Dynamics of Memory T Cell Proliferation Under Conditions of Heterologous Immunity and Bystander Stimulation

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1 This information is current as of September 16, Dynamics of Memory T Cell Proliferation Under Conditions of Heterologous Immunity and Bystander Stimulation Sung-Kwon Kim, Michael A. Brehm, Raymond M. Welsh and Liisa K. Selin J Immunol 2002; 169:90-98; ; doi: /jimmunol References Subscription Permissions Alerts This article cites 35 articles, 21 of which you can access for free at: Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on September 16, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2002 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Journal of Immunology Dynamics of Memory T Cell Proliferation Under Conditions of Heterologous Immunity and Bystander Stimulation 1 Sung-Kwon Kim, Michael A. Brehm, Raymond M. Welsh, 2 and Liisa K. Selin By examining adoptively transferred CSFE-labeled lymphocytic choriomeningitis virus (LCMV)-immune donor T cells in Thy-1 congenic hosts inoculated with viruses or with the cytokine inducer poly(i:c), strikingly different responses of bona fide memory T cells were found in response to different stimuli. Poly(I:C) (cytokine) stimulation caused a limited synchronized division of memory CD8 T cells specific to each of five LCMV epitopes, with no increase and sometimes a loss in number, and no change in their epitope hierarchy. Homologous LCMV infection caused more than seven divisions of T cells specific for each epitope, with dramatic increases in number and minor changes in hierarchy. Infections with the heterologous viruses Pichinde and vaccinia (VV) caused more than seven divisions and increases in number of T cells specific to some putatively cross-reactive but not other epitopes and resulted in substantial changes in the hierarchy of the LCMV-specific T cells. Hence, there can be memory T cell division without proliferation (i.e., increase in cell number) in the absence of Ag and division with proliferation in the presence of Ag from homologous or heterologous viruses. Heterologous protective immunity between viruses is not necessarily reciprocal, given that LCMV protects against VV but VV does not protect against LCMV. VV elicited proliferation of LCMV-induced CD8 and CD4 T cells, whereas LCMV did not elicit proliferation of VV-induced T cells. Thus, depending on the pathogen and the sequence of infection, a heterologous agent may selectively stimulate the memory pool in patterns consistent with heterologous immunity. The Journal of Immunology, 2002, 169: Memory CD8 T cells were once thought to be resting nondividing cells lying in wait until re-exposure to the Ag that initially caused their differentiation into memory cells. It is now clear, however, that memory CD8 T cells continuously undergo a low level of homeostatic division long after Ag is cleared (1 4). This homeostatic division can be augmented by type I IFN, IL-15, and IFN inducers such as poly(i:c) or viruses, as demonstrated by the incorporation of the nucleotide analog BrdU into CD44 high CD8 T cells, which have a memory cell antigenic phenotype (5, 6). Recent studies have indicated that memory T cells specific to one virus may become activated and participate in protective immunity and immunopathology during infection with putatively unrelated viruses, and this phenomenon has been termed heterologous T cell immunity (7, 8). What is not clear is how or whether the proliferative expansion of memory CD8 T cells differs when these T cells are stimulated by homologous Ag, by heterologous infections, or by cytokines alone. Naive CD8 T cells undergo many cycles of division and expansion in cell number after only a brief contact with their cognate ligand expressed on an appropriate APC (9 11). This has been shown in vitro and in vivo by labeling cells with CFSE, a cytoplasmic dye that is diluted equally among daughter cells on cell division, and by following the expansion in cell number and the loss of CFSE label (12, 13). It appears that a multiple cycle proliferative expansion is a programmed event that does not require a Department of Pathology, University of Massachusetts, Worcester, MA Received for publication March 6, Accepted for publication April 29, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Research Grants AR (to R.M.W.) and (to L.K.S.). The contents of this publication are solely the authors and do not represent the official view of the National Institutes of Health. 2 Address correspondence and reprint requests to Dr. Raymond M. Welsh, University of Massachusetts, 55 Lake Avenue North, Worcester, MA address: Raymond.welsh@umassmed.edu continuous exposure of the naive T cells to their Ag (9 11). How then do memory T cells respond to homologous Ag, to potentially cross-reactive heterologous Ags, and to cytokines induced by viral infections or by an IFN inducer such as poly(i:c)? Additionally, when a host is infected with a heterologous virus expressing many potential T cell epitopes, how does the combination of viral Ags and virus-induced cytokines influence the activation and division of the host s memory T cells? We address these issues here by using mice adoptively reconstituted with CFSE-labeled splenocytes derived from lymphocytic choriomeningitis virus (LCMV) 3 -immune mice bearing a Thy alloantigen that allows for distinguishing donor from host T cells. We then examine the fate of the LCMV-specific memory T cells in these mice after exposure to poly(i:c), LCMV, or the heterologous viruses Pichinde (PV) and vaccinia (VV). We have previously documented that mice immune to LCMV are partially resistant to PV and VV and that splenocytes from LCMV-immune mice can be transferred into naive mice and provide resistance to PV and VV. This resistance is lost if CD8 T cells are depleted from these donor populations (7). We also have recently identified two cross-reactive CD8 T cell epitopes between LCMV and PV (LCMV-NP205 and PV-NP205) (36) and have evidence that VV may be crossreactive with two LCMV epitopes (NP205 and gp33) (8, 14) (our unpublished data). Heterologous immunity is not necessarily reciprocal, given that immunity to VV does not provide mice with resistance to LCMV and PV. Therefore, we are poised to examine memory CD8 T cell activation under conditions where effective heterologous immunity occurs and where it does not occur. We find here that heterologous viruses have the capacity to induce a many cycled proliferative expansion of memory CD8 T cells, altering the Ag-specific hierarchies of T cells specific to previously encountered pathogens. In contrast, poly(i:c) drives a limited cell division without increases in cell number or changes in hierarchies. 3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; PV, Pichinde virus; VV, vaccinia virus; PEC, peritoneal cavity leukocyte. Copyright 2002 by The American Association of Immunologists, Inc /02/$02.00

