Immune checkpoint therapy (ICT) has the potency to eradicate cancer but the mechanisms that determine effective versus non-effective therapy-induced immune responses are not fully understood. Here, using high-dimensional single-cell profiling we examined whether T cell states in the blood circulation could predict responsiveness to a combined ICT, sequentially targeting OX40 costimulatory and PD-1 inhibitory pathways, which effectively eradicated syngeneic mouse tumors. Unbiased assessment of transcriptomic alterations by single-cell RNA sequencing and profiling of cell-surface protein expression by mass cytometry revealed unique activation states for therapy-responsive CD4 + and CD8 + T cells. Effective ICT elicited T cells with dynamic expression of distinct NK cell and chemokine receptors, and these cells were systemically present in lymphoid tissues and in the tumor. Moreover, NK cell receptor-expressing CD8 + T cells were also present in the peripheral blood of immunotherapy-responsive cancer patients. Targeting of the NK cell and chemokine receptors in tumor-bearing mice showed their functional importance for therapy-induced anti-tumor immunity. These findings provide a better understanding of ICT and highlight the use of dynamic biomarkers on effector CD4 + and CD8 + T cells to improve cancer immunotherapy.

Background CD8+ T cells play a central role in immune protection against infectious and malignant disease, and their activation and differentiation are regulated by the spatio-temporal engagement of co-stimulatory and inhibitory receptors. These receptors constitute key targets for the therapeutic modulation of T cell responses, as evidenced by the clinical success of immune checkpoint inhibitors (ICI) in some cancer patients. Strategies aiming improve the efficacy and breadth of ICI therapy include the combination with agonistic targeting of co-stimulatory receptors. However, it remains incompletely understood how different co-stimulatory pathways interact to induce efficacious T cell responses. Methods and Results By interrogating the co-stimulatory requirements for effective CD8+ T cell activation using kinase activity profiling, we found that engagement of the co-stimulatory receptors CD27 and CD28, individually or in combination, had differential impacts on intracellular signaling pathways. Moreover, CD27-CD70 and CD28-CD80/86 co-stimulatory pathways differentially impacted the transcriptional program of CD8+ T cells following T cell receptor engagement, including Il2 expression. Importantly, signaling via CD27 and CD28 distinctly contributed to de novo transcription and post-transcriptional regulation of Il2 mRNA, respectively. Expression and nuclear translocation of the transcription factor c-Rel, a NF-kB family member critical for IL-2 transcription, was differently regulated by CD27- and CD28-mediated co-stimulatory signals, with full induction requiring collective signaling. Using co-stimulation-deficient systems, we found that cytokine production, particularly IL-2 secretion, by virus-specific CD8+ T cells was collectively dependent on both the CD27-CD70 and CD28-CD80/86 co-stimulatory pathways. This co-stimulation-dependent effect on the quality of antigen-specific CD8+ T cells was programmed early after viral infection, and persisted long-term. In line with the autocrine role of IL-2, deficient co-stimulation via CD27 and CD28 severely impaired T cell expansion during primary and memory responses, which could be partially rescued by constitutive IL-2 expression. Conclusions Collectively, our results demonstrate that the CD27-CD70 and CD28-CD80/86 co-stimulatory pathways act in a distinct, yet collaborative manner to instruct effective cytokine and proliferative responses of CD8+ T cells. These findings may inform the conception and development of immunotherapies harnessing the protective capacity of CD8+ T cells, including novel vaccine strategies, agonistic targeting of co-stimulatory receptors, and adoptive cell therapies.

Human cytomegalovirus (HCMV) is an ubiquitous herpesvirus that can cause serious morbidity and mortality in immunocompromised or immune-immature individuals. A vaccine that induces immunity to CMV in these target populations is therefore highly needed. Previous attempts to generate efficacious CMV vaccines primarily focused on the induction of humoral immunity by eliciting neutralizing antibodies. Current insights encourage that a protective immune response to HCMV might benefit from the induction of virus-specific T cells. Whether addition of antiviral T cell responses enhances the protection by antibody-eliciting vaccines is however unclear. Here, we assessed this query in mouse CMV (MCMV) infection models by developing synthetic vaccines with humoral immunity potential, and deliberately adding antiviral CD8+ T cells. To induce antibodies against MCMV, we developed a DNA vaccine encoding either full-length, membrane bound glycoprotein B (gB) or a secreted variant lacking the transmembrane and intracellular domain (secreted (s)gB). Intradermal immunization with an increasing dose schedule of sgB and booster immunization provided robust viral-specific IgG responses and viral control. Combined vaccination of the sgB DNA vaccine with synthetic long peptides (SLP)-vaccines encoding MHC class I-restricted CMV epitopes, which elicit exclusively CD8+ T cell responses, significantly enhanced antiviral immunity. Thus, the combination of antibody and CD8+ T cell-eliciting vaccines provides a collaborative improvement of humoral and cellular immunity enabling enhanced protection against CMV.

Comprehensive analysis of CD8(+) T cell populations specific to cytomegalovirus reveals that the evolution of the T cell antigen receptor repertoire during chronic infections is characterized by the expansion of low-affinity clones.

The relationship between human cytomegalovirus (HCMV) infections and accelerated immune senescence is controversial. Whereas some studies reported a CMV-associated impaired capacity to control heterologous infections at old age, other studies could not confirm this. We hypothesized that these discrepancies might relate to the variability in the infectious dose of CMV occurring in real life. Here, we investigated the influence of persistent CMV infection on immune perturbations and specifically addressed the role of the infectious dose on the contribution of CMV to accelerated immune senescence. We show in experimental mouse models that the degree of mouse CMV (MCMV)-specific memory CD8+ T cell accumulation and the phenotypic T cell profile are directly influenced by the infectious dose, and data on HCMV-specific T cells indicate a similar connection. Detailed cluster analysis of the memory CD8+ T cell development showed that high-dose infection causes a differentiation pathway that progresses faster throughout the life span of the host, suggesting a virus-host balance that is influenced by aging and infectious dose. Importantly, short-term MCMV infection in adult mice is not disadvantageous for heterologous superinfection with lymphocytic choriomeningitis virus (LCMV). However, following long-term CMV infection the strength of the CD8+ T cell immunity to LCMV superinfection was affected by the initial CMV infectious dose, wherein a high infectious dose was found to be a prerequisite for impaired heterologous immunity. Altogether our results underscore the importance of stratification based on the size and differentiation of the CMV-specific memory T cell pools for the impact on immune senescence, and indicate that reduction of the latent/lytic viral load can be beneficial to diminish CMV-associated immune senescence.

