Attrition of Virus-Specific Memory CD8+ T Cells During Reconstitution of Lymphopenic Environments

Craig D. Peacock, Sung-Kwon Kim and Raymond M. Welsh
J Immunol July 15, 2003, 171 (2) 655-663; DOI: https://doi.org/10.4049/jimmunol.171.2.655

Abstract

Viruses can cause a severe lymphopenia early in infection and a subsequent, lasting loss of pre-existing CD8+ memory T cells. We therefore questioned how well virus Ag-specific memory CD8+ T cells could reconstitute mice rendered lymphopenic as a consequence of genetics, irradiation, or viral or poly(I:C)-induced cytokines. In each case, reconstitution of the CD8+ compartment was associated with limited division of virus-specific memory T cells and a reduction in their proportion. This indicates that foreign Ag-experienced CD44highCD8+ memory T cells may respond differently to homeostatic signals than other CD44highCD8+ cells, and that events inducing lymphopenia may lead to a permanent reduction in T cell memory.

The peripheral CD8+ T cell pool consists of CD44low and CD44high compartments, commonly thought to represent naive and memory subsets, respectively. These subsets appear to be regulated by independent homeostatic mechanisms (1) allowing for the generation and maintenance of a diverse repertoire of naive cells, while maintaining a separate pool of Ag-experienced memory T cells. Virus-specific memory CD8+ T cells can be maintained at stable frequencies for long periods of time, associated with a background rate of cell division that appears to be independent of class I MHC (2, 3, 4). However, the immune response to a novel virus infection results in the attrition of memory CD8+ T cells specific to previously encountered viruses (5, 6).

Division of resting naive CD8+ T cells is negligible, and to survive in the periphery, they must maintain continuous contact with self-peptide/class I MHC complexes (7), perhaps similar to those responsible for their positive selection in the thymus (8). However, under conditions of lymphopenia, this signal becomes stimulatory and leads to T cell activation and proliferation until total cell counts return to near-normal levels (9, 10). This homeostatic proliferation of naive CD8+ T cells seems to be dependent on the presence of IL-7 (11, 12, 13) and is associated with the up-regulation of activation/memory markers, including CD44 (14). Hence, it is probable that many memory CD8+ T cells defined by CD44 expression are not the progeny of cells that have responded to foreign Ag, but instead have emerged following homeostatic stimulation by self-Ag during competition for space in the peripheral immune compartment.

With the possible exception of the irradiation used in some clinical settings, model systems used in recent studies of homeostatic proliferation (15, 16, 17) provide only theoretical insights regarding the repopulation of a hole in a peripheral immune compartment. Where, then, would repopulation of a lymphopenic environment with memory CD8+ T cells be a naturally occurring event in a human condition? The answer, we believe, is during the lymphopenia that is a common feature of severe human and animal viral infections, including measles (18), influenza (19), Ebola (20), Venezuelan equine encephalitis (21), varicella zoster (22), and lymphocytic choriomeningitis viruses (LCMV)4 (23), among many others. Studies with LCMV have shown that a combination of type I IFN and TNF-α can shut down bone marrow function early in infection (24), and there is also a substantial type I IFN-dependent apoptosis and loss of mature peripheral CD8+ T cells early in infection, particularly within the CD44high subset (23). This early apoptosis is mimicked within 24 h of treatment with the IFN-inducing agent poly(I:C) (23) and precedes a sharp increase in the background division of CD44high cells normally found in mice (25), apparently via the subsequent induction of IL-15 (26).

Because memory CD8+ T cells specific to previously encountered pathogens are lost during viral infections (5, 6), we asked whether homeostatic reconstitution occurring in response to lymphopenia, such as that induced during virus infection (18, 19, 20, 21, 22, 23), might select against pre-existing virus-specific memory cells and result in a reduction in their frequency.

In this study, we examine the nature of lymphopenia-induced division of CD44highCD8+ cells using an adoptive transfer system, where the participation of bona fide virus-specific memory CD8+ T cells could be monitored. Using virus-, genetically, irradiation-, and cytokine-induced models of lymphopenia, we report on a pattern of CD44highCD8+ T cell division that dilutes the LCMV-specific memory population.

