Michael J. Molloy

CD4+ T cell regulation of CD25 expression controls development of short-lived effector CD8+ T cells in primary and secondary responses

Both CD4+ T cell help and IL-2 have been postulated to “program” activated CD8+ T cells for memory cell development. However, the linkage between these two signals has not been well elucidated. Here we have studied effector and memory CD8+ T cell differentiation following infection with three pathogens (Listeria monocytogenes, vesicular stomatitis virus, and vaccinia virus) in the absence of both CD4+ T cells and IL-2 signaling. We found that expression of CD25 on antigen-specific CD8+ T cells peaked 3–4 days after initial priming and was dependent on CD4+ T cell help, likely through a CD28:CD80/86 mediated pathway. CD4+ T cell or CD25-deficiency led to normal early effector CD8+ T cell differentiation, but a subsequent lack of accumulation of CD8+ T cells resulting in overall decreased memory cell generation. Interestingly, in both primary and recall responses KLRG1high CD127low short-lived effector cells were drastically diminished in the absence of IL-2 signaling, although memory precursors remained intact. In contrast to previous reports, upon secondary antigen encounter CD25-deficient CD8+ T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. Thus, these results demonstrated that CD4+ T cell help and IL-2 signaling were linked via CD25 up-regulation, which controls the expansion and differentiation of antigen-specific effector CD8+ T cells, rather than “programming” memory cell traits.

Distinction of the memory B cell response to cognate antigen versus bystander inflammatory signals

The hypothesis that bystander inflammatory signals promote memory B cell (BMEM) self-renewal and differentiation in an antigen-independent manner is critically evaluated herein. To comprehensively address this hypothesis, a detailed analysis is presented examining the response profiles of B-2 lineage B220+IgG+ BMEM toward cognate protein antigen in comparison to bystander inflammatory signals. After in vivo antigen encounter, quiescent BMEM clonally expand. Surprisingly, proliferating BMEM do not acquire germinal center (GC) B cell markers before generating daughter BMEM and differentiating into plasma cells or form structurally identifiable GCs. In striking contrast to cognate antigen, inflammatory stimuli, including Toll-like receptor agonists or bystander T cell activation, fail to induce even low levels of BMEM proliferation or differentiation in vivo. Under the extreme conditions of adjuvanted protein vaccination or acute viral infection, no detectable bystander proliferation or differentiation of BMEM occurred. The absence of a BMEM response to nonspecific inflammatory signals clearly shows that BMEM proliferation and differentiation is a process tightly controlled by the availability of cognate antigen.

Recall responses by helpless memory CD8+ T cells are restricted by the up-regulation of PD-1.

CD4 help is crucial for memory CD8+ T cell development, yet the mechanisms of CD4 help and why (CD4) helpless memory CD8+ T cells elicit poor recall responses are currently not well understood. In this study we investigated these questions using an in vivo acute virus infection model. We show herein that CD4 help during priming is required for memory CD8+ T cell differentiation, and that stimulation of CD40 during priming rescues the helpless defects in the absence of CD4+ T cells. The defective recall response by helpless memory cells did not correlate with the amount of cell death and was independent of TRAIL. However, helpless memory cells excessively up-regulated the inhibitory receptor PD-1 (programmed cell death-1), and PD-1 blockade enhanced the recall response of helpless memory cells. Furthermore, providing IL-2 signaling in vivo during the recall response reduced PD-1 expression and rescued the recall response of helpless memory cells. Our study identifies molecular pathways involved in CD4 help for memory CD8+ T cell generation that are independent of TRAIL, and it provides therapeutic implications that helpless memory cell function can be restored at multiple stages through various immunological interventions.

Suppressive CD8+ T Cells Arise in the Absence of CD4 Help and Compromise Control of Persistent Virus

There is an urgent need to develop novel therapies for controlling chronic virus infections in immunocompromised patients. Disease associated with persistent γ-herpesvirus infection (EBV, human herpesvirus 8) is a significant problem in AIDS patients and transplant recipients, and clinical management of these conditions is difficult. Immune surveillance failure followed by γ-herpesvirus recrudescence can be modeled using murine γ-herpesvirus (MHV)-68 in mice lacking CD4+ T cells. In contrast with other chronic infections, no obvious defect in the functional capacity of the viral-specific CD8+ T cell response was detected. We show in this article that adoptive transfer of MHV-68–specific CD8+ T cells was ineffective at reducing the viral burden. Together, these indicate the potential presence of T cell extrinsic suppressive factors. Indeed, CD4-depleted mice infected with MHV-68 express increased levels of IL-10, a cytokine capable of suppressing the function of both APCs and T cells. CD4-depleted mice developed a population of CD8+ T cells capable of producing IL-10 that suppressed viral control. Although exhibiting cell surface markers indicative of activation, the IL-10–producing cells expressed increased levels of programmed death-1 but were not enriched in the MHV-68–specific compartment, nor were they uniformly CD44hi. Therapeutic administration of an IL-10R blocking Ab enhanced control of the recrudescent virus. These data implicate IL-10 as a promising target for the restoration of immune surveillance against chronic γ-herpesvirus infection in immunosuppressed individuals.

