The transcription factor KLF2 restrains CD4⁺ T follicular helper cell differentiation.

T follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4(+) T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4(+) T cells led to increased Tfh cell generation and B cell priming, whereas KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4(+) T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization and by regulation of lineage-defining transcription factors.

Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells.

Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (T(RM) cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of T(RM) cells is unknown. We found that CD8(+) T(RM) cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P1, a receptor for sphingosine 1-phosphate). Forced expression of S1P1 prevented the establishment of T(RM) cells. Cytokines that induced a T(RM) cell phenotype (including transforming growth factor-ß (TGF-ß), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P1 provides a switch that dictates whether CD8(+) T cells commit to recirculating or tissue-resident memory populations.

Cutting edge: intravascular staining redefines lung CD8 T cell responses.

Nonlymphoid T cell populations control local infections and contribute to inflammatory diseases, thus driving efforts to understand the regulation of their migration, differentiation, and maintenance. Numerous observations indicate that T cell trafficking and differentiation within the lung are starkly different from what has been described in most nonlymphoid tissues, including intestine and skin. After systemic infection, we found that >95% of memory CD8 T cells isolated from mouse lung via standard methods were actually confined to the pulmonary vasculature, despite perfusion. A respiratory route of challenge increased virus-specific T cell localization within lung tissue, although only transiently. Removing blood-borne cells from analysis by the simple technique of intravascular staining revealed distinct phenotypic signatures and chemokine-dependent trafficking restricted to Ag-experienced T cells. These results precipitate a revised model for pulmonary T cell trafficking and differentiation and a re-evaluation of studies examining the contributions of pulmonary T cells to protection and disease.

Chemokines control naive CD8+ T cell selection of optimal lymph node antigen presenting cells.

Naive antiviral CD8(+) T cells are activated in the draining LN (DLN) by dendritic cells (DCs) presenting viral antigens. However, many viruses infect LN macrophages, which participate in initiation of innate immunity and B cell activation. To better understand how and why T cells select infected DCs rather than macrophages, we performed intravital microscopy and ex vivo analyses after infecting mice with vaccinia virus (VV), a large DNA virus that infects both LN macrophages and DCs. Although CD8(+) T cells interact with both infected macrophages and DCs in the LN peripheral interfollicular region (PIR), DCs generate more frequent and stable interactions with T cells. VV infection induces rapid release of CCR5-binding chemokines in the LN, and administration of chemokine-neutralizing antibodies diminishes T cell activation by increasing T cell localization to macrophages in the macrophage-rich region (MRR) at the expense of PIR DCs. Similarly, DC ablation increases both T cell localization to the MRR and the duration of T cell-macrophage contacts, resulting in suboptimal T cell activation. Thus, virus-induced chemokines in DLNs enable antiviral CD8(+) T cells to distinguish DCs from macrophages to optimize T cell priming.

Fox factors fight over T cell quiescence.

The transcription factor Foxp1 helps maintain the quiescence of naive T cells by inhibiting IL-7Rα expression and diminishing signaling by the kinase Erk.

Unexpected role for the immunoproteasome subunit LMP2 in antiviral humoral and innate immune responses.

Proteasomes are multisubunit proteases that initiate degradation of many Ags presented by MHC class I molecules. Vertebrates express alternate forms of each of the three catalytic proteasome subunits: standard subunits, and immunosubunits, which are constitutively expressed by APCs and are induced in other cell types by exposure to cytokines. The assembly of mixed proteasomes containing standard subunits and immunosubunits is regulated in a tissue specific manner. In this study, we report that the presence of mixed proteasomes in immune cells in LMP2(-/-) mice compromises multiple components that contribute to the generation of antiviral Ab responses, including splenic B cell numbers, survival and function of adoptively transferred B cells, Th cell function, and dendritic cell secretion of IL-6, TNF-alpha, IL-1beta, and type I IFNs. These defects did not result from compromised overall protein degradation, rather they were associated with altered NF-kappaB activity. These findings demonstrate an important role for immunoproteasomes in immune cell function beyond their contribution to Ag processing.

KLF2 transcription-factor deficiency in T cells results in unrestrained cytokine production and upregulation of bystander chemokine receptors.

The transcription factor KLF2 regulates T cell trafficking by promoting expression of the lipid-binding receptor S1P(1) and the selectin CD62L. Recently, it was proposed that KLF2 also represses the expression of chemokine receptors. We confirmed the upregulation of the chemokine receptor CXCR3 on KLF2-deficient T cells. However, we showed that this was a cell-nonautonomous effect, as revealed by CXCR3 upregulation on wild-type bystander cells in mixed bone-marrow chimeras with KLF2-deficient cells. Furthermore, KLF2-deficient T cells overproduced IL-4, leading to the upregulation of CXCR3 through an IL-4-receptor- and eomesodermin-dependent pathway. Consistent with the increased IL-4 production, we found high concentrations of serum IgE in mice with T cell-specific KLF2 deficiency. Our findings support a model where KLF2 regulates T cell trafficking by direct regulation of S1P(1) and CD62L and restrains spontaneous cytokine production in naive T cells.

Direct priming of antiviral CD8+ T cells in the peripheral interfollicular region of lymph nodes.

It is uncertain how antiviral lymphocytes are activated in draining lymph nodes, the site where adaptive immune responses are initiated. Here, using intravital microscopy we show that after infection of mice with vaccinia virus (a large DNA virus) or vesicular stomatitis virus (a small RNA virus), virions drained to the lymph node and infected cells residing just beneath the subcapsular sinus. Naive CD8+ T cells rapidly migrated to infected cells in the peripheral interfollicular region and then formed tight interactions with dendritic cells, leading to complete T cell activation. Thus, antigen presentation at the lymph node periphery, not at lymphocyte exit sites in deeper lymph node venules, as dogma dictates, has a dominant function in antiviral CD8+ T cell activation.

Role for Msh5 in the regulation of Ig class switch recombination.

Ig class switch recombination (CSR) and somatic hypermutation serve to diversify antibody responses and are orchestrated by the activity of activation-induced cytidine deaminase and many proteins involved in DNA repair and genome surveillance. Msh5, a gene encoded in the central MHC class III region, and its obligate heterodimerization partner Msh4 have a critical role in regulating meiotic homologous recombination and have not been implicated in CSR. Here, we show that MRL/lpr mice carrying a congenic H-2(b/b) MHC interval exhibit several abnormalities regarding CSR, including a profound deficiency of IgG3 in most mice and long microhomologies at Ig switch (S) joints. We found that Msh5 is expressed at low levels on the H-2(b) haplotype and, importantly, a similar long S joint microhomology phenotype was observed in both Msh5 and Msh4-null mice. We also present evidence that genetic variation in MSH5 is associated with IgA deficiency and common variable immune deficiency (CVID) in humans. One of the human MSH5 alleles identified contains two nonsynonymous polymorphisms, and the variant protein encoded by this allele shows impaired binding to MSH4. Similar to the mice, Ig S joints from CVID and IgA deficiency patients carrying disease-associated MSH5 alleles show increased donor/acceptor microhomology, involving pentameric DNA repeat sequences and lower mutation rates than controls. Our findings suggest that Msh4/5 heterodimers contribute to CSR and support a model whereby Msh4/5 promotes the resolution of DNA breaks with low or no terminal microhomology by a classical nonhomologous end-joining mechanism while possibly suppressing an alternative microhomology-mediated pathway.