Cytomegalovirus-Specific T Cells Persist at Very High Levels during Long-Term Antiretroviral Treatment of HIV Disease

Background

In healthy, HIV seronegative, CMV seropositive adults, a large proportion of T cells are CMV-specific. High-level CMV-specific T cell responses are associated with accelerated immunologic aging (“immunosenesence”) in the elderly population. The impact of untreated and treated HIV infection on the frequency of these cells remains undefined.

Methodology/Principal Findings

We measured the proportion of CD4+ and CD8+ T cells responding to CMV pp65 and IE proteins was measured using flow cytometry in 685 unique HIV seronegative and seropositive individuals. The proportion of CMV-specific CD8+ T cells was consistently higher in the HIV-seropositive subjects compared to the HIV-seronegative subjects. This HIV effect was observed even in patients who lacked measurable immunodeficiency. Among the HIV-seropositive subjects, CMV-specific CD8+ T cell responses were proportionately lower during recent infection, higher during chronic untreated infection and higher still during long-term antiretroviral treated infection. The CD8+ T cell response to just two CMV proteins (pp65 and IE) was approximately 6% during long-term therapy, which was over twice that seen in HIV-seronegative persons. CMV-specific CD4+ T cell responses followed the same trends, but the magnitude of the effect was smaller.

Conclusions/Significance

Long-term successfully treated HIV infected patients have remarkably high levels of CMV-specific effector cells. These levels are similar to that observed in the elderly, but occur at much younger ages. Future studies should focus on defining the potential role of the CMV-specific inflammatory response in non-AIDS morbidity and mortality, including immunosenescence.     

Differential CMV-specific CD8+ effector T cell responses in the lung allograft predominate over the blood during human primary infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient- (D+R-) individuals. In 15 D+R- LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA- during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA- phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.     

Effect of ageing on CMV-specific CD8 T cells from CMV seropositive healthy donors

Background  

Ageing is associated with changes in the immune system with substantial alterations in T-lymphocyte subsets. Cytomegalovirus (CMV) is one of the factors that affect functionality of T cells and the differentiation and large expansions of CMV pp65-specific T cells have been associated with impaired responses to other immune challenges. Moreover, the presence of clonal expansions of CMV-specific T cells may shrink the available repertoire for other antigens and contribute to the increased incidence of infectious diseases in the elderly. In this study, we analyse the effect of ageing on the phenotype and frequency of CMV pp65-specific CD8 T cell subsets according to the expression of CCR7, CD45RA, CD27, CD28, CD244 and CD85j.     

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA-peptide complexes

BACKGROUND: Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS: CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT: Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION: This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.     

Infrequent detection of HIV-1-specific, but not cytomegalovirus-specific, CD8(+) T cell responses in young HIV-1-infected infants.

Early potent combination antiretroviral therapies (ART) for HIV-1 infection can preserve or restore immune function, but control of viral replication early in infection may interfere with the development of HIV-1-specific immune responses. Using an IFN-gamma ELISPOT assay, we evaluated the breadth and intensity of HIV-1-specific CD8(+) T cell responses in 17 vertically infected infants who began ART at 1-23 mo of age. CMV-specific responses were also characterized in three infants coinfected with HIV-1 and CMV. Before ART, HIV-1-specific CD8(+) T cell responses were detected in two of 13 (15%) infants <6 mo of age. HIV-1-specific CD8(+) T cells became undetectable in these two infants after the control of viral replication. Intermittent HIV-1-specific responses were noted in six infants who did not experience durable control of viral replication. In contrast, HIV-1-specific responses were detected before ART in four of four infants >6 mo of age and became persistently undetectable in only one child. CMV-specific CD8(+) T cell responses were persistently detected in all HIV-1 and CMV coinfected infants. In conclusion, HIV-1-specific CD8(+) T cell responses were less commonly detected before therapy in young infants than in older infants. Suppression of viral replication appeared to interfere with the development and maintenance of HIV-1-specific CD8(+) T cell responses. The detection of CMV-specific responses in HIV-1 and CMV coinfected infants suggests a selective defect in the generation or maintenance of HIV-1-specific CD8(+) T cell responses. Therapeutic HIV-1 vaccine strategies in young infants may prolong the clinical benefit of ART by expanding the HIV-1-specific CD8(+) T cell pool.     

