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Published in final edited form as: J Immunol. 2016 Mar 21;196(9):3920–3926. doi: 10.4049/jimmunol.1502337

IL-15 independent maintenance of tissue resident and boosted effector memory CD8 T cells

Jason M Schenkel 1,*, Kathryn A Fraser 1,*, Kerry A Casey 1, Lalit K Beura 1, Kristen E Pauken 2, Vaiva Vezys 1, David Masopust 1
PMCID: PMC5145194  NIHMSID: NIHMS764390  PMID: 27001957

Abstract

IL-15 regulates central (TCM) and effector (TEM) memory CD8 T cell homeostatic proliferation, maintenance and longevity. Consequently, IL-15 availability hypothetically defines the carrying capacity for total memory CD8 T cells within the host. In conflict with this hypothesis, previous observations demonstrated that boosting generates preternaturally abundant TEM that increases the total quantity of memory CD8 T cells in mice. Here we provide a potential mechanistic explanation by reporting that boosted circulating TEM do not require IL-15 for maintenance. We also investigated tissue resident memory CD8 T cells (TRM), which protect nonlymphoid tissues from reinfection. We observed up to a 50-fold increase in the total magnitude of TRM in mouse mucosal tissues after boosting, suggesting that the memory T cell capacity in tissues is flexible and that TRM may not be under the same homeostatic regulation as primary TCM and TEM. Further analysis identified distinct TRM populations that either 1) depended on IL-15 for homeostatic proliferation and survival, 2) depended on IL-15 for homeostatic proliferation but not for survival, or 3) did not depend on IL-15 for either process. These observations on the numerical regulation of T cell memory indicate that there may be significant heterogeneity among distinct TRM populations and also argues against the common perception that developing vaccines that confer protection by establishing abundant TEM and TRM will necessarily erode immunity to previously encountered pathogens due to competition for IL-15.

Introduction

After clearance of an acute infection, expanded populations of pathogen specific memory CD8 T cells are maintained and can be broadly divided into three distinct subsets, each with discrete trafficking properties and roles in immunosurveillance (1). Central memory CD8 T cells (TCM) are recirculating T cells that migrate between blood and lymph nodes by crossing high endothelial venules (HEVs) and are typically poised to proliferate should they re-encounter antigen. Effector memory CD8 T cells (TEM) patrol blood and certain nonlymphoid tissues, constitutively express markers of effector differentiation, but lack the ability to cross high endothelial venules. Resident memory CD8 T cells (TRM) comprise a third, more recently appreciated subset that do not leave and reenter tissues (and thus do not cross HEVs). TRM can be found in nonlymphoid tissues, certain vascular compartments, and secondary lymphoid organs (SLOs), and accelerate control of local infections (2, 3). Recent work has highlighted the collaboration between both resident and recirculating CD8 T cells in the event of reinfection, however the factors that regulate the size of these populations and how subsequent unrelated infections alter T cell numbers is not completely understood.

Interleukin-15 (IL-15) is a common gamma chain (γc) cytokine that promotes homeostatic proliferation and survival of circulating TCM and TEM after primary infection (4–6). IL-15 is also required for the development and maintenance of CD103+ CD8 TRM in the skin epidermis following HSV infection (7). Consequently, one hypothesis is that IL-15 defines the host’s carrying capacity for memory CD8 T cells within both lymphoid and nonlymphoid tissues, suggesting that subsequent infection may result in the displacement of pre-existing memory CD8 T cells (8) in part due to competition for IL-15 (9). However, it is unclear whether all memory CD8 T cells require IL-15, and IL-15 independent CD8 T cells have been observed after local lung infection, by TRM in secondary lymphoid organs, and during persistent infection (10–13).

Heterologous prime-boost-boost (HPBB) vaccination involves immunizing a host with three serologically distinct vectors that all carry the same conserved CD8 T cell epitope. Iterative T cell stimulation with potent HPBB vaccines can generate amplified populations of recirculating memory CD8 T cells that undergo less contraction with each subsequent boost (14). Most memory CD8 T cells generated by HPBB are TEM, as defined by a lack of CD62L expression (15, 16). However, TEM generated by HPBB are phenotypically distinct from TEM generated after a primary infection (17). Of note, we previously demonstrated that HPBB vaccination did not induce substantial attrition of pre-existing CD8+ TCM specific for other pathogens, and thus HPBB immunization substantially increased the overall magnitude of the memory CD8 T cell compartment (15). How HPBB is able to introduce a large new pool of TEM without causing coordinate erosion of pre-existing memory CD8 T cells is unknown.

