-2Rß
P. Burkett and R. Koka contributed equally to this work.
Abbreviation used in this paper:
c,
chain.
 |
Introduction
|
---|
NK cells and memory CD8+ T cells both play vital roles in protecting the host from intracellular pathogens. Understanding how the survival and maintenance of these populations is regulated in vivo has recently been a field of intensive investigation. Memory CD8+ T cell maintenance is a dynamic process that is critically dependent upon two common
chain (
c)-dependent cytokines, IL-7 and IL-15. Whereas IL-7 promotes the survival of both naive and memory CD8+ T cells, IL-15 uniquely supports basal memory CD8+ T cell proliferation (13). Thus, in the absence of proliferative IL-15 signals, memory CD8+ T cells undergo a slow atrophy in number, until they become essentially undetectable (24). In addition to maintaining memory CD8+ T cells, IL-15 and IL-15R
are also critical for the maintenance of peripheral NK cells (5, 6). Whereas the maintenance of memory CD8+ T cells by IL-15 is mediated by both proliferation and survival, NK cell numbers are primarily maintained by regulating survival (5, 6). Thus, IL-15 plays important, nonredundant roles in maintaining the numbers of both memory CD8+ T cells and NK cells in the periphery.
Earlier studies suggested that IL-15 mediates its biological effects by binding to a high affinity, heterotrimeric receptor complex comprised of IL-15R
, IL-2/15Rß, and
c. IL-15R
, which uniquely binds IL-15, is widely expressed by both hematopoietic and parenchymal cell types and has a high affinity for IL-15 (Kd
1011 M; reference 7). Although IL-15R
may play a role in intracellular signal transduction in certain cell types, other studies suggest that the cytoplasmic tail of IL-15R
is not critically necessary for enhancing IL-15induced proliferation (811). By contrast, IL-2/15Rß and
c heterodimers exhibit lower affinity binding for IL-15 (Kd
109 M) in the absence of IL-15R
, but are clearly essential for transducing IL-15induced intracellular signals (12, 13). Gene targeting experiments have further demonstrated that all three receptor chains are required to support IL-15dependent cell populations in vivo (1416). Importantly, the phenotypes of IL-15 (IL-15/) and IL-15R
(IL-15R
/) deficient mice are indistinguishable, suggesting that physiologically relevant IL-15 signals require IL-15R
(16, 17).
Although the studies above are consistent with the idea that soluble IL-15 binds to heterotrimeric IL-15R
, IL-2/15Rß, and
c receptors on responsive lymphocytes (e.g., NK cells and memory CD8+ T cells) and stimulates their survival and proliferation, more recent studies have demonstrated that IL-15R
is required in a non-cellautonomous manner, i.e., not on responding lymphocytes, but rather on a variety of accessory cell types in the mouse (4, 5, 18). IL-15R
expression on hematopoietic cells other than the CD8+ T cell is required for CD8+ T cell bystander proliferation and preferentially supports the basal maintenance of memory CD8+ T cells (4, 18). Moreover, IL-15R
expression by both radiation-sensitive and radiation-resistant cells, but not by responding NK cells, is required for the peripheral survival of NK cells (5 and unpublished data). Finally, IL-15R
expression by radiation-resistant cells, likely intestinal epithelial cells, is critical for the development of TCR-
/
intraepithelial lymphocytes (19). Therefore, as far as the development and subsequent support of several distinct IL-15dependent cell types is concerned, the critical in vivo functions of IL-15R
do not appear to be mediated by IL-15R
expression on IL-15dependent cell types.
IL-15R
's non-cellautonomous role in supporting NK and memory CD8+ T cells is consistent with multiple indirect mechanisms by which IL-15 might signal through IL-15R
on accessory cells to induce the production of proteins that subsequently support NK and memory CD8+ T cells. It is also consistent with a novel mechanism described in vitro by which IL-15R
on accessory cells can present IL-15 in trans to IL-2/15Rß and
c-bearing lymphocytes (20). Which of these multiple mechanisms is physiologically relevant has not been addressed in vivo, and the molecular mechanisms underlying this novel cellular physiology have not been investigated. Accordingly, we have used a variety of mixed radiation chimera to examine the in vivo roles of IL-15 and its various receptor chains in supporting NK and memory CD8+ T cell homeostasis.
 |
Materials and Methods
|
---|
Mice, Adoptive Transfers, and Immunization.
C57BL/6J IL-15R
and congenic Ly5.2+ C57BL/6J/SJL IL-15R
mice, and OT-1 RAG-1 and IL-15R
OT-1 RAG-1 mice were generated and interbred as described previously (4, 5, 16). All strains were backbred to a C57Bl/6J background for at least nine generations. IL-2/15Rß/ and Ly5.2+ C57BL/6J/SJL mice on a C57BL/6J background were purchased from The Jackson Laboratory. IL-15/ mice on a C57BL/6J background were purchased from Taconic Laboratories. Radiation bone marrow chimeras were produced as described previously (4), except that mixed radiation chimera were generated using mixtures of bone marrow cells from congenic donors of distinct genotypes of mice. All mice were housed and bred in specific pathogen-free facilities according to University of Chicago and University of California, San Francisco Institutional and Animal Care Use Committee guidelines. Adoptive transfers of naive OT-1+ CD8+ T cells and subsequent immunizations were performed as described previously (4). NK cells were isolated and adoptively transferred as described previously, except that NK cells were purified from RAG-1/ mice (5).
