T-cell help permits memory CD8+ T-cell inflation during cytomegalovirus latency

Authors


Abstract

CD4+ T cells are implied to sustain CD8+ T-cell responses during persistent infections. As CD4+ T cells are often themselves antiviral effectors, they might shape CD8+ T-cell responses via help or via controlling antigen load. We used persistent murine CMV (MCMV) infection to dissect the impact of CD4+ T cells on virus-specific CD8+ T cells, distinguishing between increased viral load in the absence of CD4+ T cells and CD4+ T-cell-mediated helper mechanisms. Absence of T-helper cells was associated with sustained lytic MCMV replication and led to a slow and gradual reduction of the size and function of the MCMV-specific CD8+ T-cell pool. However, when virus replication was controlled in the absence of CD4+ T cells, CD8+ T-cell function was comparably impaired, but in addition CD8+ T-cell inflation, a hallmark of CMV infection, was completely abolished. Thus, CD8+ T-cell inflation during latent CMV infection is strongly dependent on CD4+ T-cell helper functions, which can partially be compensated by ongoing lytic viral replication in the absence of CD4+ T cells.

Introduction

Persistent viral infections shape their cognate CD8+ T cells markedly. This is most apparent in chronic infections with continuous productive viral replication such as chronic lymphocytic choriomeningitis virus (LCMV) infection in the mouse, where permanent activation of CD8+ T cells drives these cells into exhaustion, 1–4 with the antigen load and/or duration of antigen exposure being major promoters of CD8+ T-cell exhaustion 5.

Low level of antigen persistence as seen during chronic infection with latent, reactivating viruses also shapes antigen-specific CD8+ T-cell responses, but to a less severe extent than in situations with continuous productive viral replication. Murine CMV (MCMV) is one of the best studied examples of a chronic latent infection. Two types of virus-specific CD8+ T-cell responses, an inflationary and a non-inflationary, can be distinguished during the course of MCMV infection 6–9. CD8+ T cells of the non-inflationary type resemble CD8+ T cells that develop during acute resolved infections with respect to their kinetics, function and phenotype 7, 9–12. In contrast, inflationary CD8+ T cells show continued expansion after control of lytic replication, eventually stabilizing at high frequencies 6–9. At late stages of infection, these cells are partially exhausted as they lack IL-2 secretion capacity and have reduced TNF-α production potential 7. MCMV-specific inflationary CD8+ T cells, in contrast to non-inflationary ones, are dependent on cognate antigen for their maintenance, are unresponsive to homeostatic cytokines 12, and exhibit an effector/effector memory phenotype. In contrast, non-inflationary CD8+ T cells display a central memory phenotype 12, 13.

CD4+ helper T cells were shown to promote priming and/or memory differentiation of CD8+ T cells in various immunizations and acute infections 10, 14. However, whether and how CD4+ T cells affect CD8+ T-cell responses are less well understood during latent viral infections. As CD4+ T cells are often themselves important effectors in controlling chronic viral infections, it is often difficult to assign differences in CD8+ T-cell numbers, phenotype and function to a lack of CD4+ T-cell help. Indeed, in CD4+ T-cell-deficient mice, control of MCMV is severely hampered, especially in the salivary gland (SG) where lytic virus replication is not controlled 15. Hence, it is difficult to dissect if differences in CD8+ T-cell responses in CD4+ T-cell-deficient animals during MCMV infection are due to impaired pathogen control, leading to increased antigen load, or absence of CD4+ helper functions. Here, we compared the impact of CD4+ T-cell deficiency on MCMV-specific CD8+ T-cell responses over a very long observation period in two different settings: (i) in MHC class II knock out (MHCII−/−) mice infected with MCMV exhibiting uncontrolled MCMV replication in the SG due to the absence of anti-virally active CD4+ T cells and (ii) in MHCII−/− mice infected with a thymidine kinase (TK) recombinant MCMV whose lytic replication can be controlled by famcyclovir administration, thereby normalizing viral loads to CD4+ T-cell-sufficient mice. Intriguingly, when viral replication was experimentally controlled in the absence of CD4+ T cells, CD8+ T-cell inflation was completely abolished, indicating a fundamental role of T-cell help in driving CD8+ T-cell inflation during true MCMV latency.

Results

Functional and phenotypical features of CD8+ T cells in the presence or absence of CD4+ T cells

To investigate if CD4+ T cells are needed for the development of fully functional MCMV-specific CD8+ T-cell responses, C57BL/6 (B6) WT and MHCII−/− mice were infected with a mutant MCMV strain lacking the m157 gene (MCMV-Δm157). m157 is expressed on the surface of MCMV-infected host cells and interacts with the activating receptor Ly49H on NK cells of B6 mice, thereby leading to an increase in viral control 16. We deliberately used MCMV-Δm157 to circumvent this B6 strain-specific phenomenon. Comparable to published results 15, virus titers were very high in the SG even >1 year post infection in MHCII−/− mice and infectious virus was occasionally detectable in other organs in a few mice, although at very low levels (Fig. 1A and Supporting Information Fig. 1). To investigate if the concomitant absence of CD4+ T-cell help and increased viral load influenced the MCMV-specific CD8+ T-cell response, the kinetics of different CD8+ T-cell populations, specific for the epitopes M45 and M57 (non-inflationary) or M38, m139 and IE3 (inflationary), were analyzed in the blood and in peripheral organs such as the lung, spleen and liver by tetramer staining (Fig. 1B and 1C and data not shown) and IFN-γ secretion (Fig. 1D) over more than 1 year of infection.