Here, we evaluated the role of IL-15 in different contexts of memory T cell differentiation including HPBB vaccination and a primary viral infection that establishes broadly distributed TRM.

Materials and Methods

Mice and Infections

C57BL/6 mice were purchased from The Jackson Laboratory. All mice were used in accordance with National Institutes of Health and the University of Minnesota Institutional Animal Care and Use Committee guidelines. For experiments analyzing CD8 T cell generated by heterologous prime-boost-boost system, N52–59 specific 1° memory CD8 T cells were generated by an i.v. infection of CD45.2+ naive C57BL/6 mice (age matched to 3° C57BL/6 mice) with 1×107 pfu of vesicular stomatitis virus Indiana strain (VSVind). For the generation of 3° memory CD8 T cells, CD45.1/CD45.1+ 8–10 week old naive C57BL/6 mice were primed i.v. 5×105 pfu VSVnj, rested 60–90 days, then infected i.v. with 2×106 pfu recombinant vaccinia virus expressing the N protein of VSV (Yewdell et al., 1986), rested an additional 60–90 days, and then challenged i.v. with 1×107 pfu VSVind. For homeostatic maintenance experiments, 5×105 1° P14 or H-2Kb-N52–59 specific 3° memory CD8 T cells were co-transferred into naïve C57Bl/6J or IL-15−/− recipients. The number of donor cells in spleen were then quantified 60 days post transfer. To measure homeostatic proliferation, donor cells from 1° P14 or H-2Kb-N52–59 specific 3° memory CD8 T cells were first labeled with either CFSE or CellTrace Violet according to manufacturer’s protocol (Invitrogen). 5×105 labeled cells were then transferred into naïve C57Bl/6J or IL15−/− mice. Turnover was then analyzed in spleen 60 days post transfer.

For generation of memory P14 CD8 T cells, 5×104 naive Thy1.1 P14 CD8+ T cells were transferred i.v. into naïve C57BL/6 or IL15−/− recipients. Mice were infected the next day ip with 2×105 PFU of LCMV Armstrong. For BrdU experiments, 52 days post infection, P14 memory WT or IL15−/− chimeras were given 0.8 mg/mL BrdU in a solution of 2% sucrose/drinking water for 8 days. For dual infection studies, P14 immune chimeras were generated as above. 30 days after LCMV infection, 5×104 naive CD45.1+ OT-I CD8 T cells were adoptively transferred into P14 memory mice. The next day, animals were infected i.v. with 2×106 pfu of VSV-OVA. 30 days later, total numbers of memory P14 and OT-I CD8 T cells in the indicated organs and were compared to the number of P14 CD8 T cells in P14 immune chimeras that were not infected with VSV-OVA.

Isolation of lymphocytes, in vitro stimulation, flow cytometry and immunofluorescence

Lymphocytes were isolated from peripheral tissues as previously described (23). Isolated lymphocytes were stimulated with LCMV gp33–41 peptide (1µg/ml) for 4hrs at 37°C and cytokine secretion was evaluated as described earlier (35). Single cell suspensions were stained with antibodies against CD69 (H1.2F3), CD44 (IM7), Thy1.1 (OX-7), CD62L (MEL-14) from Biolegend, CD8a (53–6.7), CD103 (M290), CD45.1 (A20), CD45.2 (104), IFNγ (XMG1.2), TNFα (MP6-XT22), IL2 (JES6-5H4) from eBioscience and BrdU (B44) from BDBiosciences. Samples were acquired on a Fortessa flow cytometer (BD Biosciences). Immunofluorescence was performed as described(36). Antibodies against Thy1.1 (OX-7), CD45.1 (A20), and CD8β (YTS156.7.7) from Biolegend were used to stain sections.

For in situ tetramer staining, mouse female reproductive tracts were manually cut into ~500 µm sections using a surgical blade. Tissue pieces were incubated with PE conjuagted tetramers in a 2% fetal bovine serum in phosphate buffered saline (PBS) overnight at 4°C. The next day, tissues were washed with ice cold PBS three times on ice. Tissue pieces were then fixed in 2% paraformaldehyde in PBS for 2 hours, then washed in PBS and then placed in a 30% sucrose PBS solution overnight. The next day tissue pieces were frozen in OCT.