3 The Journal of Immunology 91 Further, we show that heterologous viruses stimulate memory T cells poorly when heterologous immunity is weak. Materials and Methods Viruses and poly(i:c) treatment LCMV, strain Armstrong, an RNA virus in the Old World arenavirus family, was propagated in BHK21 baby hamster kidney cells (15). The WR strain of VV, a DNA virus in the poxvirus family, was propagated in NCTC 929 cells (16), and the AN3739 strain of PV, an RNA virus in the New World arenavirus family only distantly related to LCMV, was propagated in BHK21 cells (15, 17). Mice were inoculated i.p. with PFU of LCMV, PFU of PV, or PFU of VV. These doses were selected based on our previous experience in our studies on heterologous immunity between these viruses (7). The IFN- inducer, poly(i:c) (Sigma-Aldrich, St. Louis, MO), was injected i.p. at a dose of 200 g/200 l HBSS (Life Technologies, Gaithersburg, MD) per mouse, 1 day after adoptive cell transfer (18). Splenocytes were harvested from host mice 6 days after poly(i:c) injection or virus infection, unless indicated otherwise, and assessed for donor cell number, cell division, and peptide-specific IFN- production. Adoptive transfer of virus-immune splenocytes Congenic male C57BL/6 (B6, Thy-1.2) and B6.PL Thy-1a/Cy (Thy-1.1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 4 5 wk of age and maintained under specific-pathogen-free conditions within the Department of Animal Medicine at the University of Massachusetts Medical School. These Thy-1.1 and Thy-1.2 mice are genetically identical mice differing only in their expression of the Thy-1 allele. Thy-1.1 (or Thy-1.2) mice infected i.p with LCMV (19) were considered LCMV immune after 6 wk or longer of infection. Either Thy-1.1 or Thy-1.2 LCMV-immune splenocytes pooled from four to five mice were adoptively transferred via the tail vein into the congenic strain at 2 or cells in a 200- l volume of HBSS. Both strain combinations were used in these studies based on the availability of LCMV-immune mice, and the results were comparable. Flow cytometry analysis of donor cells before transfer revealed that the LCMV-immune donor lymphocyte pool was composed of 10% CD8 T cells and 20% CD4 T cells. The CD8 T cell compartment was composed of 60% CD44 high and 40% CD44 low subpopulations. Further analysis via peptide-induced intracellular IFN- assays showed that 15 20% of donor CD8 T cells were LCMV specific and had the usual hierarchy of epitope-specific responses to the five known LCMV-specific peptides (gp33 NP396 gp276 NP205 gp92) (8, 36). VV-immune mice were generated in a similar manner by infecting mice with VV. Although VV epitopes are not known, the frequencies of VV-specific CD8 T cells in the memory state are comparable with that of LCMV (20). This type of technique was adapted from previous studies (21 23). Adoptively reconstituted mice were challenged with virus or poly(i:c) between 24 and 48 h after transfer. Data presented in the figures are from a single representative individual host mouse. CFSE labeling Splenocytes from donor mice were harvested and isolated as previously described (7). The donor splenocytes were labeled with CFSE using a modification of previously described techniques (12, 13). Briefly, the splenocytes were suspended in HBSS at cells/ml and incubated in 2 M CFSE (Molecular Probes, Eugene, OR) solution for 15 min at 37 C. After incubation, donor cells were washed twice with HBSS. Flow cytometry and intracellular IFN- staining Single-cell lymphocyte suspensions were prepared from spleens and the peritoneal cavity. The erythrocytes were lysed using a 0.84% NH 4 Cl solution. After preincubation with 1 l of Fc Block (2.4G2) in 96-well plates containing 100 l of FACS buffer (HBSS, 2% FCS, 0.1% NaN 3 ), the cells were stained for 30 min at 4 C with combinations of fluorescently labeled mabs specific for CD8 (53-6.7, FITC, or PerCP), Thy-1.1 (OX-7, PE), and IFN- (XMG1.2, APC), all purchased from BD PharMingen (San Diego, CA). LCMV peptide-specific, IFN- -secreting CD8 T cells were detected using the Cytofix/Cytoperm Kit Plus (with GolgiPlug; BD Phar- Mingen), as described previously (24). Briefly, cells were incubated with 5 M synthetic peptide, 10 U/ml human ril-2 (BD PharMingen), and 0.2 l GolgiPlug for 5 h at 37 C. Freshly stained samples were analyzed using a BD Biosciences FACSCalibur and CellQuest software (BD Biosciences, San Diego, CA). Peptides used here were originally defined by Whitton et al. (25) and van der Most et al. (26). They include NP (FQPQNGQFI), gp33 41 (KAVYNFATC), gp (SGVENPG- GYCL), NP (YTVKYPNL), and gp (CSANNSHHYI) and were purchased from American Peptide (Sunnyvale, CA) at 90% HPLC purity. Statistical analyses Results were expressed as the arithmetic mean SEM. An unpaired Student t test was used for data analysis where indicated. Results Donor CD8 T cells from LCMV-immune mice increase in number after homologous and heterologous virus infections but not after poly(i:c) treatment We have previously demonstrated that adoptively transferred LCMV-immune splenocytes can provide hosts with a moderate degree of protective immunity against the heterologous viruses, PV and VV (7). We wished to analyze the fate of the adoptively transferred donor T cells under these conditions of heterologous immunity and compare it with their fate on rechallenge with homologous LCMV Ags or on stimulation by cytokines induced by poly(i:c). To examine this