Figure 1.

CD4+ T cells are crucial to sustain frequencies and function of MCMV-specific CD8+ T cells. MHCII−/− (open circles) and B6 WT (filled circles) mice were infected with MCMV-Δm157. (A) Virus titers in the SG at > 1 year infection. (B and C) Kinetics of M45- (B, left panel) and M38- (B, right panel) specific CD8+ T-cell frequencies were assessed (B) in blood (first time point of analysis: day 8) or (C) in the lung at >1 year infection by direct ex vivo tetramer staining. (D) IFN-γ staining after short-term restimulation of lung lymphocytes with the indicated peptide epitopes at >1 year infection (similar results were observed in spleen and liver, data not shown). (E) Lung lymphocytes were restimulated with a panel of CD8+ T-cell epitopes and costained for CD107a and IFN-γ. Frequencies of IFN-γ+ cells within CD107a+ cells are shown (data are only shown if frequencies of epitope-specific CD8+ T cells were above 0.1%). (F) M45- and M38-tetramer positive CD8+ T cells were stained with antibodies against the surface markers CD62L and IL-7Rα. Single and double positive CD8+ T cells are displayed as frequencies of total M45- and M38-tetramer CD8+ T cells. Statistically significant differences were observed between CD8 T-cell phenotypes of B6 and MHCII−/− mice for M45: CD62L+IL-7Rα+; CD62LIL-7Rα and for M38: CD62LIL-7Rα+; CD62LIL-7Rα. Representative data from one out of at least three comparable experiments are shown. Bars represent mean values. (n.d., below detection limit; *p<0.05, **p<0.01, ***p<0.001, two-tailed unpaired Student's t-test).

At 4 wks post infection, the absence of CD4+ T cells did not influence the frequencies of non-inflationary (M45 and M57) or inflationary (M38, m139, IE3) CD8+ T cells (Fig. 1B and Supporting Information Fig. 2B–D). In some experiments, even an increase of MCMV-specific CD8+ T cells was detected in MHCII−/− mice during the acute phase of infection. As reported previously 17, non-inflationary MCMV-specific CD8+ T cells were steadily decreasing in MHCII−/− mice and were hardly detectable 1 year after infection (Fig. 1B–D). Interestingly, the accumulation of inflationary CD8+ T cells was also reduced in all organs of MHCII−/− animals (Fig. 1B–D and data not shown).

To investigate whether absence of CD4+ T cells influenced the functional capacities of epitope-specific CD8+ T cells, we co-stained stimulated cells with anti-IFN-γ and anti-CD107a antibodies. As degranulation (i.e. CD107a surface staining) is largely preserved in CD8+ T cells during chronic viral infections 4, co-staining of CD107a and IFN-γ allows the assessment of the relative function of antigen-specific CD8+ T cells. Based on this analysis, the ability of MCMV-specific CD8+ T cells to produce IFN-γ was comparable between MHCII−/− and WT animals at 4 wks post infection (Supporting Information Fig. 2D). However, more than 1 year after primary MCMV encounter, there was a clear functional impairment of CD8+ T cells of the inflationary type, both in the presence or absence of CD4+ T cells (Fig. 1E), though in the absence of CD4+ T cells this deficit was even more substantial as less than 20% and less than 40% of M38- and m139-specific CD8+ T cells respectively were able to produce IFN-γ (Fig. 1E). This is in contrast to non-inflationary CD8+ T cells, where most of the CD107a positive CD8+ T cells were able to produce IFN-γ in WT mice.

Inflationary, in contrast to non-inflationary CD8+ T-cell subsets, have a more activated phenotype, most likely due to occasional reencounter with their cognate antigen 7, 9, 12. Increased viral replication in the organs of MHCII−/− mice compared with B6 mice apparently leads to higher antigen loads in various organs, and should consequently result in an augmented activation status of MCMV-specific T cells. This was indeed the case. Early during MCMV infection, the M45-specific CD8+ T-cell population of MHCII−/− mice was mostly IL-7Rα CD62L, a phenotype compatible with effector T cells, whereas the counterpart population in WT mice showed considerably increased surface expression of IL-7Rα and CD62L, indicative of a larger proportion of effector memory/central memory cells (Supporting Information Fig. 2E). M38-specific CD8+ T cells were mostly IL-7Rα CD62L at 4 wks post infection, irrespective of the presence or absence of CD4+ T cells (Supporting Information Fig. 2E). However, during late stages of infection, MCMV-specific CD8+ T cells of both the inflationary and non-inflationary type displayed a more activated phenotype in the absence of CD4+ T cells (Fig. 1F).

