International Immunology Advance Access originally published online on June 1, 2007
International Immunology 2007 19(6):733-743; doi:10.1093/intimm/dxm039
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The CD8 T cell response to vaccinia virus exhibits site-dependent heterogeneity of functional responses
Center for Immunology and Department of Laboratory Medicine and Pathology, University of Minnesota, 420 Delaware Street S.E., Minneapolis, MN, USA
Correspondence to: M. F. Mescher; E-mail: mesch001{at}umn.edu
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Abstract |
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CD8 T cell responses to vaccinia virus (VV) and a virus-encoded ovalbumin peptide (OVAP) epitope were examined using adoptively transferred OT-I T cells. The results demonstrate that upon intra-peritoneal challenge with ovalbumin-expressing VV (VV-OVAP), OT-I T cell proliferation occurs initially in lymph nodes and spleens followed by migration of the divided cells to the peritoneal cavity. Massive clonal expansion occurs in response to both the virus and the virus-encoded ovalbumin (OVA) epitope, as demonstrated using low numbers of adoptively transferred cells, and the responding OT-I cells display marked site-dependent functional heterogeneity with respect to IFN-
and tumor necrosis factor-
(TNF-) production and granzyme B expression. OT-I cells responding to VV-OVAP develop the capacity to produce IFN- in response to antigen as they proliferate and differentiate. In marked contrast, naive OT-I cells rapidly produce TNF- upon antigen recognition, and this capacity declines as the cells proliferate in response to the virus, suggesting that this potent inflammatory cytokine may be important primarily during initiation of the response. At the peak of clonal expansion, a large fraction (30–60%) of the OT-I cells responding to the virus express high IL-7R levels, and the majority of these cells is subsequently lost. While high IL-7R expression may be necessary for a CD8 T cell to transition to memory, it is clearly not sufficient. Thus, OT-I cells responding to VV infection exhibit a high degree of heterogeneity within the responding population that differs depending on their anatomical location, despite the specificity and affinity of the TCR being identical on all of the cells.
Keywords: cell activation, cytotoxic T cells, viral infection
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Introduction |
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Vaccinia virus (VV), a large DNA virus, is closely related to the other orthopoxviruses including variola virus, the cause of smallpox. Immunization with VV protects against variola virus infection (1), and use of VV as a vaccine has played a key role in the eradication of smallpox. Infection with VV induces strong CD8 T cell responses in Balb/c (2) and C57BL/6 mice (3), but the CD8 T cell response is not required for protection in normal mice. The absence of perforin- or Fas-dependent cytotoxicity (4), or the almost complete absence of CD8 T cells in B2m–/– mice (5), do not affect viral clearance or survival. In a more detailed study of the basis for protective immunity, Xu et al. (3) recently showed that CD4 T cell-dependent antibody production plays the major role in viral clearance following acute infection, but that the CD8 T cell response could mediate protection in the absence of an antibody response. This study also demonstrated that VV-specific memory CD8 T cells could protect against secondary infection.
In addition to providing an important vaccine for protection against smallpox, VV has been widely used as an expression vector for foreign genes and as a vaccine for other infectious diseases and cancers (6). Despite this, CD8 T cell responses to VV and epitopes expressed by recombinant VVs have not been extensively studied. Harrington et al. (2) characterized the time course for the CD8 T cell response in the spleen to a recombinant VV and showed that there was a coordinate response to the VV vector and the foreign epitope, and that the response was comparable to that induced by lymphocyte choriomeningitis virus (LCMV) infection. They further showed that the cells responding to the foreign epitope developed potent function, as assessed by production of effector cytokines, and established a long-term, responsive memory population.