Cellular Analyses by Flow Cytometry.
Single cell suspensions from peripheral blood or tissues were prepared, incubated with monoclonal antibodies or dimers of H-2KbOVA, and analyzed by flow cytometry using a FACSCalibur and CELLQuest software as described previously (4). Antibodies specific for CD3, CD4, CD8, Ly5.1, Ly5.2, CD44, CD122 (IL-2/15Rß), NK1.1, IFN-
(BD Biosciences), and IL-15R
(R&D Systems) were used at 5 µg/ml. Dimers of H-2Kb (BD Biosciences) were incubated with SIINFEKL peptide, and then used to detect OT-1+ cells as described previously (4).
 |
Results
|
---|
IL-15R
Expression by Hematopoietic Cells, But Not NK Cells, Supports Development and Maintenance of NK Cells in a Non-cellautonomous Manner.
Our previous studies indicated that IL-15R
competent, radiation-sensitive cells appear to play a greater role than radiation-resistant cells in supporting the development and maintenance of NK cells. Therefore, we examined the ability of IL-15R
competent hematopoietic cells to rescue NK cell development in chimeric IL-15R
/ mice. Consistent with previous studies, significant numbers of NK cells were observed in the peripheral blood of lethally irradiated IL-15R
/ mice reconstituted with IL-15R
+/, but not IL-15R
/, bone marrow stem cells (Fig. 1 A, top). Although recent studies indicated that IL-15R
expression on NK cells was not absolutely required for their development (5, 21), it was unclear from these experiments whether IL-15R
expression on NK cells might partly contribute to their development and survival. To examine this question, IL-15R
+/ and IL-15R
/ bone marrow stem cells from congenic backgrounds were mixed and coinjected into irradiated IL-15R
/ mice. Analyses of the resulting mixed chimera revealed that significant numbers of NK cells were obtained from these chimera. Moreover, the ratio of NK cells derived from each genotype was virtually identical to the ratio of nonIL-15R
dependent B and CD4+ T lymphocytes (Fig. 1 A, bottom left). Thus, IL-15R
/ NK cells differentiate as well as IL-15R
+/ NK cells in the presence of other IL-15R
+/ hematopoietic cells, suggesting that IL-15R
expression on NK cells does not play an essential role in their differentiation or peripheral maintenance.
IL-15R
Expression by RAG-1independent Hematopoietic Cells Supports NK Cell Development.
As IL-15R
is expressed by many types of hematopoietic cells, including macrophages and dendritic cells, we investigated whether IL-15R
expression by RAG-1independent cell types could support NK cell development. Therefore, radiation chimera were generated by reconstituting lethally irradiated IL-15R
mice with a mixture of congenic bone marrow stem cells from RAG-1/ and IL-15R
2/ mice (RAG/R
KO
R
KO), or WT and IL-15R
mice (WT/R
KO
R
KO). After 8 wk, analyses of these mixed chimera revealed that similar numbers of NK cells were present in WT/R
KO
R
KO compared with RAG/R
KO
R
KO chimera (Fig. 1 A, top). Moreover, NK cells in RAG/R
KO
R
KO chimera were derived from both RAG-1/ and IL-15R
/ bone marrow progenitors in approximately equal proportions (Fig. 1 A, bottom right). Thus, RAG-1independent hematopoietic cells support NK cell development as well as WT hematopoietic cells.
IL-15R
Expression by RAG-1independent Cells Supports Development and Maintenance of Memory CD8+ T Cells.
Recent studies indicated that non-cellautonomous expression of IL-15R
is important for the maintenance of memory CD8+ T cells (4). To further define the hematopoietic cells that perform this function, we assessed whether IL-15R
expression by RAG-1independent cells is sufficient to support memory CD8+ T cells. At least 8 wk after irradiation and reconstitution, we adoptively transferred transgenic OT-1+ RAG-1/ CD8+ T cells into WT
R
KO, R
KO
R
KO, WT/R
KO
R
KO, and RAG/R
KO
R
KO chimera. 2 d after adoptive transfer of OT-1+ CD8+ T cells, these chimera were immunized with OVA and poly I:C, and the initial expansion, memory generation, and maintenance of OT-1+ CD8+ T cells were quantitated by analyzing the numbers of H2Kb-OVA+reactive CD8+ T cells in serial peripheral blood samples. At all time points examined after immunization, WT
R
KO, WT/R
KO
R
KO, and RAG/R
KO
R
KO chimera possessed similar frequencies of H2Kb-OVA+ CD8+ T cells (Fig. 1 B). In contrast, despite similar primary responses (i.e., day 4 after immunization), the population of H-2Kb-OVA+ CD8+ T cells declined progressively after 30 d in R
KO
R
KO chimera (Fig. 1 B). Moreover, normal frequencies of functional memory H-2Kb-OVA+ CD8+ T cells were observed in the spleens and lymph nodes of RAG/R
KO
R
KO, but not R
KO
R
KO, chimera up to 90 d after immunization (not depicted). Therefore, IL-15R
expression on RAG-1independent hematopoietic cells supports memory CD8+ T cell generation and maintenance.
IL-2/15Rß/ Hematopoietic Cells Support IL-15dependent Cell Types in Trans; In Vivo Evidence for Trans Presentation as the Exclusive Mechanism of IL-15R
mediated Lymphoid Homeostasis.