Results

Tertiary memory CD8 T cells undergo less homeostatic proliferation than primary memory CD8 T cells

HPBB vaccination has been shown to establish abundant TEM with minimal corresponding erosion in pre-existing TCM, leading to a net increase in total host memory CD8 T cells (15). How additional memory CD8 T cells are accommodated is unknown, but led us to investigate whether HPBB CD8 TEM are controlled by different homeostatic mechanisms compared to primary circulating memory CD8 T cells. We immunized CD45.1+ C57BL/6 mice on three occasions with serologically distinct viruses (VSVnj, followed by rVVn, then VSVind, see methods) that each stimulates an H-2Kb-N52–59 specific CD8+T cell response (hereafter referred to as HPBB vaccination). Mice were rested 60–90 days between immunizations and HPBB vaccination resulted in abundant CD45.1/CD45.1+ tertiary memory CD8 T cells specific for H-2Kb-N52–59 (14). We generated primary memory CD8 T cells by adoptively transferring naive Thy1.1+ P14 CD8 T cells that are specific for the glycoprotein of lymphocytic choriomeningitis virus (LCMV) to naïve C57BL/6 mice. The next day mice were infected with LCMV. Sixty five days after primary LCMV or tertiary HPBB immunization, lymphocytes were isolated from the spleens of both mice, labeled with CFSE, and then were co-transferred into C57BL/6 recipients to monitor basal turnover within the same mice (figure 1a). Sixty days after transfer, splenocytes were isolated and CFSE dilution was assessed on primary P14 and tertiary H-2Kb/N MHC I tetramer+ memory CD8 T cells by flow cytometry. As shown in figure 1b&c, tertiary memory CD8 T cells underwent less homeostatic divisions than primary CD8 T cells. As CD62L- memory CD8 T cells undergo less HP than CD62L+ memory CD8 T cells, this may be explained by the enrichment of CD62L- cells in tertiary immune populations (figure 1d and (17, 18)). Therefore we segregated our analysis based on CD62L expression and found that, regardless of CD62L expression, tertiary memory CD8 T cells underwent less homeostatic proliferation compared to their primary memory CD8 T cell counterparts (figure 1e&f). Thus, primary and tertiary memory CD8 T cells exhibited fundamental differences in their rate of homeostatic proliferation that was not completely defined by CD62L expression.

Figure 1. Tertiary memory CD8 T cells undergo less homeostatic proliferation than primary memory CD8 T cells.

Figure 1

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a) 5×105 primary Thy1.1+ P14 or CD45.1+ H-2Kb-N52–59 specific tertiary memory CD8 T cells were labeled with CFSE and transferred into naïve C57BL/6. Sixty days after transfer CFSE dilution was evaluated by flow cytometry. b) Cumulative frequency graph and c) representative histograms showing CFSE dilution of primary P14 or H-2Kb-N52–59 specific tertiary memory CD8 T cells. d) Expression of CD62L in primary P14 or H-2Kb-N52–59 specific tertiary memory CD8 T cells sixty days after transfer into naïve C57BL/6 mice. e) Representative histograms and f) cumulative frequency showing CFSE dilution in CD62L+ and CD62L- P14 or H-2Kb-N52–59 specific tertiary memory CD8 T cells. *=p<.05,**=p<.01,***=p<.001. Data are from 6 mice from 2 independent experiments.

Tertiary CD8 TEM are maintained without IL-15

The common gamma chain cytokine interleukin-15 (IL-15) is required for the homeostatic proliferation and maintenance of primary memory CD8 T cells after acute infection (19). To determine if IL-15 is required for maintaining tertiary memory CD8 T cells, we co-transferred Thy1.1+ primary and CD45.1+ tertiary memory CD8 T cells into either CD45.2+ wild type or IL-15−/− naive recipients. After 60 days, we enumerated Thy1.1+ P14 and H-2Kb/N52–59 MHC I tetramer+ CD8 T cells in the spleen. As shown in figure 2, recovery of both primary and tertiary CD62L+ memory CD8 T cells was decreased 60 days after transfer into IL-15−/− mice compared to controls. In contrast, maintenance of tertiary CD62L- N52–59-specific CD8 T cells was not observably impaired in IL-15−/− mice. These data indicate that tertiary CD8 TEM are less stringently dependent on IL-15 for maintenance, which could explain how they exceed IL-15-defined carrying capacities that may dictate the size of CD8 TCM and primary CD8 TEM populations.

Figure 2. Tertiary memory CD8 T cells do not require IL-15 for maintenance.

