Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.     

4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses.

4-1BB (CD137) is a costimulatory molecule expressed on activated T cells and interacts with 4-1BB ligand (4-1BBL) on APCs. To investigate the role of 4-1BB costimulation for the development of primary immune responses, 4-1BBL-deficient (4-1BBL-/-) mice were infected with lymphocytic choriomeningitis virus (LCMV). 4-1BBL-/- mice were able to generate CTL and eliminate acute LCMV infection with normal kinetics, but CD8 T cell expansion was 2- to 3-fold lower than in wild-type (+/+) mice. In the same mice, virus-specific CD4 Th and B cell responses were minimally affected, indicating that 4-1BB costimulation preferentially affects CD8 T cell responses. This result contrasts with our earlier work with CD40L-deficient (CD40L-/-) mice, in which the CD8 T cell response was unaffected and the CD4 T cell response was markedly impaired. When both 4-1BBL- and B7-dependent signals were absent, CD8 T cell expansion was further reduced, resulting in lower CTL activity and impairing their ability to clear LCMV. Altogether, these results indicate that T cells have distinct costimulatory requirements: optimal CD8 responses require 4-1BBL-dependent interactions, whereas CD4 responses are minimally affected by 4-1BB costimulation, but require CD40-CD40L and B7-dependent interactions.     

Costimulation requirements of induced murine systemic autoimmune disease

Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl2-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L-/- mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.     

Memory CD8+ T cells require CD28 costimulation

CD8(+) T cells are a critical component of the adaptive immune response against infections and tumors. A current paradigm in immunology is that naive CD8(+) T cells require CD28 costimulation, whereas memory CD8(+) T cells do not. We show here, however, that during viral infections of mice, costimulation is required in vivo for the reactivation of memory CD8(+) T cells. In the absence of CD28 costimulation, secondary CD8(+) T cell responses are greatly reduced and this impairs viral clearance. The failure of CD8(+) T cells to expand in the absence of CD28 costimulation is CD4(+) T cell help independent and is accompanied by a failure to down-regulate Bcl-2 and by cell cycle arrest. This requirement for CD28 costimulation was shown in both influenza A and HSV infections. Thus, contrary to current dogma, memory CD8(+) T cells require CD28 costimulation to generate maximal secondary responses against pathogens. Importantly, this CD28 requirement was shown in the context of real infections were multiple other cytokines and costimulators may be up-regulated. Our findings have important implications for pathogens, such as HIV and measles virus, and tumors that evade the immune response by failing to provide CD28 costimulation. These findings also raise questions about the efficacy of CD8(+) T cell-based vaccines against such pathogens and tumors.     

Costimulation requirements for antiviral CD8+ T cells differ for acute and persistent phases of polyoma virus infection

The requirement for costimulation in antiviral CD8+ T cell responses has been actively investigated for acutely resolved viral infections, but it is less defined for CD8+ T cell responses to persistent virus infection. Using mouse polyoma virus (PyV) as a model of low-level persistent virus infection, we asked whether blockade of the CD40 ligand (CD40L) and CD28 costimulatory pathways impacts the magnitude and function of the PyV-specific CD8+ T response, as well as the humoral response and viral control during acute and persistent phases of infection. Costimulation blockade or gene knockout of either CD28 or CD40L substantially dampened the magnitude of the acute CD8+ T cell response; simultaneous CD28 and CD40L blockade severely depressed the acute T cell response, altered the cell surface phenotype of PyV-specific CD8+ T cells, decreased PyV VP1-specific serum IgG titers, and resulted in an increase in viral DNA levels in multiple organs. CD28 and CD40L costimulation blockade during acute infection also diminished the memory PyV-specific CD8+ T cell response and serum IgG titer, but control of viral persistence varied between mouse strains and among organs. Interestingly, we found that CD28 and CD40L costimulation is dispensable for generating and/or maintaining PyV-specific CD8+ T cells during persistent infection; however, blockade of CD27 and CD28 costimulation in persistently infected mice caused a reduction in PyV-specific CD8+ T cells. Taken together, these data indicate that CD8+ T cells primed within the distinct microenvironments of acute vs persistent virus infection differ in their costimulation requirements.     

Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus.

4-1BB ligand (4-1BBL) is a member of the TNF family expressed on activated APC. 4-1BBL binds to 4-1BB (CD137) on activated CD4 and CD8 T cells and in conjunction with strong signals through the TCR provides a CD28-independent costimulatory signal leading to high level IL-2 production by primary resting T cells. Here we report the immunological characterization of mice lacking 4-1BBL and of mice lacking both 4-1BBL and CD28. 4-1BBL-/- mice mount neutralizing IgM and IgG responses to vesicular stomatitis virus that are indistinguishable from those of wild-type mice. 4-1BBL-/- mice show unimpaired CTL responses to lymphocytic choriomeningitis virus (LCMV) and exhibit normal skin allograft rejection but have a weaker CTL response to influenza virus than wild-type mice. 4-1BBL-/-CD28-/- mice retain the CTL response to LCMV, respond poorly to influenza virus, and exhibit a delay in skin allograft rejection. In agreement with these in vivo results, allogeneic CTL responses of CD28-/- but not CD28+/+ T cells to 4-1BBL-expressing APC are substantially inhibited by soluble 4-1BB receptor as is the in vitro secondary response of CD28+ T cells to influenza virus peptides. TCR-transgenic CD28-/- LCMV glycoprotein-specific T cells are insensitive to the presence of 4-1BBL when a wild-type peptide is used, but the response to a weak agonist peptide is greatly augmented by the presence of 4-1BBL. These results further substantiate the idea that different immune responses vary in their dependence on costimulation and suggest a role for 4-1BBL in augmenting suboptimal CTL responses in vivo.     

Suppression of antigen-specific T cell responses by the Kaposi's sarcoma-associated herpesvirus viral OX2 protein and its cellular orthologue, CD200

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.     

Evaluation of OX40 ligand as a costimulator of human antiviral memory CD8 T cell responses: comparison with B7.1 and 4-1BBL

CTL are important effectors of antiviral immunity. Designing adjuvants that can induce strong cytotoxic T cell responses in humans would greatly improve the effectiveness of an antiviral vaccination or therapeutic strategy. Recent evidence suggests that, in addition to its well-established role in costimulation of CD4 T cell responses, OX40L (CD134) can directly costimulate mouse CD8 T cells. In this study, we evaluated the role of OX40L in costimulation of human antiviral CD8 T cell responses and compared it with two other important costimulators, B7.1 (CD80) and 4-1BBL (CD137L). Delivery of OX40L to human monocytes using a recombinant replication-defective adenovirus led to greater expansion, up-regulation of perforin, enhanced cytolytic activity, and increased numbers of IFN-gamma- and TNF-alpha-producing antiviral memory CD8 T cells in cultures of total T cells. Synergistic or additive effects were observed when OX40L costimulation was combined with 4-1BBL (CD137L) or B7.1 (CD80) costimulation. In total T cell cultures, at low Ag dose, 4-1BBL provided the most potent costimulus for influenza-specific CD8 T cell expansion, followed by B7.1 (CD80) and then OX40L. For isolated CD8 T cells, 4-1BBL was also the most consistent costimulator, followed by B7.1. In contrast, OX40L showed efficacy in direct activation of memory CD8 T cells in only one of seven donors. Thus, OX40L costimulates human antiviral memory CD8 T cell responses largely through indirect effects and can enhance anti-influenza, anti-EBV, and anti-HIV responses, particularly in combination with 4-1BBL or B7.     