Materials and Methods

Adoptive transfer of LCMV-immune splenocytes

Male C57BL/6 (B6), B6.PL Thy1a/Cy (Thy1.1+), B6.129P2 TcrbtmlMomTcrdtmlMom (αβγδKO), and B6.CB17-Prkdcscid/SzJ (SCID) mice (all H2b) were purchased from The Jackson Laboratory (Bar Harbor, ME) at 5–6 wk of age and maintained under specific pathogen-free conditions within the Department of Animal Medicine at the University of Massachusetts Medical School. Thy1.1+ mice infected i.p. with 5 × 104 PFU of the Armstrong strain of LCMV (27) or 2 × 107 PFU of Pichinde virus (PV) were considered immune 6 wk or longer after infection. Single-cell suspensions were prepared from spleens, and erythrocytes were removed by lysis using a 0.84% NH4Cl solution. CFSE-labeled LCMV- or PV-immune Thy1.1+ splenocytes (2–4 × 107) were adoptively transferred via the tail vein into Thy1.2+ αβγδKO, SCID, B6-irradiated, or B6 mice in a 200-μl volume of HBSS (Life Technologies, Grand Island, NY). Irradiated naive and LCMV-immune B6 mice were created by exposure to 6 Gy of whole body gamma irradiation in a Gammacell 40 irradiator (Atomic Energy of Canada, Ottawa, Canada), and where applicable, cell transfers were performed 24 h later. PV (2 × 107 PFU) and the IFN-αβ inducer poly(I:C) (200 μg/200 μl HBBS; Sigma-Aldrich, St. Louis, MO) were delivered to mice i.p. 1 day after adoptive cell transfer. Multiple poly(I:C) inoculations were administered at 7-day intervals.

Adoptive transfer of FACS-purified CD8+ T cell subsets

LCMV-immune splenocytes were preincubated with Fc Block (2.4G2) and then stained (30 min; 4°C) with combinations of fluorescently labeled mAbs specific for CD8α (53-6.7) and CD44 (IM7). All reagents were purchased from BD PharMingen (San Diego, CA). Purified (>95%) populations of CD8+CD44low and CD8+CD44high cells were sorted using a FACStarPlus and CellQuest Software (BD Biosciences, Mountain View, CA). CFSE-labeled Thy1.1+CD8+CD44low or CD8+CD44high cells (1–2 × 106) were adoptively transferred into Thy1.2+ αβγδKO or B6 mice as described in the preceding section above.

Intracellular IFN-γ staining

LCMV-specific memory CD8+ T cells were detected by measuring IFN-γ secretion to immunodominant LCMV peptides using the Cytofix/Cytoperm Kit Plus (with GolgiPlug; BD PharMingen), as described previously (28). Briefly, 1–2 × 106 cells were incubated in 96-well plates (5 h; 37°C) with 5 μM synthetic peptide, 10 U/ml human rIL-2 (BD PharMingen), and 0.2 μl of GolgiPlug. Cells were then washed in FACS buffer (HBBS, 2% FCS, and 0.1% NaN3), blocked with Fc Block, and incubated (30 min; 4°C) with combinations of fluorescently labeled mAbs specific for Thy1.1 (OX-7), CD8α, and CD44 (BD PharMingen). Subsequent fixation and permeabilization of the cells was performed to allow intracellular access to the anti-IFN-γ mAb (XMG1.2; BD PharMingen). Freshly stained samples were analyzed using a BD Biosciences FACSCalibur and CellQuest software.

Statistical analyses

Results are expressed as arithmetic mean ± SEM. An unpaired Student’s t test was used for data analysis where indicated.

Results

Limited homeostatic reconstitution by LCMV-specific memory cells

We examined the impact of reconstitution of a vacant CD8+ T cell compartment on a pre-existing, LCMV-specific memory cell population using a conventional model of homeostatic proliferation. The profound deficiency in αβ+ and γδ+ T lymphocytes in αβγδKO mice constituted an empty vessel in which we could follow CD8+ T cell replenishment after reconstitution with CFSE-labeled splenocytes from LCMV-immune donor (Thy1.1+) B6 mice. The spleens of αβγδKO mice contained 4.5 ± 0.32 × 105 and 15 ± 1.6 × 105 donor CD8+ T cells on days 6 and 13 after adoptive transfer, respectively. By comparison, syngeneic B6 control mice at each time point contained only ∼1 × 105 donor CD8+ T cells, reflecting the fact that they have full CD8+ T cell compartments that permitted negligible division of adoptively transferred donor cells (Fig. 1⇓A).

FIGURE 1.
FIGURE 1.

LCMV-specific memory cells are diluted during the homeostatic reconstitution of T cell-deficient hosts. CFSE-labeled splenocytes (2 × 107) from donor (Thy1.1+) LCMV-immune mice were adoptively transferred into B6 or αβγδKO hosts (Thy1.2+). Six or 13 days after transfer, lymphocytes were isolated from the spleen, and donor CD8+ T cells were electronically gated for FACS analysis. A, The percentage of donor CD8+ T cells in the lymphoid compartment of the host spleen was calculated, and the loss of CFSE labeling was used to ascertain the cell division patterns of CD44high and CD44low subsets. B, LCMV-specific memory cells within the donor CD8+ population were visualized by measuring IFN-γ+ production against LCMV peptides. The percentage of the total donor and LCMV-specific memory (IFN-γ+) CD8+ T cells in each of the CFSElow and CFSEhigh subsets is indicated on the dot plot analysis of a representative animal. C, Total LCMV-specific memory cell percentage was determined by calculating the donor CD8+ T cell population capable of producing IFN-γ to the three LCMV epitopes examined, averaged from three individual mice in the experimental groups from a representative experiment. Statistical variation from B6 control mice was calculated (t test; ∗, p < 0.005, ∗∗, p < 0.001).