Homeostatic maintenance of NK cells and memory CD8+ T cells requires IL-15R
expression in a non-cellautonomous fashion, and in vitro studies suggest that IL-15R
may function by presenting IL-15 in trans to these cells (4, 5, 19, 20). Taken together, these studies suggest that trans presentation of IL-15 may support lymphocytes in vivo. However, no direct evidence exists to rule out the possibility that IL-15R
dependent secondary signals on accessory cells lead to the production of unidentified proteins that in turn support NK and memory CD8 T cells in vivo. To distinguish IL-15R
mediated trans presentation of IL-15 from IL-15R
mediated signaling on accessory cells, we used IL-2/15Rß/ bone marrow stem cells to generate chimeric mice in which hematopoietic cells expressed IL-15R
, but were unable to signal through IL-15R complexes (Fig. 2 A). Because IL-2/15Rß/ bone marrow cells are unable to generate regulatory T cells, leading to spontaneous autoimmunity and inflammation, we coinjected IL-15R
/ bone marrow stem cells with IL-2/15Rß/ bone marrow cells to generate mixed chimera. Intact IL-15R
/ mice and chimera derived from IL-15R
/ bone marrow stem cells possess normal numbers of CD4+ CD25+ regulatory T cells and do not develop spontaneous autoimmunity (not depicted). Mixed chimera generated from IL-15R
/ and IL-2/15Rß/ bone marrow stem cells (R
KO/RßKO) were then examined for their ability to support NK and CD8+ T cell homeostasis.

View larger version (28K):
[in this window]
[in a new window]
|
Figure 2. IL-15R , but not IL-2/15Rß, expression is required by hematopoietic cells to support NK cell development and survival. (A) Model illustrating two distinct mechanisms by which non-cellautonomous IL-15R expression is required to support IL-15dependent cell types. Mechanism 1 requires IL-2Rß expression on accessory cells to mediate signal transduction, whereas mechanism 2 (trans presentation) does not. (B) Flow cytometric analysis of NK cell reconstitution in indicated chimeric mice depends upon IL-15R , but not IL-2/15Rß, competent cells. IL-15R / mice were lethally irradiated and reconstituted with either IL-15R +/ (Wt) or IL-15R / (R KO) bone marrow cells, or a mixture of IL-15R +/ and IL-15R / bone marrow cells (Wt/R KO), or a mixture of IL-2/15Rß/ and IL15R / bone marrow cells (RßKO/R KO). NK cell populations in chimeric mice were assessed 8 wk after reconstitution. The percentages of total lymphocytes that are NK cells (NK1.1+ CD3 cells) are indicated in each of the top panels. The bottom plots are gated on NK1.1+ CD3 cells in WT/R KO and RßKO/R KO chimera. The percentages of total lymphocytes expressing either Ly5.1 or Ly5.2 in WT/R KO and RßKO/R KO chimera are shown in the top right corner of the bottom panels. Note that NK cell reconstitution in chimeric mice depends upon IL-15R , but not IL-2/15Rß, competent cells. (C) The frequency of IL-15R competent cells controls the frequency of peripheral NK cells. IL-15R / mice were lethally irradiated and reconstituted with bone marrow from either IL-15R +/ or IL-15R / mice, or with various ratios of IL-15R +/ and IL-15R / bone marrow, or assorted ratios of IL-2/15Rß/ and IL-15R / bone marrow. Mice were bled and NK cell populations in chimeric mice were assessed 8 wk after reconstitution. The percentage of total lymphocytes that are NK1.1+ CD3 cells is shown. Plots are representative of at least two mice per condition, and all experiments were performed three times with similar results.
| |
As previously observed, significant numbers of NK cells were observed in the periphery of WT
R
KO chimera, but not R
KO
R
KO chimera (Fig. 2 B, top). Importantly, comparable numbers of NK cells were readily observed in the spleens and peripheral blood of both WT/R
KO
R
KO and RßKO/R
KO
R
KO chimera (Fig. 2 B, top). The presence of IL-15R
competent cells, regardless of their ability to express IL-2/15Rß, is therefore sufficient to support the development and survival of peripheral NK cells. This finding indicates that hematopoietic cells do not need to transduce IL-2/15Rßdependent signals to support NK cells in trans.
We then investigated the cell-autonomous role of IL-2/15Rß expression in supporting NK cells by examining the genotype of surviving NK cells in WT/R
KO
R
KO and RßKO/R
KO
R
KO chimera via congenic markers. Consistent with the data above, the percentages of NK cells derived from IL-15R
/ and IL-15R
+/ bone marrow stem cells were similar to the percentages of nonIL-15R
dependent lymphocytes (B and CD4+ T cells) in WT/R
KO
R
KO chimera (Fig. 2 B, bottom left). By contrast, the percentage of total NK cells that were derived from Ly5.1+ IL-2Rß/ cells (8.4%) was dramatically reduced when compared with the percentage of other Ly5.1+ lymphocytes (41%) reconstituted in RßKO/R
KO
R
KO chimera (Fig. 2 B, bottom right). These data indicate that IL-2/15Rß expression is required on NK cells for their development and maintenance.