Adaptive immunity is initiated by T cell recognition of specific antigens presented by major histocompatibility complexes (MHCs). MHC multimer technology has been developed for the detection, isolation, and characterization of T cells in infection, autoimmunity, and cancer. Here, we present a simple, fast, flexible, and efficient method to generate many different MHC class I (MHC I) multimers in parallel using temperature-mediated peptide exchange. We designed conditional peptides for HLA-A*02:01 and H-2Kb that form stable peptide–MHC I complexes at low temperatures, but dissociate when exposed to a defined elevated temperature. The resulting conditional MHC I complexes, either alone or prepared as ready-to-use multimers, can swiftly be loaded with peptides of choice without additional handling and within a short time frame. We demonstrate the ease and flexibility of this approach by monitoring the antiviral immune constitution in an allogeneic stem cell transplant recipient and by analyzing CD8+ T cell responses to viral epitopes in mice infected with lymphocytic choriomeningitis virus or cytomegalovirus.

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti-tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide-peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MTTE antibodies with MTTE-containing conjugates are able to induce DC and T cell activation using model antigens.

Primary cytomegalovirus (CMV) infection leads to strong innate and adaptive immune responses against the virus, which prevents serious disease. However, CMV infection can cause serious morbidity and mortality in individuals who are immunocompromised. The adaptive immune response to CMV is characterized by large populations of effector-memory (EM) T cells that are maintained lifelong, a process termed memory inflation. Recent findings indicate that infection with CMV leads to continuous differentiation of CMV-specific EM-like T cells and that high-dose infection accelerates this progression. Whether measures that counteract CMV infection, such as anti-viral drugs, targeting of latently infected cells, adoptive transfer of CMV-specific T cells, and vaccination strategies, are able to impact the progressive differentiation of CMV-specific EM-like cells is discussed.

Cytomegalovirus (CMV)-based vaccine vectors are promising vaccine platforms because they induce strong and long-lasting immune responses. Recently it has been shown that vaccination with a mouse CMV (MCMV) vector expressing the melanoma-specific antigen TRP2 (MCMV-TRP2) protects mice against outgrowth of TRP2-positive B16 melanoma tumors, and this protection was dependent on the induction of IgG antibodies. Here we demonstrate that, although mice lacking all receptors for the Fc part of IgG (FcγRs) develop normal IgG responses after MCMV-TRP2 vaccination, the protection against B16 melanoma was completely abrogated, indicating that FcγRs are indispensable in the downstream effector pathway of the polyclonal anti-TRP2 antibody response. By investigating compound FcγR-deficient mouse strains and by using immune cell type-specific cell ablation we show that the IgG antibody-mediated tumor protection elicited by MCMV-TRP2 mainly depends on FcγRI expression on macrophages, whereas FcγRIV plays only a modest role. Thus, tumor-specific antibody therapy might benefit from combination therapy that recruits FcγRI-expressing pro-inflammatory macrophages to the tumor micro-environment.

Protection against a malaria infection can be achieved by immunization with live-attenuated Plasmodium sporozoites and while the precise mechanisms of protection remain unknown, T cell responses are thought to be critical in the elimination of infected liver cells. In cancer immunotherapies, agonistic antibodies that target T cell surface proteins, such as CD27, OX40 (CD134) and 4-1BB (CD137), have been used to enhance T cell function by increasing co-stimulation. In this study, we have analyzed the effect of agonistic OX40 monoclonal antibody treatment on protective immunity induced in mice immunized with genetically attenuated parasites (GAPs). OX40 stimulation enhanced protective immunity after vaccination as shown by an increase in the number of protected mice and delay to blood-stage infection after challenge with wild-type sporozoites. Consistent with the enhanced protective immunity enforced OX40 stimulation resulted in an increased expansion of antigen-experienced effector (CD11ahiCD44hi) CD8+ and CD4+ T cells in the liver and spleen and also increased IFN-γ and TNF producing CD4+ T cells in the liver and spleen. In addition, GAP immunization plus α-OX40 treatment significantly increased sporozoite-specific IgG responses. Thus, we demonstrate that targeting T cell costimulatory receptors can improve sporozoite-based vaccine efficacy.

Plasmacytoid dendritic cells (pDCs) are antigen presenting cells specialized in viral recognition through Toll-like receptor (TLR)7 and TLR9, and produce vast amounts of interferon alpha upon ligation of these TLRs. We had previously demonstrated a strong influx of pDCs in the tubulointerstitium of renal biopsies at the time of acute rejection. However, the role of human pDCs in mediating acute or chronic allograft rejection remains elusive. pDCs are thought to have a limited capacity to ingest apoptotic cells, critical for inducing CD4+ T cell activation via indirect antigen presentation and subsequent activation of antibody producing B cells. Here we tested whether the function of pDCs is affected by their presence within the graft. Maturation and interferon alpha production by pDCs was enhanced when cells were activated in the presence of viable HK2 renal epithelial cells. Importantly, soluble factors produced by cytomegalovirus-infected (primary) epithelial or endothelial cells enhanced pDC activation and induced their capacity to phagocytose apoptotic cells. Phagocytosis was not induced by free virus or soluble factors from non-infected cells. Activated pDCs showed an enhanced CD4+ and CD8+ T cell allostimulatory capacity as well as a potent indirect alloantigen presentation. Granulocyte Macrophage-Colony Stimulating Factor is one of the soluble factors produced by renal epithelial cells that, combined with TLR9 ligation, induced this functional capacity. Thus, pDCs present in the rejecting allograft can contribute to alloimmunity and potentially act as important orchestrators in the manifestation of acute and chronic rejection.