The expanded donor CD8+ population in host αβγδKO mice spleens on days 6 and 13 was associated with a CD44high subset in which the vast proportion of cells had undergone at least the seven to eight rounds of division that is the limit of detection for CFSE staining (CFSElow; Fig. 1⇑B). In contrast, LCMV-specific memory cells, identified by their production of IFN-γ in response to the LCMV peptides NP396396–404, gp3333–41, and gp276276–286, were concentrated in the CFSEhigh subset of CD44high cells that had participated in only limited rounds of division (Fig. 1⇑B). On day 6, only 6.7 ± 0.9% of LCMV-specific memory cells were CFSElow, compared with 65 ± 5.9% of the total donor CD8+ T cell population. We should point out that, despite their limited division, the total number of LCMV-specific cells in the reconstituted spleens of αβγδKO mice on days 6 and 13 (∼4.5 and 5.9 × 104, respectively) is slightly higher than in B6 hosts, in which they do not divide (∼3.2 × 104; Fig. 1⇑B). However, whereas the total number of donor CD8+ T cells in the spleens of αβγδKO mice has increased 4.5-fold and 15-fold compared with B6 controls on days 6 and 13, respectively, the number of the LCMV-specific memory cells has less than doubled at either time point. This reflects the fact that the frequency of LCMV-specific memory cells present in the donor CD8+ T cell population of αβγδKO mice was significantly reduced, relative to that in B6 hosts (Fig. 1⇑C). Thus, the CD8+ T cell population from the spleens of αβγδKO mice 13 days after adoptive transfer, if equal in size to that present in the LCMV-immune donor mice, would actually contain ∼90% fewer LCMV-specific memory cells, demonstrating a loss of virus-specific memory in response to homeostatic reconstitution of a lymphopenic environment. Because our studies are concerned with the relative changes in virus-specific memory cells, our data are presented as percentages within the total donor CD8+ T cell population and not absolute numbers of cells.

The relative ratios of CD8+ T cells specific for each of the three LCMV epitopes examined were similar in the spleens of αβγδKO and B6 mice (Fig. 1⇑B), suggesting that the decrease in frequency of bona fide memory cells that occurs following homeostatic proliferation appears to be independent of specificity. The reduced frequency of LCMV-specific memory cells in the spleen did not appear to be accounted for by migration to peripheral organs, because proportions of gp33-specific memory cells in the donor CD8+ T cell population were also lower in the peritoneal cavities of αβγδKO mice on day 13 (2.3%) than B6 controls (14%; Fig. not shown).

Results similar to those described above for αβγδKO mice were obtained following adoptive transfer of LCMV-immune B6 splenocytes into T and B cell-deficient SCID mice. That is, 6 days after adoptive transfer, LCMV-specific memory cells within the donor CD8+ subset of SCID mice were concentrated in the CFSEhigh subset and reduced in frequency compared with B6 recipients (Fig. 2⇓).

FIGURE 2.
FIGURE 2.

Dilution of LCMV-specific memory cells persists after homeostatic reconstitution of T cell-deficient hosts. CFSE-labeled splenocytes (2 × 107) from donor (Thy1.1+) LCMV-immune mice were adoptively transferred into B6 or SCID hosts (Thy1.2+). Six or 140 days after transfer, lymphocytes were isolated from the spleen, and donor CD8+ T cells were electronically gated for FACS analysis. A, The loss of CFSE labeling was used to ascertain the cell division patterns of CD44high and CD44low subsets. B, LCMV-specific memory cells within the donor CD8+ population were visualized by measuring IFN-γ+ production against LCMV peptides, and their percentage is indicated on the dot plot analysis of a representative animal. C, Total LCMV-specific memory cell percentage was determined by calculating the donor CD8+ T cell population capable of producing IFN-γ to the three LCMV epitopes examined, averaged from three individual mice in the experimental groups from a representative experiment. Statistical variation from B6 control mice was calculated (t test; ∗, p < 0.005, ∗∗, p < 0.001).