Persistent cytomegalovirus-specific memory responses in the lung allograft and blood following primary infection in lung transplant recipients

Primary CMV infection in lung transplant recipients (LTRs) is associated with increased mortality. We studied 22 donor CMV-positive, recipient-negative (D(+)R(-)) LTRs for the development of posttransplant CMV-specific immunity. We found that 13 of 22 D(+)R(-) LTRs (59.1%) seroconverted (CMV IgG Ab(+)). Using pooled peptides of the immunodominant CMV Ags pp65 and IE1, we detected CMV-specific CD8(+)IFN-gamma(+) T cells in the PBMC of 90% of seroconverted individuals following primary infection by intracellular cytokine staining. In contrast, few seroconverters had detectable CMV-specific CD4(+)IFN-gamma(+) T cells during viral latency. However, the majority of IgG(+) LTRs demonstrated CMV-specific CD4(+) and CD8(+) T cell proliferative responses from PBMC, with CD4(+)IFN-gamma(+) T cells detectable upon re-expansion. Examination of lung allograft mononuclear cells obtained by bronchoalveolar lavage revealed both CMV-specific CD4(+) and CD8(+)IFN-gamma(+) T cells, including patients from whom CD4(+)IFN-gamma(+) T cells were simultaneously undetectable in the PBMC, suggesting differential effector memory populations between these compartments. Moreover, both responses in the PBMC and lung allograft were found to persist, despite substantial immunosuppression, long after primary infection. Clinical correlation in this cohort demonstrated that the acquisition of CMV immunity was associated with freedom from CMV disease (p < or = 0.009) and preservation of allograft function (p < or = 0.02) compared with those who failed to develop CMV immunity. Together, our data reveal immunologic heterogeneity in D(+)R(-) LTRs, with the development and persistence of primary CMV responses that may provide clinical benefit.     

HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.     

Clonotypic structure of the human CD4+ memory T cell response to cytomegalovirus

High steady-state frequencies of CMV-specific CD4(+) memory T cells are maintained in CMV-exposed subjects, and these cells are thought to play a key role in the immunologic control of this permanent infection. However, the essential components of this response are poorly defined. Here, we report the use of a step-wise application of flow cytometric and molecular techniques to determine the number and size of the TCR Vbeta-defined clonotypes within freshly obtained CMV-specific CD4(+) memory T cell populations of four healthy, CMV-exposed human subjects. This analysis revealed a stable clonotypic hierarchy in which 1-3 dominant clonotypes are maintained in concert with more numerous subdominant and minor clonotypes. These dominant clonotypes accounted for 10-50% of the overall CMV response, and comprised from 0.3 to 4.0% of peripheral blood CD4(+) T cells. Two subjects displayed immunodominant responses to single epitopes within the CMV matrix phosphoprotein pp65; these single epitope responses were mediated by a single dominant clonotype in one subject, and by multiple subdominant and minor clonotypes in the other. Thus, the CMV-specific CD4(+) T cell memory repertoire in normal subjects is characterized by striking clonotypic dominance and the potential for epitope focusing, suggesting that primary responsibility for immunosurveillance against CMV reactivation rests with a handful of clones recognizing a limited array of CMV determinants. These data have important implications for the understanding of mechanisms by which a genetically stable chronic viral pathogen such as CMV is controlled, and offer possible insight into the failure of such control for a genetically flexible pathogen like HIV-1.     

Differential usage of cellular niches by cytomegalovirus versus EBV- and influenza virus-specific CD8+ T cells

Immunological memory provides long-term protection against reinfection or reactivation of pathogens. Murine memory T cell populations may be compressed following infections with new pathogens. Humans have to retain memory T cells directed against a variety of microbes for many decades. Under these circumstances, the effect of pathogens that mount robust T cell reactivity on the pre-existing memory directed against unrelated microbes is unknown. In this study, we studied peripheral blood memory CD8+ T cells directed against different viruses following primary CMV infection in renal transplant recipients. The entrance of CMV-specific CD8+ T cells expanded the Ag-primed CD8+ T cell compartment rather than competing for space with pre-existing memory T cells specific for persistent or cleared viruses. Neither numbers nor phenotype of EBV- or influenza-specific CD8+ T cells was altered by primary CMV infection. CMV-specific CD8+ T cells accumulated over time, resulting in increased total CD8+ T cell numbers. Additionally, they acquired a highly differentiated cytolytic phenotype that was clearly distinct from EBV- or influenza-reactive T cells. Thus, the human immune system appears to be flexible and is able to expand when encountering CMV. In view of the phenotypic differences between virus-specific T cells, this expansion may take place in cellular niches different from those occupied by EBV- or influenza-specific T cells, thereby preserving immunity to these pathogens.     