Generation of proliferation-competent CD8+ T cells is often dependent on CD4+ T-cell help 14, 18, 19. We therefore assessed the proliferative capacities of MCMV-specific CD8+ T cells, which had developed in a CD4+ T-cell-deficient environment. Splenic CD8+ T cells of infected Ly5.2+ MHCII−/− and Ly5.2+ B6 mice were MACS purified and subsequently equal numbers of M38-Tetramer-positive CD8+ T cells were transferred into naïve, congenic Ly5.1+ WT animals (experimental setup shown in Fig. 2A). By doing so, we eliminated differences in viral load present in MHCII−/− versus B6 animals during the recall response and provided comparable T-cell help during secondary expansion. One day post transfer, mice were infected with MCMV-Δm157. After 1 wk, exogenous and endogenous CD8+ T-cell responses were analyzed. MCMV-specific CD8+ T cells, isolated from B6 mice 4 wks after MCMV infection, had proliferated vigorously and to a greater extent than their counterparts isolated from MHCII−/− mice (Fig. 2B). However, this was even more pronounced when CD8+ T cells were isolated >1 year after MCMV infection (B6 versus MHCII−/−: 1.5 times increased if isolated at 4 wks post infection; B6 versus MHCII−/−: 9.7 times increased if isolated at >1 year). To compare proliferative capacities of M45-specific CD8+ T cells, frequencies of proliferated cells were normalized to the amount of M45-specific CD8+ T cells that had been transferred. By doing so, M45-specific CD8+ T cells isolated from B6 mice proliferated twice better than the ones isolated from MHCII−/− mice at >1 year of MCMV infection (Fig. 2B).

Figure 2.

MCMV-specific CD8+ T cells from CD4+ T-cell-deficient mice exhibit impaired recall responses. (A) Experiment layout: Spleens of Ly5.2+ MCMV-infected B6 and MHCII−/− mice were isolated and spleens of the same experimental group were pooled. CD8+ T cells were MACS purified and a small sample was stained by tetramers in order to adoptively transfer equal numbers of M38-specific CD8+ T cells into congenic Ly5.1+ B6 WT animals. One day post transfer, mice were infected with MCMV-Δm157 and 7 days later lymphocytes of the spleen, lung and liver were stimulated with a panel of MCMV-specific CD8 T-cell epitopes. (B) Lung lymphocytes are shown as representative data. Donor animals were either infected for 4 wk (left plot) or for >1 year (right plot). FACS plots were pre-gated on CD8+ T cells. Frequencies of donor-derived IFN-γ-secreting CD8+ T cells are displayed. The number of M45-specific CD8+ T cells transferred differed between B6 and MHCII−/− mice; the ratio of M45-specific CD8+ T cells between B6 and MHCII−/− mice at transfer was determined and was used to normalize the frequencies of M45-specific CD8+ T cells originating from MHCII−/− mice at day 8 post transfer (“M45 norm”). Representative data from one out of a minimum of three similar experiments are shown. Bars represent mean values. (*p<0.05, **p<0.01, ***p<0.001, two-tailed unpaired Student's t-test).

New thymic emigrants can be primed during chronic stages of MCMV and LCMV infection 12, 20. However, it is unclear to which extent this contributes to the overall virus-specific CD8+ T-cell pool during MCMV infection. Presence of CD4+ T cells was necessary not only during the acute phase but also during later stages for the maintenance of both inflationary and non-inflationary CD8+ T-cell responses (Supporting Information Fig. 3). Hence, CD4+ T cells might provide help to prime new thymic emigrants, which then contribute to stabilize the MCMV-specific CD8+ T-cell pool during late stages of infection. To test this hypothesis, we infected control and thymectomized B6 mice (Supporting Information Fig. 4) with MCMV-Δm157 and analyzed the kinetics and functional capacities of virus-specific CD8+ T cells. Inflation as well as maintenance, cytokine production and recall proliferation of M38- or M45-specific CD8+ T cells were not impaired in thymectomized animals (Supporting Information Fig. 5), indicating that priming of new thymic emigrants was not a prerequisite for the physical and functional maintenance of the inflationary or non-inflationary CD8+ T-cell pools.

Taken together, MCMV-specific CD8+ T cells differentiated and maintained in the absence of CD4+ T cells were not only decreased in their frequencies at late stages of infection, but inflationary CD8+ T cells exhibited also more pronounced functional deficits. Further, CD8+ T cells in MHCII−/− mice displayed a more activated phenotype and exhibited an impaired proliferative capacity following a challenge infection. Finally, priming of new thymic emigrants was not required for MCMV-specific CD8+ T-cell inflation but could occur during late stages of MCMV infection, which was independent of CD4+ T cells (data not shown), suggesting that the reduced MCMV-specific CD8+ T-cell responses in the absence of CD4+ T cells is not due to impaired recruitment and priming of naïve CD8+ T cells during late stages of infection.

Lytic MCMV replication maintains inflationary CD8+ T cells in the absence of CD4+ T cells

So far we had compared MCMV-specific CD8+ T-cell responses in B6 and MHCII−/− mice with the important caveat that in the latter not only T-cell help is lacking but also lytic MCMV replication is uncontrolled. Next, we assessed whether ongoing viral replication and thus increased antigen load in MHCII−/− mice was the main factor leading to impaired MCMV-specific CD8+ T-cell responses. To this end, we infected MHCII−/− and WT mice with an MCMV-Δm157 mutant expressing TK under the control of the m157 promoter (MCMV-Δm157-TK). By treating animals with famcyclovir beginning at day 28 and continuing throughout later stages of infection, we were able to completely suppress lytic MCMV replication in MHCII−/− mice in the SG, spleen, liver and lung (Fig. 3B and data not shown).