Finally, as both WT/R
KO
R
KO and RßKO/R
KO
R
KO chimera generally contained reduced percentages of NK cells compared with WT
R
KO chimera, we hypothesized that the percentage of peripheral NK cells might be a function of the relative percentage of IL-15R
competent hematopoietic cells. To investigate this possibility, we reconstituted IL-15R
/ mice with either 1:1 or 1:4 mixtures of IL-15R
/ and either WT or IL-2/15Rß/ bone marrow stem cells. Examination of the percentage of NK cells in these chimera revealed that the numbers of NK cells decreased as the proportion of IL-15R
competent (either WT or IL-2/15Rß/) bone marrow stem cells decreased (Fig. 2 C). These findings suggest that the relative frequency of IL-15R
competent hematopoietic cells regulates the size of the peripheral NK cell pool.
IL-2/15Rß/ Hematopoietic Cells Support Memory CD8+ T Cells.
Memory phenotype CD8+ T cells are dependent upon both IL-15 and IL-15R
for their development and peripheral survival (16, 17, 22). To investigate whether IL-2/15Rß/ cells could support memory phenotype CD8+ T cells in a non-cellautonomous fashion, we examined the reconstitution of this population in RßKO/R
KO
R
KO chimera. CD44hi IL-2/15Rßhi CD8+ T cells were readily observed in both WT
R
KO and RßKO/R
KO
R
KO chimera, but not in R
KO
R
KO chimera (Fig. 3 A). Notably, there was no obvious population of IL-2/15Rß/ CD44hi CD8+ T cells present in RßKO/R
KO
R
KO chimera, suggesting that IL-2/15Rß, but not IL-15R
, expression by CD44hi CD8+ memory phenotype cells is critical for their peripheral maintenance (Fig. 3 A and not depicted).
CD44hi IL-2/15Rßhi CD8+ T cells include antigen-experienced memory cells as well as cells that may have been activated via alternate mechanisms (e.g., homeostatic expansion). To directly assess the ability of IL-2/15Rß/ hematopoietic cells to support antigen-experienced memory CD8+ T cells, we adoptively transferred naive OT-1+ RAG-1/ CD8+ T cells into WT
R
KO, R
KO
R
KO, WT/R
KO
R
KO, or RßKO/R
KO
R
KO chimera 8 wk after reconstitution. These chimera were then immunized with OVA and poly I:C, and the frequency of OT-1+ CD8+ T cells was serially examined as described above. Similar primary responses of OT-1+ CD8+ T cells were observed in all types of chimera during the first 2030 d after immunization. However, after
50 d, progressive loss of these cells was noted in R
KO
R
KO chimera, but not in the other types of chimera, all of which contained IL-15R
competent hematopoietic cells (Fig. 3 B). Thus, in parallel to our findings with NK cells, IL-2/15Rß/ hematopoietic cells are capable of supporting memory CD8+ T cells in a non-cellautonomous fashion, and this finding suggests that hematopoietic cells use IL-15R
to support memory CD8+ T cells exclusively by a trans presentation mechanism (Fig. 2 A).
Coordinate Expression of IL-15 and IL-15R
by Hematopoietic Cells Is Required for Supporting NK Cells In Vivo.
The findings described above support a model whereby RAG-1independent hematopoietic cells use IL-15R
, but not IL-2/15Rß, to present IL-15 in trans to IL-2/15Rß, but not IL-15R
, dependent receptors on NK and memory CD8+ T cells. However, it remained unclear why NK and memory CD8+ T cells are able to respond to soluble or plate-bound IL-15 in vitro, but apparently fail to receive IL-15 signals in mice that are IL-15R
deficient but IL-15 competent. One possible explanation would be that IL-15R
is not only required on accessory cells for trans presenting IL-15, but is also required for making IL-15 bioavailable for trans presentation. In this scenario, IL-15 would not be freely available in the serum of mice to be bound by IL-15R
presenting cells, but might need to be produced by the same cells that produce IL-15R
. To test this hypothesis, we investigated whether coordinate expression of IL-15 and IL-15R
was required to support NK and memory CD8+ T cells in vivo (Fig. 4). Lethally irradiated IL-15R
/ mice were reconstituted with WT (WT
R
KO), IL-15R
/ (R
KO
R
KO), IL-15/ (15KO
R
KO), a mixture of WT and IL-15R
/ (WT/R
KO
R
KO), or a mixture of IL-15/ and IL-15R
/ bone marrow stem cells (15KO/R
KO
R
KO). Roughly half of the hematopoietic cells in 15KO/R
KO
R
KO chimera should express IL-15R
, but not IL-15, whereas the other half of hematopoietic cells and all residual stromal cells should express IL-15, but not IL-15R
. Thus, if IL-15 can be secreted from IL-15R
/ cells to bind to IL-15R
expressed on IL-15/ cells, then 15KO/R
KO
R
KO chimera should support IL-15dependent lymphocytes as well as WT/R
KO
R
KO chimera. Alternatively, if IL-15R
is required for IL-15 elaboration, then 15KO/R
KO
R
KO chimera would not be able to support NK and CD8+ memory T cells. Analyses of NK cells in these various chimera 8 wk after reconstitution revealed that WT
R
KO and WT/R
KO
R
KO chimera contained significant numbers of peripheral NK cells, whereas 15KO
R
KO, R
KO
R
KO, and 15KO/R
KO
R
KO chimera failed to reconstitute these cells (Fig. 5 A). Importantly, IL-15R
was readily observed on the surface of a variety of IL-15/ cell types, including IL-15/ dendritic cells (Fig. 5 B). Taken together, these findings suggest that IL-15 and IL-15R
must be coordinately expressed by hematopoietic cells to support the development of NK cells via trans presentation.