Regulation of follicular dendritic cell networks by activated T cells: the role of CD137 signaling

B cells, but not T cells, are considered to be important for the formation of follicular dendritic cell (FDC) clusters. Stimulation with agonist mAbs against CD137 (4-1BB), a TNFR family member primarily expressed on activated T cells, was effective in promoting T cell responses, but paradoxically suppressed T-dependent humoral immunity and autoantibody production in autoimmune disease models. Our present study shows that agonistic anti-CD137 treatment activates T cells, resulting in diminished FDC networks in B cell follicles, which are important components in T-dependent humoral immune responses both before and after the initiation of an immune response. Pretreatment with anti-CD137 before the secondary immunization inhibited memory Ab responses. Interestingly, CD137 costimulation-induced diminishment of FDC is T cell dependent. In addition, both CD4(+) and CD8(+) T cells are recruited into FDC area and are able to regulate FDCs by CD137 costimulation through a direct or indirect mechanism. These studies have revealed a previously unappreciated role of T cells in the regulation of FDC networks.     

Opposing effects of CD70 costimulation during acute and chronic lymphocytic choriomeningitis virus infection of mice

T cell costimulation is important for T cell activation. The CD27/CD70 pathway contributes to effector and memory T cell development and is involved in T cell and B cell activation. CD27/CD70 is known for having opposing roles during different models of antigenic challenges. During primary T cell responses to influenza virus infection or during tumor challenges, CD27/CD70 costimulation has a positive role on T cell responses. However, during some chronic infections, constitutive triggering of this signaling pathway has a negative role on T cell responses. It is currently unclear what specific characteristic of an antigen determines the outcome of CD27/CD70 costimulation. We investigated the effect of a transient CD70 blockade during an acute or a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Blockade of this pathway during acute LCMV infection (Armstrong strain) resulted in delayed T cell responses and decreased CD127 (interleukin-7 receptor α [IL-7Rα] chain) conversion. Upregulation of CD127 is an important event in T cell differentiation that heralds the passage of an effector T cell to a long-lived memory T cell. In contrast to the reduced CD8 T cell responses after CD70 blockade during acute infection, CD70 blockade during chronic LCMV infection resulted in increased CD8 T cell responses. Our data show the dual roles of this costimulatory pathway in acute versus persistent antigen challenge. Our findings suggest that antigen persistence may determine the effect of CD27/CD70 signaling on CD8 T cell responses. Tailored triggering or blockade of this costimulatory pathway may be important in vaccination regimens against acute or chronic pathogens.     

CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses.

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (-/-) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L-/- mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L-/- mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.     

Induction of a VLA-2 (CD49b)-expressing effector T cell population by a cell-based neuroblastoma vaccine expressing CD137L

In malignancies where no universally expressed dominant Ag exists, the use of tumor cell-based vaccines has been proposed. We have modified a mouse neuroblastoma cell line to express either CD80 (B7.1), CD137L (4-1BBL), or both receptors on the tumor cell surface. Vaccines expressing both induce a strong T cell response that is unique in that among responding CD8 T cells, a T effector memory cell (T(EM)) response arises in which a large number of the T(EM) express the alpha-chain of VLA-2, CD49b. We demonstrate using both in vitro and in vivo assays that the CD49b(+) CD8 T cell population is a far more potent antitumor effector cell population than nonfractionated CD8 or CD49b(-) CD8 T cells and that CD49b on vaccine-induced CD8 T cells mediates invasion of a collagen matrix. In in vivo rechallenge studies, CD49b(+) T cells no longer expanded, indicating that CD49b T(EM) expansion is restricted to the initial response to vaccine. To demonstrate a mechanistic link between the expression of costimulatory molecules on the vaccine and CD49b on responding T cells, we stimulated naive T cells in vitro with artificial APC expressing different combinations of anti-CD3, anti-CD28, and CD137L. Although some mRNA encoding CD49b was induced by combining anti-CD3 with anti-CD28 or CD137L, the highest level was induced when all three signals were present. This indicates that CD49b expression results from additive costimulation and that the level of CD49b message serves as an indicator of the effectiveness of T cell activation by a cell-based vaccine.     

Characterization of rat OX40 ligand by monoclonal antibody

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.     

OX40 ligand-transduced tumor cell vaccine synergizes with GM-CSF and requires CD40-Apc signaling to boost the host T cell antitumor response

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-gamma or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.     

