Fully functional memory CD8 T cells in the absence of CD4 T cells

The role of CD4 T cells in providing help to CD8 T cells in primary and secondary responses to infection remains controversial. Using recombinant strains of virus and bacteria expressing the same Ag, we determined the requirement for CD4 T cells in endogenous CD8 T cell responses to infection with vesicular stomatitis virus and Listeria monocytogenes (LM). Depletion of CD4 T cells had no effect on the frequency of primary or secondary vesicular stomatitis virus-specific CD8 T cells in either lymphoid or nonlymphoid tissues. In contrast, the primary LM-specific CD8 T cell response was CD4 T cell dependent. Surprisingly, the LM-specific CD8 T cell recall response was also CD4 T cell dependent, which correlated with a requirement for CD40/CD40L interactions. However, concomitant inhibition of CD40L and CD4 T cell removal revealed that these pathways may be operating independently. Importantly, despite the absence of CD4 T cells during the recall response or throughout the entire response, CD8 memory T cells were functional effectors and proliferated equivalently to their "helped" counterparts. These data call into question the contention that CD4 T cells condition memory CD8 T cells during the primary response and indicate that the principal role of CD4 T cells in generating CD8 memory cells after infection is augmentation of proliferation or survival through costimulatory signals.     

Abrogation of anti-Pichinde virus cytotoxic T cell memory by cyclophosphamide and restoration by coinfection or interleukin 2

Previously, we demonstrated that memory cell-mediated immune responses can be generated in Pichinde virus (PV)-primed mice after secondary challenge in vivo with homologous virus. Further, treatment of mice with cyclophosphamide (CY) before primary infection with PV abrogated the generation of H-2-restricted, virus-specific cytotoxic T lymphocytes (CTL), and rechallenge of these mice was followed by neither a primary nor a secondary CTL response. Here, we demonstrate that this CY-induced block in memory anti-PV CTL generation was not due to establishment of a persistent infection. Interestingly, this CY-induced block in memory anti-PV CTL generation was overcome by secondarily coinfecting mice with PV and lymphocytic choriomeningitis virus (LCMV) or PV and Tacaribe virus. Secondary infection with LCMV or Tacaribe virus alone did not elicit anti-PV CTL. Coinfection resulted in the generation of a PV-specific memory CTL response as judged by maximal activity on day 4 after rechallenge. Co-infection with PV and vesicular stomatitis virus, an unrelated rhabdovirus, did not efficiently restore memory anti-PV CTL responses. Memory anti-PV CTL responses were also restored when interleukin 2 (IL 2)-containing supernatants were injected i.p. after rechallenge of CY-treated mice with PV. To demonstrate that IL 2 was the responsible lymphokine in these preparations, highly purified IL 2 was added to in vitro cultures of spleen cells from CY-treated PV-primed mice. In the presence of PV-infected syngeneic macrophages, addition of purified IL 2 resulted in a dose-dependent restoration of H-2-restricted anti-PV CTL activity. The CTL precursor (CTLp) frequency of CY-treated PV-primed mice was markedly decreased from that of normal PV-primed mice. Thus, the long-lasting block in the ability to generate a PV-specific memory CTL response after CY treatment appears to be due to both a lack of helper T cell activity and a significant reduction of CTLp. However, this block may be overcome by coinfecting with viruses that cross-react at the helper T cell level or by exogenous treatment with highly purified IL 2.     

Exhaustion of type I interferon response following an acute viral infection

Viral infections often cause a period of heightened susceptibility to a secondary infection but the cause of this phenomenon is unknown. We found that a primary viral infection in mice rapidly triggers an IFN-I-dependent partial activation state in the majority of B and T lymphocytes, which reverts to a resting phenotype within 5 days. When a secondary infection with an unrelated virus occurred 5 to 9 days after the primary infection, no recurrence of marked activation of lymphocytes was observed. This was not due to an inherent inability of the previously activated cells to undergo renewed partial activation, because they responded when challenged with virus after transfer into "naive" recipients. Instead, the failure to respond optimally resided in the original host's incapacity to mount an IFN-I response to the secondary infection during this time period. Thus, transient immunosuppression through exhaustion of IFN-I production during an acute viral infection creates a time period of enhanced susceptibility to secondary infection.     