Human cytomegalovirus (HCMV) encodes numerous proteins and microRNAs that function to evade the immune response and allow the virus to replicate and disseminate in the face of a competent innate and acquired immune system. The establishment of a latent infection by CMV, which if completely quiescent at the level of viral gene expression would represent an ultimate in immune evasion strategies, is not sufficient for lifelong persistence and dissemination of the virus. CMV needs to reactivate and replicate in a lytic cycle of infection in order to disseminate further, which occurs in the face of a fully primed secondary immune response. Without reactivation, latency itself would be redundant for the virus. It is also becoming clear that latency is not a totally quiescent state, but is characterized by limited viral gene expression. Therefore, the virus also needs immune evasion strategies during latency. An effective immune response to CMV is required or viral replication will cause morbidity and ultimately mortality in the host. There is clearly a complex balance between virus immune evasion and host immune recognition over a lifetime. This poses the important question of whether long-term evasion or manipulation of the immune response driven by CMV is detrimental to health. In this meeting report, three groups used the murine model of CMV (MCMV) to examine if the contribution of the virus to immune senescence is set by the (i) initial viral inoculum, (ii) inflation of T cell responses, (iii) or the balance between functionally distinct effector CD4+ T cells. The work of other groups studying the CMV response in humans is discussed. Their work asks whether the ability to make immune responses to new antigens is compromised by (i) age and HCMV carriage, (ii) long-term exposure to HCMV giving rise to an overall immunosuppressive environment and increased levels of latent virus, or (iii) adapted virus mutants (used as potential vaccines) that have the capacity to elicit conventional and unconventional T cell responses.

The phenotype and functionality that CD8+ T cells acquire upon encountering their cognate antigen depend on many factors that are controlled by the nature of the pathogen The acquired phenotypical and functional characteristics determine the potential of the CD8+ T cells to form bona fide memory populations that are able to expand upon secondary challenge. We investigated factors that control CD8+ T cell differentiation. We have shown that the height of the viral dose greatly impacts the immunological outcome by affecting the magnitude of the response and the phenotype and function of the (memory) CD8+ T cells that are induced. The acquired phenotype and functions determine the capacity to expand upon re-encounter with the same pathogen and we have shown that the IL-2 producing capacity of CD8+ T cells functions as a crucial determinant of CD8+ T cell expansion potential. Although we addressed only a small fraction of all variables involved in the induction and maintenance of antigen-specific T cell populations our observations and insights gathered by others already provide a basis for rational immunotherapeutic treatment design, however, for fine-tuning of therapy-induced immune responses aspects about e.g. the timing and cooperating signals still need to be understood in more detail.

Adoptive cellular therapy (ACT) is a form of immunotherapy whereby antigen-specific T cells are isolated or engineered, expanded ex vivo, and transferred back to patients. Clinical benefit after ACT has been obtained in treatment of infection, various hematological malignancies, and some solid tumors; however, due to poor functionality and persistence of the transferred T cells, the efficacy of ACT in the treatment of most solid tumors is often marginal. Hence, much effort is undertaken to improve T cell function and persistence in ACT and significant progress is being made. Herein, we will review strategies to improve ACT success rates in the treatment of cancer and infection. We will deliberate on the most favorable phenotype for the tumor-specific T cells that are infused into patients and on how to obtain T cells bearing this phenotype by applying novel ex vivo culture methods. Moreover, we will discuss T cell function and persistence after transfer into patients and how these factors can be manipulated by means of providing costimulatory signals, cytokines, blocking antibodies to inhibitory molecules, and vaccination. Incorporation of these T cell stimulation strategies and combinations of the different treatment modalities are likely to improve clinical response rates further.

There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD8+ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8+ T cells (KLRG1hi, CD44hi, CD127lo, CD62Llo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8+ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8+ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8+ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease.

ABSTRACT Antibodies are implicated in long-term immunity against numerous pathogens, and because of this property, antibody induction is the basis for many vaccines. Little is known about the influence of viral persistence on the evolving antibody response. Here, we examined the characteristics of antibody responses to persistent infection by employing the prototypic betaherpesvirus family member cytomegalovirus (CMV) in experimental mouse models. During the course of infection, mouse CMV (MCMV)-specific IgM and IgG responses are elicited; however, IgG levels gradually inflate in the persistent phase of infection while IgM levels are stably maintained. Whereas CD27-CD70 interactions are dispensable, the CD28/B7 costimulatory pathway is critical for the class switching of MCMV-specific IgM-to-IgG B cell responses, which corresponds to the CD28/B7-dependent formation of CD4 + T follicular helper cells (T FH ) and germinal center (GC) B cells. Furthermore, the initial viral inoculum dose dictates the height of the antibody levels during IgG antibody inflation and relates to the induction of long-lived plasma cells and memory B cells. Antibody avidity nonetheless is not altered after the establishment of viral persistence and occurs independently of the inoculum doses. However, repetitive challenge with intact viral particles, accompanied by increased GC reactivity, promotes the development of high-avidity IgG responses with neutralizing capacity. These insights can be used for the rational design of CMV-based vaccines aimed at inducing antibody responses. IMPORTANCE Antibodies provide long-term protection to different pathogens. However, how antibody responses develop during persistent virus infection is not entirely clear. Here, we characterize factors that influence the virus-specific antibody response to persistent CMV. This study describes that during persistent infection, CMV-specific IgM antibody levels are stably maintained while IgG2b and IgG2c levels gradually inflate over time. In contrast, the IgG avidity remains similar after the establishment of viral persistence. The induction of T follicular helper cells and GC B cells requires CD4 + T cell help and CD28/B7 costimulation signals and is essential for the development of CMV-specific IgG antibody responses. Furthermore, neutralizing CMV-specific antibodies appear to develop late after infection, yet the neutralizing capacity can be improved upon repetitive viral challenge that is associated with increased GC reactivity. The results described here could inform the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.