Multiple layers of CD80/86-dependent costimulatory activity regulate primary, memory, and secondary lymphocytic choriomeningitis virus-specific T cell immunity

The lymphocytic choriomeningitis virus (LCMV) system constitutes one of the most widely used models for the study of infectious disease and the regulation of virus-specific T cell immunity. However, with respect to the activity of costimulatory and associated regulatory pathways, LCMV-specific T cell responses have long been regarded as relatively independent and thus distinct from the regulation of T cell immunity directed against many other viral pathogens. Here, we have reevaluated the contribution of CD28-CD80/86 costimulation in the LCMV system by use of CD80/86-deficient mice, and our results demonstrate that a disruption of CD28-CD80/86 signaling compromises the magnitude, phenotype, and/or functionality of LCMV-specific CD8(+) and/or CD4(+) T cell populations in all stages of the T cell response. Notably, a profound inhibition of secondary T cell immunity in LCMV-immune CD80/86-deficient mice emerged as a composite of both defective memory T cell development and a specific requirement for CD80 but not CD86 in the recall response, while a related experimental scenario of CD28-dependent yet CD80/86-independent secondary CD8(+) T cell immunity suggests the existence of a CD28 ligand other than CD80/86. Furthermore, we provide evidence that regulatory T cells (T(REG)s), the homeostasis of which is altered in CD80/86(-/-) mice, contribute to restrained LCMV-specific CD8(+) T cell responses in the presence of CD80/86. Our observations can therefore provide a more coherent perspective on CD28-CD80/86 costimulation in antiviral T cell immunity that positions the LCMV system within a shared context of multiple defects that virus-specific T cells acquire in the absence of CD28-CD80/86 costimulation.     

Enhancement of HIV-specific CD8 T cell responses by dual costimulation with CD80 and CD137L

HIV-specific CD8 T cell responses are defective in chronic HIV infection. In this study, we report that costimulation with either CD137L (4-1BBL) or CD80 (B7.1) enhanced the Ag-specific expansion and acquisition of effector function by HIV-specific memory CD8 T cells. Ag-specific T cells from recently infected donors showed maximal expansion with single costimulatory molecules. Dual costimulation of T cells from recently infected donors or from healthy donors responding to influenza epitopes led to enhanced responses when the accumulation of cytokines was measured. However, accumulation of regulatory cytokines, particularly IFN-gamma, led to inhibition of further Ag-specific CD8 T cell expansion in the cultures. This inhibition was relieved by neutralization of IFN-gamma or of IFN-gamma, TNF, and IL-10. Thus, strong costimulation of T cells in vitro can lead to induction of regulatory cytokines at levels that limit further T cell expansion. In marked contrast, T cells from long-term (>4 years) infected HIV+ donors exhibited reduced Ag-specific CD8 T cell expansion, reduced CD4 T cell responses, and minimal cytokine accumulation. Dual costimulation with both 4-1BBL and B7.1 enhanced responses of T cells from long-term infected subjects to a level similar to that obtained with T cells from early in HIV infection. Experiments with purified CD8 T cells showed that B7.1 and 4-1BBL could act directly and synergistically on CD8 T cells. Taken together, these data suggest that 4-1BBL and B7.1 have additive or synergistic effects on HIV-specific CD8 T cell responses and represent a promising combination for therapeutic vaccination for HIV.     