Virus-specific CD8 T cells respond to viral infection by undergoing multiple rounds of cell division leading to clonal expansion, and during this time the naive cells differentiate to develop effector functions, including cytolytic activity and the ability to rapidly produce effector cytokines including IFN- and tumor necrosis factor- (TNF-) upon encounter with antigen. A number of studies have shown that there is considerable heterogeneity in the effector functions expressed by CD8 T cells responding to various viruses (7–9) both within the population from a given anatomical site and between populations from different sites. A number of factors can potentially influence development of function, including levels of antigen and co-stimulation and affinities of TCRs on the responding cells. In addition, there is now considerable evidence that antigen and co-stimulation are not sufficient to drive differentiation and acquisition of effector functions, and that a ‘third’ signal is required that can be provided by either IL-12 (10, 11) or Type I IFNs (12). Thus, a number of factors may play a role in determining the effector competence of developing CTL.
In order to further investigate the functional heterogeneity that arises during virus-specific CTL responses and characterize in more detail the parameters of CD8 T cell responses to VV as an expression vector, we have examined the responses of endogenous CD8 T cells and adoptively transferred TCR transgenic OT-I CD8 T cells to ovalbumin-expressing VV (VV-OVAP). Use of an adoptive transfer approach makes it possible to examine the earliest phase of the response, before substantial clonal expansion has occurred, and assures that variations in functional status do not result from differences in TCR affinities, as might be the case when polyclonal responders are examined. The results demonstrate that despite identical TCRs, OT-I cells responding to VV-OVAP exhibit marked site-dependent heterogeneity with respect to their expression of granzyme B (grzB) and their ability to produce IFN- and TNF-. In addition, a large fraction of the OT-I cells present at the peak of the response express high levels of the IL-7R chain, but this is not sufficient to insure that they survive to become long-lived memory cells.
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Methods |
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Mice, virus and reagents
OT-I mice having a transgenic TCR specific for H-2Kb and OVA257–264 (ovalbumin) (13) were a gift from F. Carbone (University of Melbourne, Melbourne, Australia). OT-I mice were also crossed with Thy1-congenic B6.PL-Thy1a/Cy (Thy1.1) mice (Jackson ImmunoResearch Laboratories) and bred to homozygosity. The OT-I and OT-I/PL breeding colonies were maintained under specific pathogen-free conditions at the University of Minnesota. C57BL/6NCr mice were purchased from the National Cancer Institute. All directly conjugated fluorescent antibodies were purchased from BD Biosciences or eBioscience. Recombinant VV-GFP-JAW-OVA (VV-OVAP) was provided by J. Yewdell, National Institutes of Health, Bethesda, MD, USA. This recombinant virus includes the OVA257–264 epitope fused C-terminally to GFP and the transmembrane region of JAW-1. The Western Reserve vaccinia strain (VV-WR) was from M. Bevan (University of Washington, Seattle, WA, USA). Mice were infected by intra-peritoneal (i.p.) injection of 5 x 106 PFU of the indicated virus, with the viral titer determined by plaque assays performed using 143B cells. These studies were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Minnesota.
Naive T cell purification
Inguinal, axillary, brachial, cervical and mesenteric lymph nodes were harvested from OT-I/PL mice, pooled and disrupted to obtain a single-cell suspension. Cells were enriched for CD8+ CD44low cells by negative selection using MACS magnetic cells sorting (Miltenyi Biotec). In brief, cells were coated with FITC-labeled antibodies specific for CD4, B220, I-Ab, CD11c and CD44. Anti-FITC magnetic MicroBeads (Miltenyi Biotech) were added to the cells, which were then passed over separation columns attached to the MACS magnet. The cells that did not bind to the column were collected and were >95% CD8+ and <0.5% CD44high.
Adoptive transfer of OT-I/PL transgenic cells
A total of either 1.5 x 106 or 5 x 103 purified naive CD8+ cells from OT-1/PL mice in 0.3 ml PBS were transferred via tail vein injection into age- and sex-matched naive 6- to 8-week old C57BL/6 recipients. Recipient mice were rested for 24 h before virus inoculation. In some experiments, OT-I/PL cells were labeled with CFSE prior to adoptive transfer. Purified cells were re-suspended to 20 x 106 ml–1 in HBSS and warmed in a 37°C water bath for 10 min before adding an equal volume of 6 µM CFSE in HBSS and incubating for an additional 5 min at 37°C. Cells were washed once with ice-cold RP-10 and twice with ice-cold PBS before adoptive transfer.