Adoptive cellular therapy (ACT) is a form of immunotherapy whereby antigen-specific T cells are isolated or engineered, expanded ex vivo, and transferred back to patients. Clinical benefit after ACT has been obtained in treatment of infection, various hematological malignancies, and some solid tumors; however, due to poor functionality and persistence of the transferred T cells, the efficacy of ACT in the treatment of most solid tumors is often marginal. Hence, much effort is undertaken to improve T cell function and persistence in ACT and significant progress is being made. Herein, we will review strategies to improve ACT success rates in the treatment of cancer and infection. We will deliberate on the most favorable phenotype for the tumor-specific T cells that are infused into patients and on how to obtain T cells bearing this phenotype by applying novel ex vivo culture methods. Moreover, we will discuss T cell function and persistence after transfer into patients and how these factors can be manipulated by means of providing costimulatory signals, cytokines, blocking antibodies to inhibitory molecules, and vaccination. Incorporation of these T cell stimulation strategies and combinations of the different treatment modalities are likely to improve clinical response rates further.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Signals delivered by costimulatory molecules are implicated in driving T cell expansion. The requirements for these signals, however, vary from dispensable to essential in different infections. We examined the underlying mechanisms of this differential T cell costimulation dependence and found that the viral context determined the dependence on CD28/B7-mediated costimulation for expansion of naive and memory CD8+ T cells, indicating that the requirement for costimulatory signals is not imprinted. Notably, related to the high-level costimulatory molecule expression induced by lymphocytic choriomeningitis virus (LCMV), CD28/B7-mediated costimulation was dispensable for accumulation of LCMV-specific CD8+ T cells because of redundancy with the costimulatory pathways induced by TNF receptor family members (i.e., CD27, OX40, and 4-1BB). Type I IFN signaling in viral-specific CD8+ T cells is slightly redundant with costimulatory signals. These results highlight that pathogen-specific conditions differentially and uniquely dictate the utilization of costimulatory pathways allowing shaping of effector and memory antigen-specific CD8+ T cell responses. https://doi.org/10.7554/eLife.07486.001 eLife digest When the immune system detects a virus in the body it mounts a response to eliminate it. Immune cells called CD8+ T cells detect fragments of virus proteins that are presented on the surface of other immune cells. The CD8+ T cells then rapidly divide to form populations that roam the body to kill cells that are infected with the virus. Afterwards, some of the CD8+ T cells become ‘memory T cells’, which allow the immune system to respond more rapidly if the virus returns. This means that a subsequent infection of the same virus is usually stopped before it can become severe enough for an individual to feel unwell. Vaccines take advantage of the activities of CD8+ T cells to enable a person to become ‘immune’ to a virus without having to experience the disease. Vaccines contain dead or weakened viruses that can't spread in the body, but are able to activate the CD8+ T cells. However, a vaccine may not be as effective in activating the T cells as the live virus, perhaps because it fails to trigger the production of other molecules in the host that promote T cell activation. There are many of these ‘co-stimulatory molecules’ in the body, but it is not clear exactly how they work. Now, Welten et al. show that the role of co-stimulatory molecules in the activation of CD8+ T cells depends on the type of virus and how it affects cells. Mice that were genetically engineered to lack two co-stimulatory molecules called CD80 and CD86 failed to accumulate active CD8+ T cells in response to infection with a herpes-like virus. However, if these mice were infected with a different virus called LCMV—which causes swelling of the brain and spinal cord—they produced many active CD8+ T cells to fight the infection. Welten et al. found that other co-stimulatory molecules are able to compensate for the loss of CD80 and CD86 to boost the activation of T cells in response to LCMV, but not the herpes-like virus. Further experiments showed that LCMV triggers a lot more inflammation in infected cells than the other virus. This leads to the production of many different types of co-stimulatory molecules, which ensures that if one fails to boost the activation of CD8+ T cells, another molecule can do so instead. Better understanding of how these co-stimulatory molecules work could help scientists to develop more effective vaccines in future. https://doi.org/10.7554/eLife.07486.002 Introduction CD8+ T cells are critical for elimination of various intracellular pathogens. By incorporating differences in TCR signal strength and duration (signal 1), the spatiotemporal availability of costimulatory molecules (signal 2) and defined cytokines in the inflammatory environment (signal 3), CD8+ T cells are differentially programmed for expansion and effector cell formation resulting in considerable plasticity of the response (Williams and Bevan, 2007; Arens and Schoenberger, 2010). Costimulatory molecules augment TCR triggering but also qualitatively contribute to achieve optimal T cell expansion and differentiation (Croft, 2003). CD28 is considered as the most prominent costimulatory receptor for T cells, but signals provided by members of the TNF receptor (TNFR) super family such as CD27, OX40 (CD134) and 4-1BB (CD137) are known to provide crucial signals as well. T cell responses seem to be differentially and contextually dependent on costimulatory interactions but the underlying mechanisms are unknown (DeBenedette et al., 1999; Welten et al., 2013a; Wortzman et al., 2013). For example, the pathogen-specific CD8+ T cell response during vesicular stomatitis virus and vaccinia virus (VV) infection is highly driven by interactions between CD28 and the B7 molecules B7.1 (CD80) and B7.2 (CD86) (Sigal et al., 1998; Bertram et al., 2002; Fuse et al., 2008), while in lymphocytic choriomeningitis virus (LCMV) infection the viral-specific CD8+ T cells seem to bypass the requirements of the CD28/B7 costimulatory pathway for primary effector T cell expansion (Shahinian et al., 1993; Kundig et al., 1996; Andreasen et al., 2000; Grujic et al., 2010; Eberlein et al., 2012). Even within a single infection distinct requirements for costimulatory signals can be observed. In mouse cytomegalovirus (MCMV), the classical (non-inflationary) CD8+ T cell responses are more dependent on the CD28/B7 costimulatory pathway than the so-called inflationary CD8+ T cells, which gradually accumulate at high frequencies in time (Arens et al., 2011b; O'Hara et al., 2012). Here we examined the mechanisms of CD8+ T cell costimulation dependency. We found that the pathogen-induced environment and not the characteristics of the viral epitopes determined the requirements of naive and of memory CD8+ T cells for CD28/B7-mediated costimulation. Remarkably, related to the induction of high costimulatory ligand expression, LCMV-specific CD8+ T cell expansion can operate in a CD28/B7 independent fashion because of redundancy with the costimulatory members of the TNFR superfamily. Furthermore, direct type I IFN signaling in viral-specific CD8+ T cells is slightly redundant with CD28/B7 and CD27/CD70-mediated costimulation. These findings demonstrate that the