Figure 3.

In the absence of CD4+ T cells, lytic viral replication is required to sustain inflationary CD8+ T cells. MHCII−/− mice and B6 mice were infected with 106 plaque forming units of MCMV-Δm157-TK; 28 days post infection half of the experimental groups were treated with famcyclovir, thereby inhibiting MCMV replication. (A) Blood kinetics of M45- (left panel) and M38-(right panel) specific CD8+ T cells are displayed. Statistically significant differences at day 204 post infection: M45: B6 versus MHCII−/− (***); B6 fam versus MHCII−/− fam (***); B6 versus MHCII−/− fam (***). M38: B6 versus MHCII−/− fam (***); B6 fam versus MHCII−/− fam (***); MHCII−/− versus MHCII−/− fam (**). (B) Virus titer in the SG isolated 9 months post infection. (A and B) The experiment was repeated three times with at least three mice per group. (C) Lymphocytes were isolated from different organs and stained with M45- (left panel) or M38- (right panel) tetramers. Data from the lung are shown and are comparable to spleen and liver. (D) Frequencies of MCMV-specific CD8+ T cells secreting IFN-γ after short-term restimulation with the indicated epitopes. (E) Percentages of IFN-γ+ CD8+ T cells among CD107a+ CD8+ T cells are shown. (F) M45- (left diagram) and M38- (right diagram) tetramer positive CD8+ T cells were stained for CD62L and IL-7Rα. Single and double positive populations are displayed as percentages of total specific CD8+ T cells. Statistically significant differences were observed between CD8+ T-cell phenotypes of MHCII−/− and famcyclovir-treated MHCII−/− mice for M45: CD62L+IL-7Rα+ and for M38: CD62LIL-7Rα+; CD62LIL-7Rα. (C–F) Combined results of three independent experiments, terminated 7–10 months post infection, are shown. (G) As described in Fig. 2, CD8+ T cells of each experimental group were isolated, transferred into naïve congenic WT mice, which were infected 1 day later. At day 7 post infection, transferred CD8+ T cells were stained for M45- and M38-specific CD8+ T cells. Representative data from one out of two comparable experiments are shown. Bars represent mean values. (n.s. not significant; *p<0.05; **p<0.01; ***p<0.001, two-tailed unpaired Student's t-test).

As in the case of MCMV-Δm157 infection, MCMV-Δm157-TK infection in the absence of famcyclovir treatment resulted in reduced frequencies of CD8+ T cells recognizing the M45 or the M38 epitope in MHCII−/− compared with WT mice (Fig. 3A). Again M38-specific CD8+ T cells, although strongly expanded during the acute phase of infection, did not inflate during early stages of chronic infection (day 14 to day 50 post infection), but rather stabilized at high frequencies in untreated MHCII−/− mice. Interestingly, lytic viral replication was not the cause of reduced frequencies of the non-inflationary M45-specific CD8+ T-cell population in MHCII−/− mice, as frequencies of M45-specific CD8+ T cells were comparably low irrespective of famcyclovir treatment (Fig. 3A). Intriguingly, in the absence of lytic viral replication, M38-specific CD8+ T cells were decreasing continuously in MHCII−/− mice over the course of chronic infection in comparison with all other experimental groups (Fig. 3A, “MHCII−/− fam”). To exclude that M38-specific CD8+ T cells of famcyclovir-treated MHCII−/− mice has accumulated in peripheral tissues, we performed tetramer stainings and ex vivo restimulation assays of organ resident lymphocytes (Fig. 3C–E, data shown for lung). Comparable to the blood data, M45 tetramer-positive CD8+ T cells were decreased in all analyzed organs of MHCII−/− mice; however, famcyclovir treatment did not further reduce this response (Fig. 3C). In contrast, famcyclovir treatment had a significant effect on the size of the inflationary M38-specific CD8+ T-cell response: while there was no significant difference in untreated WT and MHCII−/− mice at late stages of infection (Fig. 3C), famcyclovir-treated MHCII−/− mice exhibited a severely reduced population of M38-specific CD8+ T cells. We extended and corroborated this finding by including additional non-inflationary (Fig. 3D, M45 and M57) and inflationary CD8+ T-cell responses (Fig. 3D, M38, m139, IE3).

Despite the fact that frequencies of inflationary MCMV-specific CD8+ T cells in the absence of both CD4+ T cells and viral replication were greatly reduced, the remaining cells might be fully functional. Yet, this was not the case, as CD8+ T cells isolated from both groups of MHCII−/− mice, famcyclovir treated or not, were similarly dysfunctional. Further, famcyclovir treatment did not influence the functionality of MCMV-specific CD8+ T cells generated and maintained in B6 animals (Fig. 3E, “B6” versus “B6 fam”).