issue, splenocytes from naive or LCMV-immune mice were adoptively transferred into Thy-congenic mice, in which donor and host T cells could be antigenically distinguished. On adoptive transfer, host mice were then inoculated with poly(i:c), LCMV, PV, or VV i.p., and donor and host cells were visualized on flow cytometry by staining cells with the appropriate Thy mab. By day 6 postinfection or treatment, both homologous and heterologous viral infections elicited substantial increases in the number of donor CD8 T cells when compared with unchallenged mice (Fig. 1, A and B, and Table I). Increases in the number of donor cells were generally more substantial in the peritoneal cavity (Fig. 1B and Table I) than in the spleen (Fig. 1A and Table I). In contrast, poly(i:c) induced no apparent expansion and, more often than not, caused a reduction of donor cells in the spleen. Only a marginal increase of cells in the peritoneal cavity (PEC) was detected on poly(i:c) treatment (Fig. 1, A and B, and Table I). As can be seen in Table I, the amount of expansion in the spleen on homologous or heterologous virus challenge occurring in the host population, which is composed of naive T cells, was much less than that in the donor population obtained from LCMV-immune mice. In fact, the expansions in the donor CD8 LCMV memory T cell populations formed a considerable portion of these heterologous virus CD8 T cell responses in both the spleen and the PEC. Very different results were observed if donor T cells were from naive mice that never had a virus infection (Fig. 1C). Neither poly(i:c) treatment nor any virus infection caused apparent increases in donor T cell number during this 6-day time period. These data suggested that the LCMV-specific memory T cells are participating in the responses to heterologous viruses. Poly(I:C) and virus infections elicit distinctive patterns of CD8 T cell divisions in CFSE-labeled donor cells from LCMV-immune mice Recent evidence has indicated that naive CD8 T cells undergo many cycles of division on exposure to Ag, leading to substantial increases in CD8 T cell number (9 11). We questioned whether a similar type of expansion of memory CD8 T cells would occur in response to homologous or heterologous Ags or to poly(i:c) stimulated cytokines. Here, we used the CFSE labeling technique to assess donor cell divisions (12, 13). This CFSE labeling technique enabled us to assess the extent of cell divisions on a single-cell level, albeit within a limited range (less than seven cycles). By day 6, poly(i:c) treatment elicited in donor CD8 T cells several rounds of cell division that were distributed between two and more than seven cycles (Fig. 2A). Consistent with previous findings (5), a

4 92 VISUALIZATION OF CD8 MEMORY T CELLS AFTER VIRUS INFECTIONS FIGURE 1. Increase in LCMV-immune donor CD8 T cells in the spleen (A) and the PEC (B) on homologous LCMV and heterologous PV and VV infections but not with poly(i:c) treatment. Spleen leukocytes from LCMV-immune Thy-1.2 donor mice ( ) were adoptively transferred into Thy-1.1 mice. Host mice were either treated with 200 g of poly(i:c) or challenged with homologous or heterologous viruses as described in Materials and Methods. Six days after treatment or infections, lymphocytes were isolated from the spleen and the PEC and stained with anti-cd8 and anti-thy-1.2 (donor-specific) Ab. On FACS analysis, the percentages of donor CD8 T cells (circled areas) were calculated based on the total CD8 T cells. This is representative of at least six different experiments. The poly(i:c) data and virus data are from different experiments, but the control mice from each experiment were shown to contain nearly identical levels of donor CD8 T cells, so only one is shown. C, No increase in naive donor CD8 T cells in the spleen after poly(i:c) or virus infection. One representative experiment is shown of three experiments. FL4-H, Fluorescence (height). phenotypic analysis revealed that the cell divisions occurred almost exclusively within the memory phenotype (CD44 high ) subset of the donor CD8 T cells (data not shown). Of significance is that the donor cell divisions elicited on poly(i:c) treatment did not result in an overall increase in donor cell number (Table I). In contrast, both homologous and heterologous virus infections induced many cycles of cell division of some of the donor cells (Fig. 2). These accumulating donor CD8 T cells, which had lost CFSE fluorescence intensity, were all CD44 high and accounted for the substantial increase in donor CD8 T cell numbers after virus infections revealed in Fig. 1 and Table I. About 50 66% of the undivided donor CD8 T cells observed in the spleen during viral challenge had a naive CD44 low phenotype. Virtually all of the CD8 T cells observed in the PECs were CD44 high CD8 T cells and had divided several times on virus challenge, suggesting that only the activated CD8 T cells have migrated into the PEC. Poly(I:C) treatment induces limited divisions of defined LCMVspecific memory CD8 T cells without an increase in cell number or hierarchical shift in the LCMV-specific T cell repertoire Analysis of donor cells before transfer revealed that 30% of the donor CD44 high CD8 T cells represented bona fide LCMV-specific memory CD8 T cells reactive to the five known LCMV-specific peptides. Poly(I:C) treatment induced cell divisions preferentially in the memory type CD44 high CD8 T cell compartment, consistent with the results of other investigators (5) (data not shown). We questioned whether these proliferating cells were bona fide LCMV-specific memory T cells. To visualize the Ag-specific T Table I. Effect of viruses and poly(i:c) on donor and host CD8 T cell number a Donor Host No. of cells 10 5 Fold increase No. of cells 10 5 Fold increase Spleen No virus Poly(I:C) LCMV PV VV PEC No virus Poly(I:C) LCMV PV VV a Using the same strategies described in Fig. 1, the total numbers of donor and host CD8 T cells were calculated by multiplying their percentages with the total leukocyte numbers. At least three mice from each group were used in calculations. Fold increases over the no virus control values were calculated based on the absolute numbers.