Figure 2

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5×105 primary Thy1.1+ P14 or tertiary CD45.1+/H-2Kb-N52–59 MHC I tetramer+ memory CD8 T cells were transferred into either CD57BL/6 or IL-15−/− hosts. Sixty days after transfer, total P14 and H-2Kb-N52–59 MHC I tetramer+ CD8 T cell numbers were assessed in the spleen. *=p<.001. Each graph consists of 4 mice per group representative of 2–3 independent experiments totaling 8–10 mice/group.

The number of memory CD8 T cells in the female reproductive mucosa and salivary gland is not fixed

The presence of memory CD8 T cells positioned within barrier tissues accelerates protection against local infections (20–22). It is unknown whether the total number of memory CD8 T cells within mucosal tissues is homeostatically regulated, and thus fixed at a defined overall carrying capacity, or whether mucosal memory CD8 T cell quantity is flexible in size. This issue is particularly important given the impetus to design vaccines that attempt to establish abundant memory CD8 T cells at mucosal surfaces. To address whether the female reproductive tract (FRT) and salivary gland (SG) had a defined carrying capacity for memory CD8 T cells, we compared total CD8β+ lymphocytes as well as N52–59-specific memory CD8 T cells in the FRT and SG via in situ tetramer staining and immunofluorescence microscopy among naïve, primary immune (VSVind only) and tertiary immune (HPBB, as above) mice. As shown in figure 3a&b, HPBB vaccinated mice had ~20-fold more N52–59-specific memory CD8 T cells in the FRT and SG compared to mice that received a single immunization. Both primary and tertiary memory CD8 T cells in the FRT expressed CD69 and low levels of CD62L, suggesting that they may include resident populations (figure 3c). In addition, HPBB immunization precipitated a ~30–50-fold increase in the total number of CD8β+ T cells within the FRT and SG, compared to naïve mice (figure 3d&e). These data suggested that HPBB immunization increased not only the magnitude of recirculating TEM in blood and spleen (15), but also the magnitude of mucosal TRM. Moreover, we also found that a single immunization increased the total number of CD8β+ T cells in the FRT ~6-fold as compared to naïve mice. This suggested that the size of CD8β+ TRM population within the mucosa was numerically quite flexible, and perhaps even primary memory CD8 T cells were not subject to the constraints of IL-15 mediated homeostatic regulation in this compartment.

Figure 3. The number of memory CD8 T cells in the female reproductive mucosa and salivary gland is not fixed.

Figure 3

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a) Total numbers of H-2Kb-N52–59 specific primary and tertiary memory CD8 T cells in the female reproductive tract and salivary gland were assessed by in situ tetramer staining and immunofluorescence microscopy sixty days after final immunization with VSVind. b) Representative images of H-2/Kb-N52–59 MHC I tetramer (red) and CD8β (blue) staining. Tetramer+ CD8 T cells indicated by arrows, white bar = 20 µm. c) Flow cytometric analysis of CD62L and CD69 expression on H-2Kb-N52–59 specific primary and tertiary memory CD8 T cells in peripheral blood and female reproductive tract sixty days after final immunization. d) Total numbers of CD8β+ T cells in the female reproductive tract and e) salivary gland were quantified by immunofluorescence microscopy in naïve, primary and tertiary memory mice. *=p<.01 and **=p<.001. Each graph consists of three mice per group representative of two independent experiments totaling 6 mice/group.

IL-15 is not required for TRM survival in all tissues

We next tested whether primary CD8 TRM that became established within mucosal tissues could persist in the absence of IL-15. To this end, we utilized a model system in which resident memory populations have already been extensively characterized via parabiosis (23). Naïve Thy1.1+ P14 CD8 T cells were transferred into naïve C57BL/6 wild type or IL-15−/− mice. The next day mice were infected with LCMV Armstrong and P14 CD8 T cells were enumerated at the peak of the effector response (day 7) and at a memory time point (day 60).

Seven days after infection, there was no significant difference in the number of P14 CD8 T cells between wild type and IL-15−/− mice in any tissue examined, (figure 4a), suggesting that IL-15 is not necessary for the generation of effector CD8 T cells or their migration to nonlymphoid tissues after LCMV infection. Consistent with the dependence of recirculating primary memory CD8 T cell populations on IL-15 for maintenance, we found that 60 days after infection there was a substantial reduction in memory CD8 T cells within LN and spleen, (figure 4b). However, in several tissues that are populated almost exclusively by TRM, including the FRT and the small intestine, we did not observe a reduction of memory CD8 T cells in the absence of IL-15 or a significant change in the expression of CD103 (supplemental figure 1). Nevertheless, IL-15 dependence varied demonstrably among TRM populations within different nonlymphoid tissues. Indeed, TRM in the salivary gland and kidney were almost completely lost in IL-15−/− mice. These data reveal the existence of CD8 TRM that are homeostatically maintained independently of IL-15, but also highlights potential heterogeneity amongst TRM that inhabit different tissues.