As shown in Fig. 1F, MCMV-specific CD8+ T cells isolated from MHCII−/− mice displayed a more activated phenotype compared with B6 mice. Should increased viral replication be the cause of these phenotypical changes, then suppression of lytic viral replication in the absence of CD4+ T cells should reduce the activation status of MCMV-specific CD8+ T cells. That was indeed the case (Fig. 3F): even though IL-7Rα and CD62L expression was decreased in M45-specific CD8+ T cells of famcyclovir-treated MHCII−/− mice compared with B6 mice, surface expression of those molecules was even lower in untreated MHCII−/− animals (Fig. 3F). Furthermore, M38-specific CD8+ T cells from untreated MHCII−/− showed a slight decrease in their IL-7Rα expression. Hence, suppression of MCMV replication in MHCII−/− mice leads to a decrease in antigen load, thereby limiting activation of MCMV-specific CD8+ T cells.

Would the curtailed exposure of CD8+ T cells to infectious MCMV in famcyclovir-treated MHCII−/− mice ameliorate their proliferative capacities or is the responsiveness to recall infection imprinted by CD4+ T-cell help? To address this question we repeated the challenge experiment described in Fig. 2A, this time including famcyclovir-treated groups. Virus-specific CD8+ T cells isolated from MHCII−/− mice of all specificities analyzed showed decreased capacities to respond to secondary challenge (Fig. 3G). Thus, unhelped virus-specific CD8+ T cells were greatly impaired in their proliferation potential in response to MCMV challenge even when lytic viral replication was inhibited in MHCII−/− mice.

In summary, we were able to show that CD4+ T-cell help per se and not via control of lytic viral replication was crucial (i) for the maintenance of a stable memory pool of non-inflationary CD8+ T cells, (ii) for the inflation of MCMV-specific CD8+ T cells over the course of infection and (iii) for the generation of virus-specific CD8+ T cells with recall proliferation potential.

IL-2 but not IL-21 promotes maintenance and inflation of MCMV-specific CD8+ T cells

Maintenance of antigen-experienced CD8+ T cells during active chronic infections was shown to depend on the cytokines IL-21 and IL-2, both cytokines being produced by CD4+ T cells 21–28. To gain insight into whether helper mechanisms exerted by CD4+ T cells during MCMV infection can be attributed to any of those two cytokines, we first analyzed CD8+ T-cell responses generated in MCMV-infected mice lacking the IL-21 receptor (IL-21R−/−). Frequencies of M45- and M38-specific CD8+ T cells were not reduced in MCMV-infected IL-21R−/− in comparison with WT mice (Fig. 4A), making it unlikely that CD4+ T cells support CD8+ T-cell responses by IL-21 during MCMV infection, unlike in murine LCMV or human HIV-1 infection 21–23.

Figure 4.

Relevance of IL-21 and IL-2 for MCMV-specific CD8+ T-cell responses. (A) MCMV-specific CD8+ T-cell responses in the blood of MCMV-Δm157-infected IL-21R−/− and B6 mice were analyzed with tetramers specific for the CD8+ T-cell epitopes M45 (left panel) and M38 (right panel) 3 months post infection. (B) To test if IL-2 signaling on CD8+ T cells is necessary for MCMV-specific CD8+ T-cell responses, mixed bone marrow chimeras were generated. Ly5.1+ B6 mice were lethally irradiated and reconstituted with either Ly5.1+ IL-2Rα−/− and Ly5.2+ B6 bone marrow (test chimeras) or Ly5.1+ B6 and Ly5.2+ B6 bone marrow (control chimeras). 7 months post MCMV-Δm157 infection, CD8+ T-cell responses in chimeras were analyzed in different organs. Lung responses are shown as representatives. Bars represent mean values. (*p<0.05; **p<0.01; ***p<0.001, two-tailed unpaired Student's t-test).

To test if IL-2 signaling on CD8+ T cells is needed for inflation during MCMV infection, we generated the following mixed bone marrow chimeras: irradiated Ly5.1+ B6 mice were reconstituted with Ly5.2+ IL-2Rα−/− and Ly5.1+ B6 bone marrow (Fig. 4B; test chimeras) or with Ly5.2+ B6 and Ly5.1+ B6 bone marrow (control chimeras). In this way, Ly5.2+ expressing CD8+ T cells lacked IL-2Rα expression whereas Ly5.1+ CD8+ T cells originated from a B6 background in test chimeras. Chimerism was determined in all mice shortly before MCMV infection and was around 50%. Minor deviations from the 50% ratio were recorded just prior to infection and were taken into account for normalization of epitope-specific Ly5.2+ CD8+ T cells of total CD8+ T cells (Fig. 4B, labeled “normalized”). M45-specific IL-2Rα−/− CD8+ T cells were significantly reduced in all organs analyzed 6 months post infection (Fig. 4B, lung lymphocytes). The necessity for IL-2 signaling for the inflation and maintenance of M38-specific CD8+ T cells was even more obvious; in the absence of IL-2Rα, M38-specific CD8+ T cells did not inflate at all in the blood and in the lung half a year post infection (Fig. 4B). Thus, absence of IL-2 signaling in MCMV-specific CD8+ T cells is not only detrimental for inflation, but also in the long run for the maintenance of an otherwise stable memory pool of M45-specific CD8+ T cells.