Flow cytometric analysis of OT-I cells
Mice were sacrificed at the indicated times after adoptive transfer and viral infection. Spleen cells, peritoneal cells and lymph node (LN) cells (pooled from axillary, brachial, cervical, inguinal and mesenteric nodes) were harvested, counted by trypan blue dye exclusion to determine total viable cell counts and stained with antibodies to CD8 and Thy 1.1 to detect the transferred OT-I/PL cells. Stained cells were analyzed on a FACSCaliburTM flow cytometer using CELLQuestTM software (BD Biosciences) to determine the percent and total OT-I/PL cells in the mice.
Intracellular staining for cytokine production and grzB expression
Spleen cells, peritoneal cells and LN cells were incubated at 2 x 106 cells ml–1 in RP-10 with 0.2 µM OVA257–264 and 1 µl GolgiPlug (BD Biosciences) for 3.5 h at 37°C, and then washed and stained with the antibodies to CD8 and Thy 1.1 to mark the OT-I/PL cells. The cells were then fixed in Cytofix buffer (BD Biosciences) for 15 min at 4°C, permeabilized in saponin-containing Perm/Wash buffer (BD Biosciences) for 15 min at 4°C and stained with FITC–anti-IFN- mAb and PE–anti-TNF- mAb for 30 min at 4°C. Following staining, cells were washed once with Perm/Wash buffer and once with PBS containing 2% FBS and analyzed on a FACSCaliburTM flow cytometer using CELLQuestTM software. GrzB intracellular staining was done directly ex vivo without peptide re-stimulation.
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Results |
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Clonal expansion in response to infection
When naive TCR transgenic OT-I T cells specific for H-2Kb/OVA257–264 (13) are adoptively transferred into C57BL/6 mice, they persist for weeks as resting naive cells in the LNs and spleens. Upon infection of the mice by i.p. inoculation with recombinant VV that expresses the OVA257–264 epitope (VV-OVAP), the OT-I cells undergo rapid clonal expansion (Fig. 1A). Expansion is greatest in the spleen, with OT-I numbers peaking by day 5. Numbers then rapidly decline over the next few days, and a stable population of cells having a memory phenotype (data not shown) is present by day 30. Clonal expansion is seen in the LN with a similar time course, but in much smaller numbers than in the spleen. Few, if any, naive cells are detectable in the peritoneal cavity (PC) prior to infection, but activated OT-I cells become detectable at this site by day 3, peak in number at day 5 and then decline (Fig. 1A).
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Concomitant with expansion of the OT-I cells, there is a large increase in the number of endogenous CD8 T cells in the spleen during days 5 through 7 with numbers then rapidly declining to normal resting levels (Fig. 1B). Endogenous CD8 T cells also increase in number in the PC during days 5 through 7, but no significant increase is seen in LN. Further characterization of these endogenous cells is described below. When mice received OT-I cells by transfer and were then challenged with the Western Reserve vaccinia strain (VV-WR) that does not express the OVA epitope, the OT-I cells did not increase in number and retained a naive phenotype (data not shown). In contrast, endogenous CD8 T cells in the spleen and PC increased in number comparably to when infection was with VV-OVAP (data not shown). Virus is rapidly cleared in the male mice used in these experiments, and is undetectable in the PC or other sites when plaque-forming assays are done on day 3 or 5 (data not shown).