Lasting reductions in memory cell frequencies postreconstitution

Previous studies have shown that LCMV-specific memory cells exhibit a low level of division in the absence of their specific Ag (2, 3), raising the possibility that, with time, this slow division would be sufficient to restore the original levels of bona fide memory cells in the reconstituted CD8+ pool. We examined the frequency of LCMV-specific memory cells present in SCID mice 140 days after adoptive transfer and found that it was still significantly reduced compared with B6 controls at the same time point (Fig. 2⇑C). Thus, the early dilution of LCMV-specific memory cells does not appear to be easily corrected, even when the initial, high rates of homeostatic proliferation required to correct the pre-existing lymphopenia have presumably subsided.

A CFSElowCD44lowCD8+ T cell subset appeared in SCID mice but not in B6 controls by day 140 (Fig. 2⇑A). Appearance of CD8+ T cells with a naive phenotype has been reported in other models of homeostatic proliferation and linked to the repopulation of the host thymus by functional progenitors of CD8+ T cells contained within the adoptively transferred donor population (29, 30). The absence of this population in B6 mice and the persistence of a small CFSEhighCD44low donor subset may reflect better competition by naive, host-derived thymic emigrants for space in an immunoreplete mouse.

The intracellular IFN-γ assay used to identify LCMV-specific memory cells was chosen for its sensitivity, because the frequencies of IFN-γ-producing cells in the LCMV system are usually slightly higher than those defined by MHC-dimers or MHC-tetramers, and except under conditions of overwhelming Ag excess, virtually all tetramer- or dimer-staining cells produce IFN-γ (5, 31). The possibility that the LCMV-specific memory population fails to produce intracellular IFN-γ after multiple rounds of division seems unlikely, because both PMA/ionomycin and anti-CD3 stimulated IFN-γ production in the majority of CFSElow donor CD8+ T cells (data not shown). Moreover, LCMV-specific memory cells that were parked in SCID or B6 mice for 140 days became CFSElow yet retained their capacity to produce IFN-γ in response to their cognate peptides (Fig. 2⇑B). In a similar adoptive transfer approach, LCMV infection induced the donor LCMV-specific memory CD8+ T cell population to massively expand (>300-fold) and still produce IFN-γ after undergoing more than seven rounds of division (32).

Reconstitution by CD44low- or CD44highCD8+ cells

As CD44lowCD8+ T cells up-regulate expression of CD44 during homeostatic proliferation (14), it was unclear whether the precursors of the homeostatically expanded CD44high population in our studies originated primarily from a pre-existing memory-like CD44high subset or from a naive CD44low subset. To pursue this query, we isolated FACS-purified subsets of CD44high- and CD44lowCD8+ T cells from LCMV-infected mice (Fig. 3⇓A, top) and assessed their individual abilities to reconstitute a lymphopenic environment. Both subsets of donor CD8+ T cells had participated in multiple rounds of division within αβγδKO hosts 14 days after adoptive transfer, as evidenced by the presence of expanded populations of CFSElow cells (Fig. 3⇓A, bottom). Predictably, CD44low-transferred cells that had undergone multiple rounds of division acquired a CD44high phenotype, recapitulating a surface marker change previously described in studies of homeostatic proliferation (14). More importantly, the frequency of LCMV-specific memory cells within the adoptively transferred donor CD44high subset found in αβγδKO mice on day 14 declined compared with the purified starting population (Fig. 3⇓, B and C). Thus, it seems that dilution of the LCMV-specific memory cells in the donor CD8+ T cell population can be due to both the preferential division of other CD44high cells and to the division of CD44low precursors that up-regulate CD44 expression.

FIGURE 3.
FIGURE 3.

Division of both CD44low- and CD44highCD8+ T cell subsets contributes to dilution of LCMV-specific memory cells after homeostatic reconstitution of T cell-deficient hosts. A, CD44high- and CD44lowCD8+ T cells were sorted from LCMV-immune donor (Thy1.1+) spleens and adoptively transferred (1–2 × 106) into αβγδKO (Thy1.2+) hosts for 14 days, after which lymphocytes were isolated from the host spleen. The loss of CFSE labeling was used to ascertain the cell division patterns of CD44high and CD44low subsets. B, LCMV-specific memory cells within the donor CD44highCD8+ population were visualized by measuring IFN-γ+ production against LCMV peptides, and their percentage is indicated on the dot plot analysis of a representative animal. C, Total LCMV-specific memory cell percentage was determined by calculating the donor CD44highCD8+ T cell population capable of producing IFN-γ to the three LCMV epitopes examined. This value was averaged over three individual mice from a representative experiment and compared with the presort CD44highCD8+ T cell population.