Figure 4. IL-15 is not required for TRM survival in all tissues.

Figure 4

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5×104 Thy1.1 P14 transgenic T cells were transferred into C57BL/6 or IL15−/− mice and were then infected with 2×105 pfu LCMV Armstrong. P14 CD8 T cell numbers were assessed by flow cytometry on a) Day 7 or b) Day 60 post infection. *=p<.05,**=p<.01,***=p<.001. Each graph consists of 5–9 mice per group representative of 3 independent experiments for a total of 10–12 mice/group.

IL-15 dependent and independent homeostatic proliferation reveals distinct TRM subsets

The long-term maintenance of the circulating memory CD8 T cell pool is intrinsically tied to IL-15 driven homeostatic self-renewal. We therefore posited that IL-15 dependence in NLT would dictate whether or not CD8 TRM underwent homeostatic proliferation in different tissues. To test this hypothesis, we treated WT and IL-15−/− P14 LCMV immune chimeras with BrdU in the drinking water for 8 days, and then examined BrdU incorporation to measure CD8 T cell turnover. As shown in figure 5, ~15% of circulating memory CD8 T cells in spleen or LN of wild type mice incorporated BrdU after 8 days, while incorporation of BrdU by circulating memory CD8 T cells was dramatically reduced in IL-15−/− mice. Additionally, SLO TRM, a minority population that can be identified on the basis of CD69 expression in the LCMV infection model (11), also underwent less HP in IL-15−/− compared to WT hosts. Interestingly, the CD69+ resident population became enriched relative to their recirculating CD69- counterparts in SLOs, suggesting that SLO TRM were dependent on IL-15 for homeostatic proliferation, but not for survival. CD8 TRM in the thymus exhibited a similar decrease in BrdU incorporation in IL-15−/− mice without a decrease in overall numbers (figure 4b). These data demonstrate that SLO and thymus TRM persistence does not require HP.

Figure 5. IL-15 dependent and independent homeostatic proliferation reveals distinct TRM subsets.

Figure 5

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a) Fifty two days after LCMV infection, BrdU was put in the drinking water. After 8 days, BrdU incorporation in memory P14 CD8 T cells was assessed in the indicated tissues. b) Cumulative frequency of BrdU+ P14 CD8 T cells. *=p<.05,**=p<.01,***=p<.001. Each graph consists of 5–9 mice per group representative of 3 independent experiments for a total of 10–12 mice/group.

Unlike CD8 TRM in SLO and thymus, CD69+ CD8 TRM in both the salivary gland and kidney were dependent on IL-15 for both survival and HP. This suggests that TRM are comprised of distinct subsets with heterogeneous requirements for IL-15 and homeostatic proliferation. In support of this interpretation, TRM in the FRT, SI, and pancreas underwent significant levels of HP, even though these TRM populations were maintained in the absence of IL-15. Moreover, FRT, SI and pancreas TRM incorporated equivalent BrdU in IL-15−/− mice, demonstrating IL-15 independent HP. In summary, these data reveal the existence of TRM that 1) divide and depend on IL-15 for survival, 2) divide in an IL-15 dependent manner but do not depend on IL-15 for survival, and 3) divide and survive independently of IL-15. Thus, TRM comprise a heterogeneous population composed of distinct subsets with unique maintenance requirements and patterns of self-renewal.

No observed defect in TRM cytokine secretion in the absence of IL-15

CD8 T cell derived cytokines are important mediators of host protection and the ability of CD8 T cells to secrete cytokines is oft used as a metric for assessing T cell quality (24). Previous work has demonstrated the ability of IL-15 to promote CD8 T cell secretion of cytokines (25–27), and thus we next tested whether IL-15 deficiency would have deleterious effects on CD8 TRM function. P14 immune chimeras were established in WT and IL-15−/− mice (as in figure 4). Thirty days after LCMV infection, TRM were isolated from the SI epithelium, the pancreas, and the FRT, and circulating memory CD8 T cells were recovered from spleen. Isolated memory CD8 T cells were restimulated in vitro with gp33–41 peptide for 4 hours and secretion of IFNγ, IL-2 and TNFα was assessed by flow cytometry. We specifically assessed these three cytokines as they have been shown to mediate a previously reported TRM sensing and alarm function (28, 29). In all tissues examined, we found that TRM were able to secrete all three cytokines independently of IL-15 (figure 6). Further, circulating memory CD8 T cells isolated from the spleens of IL-15−/− mice exhibited only a minor defect in the capacity to secrete IFNγ and TNFα, which is consistent with previous observations (30). Thus, we failed to detect a role for IL-15 in regulating CD8 TRM cytokine polyfunctionality.

