Peter D. Katsikis

In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses.

Given the key role CD8+ T cells play in controlling viral infection, strategies to enhance these responses may have important clinical applications. We found that in vivo CD137 stimulation with an agonistic monoclonal antibody enhanced the primary CD8+ T cell response to influenza type A viral infection in mice. Stimulation of CD137 increased the absolute number of CD8+ T cells to influenza epitopes in the lungs of infected animals, preferentially expanded CD8+ T cells that recognized nondominant epitopes and greatly enhanced direct ex vivo cytotoxicity. CD137 stimulation also restored the CD8+ T cell response to the immunodominant influenza epitope in CD28−/− mice. Thus, in vivo CD137 stimulation enhances and broadens the CD8+ T cell response to influenza virus and can restore the CD8+ T cell response when CD28 costimulation is absent. This suggests that CD137 stimulation may be useful as a strategy to enhance the CD8+ T cell response to viruses.

Increased CD95/Fas-Induced Apoptosis of HIV-Specific CD8+ T Cells

Why HIV-specific CD8(+) T cells ultimately fail to clear or control HIV infection is not known. We show here that HIV-specific CD8(+) T cells exhibit increased sensitivity to CD95/Fas-induced apoptosis. This apoptosis is 3-fold higher compared to CMV-specific CD8(+) T cells from the same patients. HIV-specific CD8(+) T cells express the CD45RA(-)CD62L(-) but lack the CD45RA(+)CD62L(-) T cell effector memory (T(EM)) phenotype. This skewing is not found in CMV- and EBV-specific CD8(+) T cells in HIV-infected individuals. CD95/Fas-induced apoptosis is much higher in the CD45RA(-)CD62L(-) T(EM) cells. However, cytotoxicity and IFNgamma production by HIV-specific CD8(+) T cells is not impaired. Our data suggest that the survival and differentiation of HIV-specific CD8(+) T cells may be compromised by CD95/Fas apoptosis induced by FasL-expressing HIV-infected cells.

The microRNA miR-155 controls CD8 + T cell responses by regulating interferon signaling

We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8+ T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8+ T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8+ T cells, as miR-155-deficient CD8+ T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8+ T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8+ T cells had enhanced type I interferon signaling and were more susceptible to interferons antiproliferative effect. Inhibition of the type I interferon–associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8+ T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8+ T cell responses to pathogens in vivo.

Surface expression patterns of negative regulatory molecules identify determinants of virus-specific CD8+ T-cell exhaustion in HIV infection

A highly complex network of coinhibitory and costimulatory receptors regulates the outcome of virus-specific CD8(+) T-cell responses. Here, we report on the expression patterns of multiple inhibitory receptors on HIV-specific, cytomegalovirus-specific, and bulk CD8(+) T-cell memory populations. In contrast to cytomegalovirus-specific CD8(+) T cells, the majority of HIV-specific CD8(+) T cells exhibited an immature phenotype and expressed Programmed Death-1, CD160 and 2B4 but not lymphocyte activation gene-3. Notably, before antiretroviral therapy, simultaneous expression of these negative regulators correlated strongly with both HIV load and impaired cytokine production. Suppression of HIV replication by antiretroviral therapy was associated with reduced surface expression of inhibitory molecules on HIV-specific CD8(+) T cells. Furthermore, in vitro manipulation of Programmed Death-1 and 2B4 inhibitory pathways increased the proliferative capacity of HIV-specific CD8(+) T cells. Thus, multiple coinhibitory receptors can affect the development of HIV-specific CD8(+) T-cell responses and, by extension, represent potential targets for new immune-based interventions in HIV-infected persons.