PV-specific memory is diluted during homeostatic reconstitution

To demonstrate that dilution of LCMV-specific memory cells during lymphopenia-induced homeostatic proliferation was not a peculiarity of LCMV-specific memory CD8+ T cells, we examined the reconstitution of αβγδKO mice by CFSE-labeled splenocytes from PV-immune donor (Thy1.1+) B6 mice. As expected, the PV-immune donor CD8+ population in host αβγδKO mice spleens was associated with expansion of a CFSElowCD44high subset by day 14 (Fig. 4⇓A). However, PV-specific memory cells, which were identified by their production of IFN-γ in response to the immunodominant PV peptides NP3838–45 and NP122122–132 (33), were concentrated in the CFSEhigh subset of CD44high cells that had participated in limited rounds of division (Fig. 4⇓B). Correspondingly, the proportion of PV-specific memory cells present in the expanded donor CD8+ T cell population of αβγδKO mice was significantly lower than that in B6 hosts (Fig. 4⇓C).

FIGURE 4.
FIGURE 4.

PV-specific memory cells are diluted during the homeostatic reconstitution of T cell-deficient hosts. CFSE-labeled splenocytes (2 × 107) from donor (Thy1.1+) PV-immune mice were adoptively transferred into B6 or SCID hosts (Thy1.2+). Fourteen days after transfer, lymphocytes were isolated from the spleen, and donor CD8+ T cells were electronically gated for FACS analysis. A, The loss of CFSE labeling was used to ascertain the cell division patterns of CD44high and CD44low subsets. B, PV-specific memory cells within the donor CD8+ population were visualized by measuring IFN-γ+ production against PV peptides, and their percentage is indicated on the dot plot analysis of a representative animal. C, Total PV-specific memory cell percentage was determined by calculating the donor CD8+ T cell population capable of producing IFN-γ to the two PV epitopes examined, averaged from three individual mice in the experimental groups from a representative experiment. Statistical variation from B6 control mice was calculated (t test; ∗, p < 0.001).

Memory dilution in irradiation-induced lymphopenia

To show that a reduction in frequency of LCMV-specific CD8+ T cells following homeostatic proliferation was not an artifact related to the particular lymphopenia provided by genetically T cell-deficient mice, we used another widely used model of homeostatic proliferation. Irradiation of wild-type B6 mice induces apoptosis of peripheral lymphocytes and creates an empty vessel for reconstitution (15, 16). The proportion of adoptively transferred LCMV-specific memory cells in the donor (Thy1.1+) CD8+ T cell subset of sublethally irradiated B6 hosts on day 6 was significantly lower than that in B6 recipients (Fig. 5⇓C). Interestingly, CFSE staining indicated that dilution of the LCMV-specific memory cell subset in irradiated hosts occurred in the absence of a substantial difference in the number of divisions of LCMV-specific and of all other CD8+ T cells (Fig. 5⇓B). This suggests that LCMV-specific CD8+ T memory cells may exhibit a rate of death that exceeds the increase in cell number resulting from division of this subset. The slower rate of donor CD8+ T cell division observed in the irradiation model of homeostatic proliferation (Fig. 5⇓A), compared with αβγδKO and SCID hosts (Figs. 1⇑A and 2⇑A), might reflect an increased competition for available space resulting from the presence of residual host CD8+ T cells.

FIGURE 5.
FIGURE 5.

LCMV-specific memory cells are diluted during the homeostatic reconstitution of sublethally irradiated hosts. CFSE-labeled splenocytes (2 × 107) from donor (Thy1.1+) LCMV-immune mice were adoptively transferred into unirradiated or irradiated (6 Gy) B6 hosts (Thy1.2+). Six days after transfer, lymphocytes were isolated from the spleen, and donor CD8+ T cells were electronically gated for FACS analysis. A, The loss of CFSE-labeling was used to ascertain the cell division patterns of CD44high and CD44low subsets. B, LCMV-specific memory cells within the donor CD8+ population were visualized by measuring IFN-γ+ production against LCMV peptides, and their percentage is indicated on the dot plot analysis of a representative animal. C, Total LCMV-specific memory cell percentage was determined by calculating the donor CD8+ T cell population capable of producing IFN-γ to the two LCMV epitopes examined, averaged from three individual mice in the experimental groups from a representative experiment. Statistical variation from unirradiated B6 control mice was calculated (t test; ∗, p < 0.01).

The above studies examined the capacity of virus-specific memory cells from a donor to repopulate the peripheral CD8+ compartment of an irradiated host. We also questioned whether LCMV-specific memory T cells already present within a sublethally irradiated host would demonstrate a similar deficiency in their ability to reconstitute a lymphopenic environment. By day 15 after irradiation, there was a proportional increase in the frequency of LCMV-specific cells within the CD8+ T cell population (Fig. 6⇓), in agreement with a recent study by Grayson et al. (34) showing that LCMV-specific memory cells are more resistant to the apoptosis-inducing effects of radiation than other CD8+ T cells. However, by day 69, there was significant dilution of LCMV-specific memory cells within the host CD8+ T population (Fig. 6⇓B). This presumably reflects greater expansion of non-LCMV-specific CD8+ T cells within the CD44high subset and the restoration of a CD44low compartment via the return of host thymic function.