5 The Journal of Immunology 93 FIGURE 2. Poly(I:C) and virus infections elicit distinctive patterns of CD8 T cell division in donor cells from LCMVimmune mice in the spleen (A) and the PEC (B). LCMV-immune donor cells were labeled with CFSE and adoptively transferred into congenic hosts, as described in Materials and Methods. In FACS analysis, both host and donor CD8 T cells were gated, and the cell division patterns of donor CD8 T cells were investigated by visualizing donor cells with an appropriate donor-specific Thy Ab. Representative data are shown of six experiments. FL 1, Fluorescence. cells, spleen leukocytes from a reconstituted host were incubated with known LCMV-encoded peptides for 5 h and subsequently stained with anti-cd8, anti-thy-1.1, and anti-ifn- Ab. In FACS analyses, donor CD8 T cells were gated, and cell division patterns were analyzed for the IFN- Ag-specific CD8 T cells. Poly(I:C) treatment induced limited cell divisions in all the LCMV epitopespecific CD8 T cells examined (Fig. 3, A and B) and did not result in an increase in the number of the Ag-specific CD8 T cells (Fig. 3, A and B). The memory CD8 T cells specific to each LCMV peptide had divided almost equally on poly(i:c) treatment and, consequently, there were no hierarchical changes within the LCMV-specific CD8 T cell repertoire (Fig. 3C). Also, the CD8 T cells that divided the most after poly(i:c) treatment were not specific to LCMV epitopes. This phenomenon is developed elsewhere. 4 Homologous LCMV infection induces many cycles of cell division in T cells specific to each LCMV-encoded epitope The fate of LCMV-specific CD8 T cells was then investigated during the TCR-mediated recall response induced by a secondary challenge with LCMV both in the spleen (Fig. 4B) and in the PEC (Fig. 5A). On LCMV infection, all epitope-specific donor CD8 T cells divided approximately five times by day 3 postinfection but with little increase in cell number (data not shown). By day 6, all the LCMV-specific CD8 T cells had completely lost CFSE, indicating that they had divided more than seven times and the cell number had substantially increased (Fig. 4B). When the frequencies of CD8 T cells specific to the known LCMV-specific epitopes were counted at day 6, their percentage added up to 65 and 80% of the expanded donor CD8 T cells in the spleen and the PEC, respectively, but never reached 100% (Figs. 4B and 5A). We consider this a very high frequency, because some of the response may be directed against undefined epitopes, and cells in early stages of apoptosis would not be expected to score as epitope positive in the IFN- assays. Homologous challenge with LCMV tended to stimulate a better NP396- than gp33-specific response in the spleen, causing slight changes in the LCMV-specific T cell hierarchy in the spleen (Fig. 4B) (27, 28). This result suggests that an NP396 response has a slight advantage in the spleen on secondary challenge with LCMV. The mechanism for this is presently unclear given that there continued to be a dominant gp33 response in the PEC (Fig. 5A). Heterologous viruses induce preferential expansions and changes in the hierarchy of LCMV-specific memory CD8 T cells Having demonstrated marked differences in the pattern of memory T cell division between secondary challenge with homologous 4 C. D. Peacock, S.-K. Kim, and R. M. Welsh. Reduced capacity of bona fide memory CD44 hi CD8 T cells to respond to homeostatic and poly(i:c)-induced proliferation. Submitted for publication. LCMV Ags, which caused a dramatic expansion of T cells specific to all LCMV epitopes, and poly(i:c), which induced limited cell division without an increase in number, we questioned what pattern of response would be induced by heterologous viral infections. Infections with heterologous viruses bring into the host new Ags that might cross-reactively stimulate the memory T cells, and they induce high levels of cytokines that might mimic the effects of poly(i:c). A history of an LCMV infection provides a level of protective immunity against both PV and VV (7), and Fig. 1 shows that both viruses caused significant increases in the number of donor T cells from LCMV-immune but not from naive mice (7). The expansion of LCMV Ag-specific donor memory T cells was thus examined in the spleen (Fig. 4, C and D) and peritoneal cavity (Fig. 5, B, and C) in mice challenged i.p. with either PV or VV. The spleen data were all derived from the same experiment with the same source of donor T cells, and the hierarchies of the T cells specific for LCMV epitopes after those responses are summarized in Fig. 6. The peritoneal cell data in Fig. 5 were derived from other representative experiments. By 6 days postinfection, PV caused more than seven cell divisions and about a 100-fold increase in the frequency