Siglec-H-Deficient Mice Show Enhanced Type I IFN Responses, but Do Not Develop Autoimmunity After Influenza or LCMV Infections.

Siglec-H is a DAP12-associated receptor on plasmacytoid dendritic cells (pDCs) and microglia. Siglec-H inhibits TLR9-induced IFN-α production by pDCs. Previously, it was found that Siglec-H-deficient mice develop a lupus-like severe autoimmune disease after persistent murine cytomegalovirus (mCMV) infection. This was due to enhanced type I interferon responses, including IFN-α. Here we examined, whether other virus infections can also induce autoimmunity in Siglec-H-deficient mice. To this end we infected Siglec-H-deficient mice with influenza virus or with Lymphocytic Choriomeningitis virus (LCMV) clone 13. With both types of viruses we did not observe induction of autoimmune disease in Siglec-H-deficient mice. This can be explained by the fact that both types of viruses are ssRNA viruses that engage TLR7, rather than TLR9. Also, Influenza causes an acute infection that is rapidly cleared and the chronicity of LCMV clone 13 may not be sufficient and may rather suppress pDC functions. Siglec-H inhibited exclusively TLR-9 driven type I interferon responses, but did not affect type II or type III interferon production by pDCs. Siglec-H-deficient pDCs showed impaired Hck expression, which is a Src-family kinase expressed in myeloid cells, and downmodulation of the chemokine receptor CCR9, that has important functions for pDCs. Accordingly, Siglec-H-deficient pDCs showed impaired migration towards the CCR9 ligand CCL25. Furthermore, autoimmune-related genes such as Klk1 and DNase1l3 are downregulated in Siglec-H-deficient pDCs as well. From these findings we conclude that Siglec-H controls TLR-9-dependent, but not TLR-7 dependent inflammatory responses after virus infections and regulates chemokine responsiveness of pDCs.

PTPN2 Deficiency Enhances Programmed T Cell Expansion and Survival Capacity of Activated T Cells.

Manipulating molecules that impact T cell receptor (TCR) or cytokine signaling, such as the protein tyrosine phosphatase non-receptor type 2 (PTPN2), has significant potential for advancing T cell-based immunotherapies. Nonetheless, it remains unclear how PTPN2 impacts the activation, survival, and memory formation of T cells. We find that PTPN2 deficiency renders cells in vivo and in vitro less dependent on survival-promoting cytokines, such as interleukin (IL)-2 and IL-15. Remarkably, briefly ex vivo-activated PTPN2-deficient T cells accumulate in 3- to 11-fold higher numbers following transfer into unmanipulated, antigen-free mice. Moreover, the absence of PTPN2 augments the survival of short-lived effector T cells and allows them to robustly re-expand upon secondary challenge. Importantly, we find no evidence for impaired effector function or memory formation. Mechanistically, PTPN2 deficiency causes broad changes in the expression and phosphorylation of T cell expansion and survival-associated proteins. Altogether, our data underline the therapeutic potential of targeting PTPN2 in T cell-based therapies to augment the number and survival capacity of antigen-specific T cells.

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

CD4+ T help promotes influenza virus-specific CD8+ T cell memory by limiting metabolic dysfunction.

There is continued interest in developing novel vaccine strategies that induce establish optimal CD8+ cytotoxic T lymphocyte (CTL) memory for pathogens like the influenza A viruses (IAVs), where the recall of IAV-specific T cell immunity is able to protect against serologically distinct IAV infection. While it is well established that CD4+ T cell help is required for optimal CTL responses and the establishment of memory, when and how CD4+ T cell help contributes to determining the ideal memory phenotype remains unclear. We assessed the quality of IAV-specific CD8+ T cell memory established in the presence or absence of a concurrent CD4+ T cell response. We demonstrate that CD4+ T cell help appears to be required at the initial priming phase of infection for the maintenance of IAV-specific CTL memory, with "unhelped" memory CTL exhibiting intrinsic dysfunction. High-throughput RNA-sequencing established that distinct transcriptional signatures characterize the helped vs. unhelped IAV-specific memory CTL phenotype, with the unhelped set showing a more "exhausted T cell" transcriptional profile. Moreover, we identify that unhelped memory CTLs exhibit defects in a variety of energetic pathways, leading to diminished spare respiratory capacity and diminished capacity to engage glycolysis upon reactivation. Hence, CD4+ T help at the time of initial priming promotes molecular pathways that limit exhaustion by channeling metabolic processes essential for the rapid recall of memory CD8+ T cells.

Distinct epigenetic signatures delineate transcriptional programs during virus-specific CD8(+) T cell differentiation.

The molecular mechanisms that regulate the rapid transcriptional changes that occur during cytotoxic T lymphocyte (CTL) proliferation and differentiation in response to infection are poorly understood. We have utilized ChIP-seq to assess histone H3 methylation dynamics within naive, effector, and memory virus-specific T cells isolated directly ex vivo after influenza A virus infection. Our results show that within naive T cells, codeposition of the permissive H3K4me3 and repressive H3K27me3 modifications is a signature of gene loci associated with gene transcription, replication, and cellular differentiation. Upon differentiation into effector and/or memory CTLs, the majority of these gene loci lose repressive H3K27me3 while retaining the permissive H3K4me3 modification. In contrast, immune-related effector gene promoters within naive T cells lacked the permissive H3K4me3 modification, with acquisition of this modification occurring upon differentiation into effector/memory CTLs. Thus, coordinate transcriptional regulation of CTL genes with related functions is achieved via distinct epigenetic mechanisms.

The influenza virus-specific CTL immunodominance hierarchy in mice is determined by the relative frequency of high-avidity T cells.

Virus-specific CTL responses typically fall into reproducible hierarchies with particular epitopes eliciting either immunodominant or subdominant responses after viral challenge. The recently acquired capacity to directly enumerate naive CTL precursors (CTLps) in both mice and humans has implicated CTLp frequency as a key predictor of immune response magnitude after Ag challenge. However, recent studies have indicated that naive CTLp frequencies do not necessarily predict the size of the Ag-driven response, indicating an important role for differential CTLp recruitment and/or expansion. This study characterizes the early emergence of various influenza epitope-specific CTL responses at multiple sites in C57BL/6 mice, and probes the role of Ag dose and TCR avidity in dictating immune response hierarchies. Despite large naive CTLp numbers, subdominance was found to arise largely as a consequence of the abrupt and premature cessation of CTL proliferation, at least for one epitope specificity. Investigation into the possible drivers of the poor proliferation observed for subdominant specificities showed that the immunodominance hierarchy endured irrespective of epitope abundance, and correlated with the prevalence of high-avidity T cells in both the naive and immune compartments. Our study strongly indicates that the quality, and not simply the quantity, of antiviral CTLs dictate response magnitude.