The microRNA cluster miR-17∼92 promotes TFH cell differentiation and represses subset-inappropriate gene expression.

Follicular helper T cells (TFH cells) are the prototypic helper T cell subset specialized to enable B cells to form germinal centers (GCs) and produce high-affinity antibodies. We found that expression of microRNAs (miRNAs) by T cells was essential for TFH cell differentiation. More specifically, we show that after immunization of mice with protein, the miRNA cluster miR-17∼92 was critical for robust differentiation and function of TFH cells in a cell-intrinsic manner that occurred regardless of changes in proliferation. In a viral infection model, miR-17∼92 restrained the expression of genes 'inappropriate' to the TFH cell subset, including the direct miR-17∼92 target Rora. Removal of one Rora allele partially 'rescued' the inappropriate gene signature in miR-17∼92-deficient TFH cells. Our results identify the miR-17∼92 cluster as a critical regulator of T cell-dependent antibody responses, TFH cell differentiation and the fidelity of the TFH cell gene-expression program.

B Cell-intrinsic TLR7 signaling is required for optimal B cell responses during chronic viral infection.

The importance for activation of innate immunity by pattern recognition receptors in forming an effective adaptive immune response is well known. TLRs were demonstrated to be critical for Ab responses to a variety of immunizations. In particular, recent evidence suggests that B cell-intrinsic TLR signaling is required for optimal responses to virus-like Ags, but the mechanisms by which TLR signaling impacts Ab responses during infection in vivo is unclear. In the current study, we demonstrate that deficiency of TLR7 in B cells alone is sufficient to significantly impact Ab responses in mice during chronic viral infection. This effect was independent of T follicular helper cells and resulted in a loss of plasma cells generated later, but not early, in the response. The defect in plasma cell formation appeared to be secondary to a qualitative effect of TLR signaling on the germinal center (GC) B cell response. GC B cells in TLR7-deficient mice proliferated to a lesser extent and had a greater proportion of cells with phenotypic characteristics of light zone, relative to dark zone, GC B cells. These results suggest that B cell-intrinsic TLR signaling in vivo likely affects plasma cell output by altered selection of Ag-specific B cells in the GC.

Differential roles for RIG-I-like receptors and nucleic acid-sensing TLR pathways in controlling a chronic viral infection.

The necessity for pathogen recognition of viral infection by the innate immune system in initiating early innate and adaptive host defenses is well documented. However, little is known about the role these receptors play in the maintenance of adaptive immune responses and their contribution to resolution of persistent viral infections. In this study, we demonstrate a nonredundant functional requirement for both nucleic acid-sensing TLRs and RIG-I-like receptors in the control of a mouse model of chronic viral infection. Whereas the RIG-I-like receptor pathway was important for production of type I IFNs and optimal CD8(+) T cell responses, nucleic acid-sensing TLRs were largely dispensable. In contrast, optimal anti-viral Ab responses required intact signaling through nucleic acid-sensing TLRs, and the absence of this pathway correlated with less virus-specific Ab and deficient long-term virus control of a chronic infection. Surprisingly, absence of the TLR pathway had only modest effects on Ab production in an acute infection with a closely related virus strain, suggesting that persistent TLR stimulation may be necessary for optimal Ab responses in a chronic infection. These results indicate that innate virus recognition pathways may play critical roles in the outcome of chronic viral infections through distinct mechanisms.

Expression of chemokine receptor CXCR3 on T cells affects the balance between effector and memory CD8 T-cell generation.

Generation of a robust immunological memory response is essential for protection on subsequent encounters with the same pathogen. The magnitude and quality of the memory CD8 T-cell population are shaped and influenced by the strength and duration of the initial antigenic stimulus as well as by inflammatory cytokines. Although chemokine receptors have been established to play a role in recruitment of effector CD8 T cells to sites of inflammation, their contribution to determination of T-cell fate and shaping of the long-lived memory T-cell population is not fully understood. Here, we investigated whether reduced access to antigen and inflammation through alterations in expression of inflammatory and homeostatic chemokine receptors has an impact on generation of effector and memory CD8 T cells. We found that in mice infected with lymphocytic choriomeningitis virus, colocalization of virus-specific CD8 T cells with antigen in spleen is dependent on expression of the inflammatory chemokine receptor, CXCR3. In addition, absence of CXCR3 expression on CD8 T cells leads to formation of fewer short-lived effector cells and more memory precursor cells. Furthermore, the memory CD8 T-cell population derived from CXCR3-deficient cells has fewer cells of the effector memory phenotype and exhibits a recall response of greater magnitude than that of WT cells. These data demonstrate that CD8 T-cell positioning relative to antigen and inflammatory cytokines in secondary lymphoid organs affects the balance of effector and memory T-cell formation and has both a quantitative and qualitative impact on the long-lived memory CD8 T-cell population.