Temporal segregation of 4-1BB versus CD28-mediated costimulation: 4-1BB ligand influences T cell numbers late in the primary response and regulates the size of the T cell memory response following influenza infection

In this report, we demonstrate that CD28(-/-) mice are severely impaired in the initial expansion of D(b)/NP366-374-specific CD8 T cells in response to influenza virus infection, whereas 4-1BB ligand (4-1BBL)(-/-) mice show no defect in primary T cell expansion to influenza virus. In contrast, 4-1BBL(-/-) mice show a decrease in D(b)/NP366-374-specific T cells late in the primary response. Upon secondary challenge with influenza virus, 4-1BBL(-/-) mice show a decrease in the number of D(b)/NP366-374-specific T cells compared to wild-type mice such that the level of the CD8 T cell expansion during the in vivo secondary response is reduced to the level of a primary response, with concomitant reduction of CTL effector function. In contrast, Ab responses, as well as secondary CD4 T cell responses, to influenza are unaffected by 4-1BBL deficiency. Thus, CD28 is critical for initial T cell expansion, whereas 4-1BB/4-1BBL signaling affects T cell numbers much later in the response and is essential for the survival and/or responsiveness of the memory CD8 T cell pool.     

Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and gammadelta T cells are involved in the protective immune response against viral challenge. We have now examined whether gammadelta T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRdelta(-/-) mice survived primary infection with WN virus compared with 80-85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of gammadelta T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, gammadelta T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8(+) T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8(+) T cells from TCRdelta(-/-) mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that gammadelta T cells directly link innate and adaptive immunity during WN virus infection.     

Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host

Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination. Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen. Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections. The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection. However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection. Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells. However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice. This situation occurred in response to lymphocytic choriomeningitis virus or L. monocytogenes infection and for CD8(+) T cell responses against multiple epitopes. The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells. Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response.     

CD8 T cell recall responses are regulated by the tissue tropism of the memory cell and pathogen

Whether memory CD8 T cells can be reactivated in nonlymphoid tissues is unclear. Using mice lacking the spleen, lymph nodes, or both, we show that the secondary T cell response, but not homeostatic maintenance of memory cells, required lymphoid tissue. Whereas primary and secondary CD8 T cell responses to vesicular stomatitis virus infection were lymph node dependent, responses to Listeria monocytogenes infection were driven primarily in the spleen. Memory cell subset reactivation was also regulated by location of the responding population and the pathogen. Thus, CD62Llow effector memory T cells (TEM) cells responded nearly as well as CD62Lhigh central memory T cells (TCM) and TCM cells after L. monocytogenes infection, and both subsets generated equivalent populations of secondary memory cells. In contrast, TCM cells, but not TEM cells, mounted a robust response to vesicular stomatitis virus infection. TCM and TEM cells also required lymphoid tissue to mount recall responses, and the bone marrow did not contribute significantly to the response of either subset. Our findings indicated that characteristics of the infectious agent and the migratory preferences of memory cells dictated the secondary lymphoid tissue requirement for the recall response to infection.     

Suppression by cyclosporin A of murine T-cell-mediated immunity against viruses in vivo and in vitro

The immunosuppressive effect of Cyclosporin A on T-cell-mediated antiviral immune responses was examined. When administered intraperitoneally CS-A abrogated anti-vaccinia virus, anti-lymphocytic choriomeningitis virus (LCMV), and anti-vesicular stomatitis virus (VSV) T-cell responses in a dose-dependent fashion. Usually 50-60 mg/kg were efficient in suppressing primary T-cell responses completely. In contrast, 10-20 mg/kg often enhanced T-cell responses significantly when compared with controls. Suppression was observed if CS-A treatment was started before virus injection and up to 12 hr after infection; CS-A given 24 hr after the virus still suppressed T-cell activity partially. A 50 mg/kg dose of CS-A suppressed secondary anti-vaccinia virus or anti-VSV T-cell responses in vivo by a factor of about 10. This dose suppressed the primary T-cell-dependent footpad swelling induced by local LCMV infection and prevented T-cell-mediated immunopathological death due to LCM when LCMV was injected intracerebrally. In addition, clearance of LCMV was delayed drastically by CS-A treatment. When added to cultures of in vivo-primed antiviral T cells that were restimulated in vitro, CS-A inhibited both proliferation as well as generation of virus-specific cytotoxic T cells in a dose-dependent way. The results show that in CS-A-treated mice primary and secondary antiviral T-cell responses are strongly inhibited; acute viral infections with cytopathic viruses may therefore be more dramatic. In contrast immunopathological T-cell-mediated disease caused by noncytopathic viruses such as LCMV may be prevented or attenuated.     