Discussion

High antigen load as well as absence of CD4+ T-cell help was shown to impair the development of antigen-independent and functional memory CD8+ T cells with the ability to undergo secondary expansion 10. In the absence of CD4+ T cells, control of MCMV replication is impaired in various organs but especially in the SG where replicating virus can be detected throughout the course of infection 15. To distinguish direct antiviral mechanisms from helper functions exerted by CD4+ T cells and to analyze their individual role for MCMV-specific CD8+ T-cell responses, we infected MHCII−/− mice with an MCMV mutant expressing TK under the control of the m157 promoter. Thus, by treatment of mice with famcyclovir, lytic viral replication could be controlled exogenously in the absence of CD4+ T cells. Using this approach, we were able to show that (i) CD4+ T-cell help was required for the maintenance of a stable memory CD8+ T-cell pool specific for non-inflationary epitopes and (ii) lytic MCMV replication in the absence of CD4+ T-cell help is crucial to stabilize inflationary CD8+ T-cell responses at high frequencies. Hence, in the concomitant absence of CD4+ T-cell help and lytic viral replication, one of the hallmarks of MCMV-specific CD8+ T-cell responses, namely, CD8+ T-cell inflation, was completely abolished. Finally, help provided by CD4+ T cells licensed MCMV-specific memory CD8+ T cells of all specificities to undergo robust secondary proliferation.

Regarding the mechanism of how CD4+ T cells promote MCMV-specific CD8+ T-cell responses and in light of the fact that the presence of CD4+ T cells is required during the entire course of infection to optimally support CD8+ T-cell responses, it is conceivable that CD4+ T cells are required for the priming of naive thymic CD8+ T-cell emigrants 20, 29. Even though we observed that new priming during chronic stages of MCMV infection can occur (data not shown), corroborating earlier results using a different experimental approach based on busulfan conditioning following bone marrow transplantation 12, provision of help to prime new thymic CD8+ T-cell emigrants was clearly not the main mechanism by which CD4+ T cells support MCMV-specific CD8+ T cells of the inflationary or the non-inflationary type. Thymectomized B6 mice, in clear contrast to MHCII−/− mice, showed CD8+ T-cell responses comparable to non-thymectomized B6 mice, making it very unlikely that priming of new thymus-derived naïve CD8+ T cells with the help of CD4+ T cells is instrumental for the maintenance of the MCMV-specific CD8+ T-cell pool during long-term MCMV infection.

Recently, a specific role was attributed to IL-21 secreted by CD4+ T cells to support the development and maintenance of functional virus-specific CD8+ T cells during active chronic viral infections 21–23, 27, 28. However, during MCMV infection, IL-21R−/− mice displayed normal CD8+ T-cell responses of either the inflationary or the non-inflationary type, arguing against IL-21 being an essential component of T-cell help for MCMV-specific CD8+ T cells. As MCMV-specific CD4+ T cells were shown to secrete IL-2 30, it is conceivable that IL-2 plays an important role in maintaining and possibly inflating MCMV-specific CD8+ T-cell responses. Our results obtained from mixed bone marrow chimeras harboring CD8+ T cells with and without IL-2Rα expression support this notion, as IL-2Rα-deficient CD8+ T cells completely failed to inflate and were present at significantly reduced frequencies in case of non-inflating CD8+ T-cell responses. These results corroborate and extend earlier results, which showed that absence of IL-2R signaling on inflating MCMV-specific CD8+ T cells leads to reduced frequencies already early after infection 24, suggesting that IL-2 may be important to “program” the inflationary capacity within CD8+ T cells already early during infection.

Maintenance of a stable memory-like CD8+ T-cell pool in case of non-inflationary CD8+ T cells was shown to be antigen-independent and dependent on homeostatic turnover during chronic/latent stages of MCMV infection 12. Further, their low activation status suggests that reencounter with their cognate antigen during chronic/latent stages of MCMV infection is presumably a very rare event. It was shown for a variety of acute resolved infections that pathogen-specific CD8+ T-cell responses during the acute phase of infection were comparable in the presence or absence of CD4+ T cells 31, 32. However, maintenance of a stable antigen-specific CD8+ T-cell memory pool and the ability to undergo robust secondary expansion was crucially dependent on the presence of CD4+ T cells 14, 18, 19, 33. Accordingly, MCMV-specific CD8+ T cells of the non-inflationary type expanded comparably in the absence of CD4+ T-cell help during acute infection. However, frequencies of non-inflationary CD8+ T cells as well as their recall proliferative potential declined steadily over time during chronic/latent stages of MCMV infection in the absence of CD4+ T cells, irrespective of the presence or absence of lytic viral replication. Inflationary CD8+ T-cell responses were also comparably induced during the acute phase of MCMV infection in the presence or absence of CD4+ T cells. However, in the absence of CD4+ T cells their inflation was curtailed, and in the concomitant absence of both CD4+ T cells and lytic viral replication (due to famcyclovir treatment), “inflationary” CD8+ T-cell responses became “deflationary” with their frequencies continuously declining over an observation period of 200 days.