FIGURE 6.
FIGURE 6.

Homeostatic reconstitution by host CD8+ T cells within LCMV-immune mice following sublethal irradiation dilutes LCMV-specific memory cells. LCMV-immune B6 mice were irradiated (6 Gy), and lymphocytes were isolated from the spleen 14 and 69 days later. CD8+ T cells were electronically gated for FACS analysis. LCMV-specific memory cells were visualized by measuring IFN-γ+ production against LCMV peptides. A, The percentage of the CD44low, CD44high, and LCMV-specific memory subsets is indicated on the dot plot analysis of a representative animal. B, Total LCMV-specific memory cell percentage was determined by calculating the CD8+ T cell population capable of producing IFN-γ to the three LCMV epitopes examined, averaged from three to four individual mice in the experimental groups from a representative experiment. Statistical variation from unirradiated B6 control mice was calculated (t test; ∗, p < 0.05, ∗∗, p < 0.001).

Virus-induced apoptosis as a natural model of lymphopenia

To demonstrate the cytokine-induced lymphopenia occurring in response to virus infection, we adoptively transferred LCMV-immune donor splenocytes into B6 hosts and infected them 1 day later with PV. There was a 50 ± 3.5% reduction in the number of donor CD8+ T cells in the host spleen 2 days after PV infection, compared with uninfected mice. This decline was more dramatic in the CD44high subset (65 ± 3.8%) than the CD44low subset (37 ± 0.7%; data not shown). At the same time, there was a significant reduction in the percentage of non-cross-reactive LCMV-specific memory cells (i.e., NP396, gp33, and gp276) within the total donor CD8+ T cell population in the spleen, and this persisted even out to day 21 postinfection (Fig. 7⇓B). This represented a loss of 79 ± 4.7% and 52 ± 12% of non-cross-reactive LCMV-specific memory cells on days 2 and 21 post-PV infection, respectively, compared with uninfected mice. These results showing CD8+ T cell memory loss during virus-induced lymphopenia are consistent with other models of lymphopenia, but the apparent dilution of LCMV-specific memory cells in these experiments was complicated by the fact that PV-specific T cells within the donor population were dramatically expanded. This was particularly apparent for NP205-specific memory cells, which cross-react between the viruses (Fig. 7⇓C) (32, 33).

FIGURE 7.
FIGURE 7.

Loss of LCMV-specific memory cells in response to a virus-induced lymphopenia. Splenocytes (4 × 107) from LCMV-immune donor (Thy1.1+) mice were adoptively transferred into B6 hosts (Thy1.2+), which were then challenged 1 day later with 2 × 107 PFU of PV. A and C, LCMV-specific memory (IFN-γ+) CD8+ T cells were visualized by measuring IFN-γ+ production against LCMV peptides, and their percentage is indicated on the dot plot analysis of a representative animal. B, Total LCMV-specific memory cell percentage was determined by calculating the CD8+ T cell population capable of producing IFN-γ to three of the four LCMV epitopes examined, averaged from four to five individual mice in the experimental groups from a representative experiment. Statistical variation from uninfected B6 control mice was calculated (t test; ∗, p < 0.001, ∗∗, p < 0.0001). A and B, Non-cross-reactive LCMV epitopes. C, Cross-reactive LCMV epitope.

Therefore, we chose to use an Ag-free poly(I:C) system, rather than a viral infection, to stimulate the cytokine-induced lymphopenia, even though the reduction in CD8+ T cells was smaller and less protracted. CFSE-labeled LCMV-immune donor (Thy1.1+) splenocytes were adoptively transferred into B6 hosts (Thy1.2+), followed 1 day later by poly(I:C) treatment. Consistent with a previous study (23), we found that poly(I:C) caused a 45 ± 2.3% reduction in donor CD8+ T cell number by day 1 after injection, compared with untreated controls. This decrease in donor CD8+ T cells occurred before a loss of CFSE label intensity took place (Fig. 8⇓A), consistent with other work showing that the poly(I:C)-induced apoptosis occurred before 5-bromo-2′-deoxyuridine incorporation (23). As with PV infection, the drop in CD8+ T cells was less apparent in the CD44low subset (19 ± 0.6%) and more profound in the CD44high subset (66 ± 4.6%; Fig. not shown). LCMV-specific memory CD8+ T cells seemed as susceptible to this early attrition as other CD44highCD8+ T cells, because the proportion of LCMV-specific memory cells within the CD44high subset was similar before (34 ± 1.7%) and 1 day after (33 ± 2.4%) poly(I:C) treatment. Because CD44low cells were less affected, this equated to a significant reduction in the proportion of LCMV-specific memory cells within the total donor CD8+ T cell population (Fig. 8⇓B). The decline in CD44highCD8+ T cells was previously demonstrated not to be due to migration, as reductions in this subset were observed in all organs examined (23).