of the LCMV NP205-specific memory T cells in the donor cell populations from 0.3% with no virus challenge to 30% with PV challenge (Figs. 4C and 5B). In contrast, 1% of host CD8 T cells were specific to NP205. PV-induced responses to NP396, gp33, or gp276 were not above background levels (Figs. 4C and 5B). In some but not all experiments, PV also induced a strong response to the LCMV subdominant epitope gp92, and this usually was more striking in the peritoneal cavity than in the spleen, as shown in the representative experiment in Fig. 5B. In contrast to either poly(i:c) or homologous LCMV challenge, the PV infection caused a substantial change in the hierarchy of LCMV-specific T cells, consistent with an Ag-specific expansion of T cells reactive with some epitopes but not others (Fig. 6). In several experiments, the frequency of the PV-expanded donor T cells that could be accounted for as LCMV specific ranged from 30 to 40%. We could account for another 6% of these donor cells as specific for three other newly identified PV immunodominant epitopes (36), which in the same mouse accounted for 65% of the host CD8 T cells at this same time point (data not shown). This makes it unlikely that there is a large primary acute response to PV in the donor cell population. The specificity of the other cells was not determined, although it should be pointed out that in the memory pool there are probably T cells specific for many undefined subdominant or cryptic epitopes the cross-reactive amplification of which would not be detected. VV induced a lower level amplification of CD8 donor T cells from LCMV-immune mice, but this was a true proliferation, in contrast to the division without cell number increase seen after poly(i:c) treatment (Figs. 1 and 2). In three experiments 5 14%

6 94 VISUALIZATION OF CD8 MEMORY T CELLS AFTER VIRUS INFECTIONS FIGURE 3. Poly(I:C) induces limited cell division of bona fide LCMV-specific memory CD8 T cells without an increase in number or shift in hierarchical order of LCMV-immune repertoire. To visualize memory CD8 T cells specific for LCMV epitopes, spleen cells from control (A) and poly(i:c)-treated (B) host mice (day 6) were incubated for 5 h in the presence of LCMVspecific peptides, and IFN- cells were detected by fixing and staining them with an anti-ifn- Ab, as described in Materials and Methods. For these experiments, higher numbers of LCMV-immune donor cells ( Thy-1.1) were transferred into hosts (Thy-1.2) to better visualize LCMV-specific CD8 T cells. In FACS analyses, donor CD8 T cells were gated as was shown in Fig. 1, and the cell division pattern of each epitopespecific CD8 T cells population was studied. The percentage and the absolute numbers were calculated based on the cells within the circles in the dot plots. C, Epitope hierarchy before and after poly(i:c) treatment. The relative ratios of the LCMV-specific CD8 T cells were calculated and graphed based on the control and the experimental (6 days after poly(i:c) treatment) data. This calculation was based on LCMV-specific responses normalized to 100%. FL 1, Fluorescence. of the VV-expanded donor cells could be accounted for as LCMVspecific (Figs. 4D and 5C). VV did not induce as massive an increase in T cells specific to LCMV epitopes as seen with homologous LCMV challenge or with PV for the NP205 epitope, but a closer look at the frequencies of the Ag-specific spleen T cells before (Fig. 4A) and after (Fig. 4D) VV infection showed a marked alteration of the hierarchy (Fig. 6). In general, VV, like PV, induced an expansion of NP205-specific cells. Fig. 4D shows that VV induced a 65-fold increase in the total number of NP205- specific T cells over the untreated control levels. In several experiments, we found that VV often induced preferential expansions in NP205- and gp33-specific T cells, but it is noteworthy that individual animals varied in the relative amount of gp33-specific vs NP205-specific T cells stimulated. Fig. 4D shows a mouse in which only the NP205 response was significantly amplified, but Fig. 5C shows PEC data from another mouse in which the gp33 FIGURE 4. Proliferation of epitopespecific donor T cells in the spleen after homologous LCMV and heterologous PV and VV infections. Spleen leukocytes ( ) from LCMV-immune Thy-1.1 mice were adoptively transferred into Thy-1.2 mice, as described in Materials and Methods. In this experiment, all the LCMV-immune donor cells were derived from the same source. In FACS analyses, donor CD8 T cells were gated as was shown in Fig. 1. After gating on donor CD8 T cells, the cell division pattern of each epitope-specific CD8 T cell population was studied as described in Fig. 3 and Materials and Methods. One set of representative data is shown of six different experiments. FL 1, Fluorescence.