OT-I cells labeled with CFSE were found to be largely undivided in the LN and spleen the first day after infection (Fig. 2). By day 2, however, the cells had undergone several divisions and by day 3 the CFSE was fully diluted, indicating that the cells had divided at least seven to eight times. The extent of CFSE dilution on day 2 was consistently greater in the spleen than in the LN. When OT-I cells appear in the PC by day 5, they have fully diluted CFSE (data not shown), indicating that they migrate to this site only after undergoing multiple rounds of division. The rapid onset of cell division in lymphoid tissue, together with the fact that division of CD8 T cells does not commence until 
24 h after initial recognition of antigen (14), suggests that antigen rapidly becomes available to most of the naive CD8 T cells following onset of infection.
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Functional heterogeneity of responding CD8 T cells
Major effector functions of activated CTL include direct lysis of virus-infected cells and production of cytokines including TNF-
and IFN-. The capacity of OT-I cells to produce TNF- and IFN- can be assessed by intracellular cytokine staining following stimulation for 3.5 h with OVA257–264 peptide, and levels of intracellular grzB expression correlate strongly with cytolytic activity of the cells (15). When effector cells responding to VV-OVAP infection were examined on day 5, there was substantial heterogeneity in expression of these proteins depending on the location of the cells. OT-I cells from the PC, the primary site of infection, expressed the highest levels of grzB and produced the highest levels of IFN- and TNF- upon in vitro re-stimulation, as assessed by both the percent of cells expressing the protein and the level of expression on a per cell basis (Fig. 3A). Expression of these proteins was substantially lower in OT-I cells in the spleen and lowest in cells from the LN (Fig. 3A).
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By day 32, when numbers have declined and a memory population has been established, there was less heterogeneity of function for OT-I cells from the different sites (Fig. 3B). The small number of cells that could be recovered from the PC, however, continued to express somewhat higher levels of grzB and produce more TNF-
than those from other sites. Memory cells at all sites had substantially higher capacity to produce IFN- and TNF- than did naive cells or day 5 effector cells, while grzB expression was decreased. Thus, long-term memory cells all have the capacity to rapidly produce high levels of effector cytokines upon reencounter with antigen.
Examination of the time course of expression of the proteins important for function showed that differences were greatest at day 5, the peak of clonal expansion, when considered as a percent of the OT-I cells that produced IFN- (Fig. 4A) and TNF- (Fig. 4B) or expressed grzB (Fig. 4C). Measured in this way, OT-I cells in the PC clearly have the greatest functional capacity during the effector phase of the response. Total functional capacity, however, presumably depends on both the capacity per cell and the number of cells at the site. When examined in this way, a different picture emerges. Thus, by far the greatest number of cells with the capacity to produce IFN- (Fig. 4A) and TNF- (Fig. 4B) and express grzB (Fig. 4C) are present in the spleen during the effector phase of the response.
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In the experiments described above, adoptive transfer was done using 1.5 x 106 OT-I cells per mouse so that there were sufficient cells to allow detection and characterization at the earliest times in the response, before clonal expansion had occurred. This clearly represents a much higher frequency of antigen-specific cells than is normally present. To determine if functional heterogeneity also occurs with precursor frequencies in a more physiological range, experiments were done that employed transfer of 5 x 103 OT-I T cells. At this level, naive OT-I cells cannot be detected in the recipient mice prior to infection. Despite this, the number of effector OT-I cells present at day 5, the peak of the response, was comparable to that seen when 1.5 x 106 cells were transferred (Fig. 5A). For both input levels, the numbers declined comparably by day 7. The OT-I cells that expanded from a low precursor frequency also showed functional heterogeneity, with the cells from the PC having the greatest capacity to produce IFN-
and TNF- in response to antigen and the highest grzB expression (Fig. 5B).