FIGURE 8.
FIGURE 8.

Homeostatic division occurring in response to a cytokine-induced lymphopenia dilutes LCMV-specific memory cells. Splenocytes (4 × 107) from LCMV-immune donor (Thy1.1+) mice were adoptively transferred into B6 hosts (Thy1.2+), which were then challenged i.p. 1 day later with a single 200-μg dose of poly(I:C), or two more doses given at weekly intervals (three times). One or 7 days later, lymphocytes were isolated from the spleen and donor CD8+ T cells were electronically gated for FACS analysis. A and C, LCMV-specific memory cells within the donor CD8+ population were visualized by measuring IFN-γ+ production against LCMV peptides, and their percentage is indicated on the dot plot analysis of representative animals. Loss of CFSE-labeling was used to ascertain the cell division patterns of total donor and LCMV-specific CD8+ T cell subsets (A). B and D, Total LCMV-specific memory cell percentage was determined by calculating the donor CD8+ T cell population capable of producing IFN-γ to the three LCMV epitopes examined, averaged from three to five individual mice in the experimental groups from a representative experiment. Statistical variation from untreated B6 control mice was calculated (t test; ∗, p < 0.01).

The poly(I:C)-induced donor CD8+ T cell lymphopenia was corrected by day 7, associated with the 39 ± 0.6% expansion of a donor CD44high subset within the spleen, consistent with previous observations (23, 25). As with homeostatic reconstitution in other models of lymphopenia, the donor CD44high cells that divided the most extensively (i.e., CFSElow) were not the LCMV-specific memory cells (Fig. 8⇑A). Hence, their proportion within the CD44high donor CD8+ T cell subset (27 ± 0.9%) was significantly decreased compared with untreated controls (34 ± 1.7%; t test, p < 0.01). However, the decline in LCMV-specific memory cells as a proportion of the total donor CD8+ population was modest (Fig. 8⇑B), reflecting the limited division of this population (only 5.3 ± 1% CFSElow) following a single poly(I:C) treatment. We hypothesized that multiple treatments with poly(I:C) might induce a more substantial division of CD8+ T cells and a more pronounced decrease in the frequency of LCMV-specific memory T cells. Indeed, this was the case, because the proportion of CFSElow donor CD8+ T cells increased to 16 ± 0.7% in host mice that received three treatments of poly(I:C) at weekly intervals (Fig. 8⇑C). This was associated with a significant decrease in the percentage of LCMV-specific memory cells within the donor CD8+ T cell population (Fig. 8⇑D). The proportion of LCMV-specific memory cells within the donor CD8+ T cell population from the peritoneal cavity, fat pads, and lungs (16, 11, and 5%, respectively) was also decreased compared with that of untreated mice (19, 17, and 13%, respectively; Fig. not shown), indicating that migration to peripheral organs could not account for the decrease in LCMV-specific memory cells observed in the spleen after triple poly(I:C) treatment.

Discussion

These studies show that virus-specific memory T cells fail to efficiently repopulate lymphopenic environments, because they are diluted out by other CD44highCD8+ T cells. Hence, this study also suggests that there may be at least two kinds of CD44high memory CD8+ T cells that there are subject to differential homeostatic regulation: memory-like CD44high cells, which account for the vast majority of CD8+ T cell division occurring in this subset in response to lymphopenia, and bona fide, foreign Ag-specific memory cells that exhibit a pattern of limited division insufficient to maintain their initial frequency. Several currently used therapies such as chemotherapy, radiation therapy, and cyclosporin A treatment have profound lymphotoxicity. In the natural setting of a virus infection, there is an early CD8+ T cell lymphopenia that is mimicked by poly(I:C) treatment (23). A reduced capacity of bona fide memory cells to reconstitute this depleted CD8+ T cell compartment may represent a mechanism for the reported loss of virus-specific memory cells in response to subsequent heterologous virus challenge (5, 6).

Limited lymphopenia-induced proliferation of bona fide memory cells may help to maintain a diverse CD8+ T cell pool capable of responding to novel pathogens. In contrast, if homeostatic proliferation that corrects a quantitative CD8+ T cell deficit provokes a generalized reduction in bona fide memory cell frequency, there may be deleterious consequences for the immunological integrity of individuals experiencing deletions in their peripheral lymphocyte pools. In this regard, a previous study found that there was a threshold number of adoptively transferred LCMV-specific memory CD8+ T cells, below which clearance of LCMV from a persistently infected host did not occur (35). Moreover, in an elegant study by Smith et al. (36), infection with a heterologous bacterial pathogen induced a loss of OVA-specific memory CD8+ T cells raised to an earlier, OVA-expressing bacterium and a subsequent loss of immunity to challenge with an OVA-expressing tumor. Finally, fewer Listeria monocytogenes-specific memory CD8+ T cells arise in mice given lower primary inoculums of this bacterium, and are associated with increased titers of this pathogen during rechallenge (37).