inflammatory environment dictates the characteristics of CD8+ T cell responses by allowing a differential utilization of stimulatory pathways. Results Differential requirements for CD28/B7-mediated costimulation in driving CD8+ T cell expansion Effector CD8+ T cell formation during LCMV infection seems not to be driven by the main costimulatory CD28/B7 pathway because wild-type (WT) mice and mice deficient in both B7.1 and B7.2 (Cd80/86−/−) mount similar antigen-specific responses in magnitude, and this phenomenon is apparent after both high and low viral inoculum dosages (Figure 1A). In contrast, during infection with VV or Listeria monocytogenes (LM), antigen-specific CD8+ T cell responses are highly reduced in the absence of B7-mediated costimulation (Figure 1B,C). CD8+ T cell responses against MCMV are dependent on B7-mediated costimulation as well, ranging from ∼sevenfold diminished responses in case of the non-inflationary M45 and M57-specific to ∼2.5-fold in case of the inflationary m139 and M38-specific responses (Figure 1D). Effector cell differentiation of virus-specific CD8+ T cells, indicated by the downregulation of CD62L and upregulation of CD44, also required B7-mediated costimulation in MCMV but not in LCMV infection (Figure 1—figure supplement 1). Thus, in various infections but not during LCMV infection the CD28/B7 costimulatory pathway is highly critical in driving T cell expansion. Figure 1 with 1 supplement see all Download asset Open asset Differential requirements for CD28/B7-mediated costimulation in driving pathogen-specific CD8+ T cell expansion. (A) Wild-type (WT) and Cd80/86−/− mice were infected with 2 × 102 (low dose) or 2 × 105 (high dose) PFU LCMV-Armstrong. The lymphocytic choriomeningitis virus (LCMV)-specific CD8+ T cell response in the spleen was determined 7 days post-infection. Representative flow cytometric plots show CD3+/CD8+ cells that were stained with CD44 antibodies and MHC class I tetramers (high dose infection). Percentages indicate tetramer+ cells within the CD8+ T cell population. Bar graph shows total number of splenic LCMV-specific CD8+ T cells. (B) Mice were infected with 2 × 105 PFU vaccinia virus (VV) WR and the percentage of tetramer+ cells within the CD8+ T cell population was determined in the blood 7 days post-infection. (C) The percentage of tetramer+ cells within the CD8+ T cell population was determined in the blood 7 days post-infection with 1 × 106 CFU LM-Quadvac. (D) Flow cytometric plots show a representative M45-specific tetramer staining of cells from WT and Cd80/86−/− mice at day 8 post-infection with 1 × 104 PFU mouse cytomegalovirus (MCMV). Cells are gated on CD3+/CD8+ and the percentages indicate tetramer+ cells within the CD3+/CD8+ T cell population. Bar graph indicates the total number of splenic MCMV-specific CD8+ T cells. Data in bar graphs are expressed as mean + standard error of the mean (SEM) (n = 5–12 mice per group) of at least two independent experiments. Fold difference and significance (*p < 0.05) is indicated. https://doi.org/10.7554/eLife.07486.003 Next, we examined if additional triggering of the CD28/B7 costimulatory pathway is able to differentially modulate effector T cell formation. Therefore, the co-inhibitory receptor CTLA-4 that binds to B7.1 and B7.2 was blocked with antibodies during infection, which increases the availability of the B7 molecules to stimulate CD28. Remarkably, CTLA-4 blockade during LCMV infection had no effect on T cell expansion, indicating that LCMV-specific CD8+ T cells are rather indifferent to enhanced B7-mediated signals (Figure 2A,B). However, CTLA-4 blockade during MCMV infection augmented MCMV-specific CD8+ T cell responses ∼threefold in a B7-dependent manner (Figure 2C,D). Thus, additional triggering of the CD28/B7 pathway is beneficial in settings in which T cell expansion is dependent on this pathway, while the enhancement of CD28/B7-mediated costimulation had no effect in conditions in which the B7 costimulatory molecules are not essential for initial T cell expansion. Figure 2 Download asset Open asset CTLA-4 blockade impacts B7-driven CD8+ T cell responses. (A) CTLA-4 blocking antibodies were administrated during infection with 2 × 105 PFU LCMV Armstrong in WT mice. At day 7 post-infection, the splenic LCMV-specific response was analyzed by intracellular cytokine staining. Representative flow cytometric plots show intracellular IFN-γ vs cell-surface CD8 staining after restimulation with GP33-41 peptide. The percentage of IFN-γ+ cells within the CD8+ T cell population is indicated. (B) Total numbers of splenic LCMV-specific CD8+ T cells are shown. (C) CTLA-4 interactions were abrogated by administration of blocking antibodies in WT and Cd80/86−/− mice upon infection with 1 × 104 PFU MCMV, and at day 8 post-infection the virus-specific response was analyzed by intracellular cytokine staining. Representative flow cytometric plots show intracellular IFN-γ vs CD8 staining after restimulation of splenocytes with M45985-993 peptide. The percentage of IFN-γ+ cells within the CD8+ T cell population is indicated. (D) Total numbers of MCMV-specific CD8+ T cells in the spleen are shown. Data in bar graphs are expressed as mean + SEM (n = 4–5 mice per group) of at least two independent experiments. Fold difference and significance (*p < 0.05) is indicated. https://doi.org/10.7554/eLife.07486.005 The context of viral epitope expression determines the requirement for CD28/B7-mediated costimulation in driving T cell expansion To determine whether the characteristics of LCMV-specific epitopes define the B7-independent activation of CD8+ T cell responses, we analyzed the response to the immunodominant epitope GP33-41 of LCMV (GP33) in the context of different pathogen infections. Therefore, recombinant MCMVs were generated in which the GP33 epitope was expressed within the immediate early 2 (IE2) protein (MCMV-IE2-GP33) or the M45 protein (MCMV-M45-GP33). The in vitro replication kinetics of MCMV-IE2-GP33 and MCMV-M45-GP33 were similar as WT virus (Figure 3—figure supplement 1A). Correspondingly, in vivo infection with MCMV-IE2-GP33 induced a GP33-specific response with inflationary characteristics, as specified by a gradual increasing GP33-specific CD8+ T cell response in time with an effector memory phenotype (Figure 3—figure supplement 1B,C). As determined by intracellular IFN-γ staining after restimulation (Figure 3A,B) or direct staining with MHC class I tetramers (data not shown), the GP33-specific CD8+ T cell response elicited by both MCMV-IE2-GP33 and MCMV-M45-GP33 was dependent on B7-mediated costimulation, albeit to a higher degree when the GP33 epitope was inserted within the M45 protein. Infection with an MCMV containing the model epitope OVA257-264 (SIINFEKL) inserted in the M45 protein (MCMV-M45-SIINFEKL) resulted also in an antigen-specific T cell response that depended on B7-mediated costimulation (Figure 3C), indicating that non-viral epitopes elicit similar costimulation dependent responses. Furthermore, LM expressing the LCMV GP33 epitope (LM-GP33) induced GP33-specific CD8+ T cell responses that were highly dependent on B7-mediated costimulation (Figure 3D). Also, upon vaccination with a synthetic long peptide (SLP) containing the GP33 epitope, Cd80/86−/− mice mounted a defective GP33-specific CD8+ T cell response in comparison with WT mice (Figure 3—figure supplement 2). To exclude possible effects related to differences in the TCR repertoire selection, TCR transgenic CD8+ T cells recognizing the LCMV GP33 epitope (referred hereafter as P14 cells) were used in different pathogenic contexts. Similar as observed for the endogenous LCMV-specific CD8+ T cell expansion, B7-mediated