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Thus, development of functional heterogeneity occurred at both high and low transfer numbers. However, clonal expansion was clearly very limited at high precursor numbers. In the experiment shown in Fig. 5A,
105 naive cells were present in the spleen prior to infection, and 17 x 106 at the peak of the response, for an expansion of 170-fold. For low transfer numbers, OT-I cells could not be reliably detected prior to infection, while 13 x 106 OT-I cells were present at the peak of the response. Even if all the transferred cells (5 x 103) were in the spleen prior to infection, this represents an expansion of at least 2600-fold. Thus, while transfer of high numbers allows assessment of the earliest activation events (Fig. 2) and exhibits comparable functional heterogeneity, it results in a large underestimate of the potential for clonal expansion. The extent of clonal expansion seen at low transfer numbers for VV-OVAP is similar to that reported by Blattman et al. (16) for antigen-specific CD8 T cells responding to LCMV.
Response of endogenous CD8 T cells to VV-OVAP
As shown in Fig. 1(B), the number of endogenous CD8 T cells increases greatly in the spleen, and to a lesser extent, in the PC, upon VV-OVAP infection. Total CD8 T cells increase from the normal level of 15 x 106–20 x 106 per spleen to 50 x 106–60 x 106 on days 5 through 7 of infection. A large fraction of these appear to be cells specifically responding to VV-OVAP based on expression levels of grzB. Endogenous CD8 T cells from control mice have almost no detectable grzB-positive cells, while grzB high populations can be readily detected in the spleen, PC and LN of VV-OVAP-infected mice on day 5 after infection (Fig. 6A). By far, the greatest numbers of grzB high cells are present in the spleen at the peak of the response on days 5 and 7, with fewer numbers in the PC and LN (Fig. 6B). On day 5, 40% of the CD8 T cells in the spleen are grzB positive (Fig. 6A and C), indicating that the increase in total CD8 T cells in the spleen at this time is largely accounted for by cells responding to the virus. The PC, however, has the highest percent of grzB high cells as a percent of the total CD8 T cells present at the site, and the majority is grzB high by day 3 (Fig. 6C). As is the case for OT-I cells (Fig. 4C), grzB high cells have declined precipitously at all sites by day 14 and beyond, but a small population of grzB-positive cells remains in the PC even at day 32 (Fig. 6C).
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Expression of IL-7R
chainIL-7R
chain expression is rapidly down-regulated on CD8 T cells when they respond to antigen (17–19). When IL-7R expression was examined on OT-I cells responding to VV-OVAP infection, levels were found to be slightly reduced on day 3 in comparison to naive cells, significantly down-regulated on day 5 and had returned to naive levels by day 7, and this was the case for cells in the LN, spleen and PC (Fig. 7A). Consistent with the evidence from grzB expression indicating that a large fraction of the endogenous CD8 cells in the spleen on day 5 are responding to the virus, a large fraction of these cells had also down-regulated expression of IL-7R in comparison to levels on CD8 T cells in uninfected mice (data not shown).
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It was previously shown that at the peak of the response to LCMV, the majority of virus-specific CD8 T cells has down-regulated IL-7R
expression, with only 5–15% of the cells expressing high levels (19). It was further shown that these IL-7R high cells survived better than the IL-7R low cells upon transfer into uninfected recipients, leading to the suggestion that expression of IL-7R marks the responding cells that are destined to become memory cells. In contrast to the LCMV model (19), >50% of the OT-I cells that have clonally expanded in response to VV-OVAP have high IL-7R expression at the peak of the response, and the proportion of IL-7R high cells increases at longer times (Fig. 7B). This is also the case for cells in the LN and PC, although the numbers of cells present at these sites is much lower (Fig. 1A). When the total number of IL-7R high cells present during the course of the response is determined (Fig. 7B), it is clear that high expression of this receptor does not insure survival of the cells to become memory cells. Thus, while high IL-7R expression may be required for survival and transition to memory, it is clearly not sufficient. |
Discussion |
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Use of adoptively transferred Kb/OVA-specific OT-I T cells has allowed us to determine the time course for CD8 T cell responses to i.p. infection with VV-OVAP with respect to sites of proliferation and clonal expansion, extent of expansion, development of effector functions at various sites and expression levels of IL-7R
on the cells. Despite the fact that the primary site of infection is the PC, the OT-I cells first proliferate in response to antigen in the spleens and draining lymph nodes (DLNs), with several rounds of cell division already occurring by day 2 after infection (Fig. 2). By day 3, CFSE is fully diluted, indicating that the cells have undergone at least seven to eight rounds of division, and significant clonal expansion is detectable at this time (Fig. 1A). Although not detectable with CFSE, cell division must continue since clonal expansion continues with the numbers of cells peaking at day 5 and then declining (Fig. 1A). OT-I cells only become detectable in the PC by day 4 or 5 (Fig. 1A), when they have fully diluted their CFSE (data not shown). Thus, it appears that the OT-I cells only migrate to the primary site of infection after undergoing multiple rounds of division in the DLN and spleen. This is in marked contrast to OT-I adoptively transferred mice challenged by i.p. injection of OVA-expressing E.G7 tumor, where the initial clonal expansion occurs in the PC, and not in the spleen or DLN (20).