4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination

Peptide vaccination induces T cell activation and cytotoxic T cell development. In an effort to understand what factors can improve immune responses to peptide vaccination, the role of 4-1BB (CD137) costimulation was examined, since 4-1BB has been shown to promote T cell responses in other systems. 4-1BBL-deficient (-/-) and wild-type (+/+) mice were immunized with a lipidated lymphocytic choriomeningitis virus (LCMV) peptide NP396-404. Analysis of peptide-specific responses early after immunization by CTL assay, intracellular IFN-gamma staining, and IFN-gamma enzyme-linked immunospot assay (ELISPOT) indicated that CD8 T cell responses were reduced 3- to 10-fold in the absence of 4-1BB costimulation. Moreover, when agonistic anti-4-1BB Ab was given, CD8 T cell responses in 4-1BBL-/- mice were augmented to levels similar to those in 4-1BBL+/+ mice. Two months after immunization, 4-1BBL+/+ mice still had epitope-specific cells and were protected against viral challenge, demonstrating that peptide vaccination can induce long-term protection. In fact, 70% of CD8 T cells were specific for the immunizing peptide after viral challenge, demonstrating that strong, epitope-specific CD8 T cell responses are generated after peptide vaccination. In contrast, peptide-immunized 4-1BBL-/- mice had fewer epitope-specific cells and were impaired in their ability to resolve the infection. These results show that immunization with a single LCMV peptide provides long term protection against LCMV infection and point to costimulatory molecules such as 4-1BB as important components for generating protective immunity after vaccination.     

Differential requirement for CD80 and CD80/CD86-dependent costimulation in the lung immune response to an influenza virus infection

The CD28 costimulatory pathway is critical to T cell activation. Blockade of the interaction of CD28 with its ligands CD80 and CD86 using CTLA4-Ig has been proposed as a therapy for a number of immune-based disorders. We have used a murine model of influenza virus infection to study the role of CD28-dependent costimulation in the development of antiviral immune responses. In vivo treatment with CTLA4-Ig to block the interaction of CD28 with CD80 and CD86 reduced virus-specific cytotoxicity and IFN-gamma production by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro. It also resulted in decreased numbers of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid, lung, and spleen and lowered virus-specific Ab titers. Mice treated with CTLA4-Ig were able to control and clear the virus infection, but this was delayed compared with controls. Treatment with Y100F-Ig, a mutant form of CTLA4-Ig which selectively binds to CD80 and blocks the CD28-CD80 interaction leaving CD28-CD86 binding intact, did not affect Ab production, spleen cytotoxic precursors, or clearance of virus. However, Y100F-Ig treatment had a clear effect on lung effector cell function. Secretion of IFN-gamma by bronchoalveolar lavage fluid CD8+ T lymphocytes in vitro was decreased, and the number of virus-specific CD8+ T lymphocytes in the bronchoalveolar lavage fluid and lungs of infected mice was reduced. These results indicate that CD28-dependent costimulation is important in the antiviral immune response to an influenza virus infection. The individual CD28 ligand, CD80, is important for some lung immune responses and cannot always be compensated for by CD86.     

CD134 Costimulation Couples the CD137 Pathway to Induce Production of Supereffector CD8 T Cells That Become IL-7 Dependent

The TNFR superfamily members 4-1BB (CD137) and OX40 (CD134) are costimulatory molecules that potently boost CD8 and CD4 T cell responses. Concomitant therapeutic administration of agonist anti-CD137 and -CD134 mAbs mediates rejection of established tumors and fosters powerful CD8 T cell responses. To reveal the mechanism, the role of CD137 expression by specific CD8 T cells was determined to be essential for optimal clonal expansion and accumulation of effector cells. Nonetheless, dual costimulation induced production of supereffector CD8 T cells when either the specific T cells or the host alone bore CD137. Perhaps surprisingly, the total absence of CD137 prevented anti-CD134 augmentation of supereffector differentiation demonstrating an unappreciated link between these related pathways. Ultimately, it was reasoned that these powerful dual costimulatory responses involved common gamma family members, and we show substantial increases of CD25 and IL-7Ralpha-chain expression by the specific CD8 T cells. To investigate this further, it was shown that IL-7 mediated T cell accumulation, but importantly, a gradual and preferential effect of survival was directed toward supereffector CD8 T cells. In fact, a clear enhancement of effector differentiation was demonstrated to be proportional to the increasing amount of IL-7Ralpha expression by the specific CD8 T cells. Therefore, dual costimulation through CD137 and CD134 drives production and survival of supereffector CD8 T cells through a distinct IL-7-dependent pathway.