For both inflationary and non-inflationary CD8+ T-cell responses, their recall proliferation potential declined with time when developing in a CD4+ T-cell-deficient environment, even when lytic viral replication was successfully controlled by famcyclovir treatment. Thus, the absence of CD4+ T-cell help during the course of MCMV infection – and not ongoing viral replication – impaired the secondary recall proliferation potential of MCMV-specific CD8+ T cells in MHCII−/− mice.

In chronic infections, studying helper mechanisms exerted by CD4+ T cells on antigen-specific CD8+ T-cell responses is often hampered by impaired pathogen control in CD4+ T-cell-deficient animals – as is the case in MCMV infection. In these situations, it is important to consider that not only the amount of viral antigen is increased in the absence of CD4+ T cells but antigen presentation most likely also occurs in additional cell types such as APCs apart from endothelial and epithelial cells where latency is mainly established in case of MCMV 34. As proposed previously in “the immune sensing hypothesis of latency control”, we assume that during MCMV latency epitopes recognized by inflationary CD8+ T cells are sporadically presented on MHC class I molecules, while the ones recognized by non-inflationary CD8+ T cells are not 35–37. Based on these presumptions, we propose the following scenario as being responsible for shaping MCMV-specific CD8+ T-cell responses in MCMV infection in presence or absence of CD4+ T cells (Fig. 5): once lytic MCMV replication has ceased in WT animals (Fig. 5A), cells of the non-hematopoietic cell lineage are the major site of MCMV latency. Sporadic reactivation events lead to selective antigen processing and presentation on these non-hematopoietic cell types. MCMV-specific CD8+ T cells of the inflationary type recognize these antigens, get activated and proliferate (memory inflation) if CD4+ T-cell help is provided. T-helper cells may promote this inflation either by provision of growth factors such as IL-2 or alternatively by modulating the quality of APCs needed for the priming via upregulation of co-stimulatory molecules such as 4-1BBL 38, OX40 38, CD80/86 39. In CD4+ T-cell-deficient animals (Fig. 5B), lytic MCMV replication is not controlled, leading to constant low level production of infectious virus or virus proteins in various organs. Antigens expressed by MCMV can then be presented additionally by professional APCs such as DCs, either via cross-presentation or via the conventional antigen presenting pathway if APCs get infected themselves. Interaction of an epitope-specific CD8+ T cell with its cognate antigen on professional APCs leads to activation and survival, but only limited proliferation. Thus, in the absence of CD4+ T-cell help, interaction with antigen on non-professional and/or professional APCs is not resulting in comparable activation, proliferation or maintenance as in the presence of T-cell help. Finally, in the concomitant absence of CD4+ T cells and lytic viral replication (Fig. 5C), inflationary CD8+ T cells can only recognize their cognate antigen on non-hematopoietic cells harboring reactivating CMV, which is insufficient for their full activation/proliferation or survival, resulting in a steady decrease during latent stages of MCMV infection.

Figure 5.

CD4+ T-cell help is crucial for the generation of functional MCMV-specific CD8+ T-cell responses of the inflationary and non-inflationary type and for inflation of MCMV-specific CD8+ T-cell responses, an overview. (A) In WT mice infected with MCMV, non-inflationary CD8+ T cells (e.g. M45) resemble pathogen-specific CD8+ T cells generated after acutely resolved infections. After an expansion phase, non-inflationary CD8+ T cells contract and are maintained at low frequencies during chronic stages of infection while maintaining full functionality. Most likely due to rare reactivation events, CD8+ T cells of the inflationary type reencounter their cognate antigen presented on latently infected non-hematopoietic cells (depicted in orange), thereby getting activated and being driven to proliferate. We propose that CD8+ T cells depend on CD4+ T-cell help to promote their proliferation or survival when they reencounter their cognate antigen presented on latently infected non-APCs. (B) In CD4+ T-cell-deficient mice infected with MCMV, a major control of lytic viral replication is missing in addition to CD4+ T-cell help. Kinetic analyses of inflationary CD8+ T-cell responses revealed the absence of a prominent inflation phase and instead a direct stabilization at high frequencies after the contraction phase. We propose that the stabilization of inflationary CD8+ T cells is due to antigen encounter not only on non-hematopoietic cells harboring reactivating MCMV but also due to spread of infectious virus or MCMV-derived antigens to professional APCs (yellow), which might facilitate the maintenance of a stable high level of inflationary CD8+ T cells due to increased proliferation or survival. (C) To dissect antiviral function from helper mechanisms exerted by CD4+ T cells, mice lacking CD4+ T cells were infected with a mutant MCMV strain expressing TK and were subsequently treated with famcyclovir to inhibit lytic virus replication (but not early reactivation events associated with transcription of early MCMV genes). In this situation, inflationary CD8+ T cells did not inflate at all but were constantly declining, presumably due to restricted antigen recognition on non-hematopoietic cells in the absence of CD4+ T-cell help, leading to decreased proliferation or survival.