Transfer of high numbers of OT-I cells (1.5 x 106) is necessary to examine the earliest events that occur during the response, including sites of proliferation (Fig. 2) and initial clonal expansion (Fig. 1), but underestimates the extent of clonal expansion that can occur. When a low number of OT-I cells is transferred, 5 x 103 cells per mouse, the transferred cells cannot be reliably detected prior to infection, but challenge with VV-OVAP yields almost as many OT-I cells in the spleen, DLN and PC at the peak of the response as when 1.5 x 106 cells are transferred (Fig. 5A). If it is assumed that all the 5 x 103 transferred cells survive and can respond, then the cells expand 2500-fold in response to the virus. This is almost certainly an underestimate of the expansion, since at higher transfer numbers only 10% of the input OT-I cells can be found in the spleens and LNs a day or two later at the time of infection. The much lower expansion that occurs at high transfer numbers (10- to 20-fold) is probably due to competition for antigen or ‘space’ limiting the response.
The response of endogenous CD8 T cells to the virus parallels that seen for OT-I cells, with expansion peaking at day 5–7 and occurring predominantly in the spleen (Fig. 1B) and with CD8 T cells increasing in number in the PC at this time. There is a large increase in the number of CD8 T cells in the spleen at the peak of the response, with 2- to 3-fold greater numbers than in spleens of uninfected mice (Fig. 1B). About 40% of the CD8 T cells present at the peak of the response express high grzB levels (Fig. 6), and an even higher percentage of the CD8 T cells in the PC express high grzB (Fig. 6C). GrzB-expressing cells were also present in the DLN at the peak of the response, but in much lower numbers than in the spleen. Thus, there is rapid, massive expansion of both the virus-specific cells [Fig. 1 and (2, 3)] and the cells responding to the foreign ovalbumin peptide (OVAP) encoded by the virus. These results agree well with those of Harrington et al. (2) who found that 30% of the CD8 cells in the spleens of i.p. infected mice on day 7, the peak of the response, made IFN- in response to stimulation with VV-infected cells. Similarly, Xu et al. (3) found that 22% of the cells in the spleens of infected mice produced IFN- when re-stimulated on day 7 with an infected cell line. These studies did not examine responding CD8 T cells at other sites or levels of grzB expression. The use of adoptively transferred OT-I T cells made it possible to examine site-dependent functional heterogeneity in our studies.
The OT-I cells responding to VV-OVAP exhibited considerable heterogeneity with respect to functional capacity, both at the level of the individual cells within the population from each anatomical site and between the populations at different sites. Few, if any, naive OT-I cells from mice that were adoptively transferred but not infected produced IFN- upon stimulation with OVAP (Fig. 4A), but they developed this capacity within 3 days of infection. Despite all of the cells having proliferated in response to antigen (Fig. 1), however, only a fraction of the cells made IFN- (Figs 3, 4A and 5B
). At all times, the population recovered from the PC, the primary site of infection, had the largest fraction of IFN--producing cells. However, although a smaller fraction of OT-I in the spleen made IFN-, the much larger number of cells in the spleen made this the site of most of the IFN--producing cells. The memory cells remaining 32 days after infection had the highest capacity for rapid IFN- production upon re-stimulation (Figs 3B and 4), and 75% of the cells from all sites produced IFN-. Even at this stage, however, the small number of memory cells present in the PC had greater function as measured by the amount of IFN- they produced on a per cell basis.