Differential evolution and stability of epitope-specific CD8(+) T cell responses in EBV infection

Murine models of lymphocytic choriomeningitis virus infection suggest that the memory CD8(+) T cell repertoire is reflective of the CD8(+) T cell repertoire generated during acute infection. Less is known regarding the evolution of CD8(+) T cell repertoires during human viral infections. We therefore examined epitope-specific CD8(+) T cell responses in a large cohort of individuals with acute through latent Epstein-Barr virus infection. Using 16 of 20 published EBV epitopes restricted by HLA-A2, HLA-A3 or HLA-B7, we showed that lytic cycle-specific CD8(+) T cell responses predominated during acute EBV infection. However, whereas HLA-A2(+)-restricted BMLF-1-specific CD8(+) T cell responses were maintained through latency, HLA-A2(+)- and HLA-B7(+)-restricted BZLF-1, as well as HLA-A3(+)-restricted BRLF-1 CD8(+) T cell responses, were generated but not readily maintained. Analyses of CD8(+) T cell responses to EBV latent cycle Ags showed delayed detection and lower frequencies of latent epitope-specific CD8(+) T cell responses during acute EBV infection, with maintenance of these responses 1 yr post-EBV infection. Early BMLF-1 and EBNA-3A epitope-specific CD8(+) T cell frequencies did not correlate with their frequencies at 1 yr postinfection. Interestingly, populations of EBV-specific CD8(+) T cells were stable during 20 mo in our long term EBV-seropositive populations, suggesting homeostasis between virus and the host immune system. This study demonstrates that CD8(+) T cell repertoires generated during persistent viral infections are not simply reflective of the initial pool of CD8(+) T cells and provides evidence that the generation of CD8(+) T cell responses to a persistent infection is a dynamic process.     

Direct relationship between suppression of virus-specific immunity and emergence of cytomegalovirus disease in simian AIDS

Although opportunistic infections like cytomegalovirus (CMV) are common sequelae of end-stage AIDS, the immune events leading to CMV reactivation in human immunodeficiency virus (HIV)-infected individuals are not well defined. The role of cellular and humoral CMV-specific immune responses in immune control of latent CMV infection was evaluated prospectively in a cohort of 11 simian immunodeficiency virus (SIV)-infected CMV-seropositive rhesus macaques, 6 of whom had histologic evidence of CMV disease at death. Macaques with CMV disease differed from macaques without CMV disease in having significantly higher levels of plasma SIV RNA and CMV DNA and significantly lower titers of anti-CMV binding antibodies (Abs) at the time of death. A significant decline in anti-CMV Abs and CMV-specific CD4(+) and CD8(+) T lymphocytes over time was observed in the macaques with CMV disease, but not in the macaques without CMV disease. Reduction in CMV-specific CD8(+) T lymphocytes and anti-CMV neutralizing Abs was significantly correlated with a decline in CMV-specific CD4(+) T lymphocytes. Although declines in CMV-specific T lymphocytes alone were sufficient for reactivation of low-level CMV viremia, high-level viremia (>1,000 copies of CMV DNA per ml of plasma) was observed when anti-CMV neutralizing and binding Abs had also declined. Thus, the occurrence of CMV reactivation-associated disease in AIDS is associated with suppression of both cellular and humoral CMV-specific immune responses. The underlying mechanism may be a dysfunction of memory B and CD8(+) T lymphocytes associated with SIV-induced impairment of CMV-specific CD4(+) T-cell help.     