IL-10 Immunomodulation of Myeloid Cells Regulates a Murine Model of Ovarian Cancer

Elevated levels of IL-10 in the microenvironment of human ovarian cancer and murine models of ovarian cancer are well established and correlate with poor clinical prognosis. However, amongst a myriad of immunosuppressive factors, the actual contribution of IL-10 to the ovarian tumor microenvironment, the mechanisms by which it acts, and its possible functional redundancy are unknown. We previously demonstrated that elimination of the myeloid-derived suppressor cell (MDSC) compartment within the ovarian tumor ascites inhibited tumor progression and, intriguingly, significantly decreased local IL-10 levels. Here we identify a novel pathway in which the tumor-infiltrating MDSC are the predominant producers of IL-10 and, importantly, require it to develop their immunosuppressive function in vivo. Importantly, we demonstrate that the role of IL-10 is critical, and not redundant with other immunosuppressive molecules, to in vivo tumor progression: blockade of the IL-10 signaling network results in alleviation of MDSC-mediated immunosuppression, altered T cell phenotype and activity, and improved survival. These studies define IL-10 as a fundamental modulator of both MDSC and T cells within the ovarian tumor microenvironment. Importantly, IL-10 signaling is shown to be necessary to the development and maintenance of a permissive tumor microenvironment and represents a viable target for anti-tumor strategies.

Cutting Edge: IL-2 Immune Complexes As a Therapy for Persistent Virus Infection

There is an urgent need to develop novel therapies for controlling recurrent virus infections in immune suppressed patients. Disease associated with persistent γ-herpesvirus infection (EBV, HHV-8) is a significant problem in AIDS patients and transplant recipients, and clinical management of these conditions is difficult. Disease occurs because of a failure in immune surveillance to control the persistent infection, which arises in AIDS patients principally because of an erosion of the CD4+ T cell compartment. Immune surveillance failure followed by γ-herpesvirus recrudescence can be modeled using murine γ-herpesvirus in CD4 T cell-depleted mice. We show that enhancement of IL-2 signaling using IL-2/anti-IL-2 immune complexes substantially improves immune surveillance in the context of suppressed immunity and enhances control of the infection. This effect was not due solely to increased numbers of virus-specific CD8 T cells but rather to enhanced cytotoxicity mediated by the perforin-granzyme pathway.

CD4+ T-cell dependence of primary CD8+ T-cell response against vaccinia virus depends upon route of infection and viral dose

CD4+ T-cell help (CD4 help) plays a pivotal role in CD8+ T-cell responses against viral infections. However, the role in primary CD8+ T-cell responses remains controversial. We evaluated the effects of infection route and viral dose on primary CD8+ T-cell responses to vaccinia virus (VACV) in MHC class II−/− mice. CD4 help deficiency diminished the generation of VACV-specific CD8+ T cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. A large viral dose could not restore normal expansion of VACV-specific CD8+ T cells in i.p. infected MHC II−/− mice. In contrast, dependence on CD4 help was observed in i.n. infected MHC II−/− mice when a small viral dose was used. These data suggested that primary CD8+ T-cell responses are less dependent on CD4 help in i.n. infection compared to i.p. infection. Activated CD8+ T cells produced more IFN-γ, TNF-α and granzyme B in i.n. infected mice than those in i.p. infected mice, regardless of CD4 help. IL-2 signaling via CD25 was not necessary to drive expansion of VACV-specific CD8+ T cells in i.n. infection, but it was crucial in i.p. infection. VACV-specific CD8+ T cells underwent increased apoptosis in the absence of CD4 help, but proliferated normally and had cytotoxic potential, regardless of infection route. Our results indicate that route of infection and viral dose are two determinants for CD4 help dependence, and intranasal infection induces more potent effector CD8+ T cells than i.p. infection.

Mononucleosis and Antigen-Driven T Cell Responses Have Different Requirements for Interleukin-2 Signaling in Murine Gammaherpesvirus Infection

ABSTRACT Interleukin-2 (IL-2) has been implicated as being necessary for the optimal formation of primary CD8+ T cell responses against various pathogens. Here we have examined the role that IL-2 signaling plays in several aspects of a CD8+ T cell response against murine gammaherpesvirus 68 (MHV-68). Exposure to MHV-68 causes a persistent infection, along with infectious mononucleosis, providing a model for studying these processes in mice. Our study indicates that CD25 is necessary for optimal expansion of the antigen-specific CD8+ T cell response but not for the long-term memory response. Contrastingly, IL-2 signaling through CD25 is absolutely required for CD8+ T cell mononucleosis.