7 The Journal of Immunology 95 FIGURE 5. Proliferation of epitope-specific donor T cells in the peritoneal cavity after homologous LCMV (A) and heterologous PV (B) and VV (C) infections. The lymphocytes from the peritoneal cavities of LCMV-, PV-, or VV-infected hosts adoptively reconstituted with LCMV-immune donor cells were collected, and intracellular IFN- assays were performed as described in Fig. 3 and Materials and Methods. Data are derived from different experiments. Data are not shown for uninfected PECs because there were too few cells present to score in the intracellular IFN- assays. A, The cells are from the PEC of LCMV-infected mouse, for which spleen data are shown in Fig. 4B. B, In PV infections, three of six experiments showed the expansion of gp92-specific CD8 T cells. C, In VV infections, four of six experiments showed the expansions of gp33- and NP205-specific CD8 T cells. The expansion of gp92-specific CD8 T cells on VV infection was observed two of six experiments. FL 1, Fluorescence. response was marginally higher than the NP205 response and considerably higher than the normally strong NP396 response; Fig. 5C also shows that some of the gp33-specific T cells divided more than seven times, but others underwent fewer divisions, similar to that seen during a poly(i:c)-type response. Despite this mouse to mouse variation, it was clear that VV consistently induced a hierarchical change in the LCMV-specific response (Fig. 6), consistent with cross-reactive expansions and significantly different from the pattern observed with poly(i:c). Lack of reciprocity of memory T cell proliferation in response to heterologous viruses An interesting aspect of heterologous immunity is that it is not necessarily reciprocal (7). For example, a history of an LCMV or PV infection protects a host against VV, but a history of a VV infection does not protect a host from LCMV or PV. No explanation for this has been forthcoming. We therefore questioned whether LCMV could elicit the expansion of VVinduced memory T cells as effectively as VV could elicit the expansion of LCMV-induced memory cells. Fig. 7 shows that if mice were adoptively reconstituted with CSFE-labeled T cells from VV-immune mice and then challenged with the homologous VV, there was a dramatic increase in the number of donor cells, which had divided more than seven times. However, if these mice were challenged with LCMV, there was a loss rather than an increase in donor T cell number (Fig. 7A), reminiscent more of a cytokine response than of an Ag-specific amplification. Therefore, LCMV does not stimulate the proliferation of VV-immune cells, even though VV stimulates the proliferation of LCMV-immune cells. FIGURE 6. Preferential expansions induced by heterologous virus infection result in changes in hierarchical orders in LCMVimmune repertoire. The relative ratio of LCMV-specific CD8 T cells were calculated and graphed based on no virus (Fig. 4A), homologous LCMV (Fig. 4B), and heterologous PV (Fig. 4C) and VV (Fig. 4D) infections.

8 96 VISUALIZATION OF CD8 MEMORY T CELLS AFTER VIRUS INFECTIONS FIGURE 7. Lack of reciprocity of memory T cell proliferation in response to heterologous viruses. A, CD8 T cells. Spleen cells from VV-immune mice were labeled with CFSE and adoptively transferred into Thy-congenic hosts. The host mice were then infected with homologous VV or heterologous LCMV. Six days postinfection, the lymphocytes were isolated from the spleens and stained with anti-cd8, anti-cd4 and anti-thy- 1.2 Ab. The absolute number of donor CD8 T cells is shown above each panel. B and C, CD4 T cells. The reciprocal effects of LCMV and VV infection on donor CD4 T cell populations. The data were analyzed in the same manner as in A, by gating on donor and host CD4 T cells. The absolute number of donor CD4 T cells is shown above each panel. Representative data are shown of four different experiments. FL 1, Fluorescence. Because protective heterologous immunity of LCMV-immune mice against VV was in part dependent on CD4 T cells (7), we also examined the reciprocal effects of VV and LCMV infections on the CD4 T cell pool. Both LCMV and VV infections caused substantial proliferation of memory CD4 T cell populations during homologous virus challenge (Fig. 7, B and C). This indicates that memory CD4 T cells, like memory CD8 T cells, can be visualized with CSFE to undergo many cycles of division and proliferative expansion during a secondary challenge. VV induced several cycles of division of donor T cells from LCMV-immune mice and a slight increase in their number (Fig. 7B). In contrast, LCMV caused a pronounced decrease in number of donor CD4 T cells from VV-immune mice (Fig. 7C). This was an absolute reduction in cell number, and not a dilutional effect caused by expansion of LCMV-specific CD8 T cells, given that no significant LCMVspecific expansion had occurred at this early time point (Table I). Overall, these experiments indicate that VV is much more capable of inducing the proliferation of LCMV-immune T cells than LCMV is of stimulating VV-immune cells. This is true for both CD4 and CD8 T cells and is reflective of the nonreciprocal nature of heterologous immunity between these two viruses. Discussion This work documents strikingly different ways in which a memory T cell population can respond to stimuli. A homologous LCMV challenge of mice seeded with an LCMV-immune memory T cell population, in which 20% of the seeded CD8 T cells could be shown to be specific for one of the LCMV epitopes, caused a multicycled (more than seven divisions) expansion of T cells specific for each of the LCMV epitopes (Figs. 4B and 5A). This resulted in a 300-fold increase in donor CD8 T cell number in the spleen, and 65% of those cells could be accounted for as being LCMV-specific (Table I and Figs. 4B and 5A). Thus, like the primary T cell response, in which T cells undergo many division cycles in a preprogrammed manner (9 11), the memory T cell response also involves a multicycled expansion rather than a more limited division pattern as has previously been seen by other