GrzB expression was similar to that of IFN- in that naive cells expressed little or none, while a large fraction of the cells expressed high levels by day 3. Here too, expression was highest in cells from the PC, slightly lower in spleen and significantly lower in LN (Figs 3A, 4C and 5B). In contrast to IFN- production, however, the fraction of cells that expressed grzB declined steadily at all sites beyond day 3 and expression was very low in memory cells by day 32. However, a significant fraction of the small number of cells present in the PC by day 32 still expressed some grzB (Figs 3B and 4C). GrzB expression correlates strongly with the ability to kill antigen-expressing cells (15), suggesting that cytolytic activity is greatest on a per cell basis early in the response and declines rapidly as antigen is cleared. Initial proliferation of OT-I cells occurs in both LN and spleen (Fig. 2), where cells may be responding to either antigen-bearing dendritic cell (DC) that have migrated from the PC or antigen presented by resident DC as a result of virus disseminating from the PC. The site of initial expansion may influence the functional heterogeneity of the resulting effector cells, but interpretation of this is complicated by the fact that the activated cells will migrate to other sites. Thus, it is likely that at later times, OT-I cells present in the spleen will include cells that initially expanded in the spleen, as well as cells that initially expanded in LN, and vice versa.
Development of most CD8 T cell effector functions, including cytolytic activity and capacity to produce IFN-, requires proliferation and differentiation subsequent to antigen encounter. However, this is not the case for production of TNF-, a potent inflammatory cytokine (21, 22). Brehm et al. (23) have recently shown that naive CD8 T cells can produce TNF- within a few hours of TCR engagement, and obtained evidence to suggest that this may influence maturation of antigen-presenting DC. We similarly found that a substantial fraction of naive OT-I cells from mice that were adoptively transferred but not infected were able to produce TNF- within hours of stimulation with OVAP (Fig. 4B). In addition, the capacity to produce TNF- decreased significantly in the effector cells at the peak of response to VV-OVAP, particularly in the cells in the spleen and LN (Figs 3 and 4B). This is in marked contrast to grzB expression and capacity to produce IFN-, which are low or absent in naive cells and high in effector cells (Fig. 4A and C). The memory OT-I cells present 32 days after virus infection again show rapid and high TNF- production upon stimulation with antigen (Figs 3A and 4B). These results suggest that TNF- production may have an important role early in a response, possibly involved in modifying antigen-presenting cell functions as suggested by Brehm et al. (23), but that this capacity is reduced in effector cells to avoid the immunopathology that can be induced by TNF- (24, 25). Although the pattern of TNF- expression in response to VV differs from that of IFN- and grzB, it also exhibits heterogeneity depending on the site examined, with the capacity to produce TNF- being highest in cells recovered from the PC in comparison to those from spleen and LN (Fig. 3).
Site-dependent heterogeneity of cytokine production by CD8 T cells responding to viral infections has been reported for other viruses, including LCMV and vesicular stomatitis virus (VSV) (7) as well as influenza virus (8) and hepatitis B virus (9). The basis for this heterogeneity has remained unclear, and is likely to be influenced by a number of factors. Differences in TCR affinities in a responding polyclonal population could potentially contribute to the heterogeneity in development of effector functions but this is clearly not the only factor, as demonstrated by the marked heterogeneity of the responses of TCR transgenic OT-I T cells responding to VV-OVAP (Figs 3 and 4). Kristensen et al. (7) reached a similar conclusion upon examining responses of TCR transgenic CD8 T cells to LCMV and VSV infections.