costimulation was dispensable for P14 cell expansion in LCMV infection. Importantly, for the expansion of P14 cells in MCMV-IE2-GP33 and LM-GP33 infection, B7-mediated signals were highly required (Figure 3E), which corroborates that the inflammatory environment is predominantly determining the costimulatory requirements. Together, these data indicate that the context of viral epitope expression, rather than the intrinsic nature of the epitope or the antigen-specific CD8+ T cell population, influences the dependence on B7-mediated signals for T cell expansion. Figure 3 with 2 supplements see all Download asset Open asset The context of viral epitope expression determines the requirements for B7-mediated costimulation in driving antigen-specific CD8+ T cell expansion. (A, B) WT and Cd80/86−/− mice were infected with 1 × 105 PFU MCMV-IE2-GP33 or MCMV-M45-GP33, and 8 days post-infection the splenic GP33-specific CD8+ T cell response was determined by intracellular IFN-γ staining. Representative flow cytometric plots are shown and the percentage of IFN-γ+ cells within the CD8+ T cell population is indicated. Graphs indicate the total number of splenic GP33-specific CD8+ T cells. (C) The splenic SIINFKEL-specific CD8+ T cell response was determined by intracellular IFN-γ staining at day 8 post-infection with 1 × 105 PFU MCMV-M45-SIINFEKL. (D) WT and Cd80/86−/− mice were infected with 1.5 × 103 CFU LM-GP33. At day 7 post-infection the splenic GP33-specific response was analyzed by intracellular IFN-γ staining upon restimulation with GP33 peptide. Representative flow cytometric plots are shown. The percentage of GP33-specific CD8+ T cells within the total CD8+ T cell population is indicated. Bar graph shows the total number of splenic GP33-specific CD8+ T cells. (E) 5 × 104 CD90.1+ Ifnar1+/+ P14 cells were adoptively transferred into WT and Cd80/86−/− mice that were subsequently infected with 2 × 105 PFU LCMV Armstrong, 1 × 105 PFU MCMV-IE2-GP33 or 1.5 × 103 CFU LM-GP33. At day 7 (LCMV, LM-GP33) or 8 (MCMV-IE2-GP33) post-infection, the magnitude of the P14 cell response in the spleen was determined. Data in bar graphs are expressed as mean + SEM (n = 3–5 mice per group) of at least two independent experiments. Fold difference and significance (*p < 0.05) is indicated. https://doi.org/10.7554/eLife.07486.006 The context of viral epitope expression during secondary expansion determines the requirement for CD28/B7-mediated costimulation independent of the priming context Next, we examined whether the viral context imprints the costimulatory requirements for the lifespan of T cells or if memory T cells undergo secondary expansion independently of the priming context. Therefore, crisscross adoptive transfer experiments were performed with memory GP33-specific CD8+ T cells generated in different viral environments. First, memory GP33-specific CD8+ T cells were primed in the context of an LCMV infection and adoptively transferred into WT or Cd80/86−/− hosts that were subsequently infected with either LCMV or MCMV-IE2-GP33. Comparing the GP33-specific CD8+ T cell responses upon antigenic re-challenge revealed a dispensable role for B7-mediated signals for secondary T cell expansion in an LCMV environment, but a strong requirement for these signals in an MCMV context even though these cells were primed in an LCMV environment (Figure 4A). Importantly, when memory GP33-specific CD8+ T cells that depended on B7-mediated signals during priming in MCMV infection were transferred and re-challenged in an LCMV or an MCMV environment, the viral context during secondary expansion again determined the requirement on costimulatory signals (Figure 4B). Together, these data indicate that during secondary T cell responses the viral context is dominant and determines the CD28/B7 costimulation dependency of virus-specific CD8+ T cells independent of the priming context. Figure 4 Download asset Open asset The infectious pathogen during antigenic re-challenge determines the requirements for CD28/B7-mediated costimulation for secondary expansion. (A) Experimental setup: CD45.1+ WT mice were infected with 2 × 105 PFU LCMV Armstrong. After 4 months GP33-specific memory CD8+ T cells were sorted and 2 × 103 cells were adoptively transferred into CD45.2+ WT and Cd80/86−/− mice that were subsequently infected with 2 × 105 PFU LCMV Armstrong or 1 × 105 PFU MCMV-IE2-GP33. The total number of transferred GP33-specific CD8+ T cells was determined 6 days post re-challenge. (B) Similar experimental setup as described in (A), except CD45.1+ WT mice were infected with 1 × 105 PFU MCMV-IE2-GP33. Data in bar graphs are expressed as mean + SEM (n = 5 mice per group). Fold difference and significance (*p < 0.05) is indicated. https://doi.org/10.7554/eLife.07486.009 Influence of type I IFN signaling on the requirement of CD28/B7-mediated costimulation As the context of a viral infection determines the dependence on CD28/B7-mediated costimulation for CD8+ T cell expansion, we compared the overall composition of inflammatory mediators in LCMV and MCMV infection. Expression of the inflammation-associated cytokines IL-2, IL-3, IL-13, IL-17, GM-CSF, and TNF was not enhanced in both infections at early time points compared to naive mice (data not shown). In contrast, serum levels of IFNα were particularly high in LCMV infected mice compared to the serum levels in MCMV infected mice (Figure 5A). Consistent with this, at 24 hr LCMV also induced higher expression of pro-inflammatory cytokines, which have been described to be downstream of type I IFN signaling (i.e., Rantes, IL-6, KC, Mip-1β and MCP-1) (Teijaro et al., 2013). However, after 48 hr the concentrations of these cytokines were comparable (Figure 5B). Thus, a divergent pro-inflammatory environment is induced early upon LCMV and MCMV infections. Figure 5 with 1 supplement see all Download asset Open asset Influence of type I IFN signaling on the requirement of CD28/B7-mediated costimulation. WT mice were infected with 1 × 104 PFU MCMV-Smith or 2 × 105 PFU LCMV Armstrong and at indicated times post-infection serum was collected. (A) Levels of IFNα in serum in time are shown (bd = below detection limit). (B) Concentrations of different pro-inflammatory cytokines as determined 24 and 48 hr post-infection. (C) Type I interferon receptor (IFNAR) blocking antibodies were administrated during LCMV infection in WT and Cd80/86−/− mice. The magnitude of the virus-specific CD8+ T cell response determined by MHC class I tetramer binding at day 7 post-infection is shown. Fold difference and significance (*p < 0.05) is indicated. (D) IFNα levels in serum are shown 3 days post LCMV infection. (E) Experimental setup: 5 × 104 CD90.1+ Ifnar1+/+ and Ifnar1−/− P14 cells were adoptively transferred in WT and Cd80/86−/− mice that were subsequently infected with 2 × 105 PFU LCMV Armstrong. 