Proline-rich tyrosine kinase-2 is critical for CD8 T-cell short-lived effector fate.

T-cell interactions with antigen-presenting cells are important for CD8 T-cell effector or memory fate determination. The integrin leukocyte function-associated antigen-1 (LFA-1) mediates T-cell adhesion but the contribution of LFA-1-induced signaling pathways to T-cell responses is poorly understood. Here we demonstrate that proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell activation by synergistic LFA-1 and T-cell receptor stimulation. Furthermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion and LFA-1 costimulation of CD8 T-cell migration. During lymphocytic choriomeningitis virus infection in vivo, PYK2 deficiency results in a specific loss of short-lived effector CD8 T cells but does not affect memory-precursor CD8 T-cell development. Similarly, lack of LFA-1 primarily impairs the generation of short-lived effector cells. Thus, PYK2 facilitates LFA-1-dependent CD8 T-cell responses and promotes CD8 T-cell short-lived effector fate, suggesting that PYK2 may be an interesting therapeutic target to suppress exacerbated CD8 T-cell responses.

Endomembrane targeting of human OAS1 p46 augments antiviral activity.

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1 p46 is an important determinant of COVID-19 severity.

Effect of T helper 1 (Th1)/Th2 cytokine on chemokine-induced dendritic cell functions.

Effects of T helper 2 (Th2) and Th1 cytokines, interleukin (IL)-4, and interferon (IFN)-gamma, respectively, on chemokine-induced DC migration and endocytosis are not well understood. We investigated herein the effects of these cytokines on chemokine-induced functions of murine myeloid DCs. As expected, immature DCs markedly migrated to CCL3 but not CCL19, while mature DCs showed vigorous migration in response to CCL19 but not CCL3. Both IL-4 and IFN-gamma significantly decreased CCL3-induced migration of immature DCs. In contrast, these cytokines exerted no significant effects on CCL19-induced migration of mature DCs. Of note, both IL-4 and IFN-gamma markedly enhanced CCL3-induced endocytosis of immature DCs. The messenger RNA level of CCR5, a CCL3 receptor, in immature DCs was slightly increased by IL-4 or IFN-gamma treatment. These results demonstrate that these Th1/Th2 cytokines can act to either inhibit or enhance chemokine-mediated DC functions, and may play a role in increasing antigen uptake by immature DCs at inflammatory sites.

CCR7-mediated c-Jun N-terminal kinase activation regulates cell migration in mature dendritic cells.

c-Jun N-terminal kinase (JNK) is generally thought to be involved in inflammation, proliferation and apoptosis. However, functional role(s) of this molecule in dendritic cells (DCs) has not been well understood. CCR7 ligands, CCL19 and CCL21, induce not only chemotaxis but also endocytosis in mature DCs. In the present study, we examined the role of JNK for inducing chemotaxis and endocytosis in murine mature DCs. CCL19 rapidly enhanced endocytosis of mature DCs within a few minutes, whereas significant migration of mature DCs to this chemokine was detected 30 min or more after incubation. CCL19 significantly activated JNK in mature DCs at 15 min. CCL19 also increased interaction between phospho-JNK and phospho-mitogen-activated protein kinase kinase (MKK) 4 but not phospho-MKK7 in mature DCs, suggesting that the JNK activation is mediated via MKK4. Blocking of this JNK activation significantly inhibited the CCL19-induced migration of mature DCs. Blocking of Rho-associated kinase also inhibited the CCL19-induced migration without affecting the JNK activation. On the other hand, the inhibition of either JNK or Rho-associated kinase showed no significant effects on CCL19-induced endocytosis by mature DCs. These findings suggest that CCL19 activates JNK via a Rho-independent pathway, thereby inducing migration of mature DCs, whereas the JNK activation is dispensable for the CCL19-induced endocytosis. It seems that at least two different pathways, JNK pathway and Rho-associated kinase pathway, are involved in the CCR7-mediated migration of mature DCs. Thus, we demonstrate herein a novel role of JNK for regulating chemokine-induced DC migration.