The failure to reconstitute peripheral NK cells in 15KO/R
KO
R
KO chimera may reflect a failure of development or peripheral maintenance of NK cells, or both. To directly investigate whether 15KO/R
KO
R
KO chimera are capable of supporting the survival of mature peripheral NK cells, congenic splenic NK cells were adoptively transferred into 15KO/R
KO
R
KO as well as control chimera. Serial peripheral blood analyses of these chimera revealed that transferred NK cells persisted for >2 d in WT
R
KO and WT/R
KO
R
KO chimera, but not in R
KO
R
KO, 15KO
R
KO, or most notably, 15KO/R
KO
R
KO chimera (Fig. 5 C). Therefore, coordinate expression of IL-15 and IL-15R
is required to support peripheral NK cell survival.
Coordinate Expression of IL-15 and IL-15R
by Hematopoietic Cells Is Required for Supporting Memory CD8+ T Cells In Vivo.
Next, we investigated whether coordinate expression of IL-15R
and IL-15 is required for the development and maintenance of memory phenotype CD8+ T cells. Analyses of tissues from various chimera revealed that IL-2/15Rßhi CD8+ T cells were readily observed in WT
R
KO chimera, but not in R
KO
R
KO, 15KO
R
KO, or 15KO/R
KO
R
KO chimera (Fig. 6 A). Therefore, coordinate expression of both IL-15R
and IL-15 is necessary for maintenance of memory phenotype CD8+ T cells.
Finally, we examined whether coordinate expression of IL-15 and IL-15R
is required for the generation and maintenance of antigen-experienced memory CD8+ T cells. We adoptively transferred OT-1+ CD8+ T cells into WT
R
KO, WT/R
KO
R
KO, R
KO
R
KO, 15KO
R
KO, and 15KO/R
KO
R
KO chimera, immunized these mice with poly I:C and OVA 2 d later, and examined the kinetics of transgenic T cell responses. Although the primary expansions of these cells were similar 4 d after immunization in the various chimera, memory OT-1+ CD8+ T cells were subsequently maintained in WT
R
KO and WT/R
KO
R
KO chimera, but not in R
KO
R
KO, 15KO
R
KO, and 15KO/R
KO
R
KO chimera (Fig. 6 B and not depicted). This selective loss of memory OT-1+ CD8+ T cells in 15KO
R
KO and 15KO/R
KO
R
KO chimera was particularly evident when the numbers of functional memory OT-1+ CD8+ T cells were assessed by analyzing IFN-
production in response to the cognate peptide SIINFEKL. Although WT
R
KO chimera had significant numbers of IFN-
+ CD44hi CD8+ T cells 90 d after immunization, R
KO
R
KO, 15KO
R
KO, and 15KO/R
KO
R
KO chimera possessed negligible numbers of SIINFEKL-responsive cells (Fig. 6 C). Thus, like NK cells, functional memory CD8+ T cells require coordinate expression of IL-15R
and IL-15 for their maintenance.
 |
Discussion
|
---|
RAG-1independent, IL-15R
competent Cells Define Homeostatic Space for NK Cell Survival and Memory CD8+ T Cell Homeostasis.
IL-15 regulates the homeostasis of NK and memory CD8+ T cells, and the high affinity IL-15R, IL-15R
, is critical for mediating IL-15's functions in vivo. These observations suggest that the bioavailability of IL-15 and IL-15R
defines a homeostatic space that regulates the numbers of these lymphocytes that an organism possesses at any one time. However, the cellular and molecular bases of these homeostatic interactions are poorly understood. In this work, we have examined the cellular mechanisms by which IL-15R
supports lymphoid homeostasis in vivo. Our experiments indicate that RAG-1independent hematopoietic cells comprise the predominant cell types that provide IL-15R
dependent homeostatic support. In this regard, myeloid cells such as macrophages and dendritic cells express low levels of both IL-15 and IL-15R
constitutively, and express higher levels in response to proinflammatory stimuli. We have also found that the proportion of IL-15R
competent hematopoietic cells in WT/R
KO
R
KO mixed chimera correlates directly with the number of NK cells maintained in these mice. Thus, the number of IL-15R
competent accessory cells might be a limiting resource for NK and CD8+ T cells in resting animals. We have separately examined both the survival of peripheral NK cells and the generation and maintenance of memory CD8+ T cells in various mixed chimera, and found similar IL-15R
requirements for these distinct populations. As the peripheral homeostasis of NK cells is largely maintained by cell survival, while memory CD8+ T cell homeostasis is supported by both survival and proliferation, these results suggest that IL-15R
regulates multiple cellular processes in a cell type and context-dependent fashion. Taken together, these experiments help define the nature of "homeostatic space" available to IL-15responsive lymphocytes.
Trans Presentation as the Dominant Physiological Mechanism by Which IL-15R
Supports NK and CD8+ Memory T Cells In Vivo.