Differentiation of naive CD8 T cells to acquire effector functions, including grzB expression and IFN- production, requires, in addition to antigen and co-stimulation, a third signal that can be provided by IL-12 (10, 11) or Type I IFN (12), and possibly other cytokines. In the case of LCMV infection, the CD8 T cell response to the virus is almost completely eliminated if the CD8 T cells lack the Type I IFN receptor (26, 27). In contrast, the response to VV was considerably less dependent on the Type I IFN receptor, suggesting that an alternative third signal was also available to support the response (27). Optimal development of effector functions requires prolonged exposure of CD8 T cells to the signal three cytokine during their interaction with antigen (28). Thus, functional heterogeneity may arise in vivo as a result of some cells receiving adequate levels and duration of both antigen and signal three cytokine, while other cells receive sub-optimal signals, which may be particularly the case for CD8 T cells recruited into the response at later times when antigen levels and inflammation are waning. Site-dependent heterogeneity of the response could arise if differing antigen and cytokine levels are available at the sites during differentiation of the cells. Alternatively, those cells that receive optimal signals for full differentiation may also be the cells that traffic most effectively from the DLN and spleen to peripheral sites. When optimal signals are present, CD8 T cells differentiate to become grzB-expressing, IFN--producing effector cells within 3 days. They may retain plasticity with respect to expression of these functions, however, so that signals received at sites they migrate into can change their functional phenotype. There is evidence for both CD4 (29) and CD8 (30) memory T cells that the tissue microenvironment can alter the phenotype of the cells.
Naive CD8 T cells express high levels of IL-7 receptor on their surface and rapidly lose IL-7R chain expression as they respond to antigen. During a response to LCMV, only 5–15% of the cells express IL-7R at high levels, and Kaech et al. (19) demonstrated that these are the cells capable of forming a long-lived memory population. These results suggested that the subset of cells that either never lost IL-7R expression or regained expression identified the memory precursors. Lacombe et al. (31) subsequently showed in a peptide immunization model that a large fraction of the effector CD8 T cells at the peak of the response expressed high IL-7R, but the majority did not survive to become memory cells. The same is true for the OT-I response to VV-OVAP. At the peak of the response 40% of the cells express high IL-7R levels, but the numbers decline rapidly over the next week and only a small fraction of persist long term as memory cells (Fig. 7). Thus, while expression of IL-7R may be required for a cell to transition to memory, it is not sufficient.
Although a CD8 T cell response is not essential for clearance of a VV infection (3, 5), our results demonstrate that a rapid and massive response to both viral epitopes and a foreign protein encoded by the virus does occur, and that the effector cells at various sites exhibit a high degree of functional heterogeneity. A long-lived population of memory cells results from the response, and primed CD8 T cells can mediate protection against the re-infection (3). This information should contribute to the development of immunization strategies to optimize generation of potent effector cells and high numbers of long-lived memory cells.
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Acknowledgements |
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We would like to thank Debra Lins for expert technical assistance and Sarah Hamilton for advice. This work was supported by National Institutes of Health grants RO1 AI34824 (M.F.M.), PO1 AI35296 (M.F.M.) and Center for Disease Control grant RO1 CI00100 (S.C.J.). M.P. was supported by National Institutes of Health Training Grant T32 AI07313.
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Abbreviations |
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| DC, dendritic cell |
| DLN, draining lymph node |
| grzB, granzyme B |
| i.p., intra-peritoneal |
| LCMV, lymphocyte choriomeningitis virus |
| LN, lymph node |
| OVA, ovalbumin |
| OVAP, ovalbumin peptide |
| VV-OVAP, ovalbumin-expressing VV |
| PC, peritoneal cavity |
| TNF-, tumor necrosis factor- |
| VSV, vesicular stomatitis virus |
| VV, vaccinia virus |
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Notes |
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Transmitting editor: M. J. Bevan
Received 3 November 2006, accepted 7 March 2007.
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