Senta M. Walton

Differential role of IL-2R signaling for CD8+ T cell responses in acute and chronic viral infections

IL‐2 is a cytokine with multiple and even divergent functions; it has been described as a key cytokine for in vitro T cell proliferation but is also essential for down‐regulating T cell responses by inducing activation‐induced cell death as well as regulatory T cells. The in vivo analysis of IL‐2 function in regulating specific T cell responses has been hampered by the fact that mice deficient in IL‐2 or its receptors develop lymphoproliferative diseases and/or autoimmunity. Here we generated chimeric mice harboring both IL‐2R‐competent and IL‐2R‐deficient T cells and assessed CD8+ T cell induction, function and maintenance after acute or persistent viral infections. Induction and maintenance of CD8+ T cells were relatively independent of IL‐2R signaling during acute/resolved viral infection. In marked contrast, IL‐2 was crucial for secondary expansion of memory CD8+ T cells and for the maintenance of virus‐specific CD8+ T cells during persistent viral infections. Thus, depending on the chronicity of antigen exposure, IL‐2R signaling is either essential or largely dispensable for induction and maintenance of virus‐specific CD8+ T cell responses.

Batf3 transcription factor-dependent DC subsets in murine CMV infection: Differential impact on T-cell priming and memory inflation

Priming of CD8+ T cells specific for viruses that interfere with the MHC class I presentation pathway is a challenge for the immune system and is believed to rely on cross‐presentation. Cytomegalovirus (CMV) infection induces vigorous CD8+ T‐cell responses despite its potent immune evasion strategies. Furthermore, CD8+ T cells specific for a subset of viral epitopes accumulate and are maintained at high levels exhibiting an activated phenotype – referred to as “inflationary T cells”. Taking advantage Batf3−/− mice in which the development of cross‐presenting CD8α+ and CD103+ DCs is severely compromised, we analyzed their role in the induction and inflation of murine (M)CMV‐specific CD8+ T‐cell responses. We found that priming of MCMV‐specific CD8+ T cells was severely impaired in the absence of cross‐presenting DCs. However, inflation of two immuno‐dominant MCMV‐specific CD8+ T‐cell populations was largely normal in the absence of cross‐presenting DCs, indicating that inflation during latency was mainly dependent on direct antigen presentation. These results highlight differential antigen presentation requirements during acute and latent MCMV infection.

Virus‐specific CD8 T cells: activation, differentiation and memory formation

CD8 T cells are pivotal for the control of many intracellular pathogens, and besides their role in immediate control of infections, CD8 T cells have the capacity to differentiate into long‐lived antigen‐independent memory CD8 T cells, at least in situations of acute and resolved infections. The population of memory cells is heterogeneous with respect to their phenotype, their anatomical localization and their functional capacities in order to afford optimal protection against secondary infections. In the past years, it has become clear that multiple in vivo parameters are involved in shaping the composition of the memory CD8 T cell population, including antigen load, duration and strength of CD8 T cell stimulation, the level of inflammation, availability of CD4 T cell help and CD8 T cell precursor frequencies. With respect to the timing when CD8 T cells are committed to become memory cells, several models have been proposed. In contrast to acute, resolved infection, the continued in vivo exposure to high levels of antigen during persistent chronic viral infection precludes the development of long‐lived antigen‐independent memory CD8 T cells and might even result in severe dysfunction of virus‐specific CD8 T cells.

Absence of Cross-Presenting Cells in the Salivary Gland and Viral Immune Evasion Confine Cytomegalovirus Immune Control to Effector CD4 T Cells

Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFNγ that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFNγ, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFNγ secretion interfering with viral replication in cells of non-hematopoietic origin.

IL-10 suppression of NK/DC crosstalk leads to poor priming of MCMV-specific CD4 T cells and prolonged MCMV persistence.

IL-10 is an anti-inflammatory cytokine that regulates the extent of host immunity to infection by exerting suppressive effects on different cell types. Herpes viruses induce IL-10 to modulate the virus-host balance towards their own benefit, resulting in prolonged virus persistence. To define the cellular and molecular players involved in IL-10 modulation of herpes virus-specific immunity, we studied mouse cytomegalovirus (MCMV) infection. Here we demonstrate that IL-10 specifically curtails the MCMV-specific CD4 T cell response by suppressing the bidirectional crosstalk between NK cells and myeloid dendritic cells (DCs). In absence of IL-10, NK cells licensed DCs to effectively prime MCMV-specific CD4 T cells and we defined the pro-inflammatory cytokines IL-12, IFN-γ and TNF-α as well as NK cell activating receptors NKG2D and NCR-1 to regulate this bidirectional NK/DC interplay. Consequently, markedly enhanced priming of MCMV-specific CD4 T cells in Il10 −/− mice led to faster control of lytic viral replication, but this came at the expense of TNF-α mediated immunopathology. Taken together, our data show that early induction of IL-10 during MCMV infection critically regulates the strength of the innate-adaptive immune cell crosstalk, thereby impacting beneficially on the ensuing virus-host balance for both the virus and the host.