Age-associated decrease in virus-specific CD8+ T lymphocytes during primary influenza infection

The mechanism of the age-associated decrease in CD8+ T cell response of mice to virus infection was examined in young adult (6 months) and aged (22 months) C57BL/6 mice during primary pulmonary influenza A virus infection. A significant age-associated decrease in both the percentage (P<0.0001) and number (P<0.05) of CD8+ T cells binding MHC Class I tetramers containing influenza A nucleoprotein (NP) epitope and in virus-specific CTL activity (P<0.05) was observed with pulmonary lymphocytes. The percentage of NP+CD8+ cells of individual mice strongly correlated with NP-specific cytotoxic activity (r(2)=0.77, P<0.02) and with the percentage of CD8+ cells that produced interferon-gamma (r(2)=0.86, P<0.002) in both young and aged mice. Comparable expression of the CD28, CD25, and the memory CD44(hi)/CD62L(lo) phenotype was detected on NP+CD8+ lymphocytes from mice of both age groups. There was a delay in the maximal expansion of NP+CD8+ cells in aged compared to young mice that paralleled a delay in maximal cytotoxic activity and in virus clearance. These data suggest that the age-related impairment of CD8+ lymphocyte activity during a primary influenza A infection is due to a defect in the expansion, rather than in effector activity, of influenza-specific CD8+ T cells.

Structural basis for autoantibody recognition of phosphatidylserine-β2 glycoprotein I and apoptotic cells

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by β2 glycoprotein I (β2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-β2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.

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.

Differential Association of Programmed Death-1 and CD57 with Ex Vivo Survival of CD8+ T Cells in HIV Infection

Recent studies have revealed the critical role of programmed death-1 (PD-1) in exhaustion of HIV- and SIV-specific CD8+ T cells. In this study, we show that high expression of PD-1 correlates with increased ex vivo spontaneous and CD95/Fas-induced apoptosis, particularly in the “effector-memory” CD8+ T cell population from HIV+ donors. High expression of PD-1 was linked to a proapoptotic phenotype characterized by low expression of Bcl-2 and IL7-Rα, high expression of CD95/Fas and high mitochondrial mass. Expression of PD-1 and CD57 was differentially associated with the maturation status of CD8+ T cells in HIV infection. CD57 was linked to higher apoptosis resistance, with cells expressing a PD-1LCD57H phenotype exhibiting lower levels of cell death. The majority of HIV-specific CD8+ T cells were found to express a PD-1HCD57L or PD-1HCD57H phenotype. No correlation was found between PD-1 expression and ex vivo polyfunctionality of either HIV- or CMV-specific CD8+ T cells. Contrary to CD57, high expression of PD-1 was characterized by translocation of PD-1 into the area of CD95/Fas-capping, an early necessary step of CD95/Fas-induced apoptosis. Thus, our data further support the role of PD-1 as a preapoptotic factor for CD8+ T cells in HIV infection.

Interleukin-15 Increases Effector Memory CD8 T Cells and NK Cells in Simian Immunodeficiency Virus-Infected Macaques

ABSTRACT Interleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+ T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+ T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 μg/kg of body weight, n = 3; high dose of IL-15, 100 μg/kg, n = 3; control [saline], n = 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3− NK cells. Furthermore, CD8+ T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA−CD62L− and CD45RA+CD62L− effector memory CD8+ T cells. Expression of Ki-67 in the CD8+ T cells indicated expansion of CD8+ T cells and not redistribution. IL-15 did not affect CD4+ T-cell, B-cell, and CD14+ macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+ T cells and NK cells by IL-15 further supports IL-15s possible therapeutic use in viral infections such as HIV infection.

Complement C5a Receptor Is Essential for the Optimal Generation of Antiviral CD8+ T Cell Responses

The complement system has been long regarded as an important effector of the innate immune response. Furthermore, complement contributes to various aspects of B and T cell immunity. Nevertheless, the role of complement in CD8+ T cell antiviral responses has yet to be fully delineated. We examined the CD8+ T cell response in influenza type A virus-infected mice treated with a peptide antagonist to C5aR to test the potential role of complement components in CD8+ T cell responses. We show that both the frequency and absolute numbers of flu-specific CD8+ T cells are greatly reduced in C5aR antagonist-treated mice compared with untreated mice. This reduction in flu-specific CD8+ T cells is accompanied by attenuated antiviral cytolytic activity in the lungs. These results demonstrate that the binding of the C5a component of complement to the C5a receptor plays an important role in CD8+ T cell responses.