Bystander virus infection prolongs activated T cell survival

In animals, T cells often die rapidly after activation, unless activation occurs in the presence of inflammatory factors. To understand how such activated cells survive to participate in immune responses, we studied the effects of viral infection on T cells responding to an unrelated superantigen. Normal T cells activated by superantigen in uninfected mice died as a result of their activation, whereas T cells that were activated during vaccinia infection survived longer in vivo and in culture. This bystander effect of viral infection on activated T cells was independent of effects on the magnitude of the initial T cell response, on induction of Bcl-2 and Bcl-x, on T cell proliferation, and on Fas killing. The failure of such effects to predict the fate of activated T cells in vivo indicates that virus infections shape T cell responses via mechanisms that differ from those described previously. These mechanisms may contribute to the ability of viral infections to induce autoimmunity.     

CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help dependent, it remains controversial when the "help" is needed. In this study, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to vaccinia virus infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to vaccinia virus infection and may help to design effective vaccine strategies.     

The role of antigenic stimulation and cytotoxic T cell activity in regulating the long-term immunopathogenesis of HIV: mechanisms and clinical implications

This paper develops a predictive mathematical model of cell infection, host immune response and viral replication that reproduces observed long-term trends in human immunodeficiency virus (HIV) pathogenesis. Cell activation induced by repeated exposure to many different antigens is proposed as the principal mechanism of providing target cells for HIV infection and, hence, of CD4+ T cell depletion, with regulation of the overall T cell pool size causing concomitant CD8 pool increases. The model correctly predicts the cross-patient variability in disease progression, the rate of which is found to depend on the efficacy of anti-HIV cytotoxic T lymphocyte responses, overall viral pathogenicity and random effects. The model also predicts a variety of responses to anti-viral therapy, including episodic residual viral replication and discordant responses and we find that such effects can be suppressed by increasing the potency of treatment.     

Ablation of CD8 and CD4 T cell responses by high viral loads

To evaluate the impact of sustained viral loads on anti-viral T cell responses we compared responses that cleared acute lymphocytic choriomeningitis virus infection with those that were elicited but could not resolve chronic infection. During acute infection, as replicating virus was cleared, CD8 T cell responses were down-regulated, and a pool of resting memory cells developed. In chronically infected hosts, the failure to control the infection was associated with pronounced and prolonged activation of virus-specific CD8 T cells. Nevertheless, there was a progressive diminution of their effector activities as their capacity to produce first IL-2, then TNF-alpha, and finally IFN-gamma was lost. Chronic lymphocytic choriomeningitis virus infection was also associated with differential contraction of certain CD8 T cell responses, resulting in altered immunodominance. However, this altered immunodominance was not due to selective expansion of T cells expressing particular TCR Vbeta segments during chronic infection. High viral loads were not only associated with the ablation of CD8 T cell responses, but also with impaired production of IL-2 by virus-specific CD4 T cells. Taken together, our data show that sustained exposure to high viral loads results in the progressive functional inactivation of virus-specific T cell responses, which may further promote virus persistence.     

Adenovirus-vectored T cell vaccine for hepacivirus shows reduced effectiveness against a CD8 T cell escape variant in rats

There is an urgent need for a vaccine to prevent chronic infection by hepatitis C virus (HCV) and its many genetic variants. The first human vaccine trial, using recombinant viral vectors that stimulate pan-genotypic T cell responses against HCV non-structural proteins, failed to demonstrate efficacy despite significant preclinical promise. Understanding the factors that govern HCV T cell vaccine success is necessary for design of improved immunization strategies. Using a rat model of chronic rodent hepacivirus (RHV) infection, we assessed the impact of antigenic variation and immune escape upon success of a conceptually analogous RHV T cell vaccine. Naïve Lewis rats were vaccinated with a recombinant human adenovirus expressing RHV non-structural proteins (NS)3-5B and later challenged with a viral variant containing immune escape mutations within major histocompatibility complex (MHC) class I-restricted epitopes (escape virus). Whereas 7 of 11 (64%) rats cleared infection caused by wild-type RHV, only 3 of 12 (25%) were protected against heterologous challenge with escape virus. Uncontrolled replication of escape virus was associated with durable CD8 T cell responses targeting escaped epitopes alone. In contrast, clearance of escape virus correlated with CD4 T cell helper immunity and maintenance of CD8 T cell responses against intact viral epitopes. Interestingly, clearance of wild-type RHV infection after vaccination conferred enhanced protection against secondary challenge with escape virus. These results demonstrate that the efficacy of an RHV T cell vaccine is reduced when challenge virus contains escape mutations within MHC class I-restricted epitopes and that failure to sustain CD8 T cell responses against intact epitopes likely underlies immune failure in this setting. Further investigation of the immune responses that yield protection against diverse RHV challenges in this model may facilitate design of broadly effective HCV vaccines.     