Materials and methods

Mice, in vivo CD4 depletion and peptides

C57BL/6, MHC class II-deficient 40, IL-21R−/− (kindly provided by M. Kopf, ETH Zurich, Switzerland, 22) and CD4-deficient 32, IL-2Rα-deficient (Jackson) mice were kept under specific pathogen-free conditions and were infected intravenously with 106 plaque forming units of MCMV between 6 and 12 wks of age. Bone marrow chimeras were generated and analyzed as described previously 24.

When indicated, mice were injected i.p. with 0.2 mg of purified anti-mouse CD4 monoclonal antibody (YTS 191.1, 41). For continuous depletion, mice were injected 3 and 1 days before infection and then weekly.

MCMV replication was inhibited in MCMV-Δm157-TK infected mice by administration of famcyclovir (kindly provided by Novartis Pharma AG, Basel, Switzerland) into the drinking water (2 mg/mL), which was exchanged every other day.

The M45aa985–993, M38aa316–323, M57816–824, m139419–426 and IE3416–423 peptides were purchased from NeoMPS (Strasbourg, France). Production of crude MCMV lysate was previously described 30.

Ethic statements

This study was carried out in strict accordance to the guidelines of the animal experimentation law (SR 455.163; TVV) of the Swiss Federal Government. The protocol was approved by Cantonal Veterinary Office of the canton of Zurich, Switzerland (Permit number 145/2008). All surgery was performed under isoflurane anesthesia and animals were treated pre- and post-surgically from day −1 to day +7 with the analgesic Buprenorphine. All efforts were made to minimize suffering.

Viruses

Generation of the recombinant MCMV-Δm157 (lacking the m157 gene of MCMV) was previously described 30. MCMV mutant expressing TK under the m157 promoter (MCMV-Δm157-TK) was a kind gift of Prof. A. Hill (Portland, OR).

MCMV was propagated on mouse embryonic fibroblasts and viral titers were determined using plaque forming assays as described in 42.

Antibodies

PE-conjugated peptide-MHC class I tetrameric complexes were generated as previously described 43. The following monoclonal antibodies were either purchased from BD Pharmingen (Allschwil, Switzerland) or from BioLegend (Lucerna Chem AG, Luzern, Switzerland) and used for stainings: anti-CD8 (FITC, PerCP, APC, PacificBlue, APC-Cy7), anti-CD4 (PE, PerCP, PacificBlue), anti-IFN-γ (APC), anti-CD45.1 (PerCP, APC, PacificBlue), anti-CD45.2 (PerCP, APC, PacificBlue), anti-CD127 (FITC), anti-CD62L (PerCP), antiCD107a (FITC).

Cell stimulation, immunofluorescent staining and analysis

Lymphocytes were isolated from spleen, lung and liver as previously described 44. After washing in FACS buffer (PBS, 2% heat-inactivated FCS, 5 mM EDTA, and 0.02% sodium azide), cells were surface stained with directly labeled Abs or peptide-MHC class I tetramer complexes for 20 min at 4°C, followed by erythrocyte lysis using 1 mL of FACSLyse (BD Biosciences) for 10 min at room temperature. For intracellular cytokine stainings, CD8 T cells were stimulated with 1 μg/mL peptide in the presence of 10 μg/mL brefeldin A (Sigma-Aldrich) and if indicated 1 μL/mL anti-CD107 (FITC) antibody at 37°C for 6 h. Cells were surface stained as described above, fixed and permeabilized using 500 μL of FIX/Perm solution (FACSLyse diluted to 2× concentration and 0.05% Tween 20) for 10 min at room temperature. After washing, cells were stained with directly labeled Abs against IFN-γ. Multiparameter flow cytometric analysis was performed using a FACS LSRII flow cytometer (BD, Allschwil, Switzerland) with FACS DIVA software (BD, Allschwil, Switzerland). List mode data were analyzed using FlowJo software (Treestar, San Carlos, CA).

Adoptive transfer

For adoptive transfer experiments, splenic CD8+ T cells isolated from infected CD45.2+ mice (B6 or MHC class II-deficient mice) were purified by MACS using positive selection according to the instructions of the manufacturer (Miltenyi Biotec). Cells were adoptively transferred into CD45.1+ naive recipient mice (104 to 5×104 cells when isolated from donors during chronic/ latent stages and 105 to 5×105 cells when isolated during early infection (day 28)). One day post transfer mice were infected with MCMV-Δm157.

Before transfer, the frequency of M38-specific CD45.2+ CD8+ T cells was determined by tetramer staining and the numbers of donor cells transferred were normalized to contain an equal number of M38-specific CD8+ T cells.

Statistical analysis

Statistical analysis was performed using two-tailed unpaired Student's t-test.

Acknowledgements

We are very grateful to Ann Hill (Portland, OR, US) for provision of the MCMV-Δm157-TK virus, to Novartis Pharma AG (Basel, Switzerland) for donation of famcyclovir and to Manfred Kopf (Zurich, Switzerland) for provision of the IL-21R−/− mice. Furthermore, we would like to thank Nathalie Oetiker for technical assistance and the members of the Oxenius lab for critical discussions and support. This work was supported by the ETH Zurich, the Roche Research Foundation and the Swiss National Science Foundation (Grant No. 310030-113947 to A.O.).

Conflict of interest: The authors declare no financial and commercial conflict of interests.

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