Previous studies indicated that IL-15R
supports NK cell and CD8+ memory T cell homeostasis in a non-cellautonomous fashion in vivo (4, 5, 18). The non-cellautonomous mechanism(s) by which IL-15R
competent hematopoietic cells support NK and CD8+ T cells could occur via two nonexclusive mechanisms. First, IL-15R
competent accessory cells could transduce signals through their heterotrimeric IL-15Rs and synthesize secondary proteins that support NK and CD8+ T cells. Alternatively, IL-15R
competent accessory cells could use IL-15R
to directly present IL-15 in trans to NK and CD8+ T cells. Previous studies have shown that IL-2/15Rß expression is required for IL-15induced proliferative responses (7, 12). Moreover, IL-2/15Rß/, but not IL-2R
/, mice lack NK cells, similar to both IL-15/ and IL-15R
/ mice. Taken together, these data suggest that IL-2/15Rß is critical for IL-15 responses. Hence, our finding that IL-2/15Rß/ hematopoietic cells perform as well as WT hematopoietic cells in supporting NK and CD8+ T cells indicates that accessory cells mediate this function without transducing IL-15 signals themselves, and without producing secondary proteins that in turn support lymphocytes. Therefore, these accessory cells use IL-15R
exclusively to directly present IL-15 in trans to NK cells and CD8+ memory T cells. This finding is consistent with recent findings that IL-2/15Rß/ hematopoietic cells can support intraepithelial lymphocyte homeostasis (19). Thus, trans presentation is likely to be the exclusive physiological mechanism by which IL-15R
supports NK and CD8+ T cells in vivo.
Our studies also shed light on the cell-autonomous requirements for IL-15R signaling in lymphoid homeostasis. Analyses of congenic NK cells recovered from RßKO/R
KO
R
KO mixed chimera indicate that IL-2/15Rß expression by IL-15responsive NK and memory CD8+ T cells is required for their homeostasis. This finding confirms previous suggestions that high expression levels of IL-2/15Rß on these cells correlates with their sensitivity to IL-15dependent signals in vivo (23, 24). Meanwhile, analyses of congenic NK cells recovered from our WT/R
KO
R
KO mixed chimera indicate that IL-15R
on IL-15dependent NK and memory CD8+ T cells is entirely dispensable for their homeostasis in vivo. These in vivo data are consistent with the fact that IL-15R
+/ and IL-15R
/ memory CD8+ T cells respond similarly to a given dose of IL-15 in vitro, regardless of whether it is provided as a plate-bound IL-15/IL-15R
c complex or as a soluble cytokine (unpublished data). Hence, despite the fact that IL-15R
can augment signaling responses to soluble IL-15 in transfected cells, it is unlikely that IL-15R
on NK and memory CD8+ T cells facilitates binding of IL-15 to IL-2/15Rß and
c receptor chains on these cells in vivo. Similarly, it is unlikely that IL-15R
on accessory cells transfers IL-15 to IL-15R
on responding NK and CD8+ memory T cells. Taken together, these studies indicate that IL-2/15Rß receptors are critical, whereas IL-15R
receptors are entirely dispensable on NK and memory CD8+ T cells for their homeostasis.
Trans presentation is a novel mechanism by which cytokine signals are transduced. Although a previous report suggested that IL-2R
could present IL-2 in trans (25), IL-2R
alone binds IL-2 with low affinity (Kd
108 M) and in vivo studies with IL-2R
/ T cells indicated that IL-2R
plays a cell-autonomous role in supporting T cells (2628). Thus, trans presentation is unlikely to be the physiological mechanism by which IL-2R
supports T cells. Signaling through the IL-6 receptors, IL-6R
and gp130, more closely resembles IL-15R signaling. Soluble IL-6R
is produced by both proteolytic cleavage of IL-6R
and alternative splicing. Soluble IL-6R
binds IL-6 in solution and IL-6sIL-6R
complexes then bind to gp130 receptors on cell surfaces to initiate signal transduction events (29, 30). Nevertheless, there might be fundamental differences between IL-6R
and IL-15R
mediated signaling. Specifically, in contrast to IL-6R
, it is unclear if IL-15 and IL-15R
can form soluble complexes that can signal to IL-2/15Rß
c receptors on responding cells (11, 31). Therefore, the ability of IL-15R
on the surface of accessory hematopoietic cells to present IL-15 in trans to IL-2/15Rß and
c low affinity dimeric receptors on NK and memory CD8+ T cells in vivo may represent a novel mechanism of cytokine signaling that may involve cell to cell contact.
Coordinate Expression of IL-15 and IL-15R
by Trans Presenting Accessory Cells.
Our studies with 15KO/R
KO
R
KO mixed chimera indicate that IL-15 and IL-15R
must be expressed by the same accessory cells to support both NK and memory CD8+ T cells in vivo. As IL-15/ cells express normal levels of cell surface IL-15R
(Fig. 5 B), and as IL-15R
/ cells express normal levels of IL-15 mRNA (5, 18), the inability of 15KO/R
KO
R
KO chimera to support NK and memory CD8+ T cells suggests that IL-15R
/ cells may not elaborate IL-15 protein. This surprising finding would explain why IL-15R
competent lymphocytes respond to heterologous IL-15 in vitro, but fail to respond to IL-15 elaborated from IL-15competent cells in vivo. Thus, IL-15R
might be essential for either the translation of IL-15 mRNA or the trafficking of IL-15 protein to the cell surface. With regards to the latter possibility, it is known that the signal sequences of IL-15 mediate protein secretion poorly (32). As IL-15R
associates with IL-15 with high affinity, one intriguing possibility is that IL-15R
may bind to IL-15 intracellularly and facilitate trafficking of IL-15/IL-15R
to the surface of accessory cells. Intracellular association of IL-15 and IL-15R
has recently been described in several contexts, including endosomal recycling of internalized IL-15R
IL-15 complexes that follow binding of extracellular IL-15 to surface IL-15R
(20, 33, 34). By contrast, our current findings suggest that the critical interactions between IL-15 and IL-15R
occur within IL-15producing cells, before IL-15's emergence on the plasma membrane. In addition, as soluble IL-15 has been difficult to document in mice, it is possible that IL-15R
recognizes and binds to IL-15 exclusively within cells that synthesize both proteins. Therefore, these experiments indicate novel cell biological requirements for the regulation of IL-15, and also provide a compelling explanation for why trans presentation is the physiological mechanism by which IL-15 supports lymphoid homeostasis.