Changes in peripheral immune cell numbers and functions in octogenarian walkers – an acute exercise study

Background

Age-related changes of the immune system, termed immunosenescence, may underlie the increased risk of infections and morbidity in the elderly. Little is known about the effects of acute exercise on peripheral immune parameters in octogenarians. Therefore, we investigated acute exercise-induced changes in phenotype and function of the immune system in octogenarians participating in the 2013 edition of the Nijmegen Four Days Marches. Blood sampling was performed at baseline and immediately after 4 days of the walking exercise (30 km/day). A comprehensive set of adaptive and innate immune traits were enumerated and analyzed by flow-cytometry. Peripheral blood mononuclear cells, isolated before and after walking were stimulated with LPS and supernatants were analysed for IL-1β, IL-6, IL-8 and TNF-α concentrations by ELISA. CMV serostatus was determined by ELISA.

Results

The walking exercise induced a clear leucocytosis with numerical increases of granulocytes, monocytes and lymphocytes. These exercise-induced changes were most profound in CMV seropositive subjects. Within lymphocytes, numerical increases of particularly CD4+ T cells were noted. Further T cell differentiation analysis revealed profound increases of naïve CD4+ T cells, including naïve Treg. Significant increases were also noted for CD4+ memory T cell subsets. In contrast, only slight increases in naïve and memory CD8+ T cell subsets were detected. Exercise did not affect markers of immune exhaustion in memory T cell subsets. NK cells demonstrated a numerical decline and a change in cellular composition with a selective decrease of the mature CD56^dim NK cells. The latter was seen in CMV seronegative subjects only. Also, a higher IL-6 and IL-8 production capacity of LPS-stimulated PBMC was seen after walking.

Conclusion

In this exceptional cohort of octogenarian walkers, acute exercise induced changes in immune cell numbers and functions. A clear response of CD4+ T cells, rather than CD8+ T cells or NK cells was noted. Remarkably, the response to exercise within the CD4+ T cell compartment was dominated by naïve CD4+ subsets.

     

Levels of CMV specific CD4 T cells are dynamic and correlate with CMV viremia after allogeneic stem cell transplantation

Cytomegalovirus (CMV) infection is the most frequent viral complication in patients after allogeneic stem cell transplantation. As CMV replication is tightly controlled by the cellular arm of specific immunity, the kinetics of CMV-specific T cells in association with individual reactivation episodes were prospectively analyzed in 40 allogeneic transplant recipients in a routine clinical setting and evaluated as determinant of impaired CMV control. Antigen-specific CD4 and CD8 T cells were quantified directly from whole blood using intracellular cytokine staining after specific stimulation and MHC class I multimers, respectively. Highly dynamic intraindividual changes of CMV-specific CD4 T cells were observed in patients experiencing CMV viremia. Episodes of CMV reactivation were associated with a drop of CMV-specific CD4 T cells that re-increased after viral clearance (p<0.0001). Furthermore, levels of CMV-specific CD4 T cells at the onset of viremia inversely correlated with peak viral load thereafter (p = 0.02). In contrast, CMV-peptide specific CD8 T cells did not show any association with viremia (p = 0.82). Interestingly, therapeutic dosages of cyclosporine A and corticosteroids led to a dose-dependent reduction of CMV-specific T-cell functions, indicating a causal link between intensified immunosuppressive treatment and CMV reactivation. In conclusion, levels of CMV-specific CD4 T cells inversely correlate with reactivation episodes and may represent a valuable measure to individually guide antiviral therapy after stem cell transplantation.     

Polyfunctional T cells accumulate in large human cytomegalovirus-specific T cell responses

Large cytomegalovirus (CMV)-specific CD8 T-cell responses are observed in both young and, somewhat more often, old people. Frequent CMV reactivation is thought to exhaust these cells and render them dysfunctional so that larger numbers of them are needed to control CMV. Expansions of CMV-specific CD4 T cells are also seen but are less well studied. In this study, we examined the T-cell response to the dominant CMV pp65 and IE-1 antigens in healthy CMV-infected people across a wide age range (20 to 84 years) by using multicolor flow cytometry. CMV-specific T cells were characterized by the activation markers CD40 ligand (CD40L), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) and the memory markers CD27 and CD45RA. The proportions of effector memory T cells increased in large responses, as did the proportions of polyfunctional CD8 (IFN-γ⁺ IL-2^+/− TNF-α⁺) and CD4 (CD40L^+/− IFN-γ⁺ IL-2⁺ TNF-α⁺) T-cell subsets, while the proportion of naïve T cells decreased. The bigger the CD4 or CD8 T-cell response to pp65, the larger was the proportion of T cells with an advanced memory phenotype in the entire (including non-CMV-specific) T-cell compartment. In addition, the number of activation markers per cell correlated with the degree of T-cell receptor downregulation, suggesting increased antigen sensitivity in polyfunctional cells. In summary, our findings show that polyfunctional CMV-specific T cells were not superseded by dysfunctional cells, even in very large responses. At the same time, however, the memory subset composition of the entire T-cell compartment correlated with the size of the T-cell response to CMV pp65, confirming a strong effect of CMV infection on the immune systems of some, but not all, infected people.     