The Dynamics of Mouse Cytomegalovirus-Specific CD4 T Cell Responses during Acute and Latent Infection

The dynamics of mouse cytomegalovirus (MCMV)-specific CD4 T cell responses and the mechanisms by which these cells contribute to viral control are not well understood, mainly due to lack of appropriate tools to characterize MCMV-specific CD4 T cells. We therefore generated MCMV-specific CD4 T cell hybridomas, then used an MCMV expression library and overlapping peptides to identify CD4 T cell epitopes. We used these novel tools to study the long-term kinetics and organ distribution of MCMV-specific CD4 T cells in comparison to MCMV-specific CD8 T cell responses. We demonstrate that the overall MCMV-specific CD4 T cell response stabilizes during the latent stage, which stands in contrast to subpopulations of MCMV-specific CD8 T cells and HCMV-specific CD4 T cells which accumulate over the course of CMV latency. Furthermore, MCMV-specific CD4 T cells displayed a Th1 phenotype, secreting high levels of IFN-γ and TNF-α and to some extent IL-2, cytokines which are involved in protection from CMV disease.

The Salivary Gland Acts as a Sink for Tissue-Resident Memory CD8+ T Cells, Facilitating Protection from Local Cytomegalovirus Infection

Tissue-resident memory T cells (TRM) reside in barrier tissues and provide local immediate protective immunity. Here, we show that the salivary gland (SG) most-effectively induces CD8(+) and CD4(+) TRM cells against murine cytomegalovirus (MCMV), which persists in and spreads from this organ. TRM generation depended on local antigen for CD4(+), but not CD8(+), TRM cells, highlighting major differences in T cell subset-specific demands for TRM development. CMV-specific CD8(+) T cells fail to control virus replication upon primary infection in the SG due to CMV-induced MHC I downregulation in glandular epithelial cells. Using intraglandular infection, we challenge this notion and demonstrate that memory CD8(+) T cells confer immediate protection against locally introduced MCMV despite active viral immune evasion, owing to early viral tropism to cells that largely withstand MHC I downregulation. Thus, we unravel a yet-unappreciated role for memory CD8(+) T cells in protecting mucosal tissues against CMV infection.

T-cell help permits memory CD8+ T-cell inflation during cytomegalovirus latency

CD4+ T cells are implied to sustain CD8+ T‐cell responses during persistent infections. As CD4+ T cells are often themselves antiviral effectors, they might shape CD8+ T‐cell responses via help or via controlling antigen load. We used persistent murine CMV (MCMV) infection to dissect the impact of CD4+ T cells on virus‐specific CD8+ T cells, distinguishing between increased viral load in the absence of CD4+ T cells and CD4+ T‐cell‐mediated helper mechanisms. Absence of T‐helper cells was associated with sustained lytic MCMV replication and led to a slow and gradual reduction of the size and function of the MCMV‐specific CD8+ T‐cell pool. However, when virus replication was controlled in the absence of CD4+ T cells, CD8+ T‐cell function was comparably impaired, but in addition CD8+ T‐cell inflation, a hallmark of CMV infection, was completely abolished. Thus, CD8+ T‐cell inflation during latent CMV infection is strongly dependent on CD4+ T‐cell helper functions, which can partially be compensated by ongoing lytic viral replication in the absence of CD4+ T cells.

Comparing the Kinetics of NK Cells, CD4, and CD8 T Cells in Murine Cytomegalovirus Infection

NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H+ NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H+ NK cells, may be not be as robust as the secondary response observed in T cells.

Spontaneous CD8 T Cell Responses against the Melanocyte Differentiation Antigen RAB38/NY-MEL-1 in Melanoma Patients

The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-150–58 exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A∗0201+ melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-150–58 peptide. Accordingly, monoclonal RAB38/NY-MEL-150–58-specific T cell populations were capable of specifically recognizing HLA-A2+ melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-150–58 multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-150–58 is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.