Impaired T cell immunity in B cell-deficient mice following viral central nervous system infection

CD8(+) T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (muMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected muMT and wild-type mice. Following viral recrudescence in muMT mice, total CD8(+) T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-gamma ELISPOT analysis. Although overall T cell recruitment into the CNS of muMT mice was not impaired, discrepancies in frequencies of virus-specific CD8(+) T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of muMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-gamma as the primary anti viral factor early in infection. Reduced virus-specific CD8(+) T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4(+) T cell help. Thus, in addition to the lack of Ab, limited CD8(+) and CD4(+) T cell responses in muMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.     

Generation of memory cell-mediated immune responses after secondary infection of mice with pichinde virus

Pichinde virus (PV), a member of the arenavirus group, was found to elicit strong cell-mediated immune responses in various strains of mice. After primary i.v. inoculation, augmentation of natural killer (NK) cell activity occurred and peaked 3 to 4 days after infection. The NK response was followed by a second peak of cytotoxic activity that was found to be H-2 restricted, virus specific, and mediated by Thy-1.2+, Lyt-2.2+ lymphocytes. This cytotoxic T lymphocyte (CTL) response peaked 7 days post infection. Neutralizing antibodies were not detectable after PV infection of the mice. In light of this, we investigated the generation and kinetics of secondary cell-mediated immune responses after reinjection of homologous virus in vivo. Slight but significant augmentation of NK activity was observed 1 day after secondary virus challenge. As in the primary response, effectors of this NK activity rapidly became sensitive to anti-Thy-1.2 and complement treatment. NK activity rapidly returned to background levels and was followed by an anamnestic CTL response that peaked 4 days after reinjection of the virus. Thus, cell-mediated immune responses appeared more rapidly after secondary challenge in vivo, and the temporal relationship between NK and CTL generation was maintained. Both secondary NK and CTL responses were generated in mice that had been pretreated with cyclophosphamide (CY), suggesting that memory cell-mediated immune responses can be reactivated in vivo without undergoing cell division. In contrast, treatment with CY before primary infection delayed the appearance of virus-induced NK activity and abrogated the generation of H-2-restricted virus-specific CTL. Rechallenge of these CY-treated NK-primed mice resulted in the rapid generation of a secondary NK response that was not followed by either a primary or secondary CTL response. The data suggest that cells mediating a nonspecific effector function may possess specific memory. We discuss our results with respect to possible NK-CTL relationships.     

Cutting edge: inflammatory signals drive organ-specific autoimmunity to normally cross-tolerizing endogenous antigen

Links have been observed between infections and the development of autoimmunity. Proposed explanations include activation of self-Ag-bearing APC. Using a model system in which transgenic OVA is expressed in enterocytes, we showed that CD8 T cell recognition of cross-presented Ag in gut-associated lymph nodes was tolerogenic. However, concomitant infection with vesicular stomatitis virus encoding OVA abrogated tolerance and induced disease. We now show that following transfer of naive OT-I T cells, the addition of wild-type vesicular stomatitis virus, oral cholera toxin, or CD40 triggering can induce intestinal disease in transgenic mice. Tissue damage accompanied dramatic increases in cytokine release by activated OT-I cells in the intestine. The data indicated that products of antigenically unrelated infections can combine with cross-presented self-Ags on APC to prime autoaggressiveness, independent of additional Ag release. These results help explain how diverse pathogens, lacking any homology to self-proteins, could be causative agents in induction of organ-specific autoimmunity.     

Hidden epitopes emerge in secondary influenza virus-specific CD8+ T cell responses

Influenza A virus-specific CD8+ T cell responses in H2(b) mice are characterized by reproducible hierarchies. Compensation by the D(b)PB1-F2(62) epitope is apparent following infection with a variant H3N2 virus engineered to disrupt the prominent D(b)NP(366) and D(b)PA(224) epitopes (a double knockout or DKO). Analysis with a "triple" knockout (TKO) virus, which also compromises D(b)PB1-F2(62), did not reveal further compensation to the known residual, minor, and predicted epitopes. However, infection with this deletion mutant apparently switched protective immunity to an alternative Ab-mediated pathway. As expected, TKO virus clearance was significantly delayed in Ab-deficient MHC class II(-/-) and Ig(-/-) mice, which were much more susceptible following primary, intranasal infection with the TKO, but not DKO, virus. CD8+ T cell compensation was detected in DKO, but not TKO, infection of Ig-deficient mice, suggestive of cooperation among CD8+ T cell responses. However, after priming with a TKO H1N1 mutant, MHC II(-/-) mice survived secondary intranasal exposure to the comparable H3N2 TKO virus. Such prime/challenge experiments with the DKO and TKO viruses allowed the emergence of two previously unknown epitopes. The contrast between the absence of compensatory effect following primary exposure and the substantial clonal expansion after secondary challenge suggests that the key factor limiting the visibility of these "hidden" epitopes may be very low naive T cell precursor frequencies. Overall, these findings suggest that vaccine approaches using virus vectors to deliver an Ag may be optimized by disrupting key peptides in the normal CD8+ T cell response associated with common HLA types.     