7 days post-infection the total numbers of splenic P14 cells was determined. Representative flow cytometric plots show gated CD3+/CD8+ T cells stained for cell surface expression of CD90.1 and Vα2. Fold difference and statistical significance (*p < 0.05) between groups is indicated in the bar graphs. (F) Similar setup as in (E) except mice were infected with 1 × 105 PFU MCMV-IE2-GP33. In addition, on day 1 and 2, half of the mice received 1 × 105 units IFNα. 8 days post-infection the magnitude of the P14 cells in the spleen was determined. Representative flow cytometric plots show gated CD3+/CD8+ cells stained for cell surface expression of CD90.1 and Vα2. Bar graph shows total number of P14 cells in WT and Cd80/86−/− mice, and fold difference and statistical significance (*p < 0.05) between groups is indicated. (G) Mice were vaccinated with 75 µg SLP containing the GP33 epitope in PBS. 1 × 105 units IFNα was administrated after 18 and 48 hr. At day 7 post-vaccination, GP33-specific CD8+ T cell responses were analyzed. Significance between groups is indicated (*p < 0.05). (H) Experimental setup: WT mice were infected with 2 × 105 PFU LCMV Armstrong and 2 days post-infection serum was collected and transferred to mice that were infected 1 day prior with 1 × 104 PFU MCMV. The MCMV-specific CD8+ T cell response was determined 8 days post-infection by MHC class I tetramer binding. (I) WT and Cd80/86−/− mice were co-infected with 2 × 105 PFU LCMV Armstrong and 1 × 104 PFU MCMV, and virus-specific responses were analyzed 7 days post-infection by MHC class I tetramer binding. Fold difference and significance (*p < 0.05) is indicated. Data in all bar graphs are expressed as mean + SEM (n = 4–8 mice per group) of at least two independent experiments. https://doi.org/10.7554/eLife.07486.010 To determine whether the high type I IFN levels that are induced during LCMV infection substitute the CD28/B7 costimulation promoting CD8+ T cell expansion, we investigated the relationship between type I IFN signaling and B7-mediated costimulation in driving LCMV-specific CD8+ T cell expansion. Blocking antibodies for the type I IFN receptor (IFNAR) were administered during LCMV infection and resulted in severely diminished LCMV-specific CD8+ T cell responses in WT mice (Figure 5C). IFNAR blocking antibodies administrated in Cd80/86−/− mice also severely hampered LCMV-specific responses (Figure 5C). Notably, the LCMV-specific CD8+ T cell responses in WT mice with abrogated IFNAR signaling were comparable to those in IFNAR blocked Cd80/86−/− mice. Furthermore, no differences in IFNα levels were detected between WT and Cd80/86−/− mice (Figure 5D). Thus, the necessity for IFNAR signaling in the induction of LCMV-specific CD8+ T cell responses does not change in the absence or presence of CD28/B7-mediated costimulation. To examine direct effects of type I IFN-mediated signaling on CD8+ T cell expansion, Ifnar1+/+ and Ifnar1−/− P14 cells were adoptively transferred in WT and costimulation deficient mice that were subsequently infected with LCMV. Ifnar1−/− P14 cells transferred to WT recipients were severely hampered in expansion compared to Ifnar1+/+ P14 cells (Figure 5E), which is consistent with previous reports (Kolumam et al., 2005; Aichele et al., 2006; Wiesel et al., 2012; Crouse et al., 2014; Xu et al., 2014) and confirms that type I IFNs drive directly LCMV-specific CD8+ T cell expansion. Ifnar1+/+ P14 cells in Cd80/86−/− mice expanded vigorously and comparable to WT host mice. Importantly, Ifnar1−/− P14 cells failed to expand in Cd80/86−/− mice as well and showed a slightly weaker expansion potential as Ifnar1−/− P14 cells in WT mice (Figure 5E). These data show that type I IFNs act directly on LCMV-specific CD8+ T cells, and that in the absence of this signal 3 cytokine the non-dependence of B7-mediated costimulation in driving LCMV-specific T cell expansion is to some extent altered, indicating that type I IFN signaling in expanding CD8+ T cells is slightly redundant with B7-mediated costimulation signals. Next, we examined the relationship between type I IFN signaling and the B7-mediated pathway during MCMV infection. First we tested whether MCMV-specific CD8+ T cell responses, which are driven by B7-mediated signals, are influenced by the type I IFN pathway. Adoptive transfer of Ifnar1+/+ and Ifnar1−/− P14 cells in WT mice that were subsequently infected with MCMV-IE2-GP33 resulted in profound expansion of the Ifnar1+/+ P14 cells but also of Ifnar1−/− P14 cells, although slightly diminished compared to Ifnar1+/+ P14 cells. Adoptive transfer of P14 cells in Cd80/86−/− mice resulted in hampered expansion of Ifnar1+/+ and even more so of Ifnar1−/− P14 cells, indicating that CD8+ T cells that develop during MCMV infection are to a small degree affected by type I IFN signaling (in a somewhat redundant manner with B7-mediated costimulation) but are most critically dependent on B7-mediated signals (Figure 5F). Next, we examined if the B7-dependent MCMV-specific CD8+ T cell response can be boosted via supplementary triggering of the type I IFN pathway. We used recombinant IFNα2 that was functional both in vitro, as determined by a cytopathic effect inhibition assay (Figure 5—figure supplement 1A), and in vivo as evidenced by increased expression of CD69 on lymphocytes at 18 hr upon i.p. administration (Figure 5—figure supplement 1B). Addition of recombinant type I IFN on day 1 and 2 during MCMV-IE2-GP33 infection in mice that received Ifnar1+/+ and Ifnar1−/− P14 cells, caused no significant increase in the expansion of the P14 cells either transferred in WT or Cd80/86−/− mice, indicating that additional type I IFN signaling has negligible impact on B7-mediated signals that drive T cell expansion in MCMV infection (Figure 5F). Administration of recombinant type I IFN during peptide vaccination, however, improved GP33-specific CD8+ T cell expansion, which indicated that IFNα is able to enhance T cell expansion in a low inflammatory context (Figure 5G). To examine if the dependence of T cell expansion on B7-mediated costimulatory signals could be changed by other soluble factors than type I IFN, serum of mice that were infected for 2 days with LCMV was transferred to MCMV-infected WT and Cd80/86−/− mice. However, no differences were found in the magnitude of the MCMV-specific CD8+ T cell response (Figure 5H), indicating that soluble factors in the LCMV environment do not enhance MCMV-specific CD8+ T cell expansion. To unequivocally demonstrate the uniqueness of the viral context to induce B7-mediated costimulation dependence, WT mice were co-infected with MCMV and LCMV. Remarkably, during this co-infection, MCMV-specific responses were still dependent on B7-mediated signals whereas LCMV-specific CD8+ T cells were not (Figure 5I). Together, these data show that during an LCMV and MCMV infection a unique local environment is induced that principally determines the costimulatory requirements of the activated antigen-specific CD8+ T cells, and that direct type I IFN signaling in CD8+ T cells is slightly redundant with B7-mediated costimulation. Costimulatory ligands are highly expressed in LCMV infection To further delineate factors that could locally contribute to the CD28/B7 costimulation independence of CD8+ T cell expansion during LCMV infection, we characterized the expression of cell surface bound molecules that could impact T cell expansion. First, we examined if B7 molecules were induced upon LCMV infection. Expression of both B7.1 and B7.2 was upregulated on CD11c+ and CD11b+ cells early in infection (Figure 6A). Strikingly, expression levels of B7.1 and B7.2 on these myeloid subsets were higher in LCMV infection as compared to MCMV infection. Thus, the non-dependence of B7-mediated costimulation for LCMV-specific CD8+ T cell expansion is not due to hampered expression of these costimulatory ligands during LCMV infection. Figure 6 with 1 supplement see all Download asset Open asset LCMV infection induces high expression of costimulatory ligands. (A) Mice were infected with 2 × 105 PFU LCMV Armstrong or 1 × 104 PFU MCMV and costimulatory ligand expression was determined in the spleen. Histograms show cell surface expression of indicated costimulatory molecules on CD11b+ or CD11c+ cells at day 2 post-infection with either MCMV or LCMV. Representative staining of CD11b+ and CD11c+ cells from naive WT and Cd70/80/86−/− mice are depicted for comparison. Staining with an isotype control antibody is indicated as well. (B) Graphs depict mean flu