Figure 6. IL-15 is dispensable for memory CD8 T cell cytokine secretion after in vitro antigen stimulation.

Figure 6

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P14 immune chimeras were generated in wild type and IL-15−/− mice, as described in figure 4. Thirty days after infection, P14 CD8 T cells were isolated from the spleen, SI IEL, FRT and pancreas and were stimulated in vitro with gp33–41 peptide for 4 hours. A) Representative FACS plots of IFNγ, IL-2 and TNFα staining on P14 CD8 T cells after peptide stimulation. B) Frequency of P14 CD8 T cells that stained positive for IFNγ, C) IL-2, or D) TNFα, or E) that contemporaneously stained positive for all three cytokines. *=p<.05 and **=p<.01. Each graph consists of 5 mice per group representative of 2 independent experiments totaling 8 mice/group.

Discussion

It has been proposed that the abundance of memory CD8 T cells positioned within barrier tissues could be a critical determinant of host protection (31). One deterrent against developing vaccines based on this objective is the concern that each newly introduced TRM would displace a TRM specific for previously encountered infections. In other words, vaccination could compromise host immunity against commonly encountered pathogens. This remains an important consideration that demands further exploration, but our data here may provide some perspective on this hypothesis. The introduction of new TRM simply resulted in more CD8 T cells within the female reproductive tract, and the total number of CD8 T cells varied over a 50-fold range depending on the infectious experience. A previous report did demonstrate that like TCM and TEM, TRM were IL-15 dependent (7). However, this analysis was based solely on CD103+ TRM in the skin epidermis. Our data revealed that TRM within different anatomic compartments varied considerably in their IL-15 dependence. Teleologically, regulating FRT TRM via IL-15 may have the unwanted consequence of inducing lymphopenic driven expansion of TRM in primary infected hosts. This may not be problematic in the skin because TRM precursors compete with a pre-established visibly saturating population of dendritic epidermal T cells that densely colonize the skin during development (32). In summary, these data support the hypothesis that distinct tissues regulate different populations of TRM, which may point to the existence of distinct TRM subsets. Moreover, the total number of TRM in nonlymphoid tissues may not be stringently regulated as they varied over at least a 50-fold range, although further studies will be needed to test whether an upper-limit is achievable under physiological conditions.

Observations of a dynamic range of total memory CD8 T cells are not limited to TRM. HPBB vaccination results in increased numbers of total memory CD8 T cells within the host, which are largely contained within the TEM pool. Many natural iterative stimulation events, either through heterosubtypic infections or persistent infections, may also lead to uncommonly large CD8+ TEM populations. Thus, it may be disadvantageous for the host to evacuate its pre-existing memory CD8 T cells in order to accommodate expanded TEM populations. Consistent with this conjecture, natural infection of humans with Epstein-Barr virus, which causes a persistent viral infection resulting in robust CD8 T cell responses and increased TEMRA, did not induce a measureable reduction in pre-existing CD8 T cells specific for cytomegalovirus or influenza virus (33). Here we demonstrate that unlike TCM and primary TEM, tertiary TEM were maintained after transfer to IL-15−/−. This provides a potential mechanistic explanation by which the iteratively stimulated TEM population may result in increases in net memory CD8 T cells and also highlights heterogeneity in the regulation of circulating memory CD8 T cell subsets. Of course, these data do not exclude an important role for IL-15 in the generation of boosted memory CD8 T cells (30), nor observations that certain infections induce attrition of pre-existing memory CD8 T cells via acute induction of type I IFN, lymph node fibrosis, or other means (8, 34).

Supplementary Material

1

Acknowledgments

We would like to acknowledge other members of the Masopust and Vezys labs for helpful discussions.

Grants support1

Footnotes

1

This study was funded by National Institutes of Health R01AI084913-01 (D.M.), T32AI007313 and F30DK100159-01 (J.M.S.), and Robertson Foundation/Cancer Research Institute Irvington Fellowship (K.E.P.)

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