Differential requirements for CD80/86-CD28 costimulation in primary and memory CD4 T cell responses to vaccinia virus

Vaccinia virus infection can confer immunity to smallpox by inducing potent T cell and antibody responses. While the CD8 T cell response to vaccinia virus has been well characterized, less is known about factors required for priming and memory for the CD4 T cells. Focusing on two recently described epitopes, we show that after intranasal infection, both I1L and L4R epitopes are co-dominant during the acute response, but the I1L epitope dominates during memory. CD4 T cell priming was intact in the absence of CD80/86, however secondary responses were reduced. This contrasts with our previous data showing CD80/86–CD28 interaction is required for optimal primary and memory CD8 T cell responses. The absence of CD80/86 also changed the immunodominance hierarchy during memory, with the I1L and L4R responses becoming co-dominant in knockout mice. These data highlight different costimulatory requirements for primary CD4 and CD8 T cell responses to vaccinia virus.     

Infection with seasonal influenza virus elicits CD4 T cells specific for genetically conserved epitopes that can be rapidly mobilized for protective immunity to pandemic H1N1 influenza virus

In recent years, influenza viruses with pandemic potential have been a major concern worldwide. One unresolved issue is how infection or vaccination with seasonal influenza virus strains influences the ability to mount a protective immune response to novel pandemic strains. In this study, we developed a mouse model of primary and secondary influenza infection by using a widely circulating seasonal H1N1 virus and the pandemic strain of H1N1 that emerged in Mexico in 2009, and we evaluated several key issues. First, using overlapping peptide libraries encompassing the entire translated sequences of 5 major influenza virus proteins, we assessed the specificity of CD4 T cell reactivity toward epitopes conserved among H1N1 viruses or unique to the seasonal or pandemic strain by enzyme-linked immunospot (ELISpot) assays. Our data show that CD4 T cells reactive to both virus-specific and genetically conserved epitopes are elicited, allowing separate tracking of these responses. Populations of cross-reactive CD4 T cells generated from seasonal influenza infection were found to expand earlier after secondary infection with the pandemic H1N1 virus than CD4 T cell populations specific for new epitopes. Coincident with this rapid CD4 T cell response was a potentiated neutralizing-antibody response to the pandemic strain and protection from the pathological effects of infection with the pandemic virus. This protection was not dependent on CD8 T cells. Together, our results indicate that exposure to seasonal vaccines and infection elicits CD4 T cells that promote the ability of the mammalian host to mount a protective immune response to pandemic strains of influenza virus.     

Rapid recruitment of virus-specific CD8 T cells restructures immunodominance during protective secondary responses

In this study we investigate the attributes of virus-specific memory CD8 T cells which most effectively control secondary infections. By rechallenging mice that had cleared primary lymphocytic choriomeningitis virus infections, we revealed that the secondary response is remarkably swift. Within 6 h following secondary infection, the production of gamma interferon becomes detectable directly ex vivo. During this protective phase of the secondary response, a very early elaboration of effector activities is preferentially exhibited by T cells specific for the viral NP396 epitope. This wave of activation contains the infection primarily before the initiation of the proliferative phase of the secondary response. Marked expansion is observed, but its magnitude differs depending on the epitope specificity of the responding cells; between 42 and 48 h following infection, approximately 70% of NP396-specific memory cells are in the S phase of the cell cycle, as assessed by bromodeoxyuridine incorporation studies. Epitope-dependent differences during the proliferative phase of the secondary response were confirmed by adoptive transfer studies with CFSE-labeled T cells. Although NP396-specific T cells typically dominate secondary responses, the broader multiepitope-specific population of antiviral T cells is beneficial for controlling a variant virus with an escape mutation in this epitope. These findings indicate that the induction and maintenance of a focused response contribute to the clearance of secondary infections; however, a more diverse pool of antiviral T cells facilitates long-term immunity to mutable pathogens.