Adequate responsiveness of CD8(+) T cell populations is of utmost importance for the efficacy of many vaccines and immunotherapeutic strategies against intracellular pathogens and cancer. In this study, we show in mouse models that the relative number of IL-2-producing cells within Ag-specific CD8(+) T cell populations predicts the population expansion capacity upon challenge. We further demonstrate that IL-2 producers constitute the best responding subset. Notably, we show that elevated production of IL-2 by CD8(+) T cells results in concomitant improved population expansion capacity and immunity. The amount of IL-2 produced on a per-cell basis essentially connects directly to the superior CD8(+) T cell population expansion. Together, our findings identified that autocrine IL-2 production operates in a dose-dependent fashion to facilitate the expansion potential of Ag-specific CD8(+) T cell populations, which may instigate ways to augment therapies depending on fit CD8(+) T cells.

Signals delivered by costimulatory molecules are implicated in driving T cell expansion. The requirements for these signals, however, vary from dispensable to essential in different infections. We examined the underlying mechanisms of this differential T cell costimulation dependence and found that the viral context determined the dependence on CD28/B7-mediated costimulation for expansion of naive and memory CD8(+) T cells, indicating that the requirement for costimulatory signals is not imprinted. Notably, related to the high-level costimulatory molecule expression induced by lymphocytic choriomeningitis virus (LCMV), CD28/B7-mediated costimulation was dispensable for accumulation of LCMV-specific CD8(+) T cells because of redundancy with the costimulatory pathways induced by TNF receptor family members (i.e., CD27, OX40, and 4-1BB). Type I IFN signaling in viral-specific CD8(+) T cells is slightly redundant with costimulatory signals. These results highlight that pathogen-specific conditions differentially and uniquely dictate the utilization of costimulatory pathways allowing shaping of effector and memory antigen-specific CD8(+) T cell responses.