During infection, the early loss of CD8+ T cells, including the virus-specific memory subset, may create space for the subsequent Ag-specific CTL effector response and ultimately their memory cell progeny. In this regard, irradiation of hosts before adoptive transfer of LCMV-specific transgenic CD8+ T cells has been shown to significantly enhance viral clearance compared with unirradiated controls (38), while prior treatment of mice with cyclophosphamide enhanced the normally limited CTL response to HSV (39). In addition, several recent studies have indicated a positive effect for radiation-, genetically, and chemically induced lymphodepletion on the tumor-specific CTL-mediated clearance of melanomas from mice (40, 41) and humans (42).

A previous study calculated that only about one-third of CD8+ T cells from nontransgenic B6 mice underwent division by 7 days after adoptive transfer into an irradiated host, and different transgenic CD8+ T cells show a range of responsiveness in their ability to divide in lymphopenic environments (43, 44). These data suggest that there are differing affinities of individual TCR for the self-peptide/class I MHC signals necessary for lymphopenia-induced proliferation. One hypothesis of why virus-specific memory cells are limited in their ability to proliferate in response to lymphopenia may relate to their relatively restricted TCR repertoire. This would reduce their probability of possessing a TCR capable of transducing a homeostatic signal from a self-peptide/class I MHC ligand. However, this explanation is complicated by the observation that, even within a monoclonotypic TCR-transgenic CD8+ population, only a proportion of cells divided in response to a lymphopenic environment (43).

An alternative hypothesis is that dilution of virus-specific memory cells during homeostatic reconstitution of a lymphopenic environment may reflect an increased rate of apoptosis of this subset compared with other CD8+ cells in the expanding donor population. There was indirect evidence for this in our reconstitution studies in irradiated mice, because reduced proportions of LCMV-specific memory cells were found even though their number of divisions was similar to the rest of the donor CD8+ T cell population. The size of the LCMV-specific memory subset in resting immunoreplete mice is stable over time (6), indicating that the slow homeostatic division described for these cells (2, 3) must be offset by an equal level of apoptosis.

A previous study described a preferential division of CD44highCD8+ T cells in response to poly(I:C) treatment and hypothesized that a cytokine-induced bystander proliferation mechanism might be important for the generation and maintenance of foreign Ag-experienced memory cells (25). Our experiments with poly(I:C) expand on this study by identifying the nature of the CD44high memory cells dividing in response to this compound, and show that, although there is division of foreign Ag-experienced memory cells in response to poly(I:C), the frequency of these cells declines within a memory-like CD44high subset that is responsible for the bulk of division. The similarity of this result to those described in the other models of homeostatic reconstitution we examined suggests that cytokine-induced bystander proliferation may occur in response to homeostatic signals directing replenishment of the earlier CD8+ T cell lymphopenia created by poly(I:C) treatment (23).

These studies should serve as a caveat for the exclusive use of phenotypic markers such as CD44 to characterize foreign Ag-experienced memory CD8+ T cells. We suggest that many CD44highCD8+ T cells are not bona fide memory cells, but naive, foreign Ag-inexperienced cells that have divided in response to previous lymphopenias. The differing origins of these two populations is highlighted by the observation that a marker for granzyme B synthesis associated with LCMV-specific memory cells was not detected in CD44highCD8+ T cells from unchallenged mice (45). The natural accumulation of CD44highCD8+ T cells in aging mice may reflect a homeostatic response to the decreased thymic output resulting from normal involution of this organ (46, 47). Whereas most previous studies of homeostatic proliferation have focused on CD8+ T cells from the naive (CD44low) compartment (14, 15, 16, 43), in this study, we provide clear evidence that pre-existing CD44high memory-like cells are also capable of dividing extensively in response to lymphopenia. It remains to be seen whether, like bona fide memory cells, these memory-like cells exhibit a similar basal-level homeostatic proliferation in immunoreplete mice, and indifference for class I MHC for their survival.

Acknowledgments

We thank Drs. Liisa Selin and Eva Szomolanyi-Tsuda for helpful discussion.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Research Grants AI-17672, AR-35506, and CA-34461 and by Center Grant DK32520. The contents of this publication are solely the authors and do not represent the official view of the National Institutes of Health.

  • ↵2 C.D.P. and S.-K.K. contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. Raymond M. Welsh, Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: Raymond.Welsh{at}umassmed.edu

  • ↵4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; PV, Pichinde virus.

  • Received February 27, 2003.
  • Accepted May 2, 2003.

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