investigators (29). A similar conclusion for stimulating influenza virus-specific memory CD8 T cells has recently been made by Turner et al. (23). The high frequency of definable LCMV-specific T cells in this expanded donor population argues against an extensive expansion of bystander T cells not specific for the virus, consistent with studies published previously for the acute LCMV response (30). The few cells not recorded as LCMV specific may either have been dying cells that would not produce IFN- in response to peptides or possibly to T cells reactive with undefined subdominant or cryptic epitopes present in the memory pool at low frequency. Even though T cells specific for all LCMV epitopes underwent sufficient division to lose all of their CSFE intensity, there was a slight hierarchical shift in the frequency of T cells specific for the immunodominant peptides, with the NP396:gp33 ratio increasing somewhat. This disproportional but reproducible increase of NP396-positive T cells has been observed previously in LCMV-immune mice receiving a secondary challenge (27, 28) and may represent a further selection of the repertoire, as also demonstrated by TCR analyses (28, 31). Interestingly, CD8 T cells in the PEC did not demonstrate this same shift (Fig. 5A), and the reason for that discrepancy is under investigation. This massive Ag-specific expansion of memory T cells after homologous viral challenge stood in marked contrast to the bystander stimulation of T cells elicited by the IFN inducer, poly(i:c) (Fig. 3B and Table I). Poly(I:C) induced a cell division pattern that did not result in an increase in spleen donor cell number. Poly(I:C) did cause a slight increase in donor cells in the peritoneal cavity, the site of the poly(i:c) inoculation, but these were considerably small numbers compared with the spleen. It remains possible that some of these cells migrated out to other organs, but our previous work showed reductions in host memory CD8 T cell numbers in all the organs tested at day 1 post-poly(i:c) treatment (18). Bona fide memory T cells defined by their specificity for LCMV epitopes underwent a limited number of divisions in response to poly(i:c), and an undefined CD44 high CD8 T cell population experienced

9 The Journal of Immunology more cell divisions and lost the CSFE label. This may mean that CD44 CD8 T cells distinct from true memory cells are better responders than bona fide memory cells to bystander stimulation; that hypothesis is currently under investigation in our laboratory. The phenomenon of poly(i:c)-induced division without an increase in cell number in part reflects the recent observation that poly(i:c) (and IFN type I) induces apoptosis in memory CD8 T cells (18). This apoptosis offsets the cell division. Thus, memory T cell division with an increase in number is characteristic of an Ag-specific stimulation, whereas division with no increase or loss in number is characteristic of cytokine-induced stimulation. The poly(i:c)-induced division of the different LCMV-specific T cells all appeared the same, and the hierarchy of the T cell response to the LCMV peptides was similar before and after poly(i:c) treatment (Fig. 3C). Given the recent observations of unanticipated T cell cross-reactivity between viruses and the fact that the pathogenesis of viral infections is greatly altered by prior exposures to heterologous viruses (heterologous immunity) (7, 8, 16, 32, 36), we wished to examine how memory T cells specific for one virus responded during heterologous viral infections. A history of an LCMV infection provides a degree of protective immunity to both PV and VV, and this heterologous immunity is at least in part due to CD8 T cells (7). CTL generated against either LCMV or PV do not lyse targets infected with the heterologous virus, indicating very little cross-reactivity between these viruses (17, 33). However, CTL generated in response to PV infection of LCMV-immune mice lyse targets infected with either virus, and limiting dilution assays have indicated that a low frequency of cross-reactive T cells is amplified by the second viral infection (16, 17). We have recently found that PV encodes a weak subdominant epitope (PV-NP205) that shares 7 of 9 aa with the LCMV NP205 epitope (36). We questioned, then, what effect PV infection would have on the LCMV memory pool in adoptively reconstituted mice. The results indicated that PV caused a substantial increase in the number of T cells specific for NP205 (Figs. 4C and 5B). This resulted in a strong skewing of the hierarchy of T cells recognizing LCMV-encoded epitopes (Fig. 6). In some but not all experiments, we found a significant increase in T cells specific for gp92 (Fig. 5B). About 30 40% of the T cells expanded by PV could be shown to be LCMV specific. It was not clear whether the other expanded cells were cross-reactive with undefined LCMV epitopes or were the products of bystander expansion, but PV caused a decline rather than an increase in cell number of donor cells from nonimmune mice (Fig. 1 and Table I), arguing for a cross-reactive mechanism. We cannot rule out that the cross-reactive T cell response may liberate cytokines capable of nonspecifically stimulating an increase in number of non-crossreactive T cells. Keeping in mind that we could account for the great majority of expanded cells in the homologous LCMV system as being LCMV-specific, we favor the explanations that the unknown population of PV-expanded cells may have been memory cells specific for undefined LCMV epitopes cross-reactive with PV or else of low affinity to the defined epitopes. Regardless of that interpretation, this experiment shows that during a heterologous virus challenge, a cross-reactive epitope can induce a multicycled expansion and dramatic increase in number of cross-reactive memory T cells specific for a heterologous virus. Vaccinia virus is a large DNA virus very different from LCMV, but it encodes over 200 proteins, thereby creating the possibility of many cross-reactive epitopes (16, 33). VV infection induces an expansion of CD8 T cells similar