In summary, we have examined the mechanism by which IL-15R
supports NK cell survival and CD8+ memory T cell proliferation in vivo. Our findings indicate that myeloid accessory cells do not use IL-15Rs to transduce signals leading to the elaboration of secondary proteins that support lymphoid homeostasis. Instead, these cells use IL-15R
to present IL-15 in trans to IL-2/15Rßbearing receptors on the surface of NK and CD8+ memory T cells. These accessory cells must coordinately synthesize IL-15 and IL-15R
to present IL-15 in trans. Therefore, the critical events regulating homeostatic niches for NK and CD8+ memory T cells in vivo can be focused upon the production and trans presentation of IL-15 by IL-15R
expressing myeloid cells.
 |
Acknowledgments
|
---|
We thank D. Boone for critically reading the manuscript.
This work was supported by National Institutes of Health (NIH) RO1 grants AI45860 and AI59827 (to A. Ma), NIH T32DK60414 (to R. Koka), and the Sandler Family Foundation.
The authors have no conflicting financial interests.
Submitted: 12 July 2004
Accepted: 9 August 2004
 |
References
|
---|
- Schluns, K.S., W.C. Kieper, S.C. Jameson, and L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. 1:426432.[CrossRef][Medline]
- Becker, T.C., E.J. Wherry, D. Boone, K. Murali-Krishna, R. Antia, A. Ma, and R. Ahmed. 2002. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells. J. Exp. Med. 195:15411548.[Abstract/Free Full Text]
- Goldrath, A.W., P.V. Sivakumar, M. Glaccum, M.K. Kennedy, M.J. Bevan, C. Benoist, D. Mathis, and E.A. Butz. 2002. Cytokine requirements for acute and basal homeostatic proliferation of naive and memory CD8+ T cells. J. Exp. Med. 195:15151522.[Abstract/Free Full Text]
- Burkett, P.R., R. Koka, M. Chien, S. Chai, F. Chan, A. Ma, and D.L. Boone. 2003. IL-15R alpha expression on CD8+ T cells is dispensable for T cell memory. Proc. Natl. Acad. Sci. USA. 100:47244729.[Abstract/Free Full Text]
- Koka, R., P.R. Burkett, M. Chien, S. Chai, F. Chan, J.P. Lodolce, D.L. Boone, and A. Ma. 2003. Interleukin (IL)-15R
deficient natural killer cells survive in normal but not IL-15R
deficient mice. J. Exp. Med. 197:977984.[Abstract/Free Full Text]
- Cooper, M.A., J.E. Bush, T.A. Fehniger, J.B. VanDeusen, R.E. Waite, Y. Liu, H.L. Aguila, and M.A. Caligiuri. 2002. In vivo evidence for a dependence on interleukin 15 for survival of natural killer cells. Blood. 100:36333638.[Abstract/Free Full Text]
- Giri, J.G., S. Kumaki, M. Ahdieh, D.J. Friend, A. Loomis, K. Shanebeck, R. DuBose, D. Cosman, L.S. Park, and D.M. Anderson. 1995. Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor. EMBO J. 14:36543663.[Abstract]
- Bulanova, E., V. Budagian, T. Pohl, H. Krause, H. Durkop, R. Paus, and S. Bulfone-Paus. 2001. The IL-15R alpha chain signals through association with Syk in human B cells. J. Immunol. 167:62926302.[Abstract/Free Full Text]
- Bulanova, E., V. Budagian, Z. Orinska, H. Krause, R. Paus, and S. Bulfone-Paus. 2003. Mast cells express novel functional IL-15 receptor alpha isoforms. J. Immunol. 170:50455055.[Abstract/Free Full Text]
- Bulfone-Paus, S.S., E. Bulanova, T. Pohl, V. Budagian, H. Durkop, R. Ruckert, U. Kunzendorf, R. Paus, and H. Krause. 1999. Death deflected: IL-15 inhibits TNF-alpha-mediated apoptosis in fibroblasts by TRAF2 recruitment to the IL-15Ralpha chain. FASEB J. 13:15751585.[Abstract/Free Full Text]
- Anderson, D.M., S. Kumaki, M. Ahdieh, J. Bertles, M. Tometsko, A. Loomis, J. Giri, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, et al. 1995. Functional characterization of the human interleukin-15 receptor alpha chain and close linkage of IL15RA and IL2RA genes. J. Biol. Chem. 270:2986229869.[Abstract/Free Full Text]
- Bamford, R.N., A.J. Grant, J.D. Burton, C. Peters, G. Kurys, C.K. Goldman, J. Brennan, E. Roessler, and T.A. Waldmann. 1994. The interleukin (IL) 2 receptor beta chain is shared by IL-2 and a cytokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Proc. Natl. Acad. Sci. USA. 91:49404944.[Abstract]
- Giri, J.G., M. Ahdieh, J. Eisenman, K. Shanebeck, K. Grabstein, S. Kumaki, A. Namen, L.S. Park, D. Cosman, and D. Anderson. 1994. Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:28222830.[Abstract]