A bivalent CMV vaccine formulated with human compatible TLR9 agonist CpG1018 elicits potent cellular and humoral immunity in HLA expressing mice

There is now convincing evidence that the successful development of an effective CMV vaccine will require improved formulation and adjuvant selection that is capable of inducing both humoral and cellular immune responses. Here, we have designed a novel bivalent subunit vaccine formulation based on CMV-encoded oligomeric glycoprotein B (gB) and polyepitope protein in combination with human compatible TLR9 agonist CpG1018. The polyepitope protein includes multiple minimal HLA class I-restricted CD8⁺ T cell epitopes from different antigens of CMV. This subunit vaccine generated durable anti-viral antibodies, CMV-specific CD4⁺ and CD8⁺ T cell responses in multiple HLA expressing mice. Antibody responses included broad T_H1 isotypes (IgG2a, IgG2b and IgG3) and potently neutralized CMV infection in fibroblasts and epithelial cells. Furthermore, polyfunctional antigen-specific T cell immunity and antiviral antibody responses showed long-term memory maintenance. These observations argue that this novel vaccine strategy, if applied to humans, could facilitate the generation of robust humoral and cellular immune responses which may be more effective in preventing CMV-associated complications in various clinical settings.     

Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8+ T cells

Recent studies using MHC class I tetramers have shown that CD8(+) T cell responses against different persistent viruses vary considerably in magnitude and phenotype. At one extreme, hepatitis C virus (HCV)-specific CD8(+) T cell responses in blood are generally weak and have a phenotype that is perforin low and CCR7 high (early memory). At the other, specific responses to CMV are strong, perforin high, and CCR7 low (mature or effector memory). To examine the potential mechanisms behind this diversity, we compared CMV-specific responses in HCV-infected and healthy individuals. We find a striking difference in the phenotype of CMV-specific CD8(+) T cells between these groups. In the HCV-infected cohort, CMV-specific CD8(+) T cells lost markers associated with maturity; they had increased expression of CCR7 and reduced expression of Fas and perforin. They nevertheless responded to Ag in vitro in a manner similar to controls, with strong proliferation and appropriate acquisition of effector memory markers. The reduction in mature CD8 T cells in HCV-infected individuals may arise through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. Whatever the mechanism, HCV has a pervasive influence on the circulating CD8(+) T cell population, a novel feature that may be a hallmark of this infection.     

Differential B7-CD28 costimulatory requirements for stable and inflationary mouse cytomegalovirus-specific memory CD8 T cell populations

CMV establishes a lifelong persistent infection, and viral immune-modulating strategies are important in facilitating this. A particularly diverse CD8 T cell response develops as a result of this host-virus détente, with the CMV-specific memory T cell pool displaying unique functions and phenotypes. To gain insight into the factors that regulate CMV-specific CD8 T cell responses, we examined the influence of the B7-CD28 costimulatory pathway on magnitude, kinetics, and phenotype. Initial expansion of mouse CMV-specific CD8 T cells that establish stable memory pools was severely lower in mice lacking B7-CD28 signaling, and the resulting memory levels also remained reduced during persistent/latent infection. In contrast, expansion of CD8 T cells that undergo memory inflation during chronic infection was less affected in the absence of B7-CD28 costimulatory signals, eventually reaching the levels seen in wild-type mice at later times. Regardless of their differential requirements for B7-CD28 signals, both stable and inflationary memory T cell populations showed normal cytotoxic capacity. These results reveal that B7-CD28 costimulation differentially regulates the magnitude and kinetics of the multifaceted CD8 T cell response that develops during CMV infection.