to that of LCMV and PV infection. Although immunodominant epitopes for VV are not defined, data from limiting dilution assays (33) and intracellular IFN- assays indicate that the frequencies of VV-specific and LCMV-specific memory CD8 T cells are comparable. (20). VV infection of mice seeded with LCMV-immune T cells caused only a moderate expansion in donor CD8 T cell number that was 10-fold less than that stimulated by PV (Table I). Nevertheless, this VV-induced expansion of T cells from LCMV-immune mice was considerably greater than that elicited from a nonimmune donor population (Fig. 1). The question we posed was whether the VV stimulation resembled an Ag-specific or a poly(i:c)-like bystander stimulation. The expansion of the LCMV epitope-specific T cell population was more subtle than that driven by homologous LCMV challenge or by PV, which encodes a strongly cross-reactive epitope, but VV did significantly alter the hierarchy of the LCMV-specific repertoire and in most cases tended to provoke a moderate expansion in T cells specific to some (NP205, gp33) but not other epitopes. For example, the spleen data depicted in Fig. 4A (no challenge) and Fig. 4D (VV challenge) showed that VV-induced an elevation of NP205-specific T cells from 700 to 13,000, a 2-fold loss in T cells specific for gp276, and about comparable numbers for NP396 and gp33. In the experiment shown in Fig. 5C, in PEC, substantial expansions of gp33-specific and NP205-specific T cells were noted. In general, we find that VV induced expansion of gp33- and NP205-specific T cells is common but that the relative amounts of expansion vary from mouse to mouse. This might reflect the fact that TCR usage is different between individuals for a specific epitope (28, 31). We had previously reported that VV infection of LCMV-immune mice elicited CTL that preferentially lysed gp33- coated targets over NP396-coated targets (14). More recently, we have shown the selective expansion of gp33 and NP205-specific memory CD8 T cells in the lung of LCMV mice challenged with VV (8). We also have identified three VV-encoded peptides, with homology to NP205, when coated onto target cells will sensitize them to lysis by T cells from LCMV-immune mice challenged with VV (our unpublished observations). Thus, the fact that VV causes an increase in donor cell number and a change in the hierarchy of LCMV-specific T cells is consistent with its acting more like an Ag-specific than a bystander stimulus. Only 5 10% of the donor T cell population after VV infection could be accounted for as LCMV specific. Some of the stimulation could be nonspecific bystander, but again, the other expanded cells may have had reactivity with undefined minor LCMV epitopes. In contrast to our results, others have concluded that VV infection does not increase the frequency of LCMV-specific T cells in the spleen or peripheral blood (21, 27). It is clear from the results reported here, however, that the influence of VV infection on the LCMV-specific memory pool is subtle, with loss of some T cells occurring while others are expanding in frequency. Certainly, VV infection causes substantial alterations in the LCMV-specific repertoire, and sometimes this can be seen more strikingly at sites of infection such as the lung (8) or the PEC. One of the perplexing aspects about heterologous immunity is that it is not necessarily reciprocal. For example, whereas both LCMV and PV infections can provide immunity to VV, VV infection does not protect a host against either LCMV or PV (7). Fig. 7A shows that this lack of protective immunity can be explained by the failure of VV-immune CD8 T cells to proliferate in response to LCMV, even though there is a strong expansion in response to homologous VV infection. Similarly, VV induces some division of donor CD4 T cells from LCMV-immune mice, but LCMV infection caused a complete loss of donor CD4 T cells from VV-immune mice (Fig. 7, B and C) Hence, there appear to be few VVspecific memory T cells for LCMV to call on. It could be that a virus like VV, that encodes many proteins, may more likely encode some epitopes that will cross-react with some T cells in a preexisting memory pool than would a virus, like LCMV or PV, 97

10 98 VISUALIZATION OF CD8 MEMORY T CELLS AFTER VIRUS INFECTIONS that encodes only four proteins. Recent work by Turner et al. (23) showed that influenza B did not expand T cells specific to influenza A virus, so strong expansions in T cells are not induced between all virus combinations. It is likely that large viruses that encode many proteins would need to interfere with class I Ag presentation, as many of them do, to keep them from being eliminated by cross-reactive T cell responses (34, 35). These results indicate that, in response to natural stimuli, memory CD8 T cells may either undergo a limited number of cell divisions or else divide many (more than seven) times and lose their CSFE label. They also show that undergoing a series of cell division events may either result in a marked increase in cell number, as would be expected, or else lead to no increase or even to a decrease in number. This less expected result is probably accounted for by IFN-induced apoptosis of memory T cells (18) and by the fact that there may be something fundamentally different between an Ag-independent cytokine-mediated homeostatic cell division and an Ag-dependent stimulation that results in the proliferation of T cells in the true sense of the word, i.e., an increase in cell number. It is important to make the distinction between true proliferation and division without an increase in number. Heterologous viral infections have the capacity to drive both an Ag-specific proliferation, as a consequence of cross-reactive epitopes, or a cytokine-mediated homeostatic division, as a consequence of their abilities to induce IFN, IL-15, and other cytokines. Either way, some form of memory cell stimulation is likely to occur whenever a host becomes infected by pathogen. References 1. Razvi, E. S., R. M. Welsh, and H. I. 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