- DiSanto, J.P., W. Muller, D. Guy-Grand, A. Fischer, and K. Rajewsky. 1995. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc. Natl. Acad. Sci. USA. 92:377381.[Abstract]
- Suzuki, H., T.M. Kundig, C. Furlonger, A. Wakeham, E. Timms, T. Matsuyama, R. Schmits, J.J. Simard, P.S. Ohashi, H. Griesser, et al. 1995. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta. Science. 268:14721476.[Medline]
- Lodolce, J.P., D.L. Boone, S. Chai, R.E. Swain, T. Dassopoulos, S. Trettin, and A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity. 9:669676.[Medline]
- Kennedy, M.K., M. Glaccum, S.N. Brown, E.A. Butz, J.L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C.R. Willis, et al. 2000. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15deficient mice. J. Exp. Med. 191:771780.[Abstract/Free Full Text]
- Lodolce, J.P., P.R. Burkett, D.L. Boone, M. Chien, and A. Ma. 2001. T cellindependent interleukin 15R
signals are required for bystander proliferation. J. Exp. Med. 194:11871194.[Abstract/Free Full Text]
- Schluns, K.S., E.C. Nowak, A. Cabrera-Hernandez, L. Puddington, L. Lefrancois, and H.L. Aguila. 2004. Distinct cell types control lymphoid subset development by means of IL-15 and IL-15 receptor alpha expression. Proc. Natl. Acad. Sci. USA. 101:56165621.[Abstract/Free Full Text]
- Dubois, S., J. Mariner, T.A. Waldmann, and Y. Tagaya. 2002. IL-15Ralpha recycles and presents IL-15 in trans to neighboring cells. Immunity. 17:537547.[Medline]
- Kawamura, T., R. Koka, A. Ma, and V. Kumar. 2003. Differential roles for IL-15R alpha-chain in NK cell development and Ly-49 induction. J. Immunol. 171:50855090.[Abstract/Free Full Text]
- Judge, A.D., X. Zhang, H. Fujii, C.D. Surh, and J. Sprent. 2002. Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8+ T cells. J. Exp. Med. 196:935946.[Abstract/Free Full Text]
- Schluns, K.S., K.D. Klonowski, and L. Lefrancois. 2003. Transregulation of memory CD8 T-cell proliferation by IL-15Ralpha+ bone marrow-derived cells. Blood. 103:988994.[CrossRef][Medline]
- Zhang, X., S. Sun, I. Hwang, D.F. Tough, and J. Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity. 8:591599.[Medline]
- Eicher, D.M., and T.A. Waldmann. 1998. IL-2R alpha on one cell can present IL-2 to IL-2R beta/gamma(c) on another cell to augment IL-2 signaling. J. Immunol. 161:54305437.[Abstract/Free Full Text]
- D'Souza, W.N., K.S. Schluns, D. Masopust, and L. Lefrancois. 2002. Essential role for IL-2 in the regulation of antiviral extralymphoid CD8 T cell responses. J. Immunol. 168:55665572.[Abstract/Free Full Text]
- Leung, D.T., S. Morefield, and D.M. Willerford. 2000. Regulation of lymphoid homeostasis by IL-2 receptor signals in vivo. J. Immunol. 164:35273534.[Abstract/Free Full Text]
- Willerford, D.M., J. Chen, J.A. Ferry, L. Davidson, A. Ma, and F.W. Alt. 1995. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity. 3:521530.[Medline]
- Kallen, K.J. 2002. The role of transsignalling via the agonistic soluble IL-6 receptor in human diseases. Biochim. Biophys. Acta. 1592:323343.[Medline]
- Rose-John, S. 2003. Interleukin-6 biology is coordinated by membrane bound and soluble receptors. Acta Biochim. Pol. 50:603611.[Medline]
- Dubois, S., F. Magrangeas, P. Lehours, S. Raher, J. Bernard, O. Boisteau, S. Leroy, S. Minvielle, A. Godard, and Y. Jacques. 1999. Natural splicing of exon 2 of human interleukin-15 receptor alpha-chain mRNA results in a shortened form with a distinct pattern of expression. J. Biol. Chem. 274:2697826984.[Abstract/Free Full Text]
- Kurys, G., Y. Tagaya, R. Bamford, J.A. Hanover, and T.A. Waldmann. 2000. The long signal peptide isoform and its alternative processing direct the intracellular trafficking of interleukin-15. J. Biol. Chem. 275:3065330659.[Abstract/Free Full Text]
- Pereno, R., J. Giron-Michel, A. Gaggero, E. Cazes, R. Meazza, M. Monetti, E. Monaco, Z. Mishal, C. Jasmin, F. Indiveri, et al. 2000. IL-15/IL-15Ralpha intracellular trafficking in human melanoma cells and signal transduction through the IL-15Ralpha. Oncogene. 19:51535162.[CrossRef][Medline]
- Ruckert, R., K. Brandt, E. Bulanova, F. Mirghomizadeh, R. Paus, and S. Bulfone-Paus. 2003. Dendritic cell-derived IL-15 controls the induction of CD8 T cell immune responses. Eur. J. Immunol. 33:34933503.[CrossRef][Medline]