A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL‐17 cytokines and whose differentiation requires the nuclear receptor, RORγt. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL‐17 production for human T cells. Activating cord blood (naïve) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9 and CXCR6. Despite these data, we found no strong correlation between the production of IL‐17 and expression of CCR9 or CXCR6. By contrast, virtually all IL‐17‐producing CD4+ T cells, either made in our in vitro or found in peripheral blood, expressed CCR6. Compared with CD4+CD45RO+CCR6− cells, CD4+CD45RO+CCR6+ cells contained at least 100‐fold more IL‐17A mRNA and secreted 100‐fold more IL‐17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORγt. CCR6 was likewise expressed on all IL‐17‐producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL‐17‐producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL‐17‐related immune responses and possible targets for preventing inflammatory injury. Research support: NIAID, NIH

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Monokine induced by IFN-γ (Mig; CXC chemokine ligand 9) is an IFN-γ-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4+ and CD8+, and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig−/− mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig−/− mice showed reductions of 50–75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-γ induced the expression of mumig in APCs, including CD8α+ and CD8α− dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.

The pathways for differentiation of human CD4 ؉ T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naïve CD4 ؉ T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-receptor ؉ cells suggest that they are very early memory CD4 ؉ T cells that have ''rested down'' before acquiring the phenotypes described for ''central'' or ''effector'' memory T cells. In addition, the chemokine-receptor ؉ ''naïve'' populations contain Th1 and Th2 cells, respectively, demonstrating that Th1͞Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-receptor ؉ subsets may be recruited and contribute to segregated, polarized microenvironments within lymphoid organs. Importantly, our data suggest that CD4 ؉ T cells do not differentiate according to a simple schema from naïve 3 CD45RO ؉ noneffector͞central memory 3 effector͞effector memory cells. Rather, developmental pathways branch early on to yield effector͞memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells.chemokines ͉ immunologic memory ͉ Th1͞Th2 cells U nderstanding how immunological memory develops and is sustained is a focus of ongoing research, in part because successful vaccination requires the production of long-lived memory (1, 2). Such an understanding depends on identifying subsets of memory cells, characterizing their functions, and mapping the pathways through which they are generated. As the molecular determinants of lymphocyte trafficking have been defined, the association between T cells' migratory capacities and their functions has been used to characterize T cell subsets. It was reported, for example, that expression of the chemokine receptor CCR7 identifies a subset of memory cells, so-called central memory (T CM ) cells, that coexpress L-selectin (CD62L) and, in addition to being able to enter lymphoid organs, are functionally distinct from the CCR7 Ϫ subset, so-called effector memory (T EM ) cells (3). The key findings were that T EM but not T CM cells could produce effector cytokines and that T CM cells could serve, in vitro, as precursors for effector cells. Sallusto et al. (4) have hypothesized a linear pathway of T cell differentiation of naïve 3 CD45RO ϩ ͞CD45RA Ϫ noneffector͞T CM 3 effector and T EM cells, driven by the strength of the activating signals. In this model, T CM cells provide ''reactive memory'' and serve as a long-lived pool of precursors for additional memory and effector cells.Our studies of how patterns of chemokine-recep...

It has been reported that host defense against pulmonary K. pneumoniae infection requires IL-22, which has been proposed to be of T cell origin. Supporting a role for IL-22, we found that Il22−/− mice had decreased survival as compared with wild type mice after intratracheal infection with K. pneumoniae. Surprisingly, however, Rag2−/− mice did not differ from wild type mice in survival or levels of IL-22 in the lungs after infection with K. pneumoniae. By contrast, K. pneumoniae-infected Rag2−/−Il2rg−/− mice failed to produce IL-22. These data suggested a possible role for NK cells or other innate lymphoid cells (ILC) in host defense and production of IL-22. Unlike NK cell-like ILCs that produce IL-22 and display a surface phenotype of NK1.1−NKp46+CCR6+, lung NK cells showed the conventional phenotype, NK1.1+NKp46+CCR6−. Mice depleted of NK cells using anti-asialo GM1 showed decreased survival and higher lung bacterial counts as well as increased dissemination of K. pneumoniae to blood and liver as compared with control-treated mice. NK cell depletion also led to decreased production of IL-22 in the lung. Within one day after infection, although there was no increase in the number of lung NK cells, a subset of lung NK cells became competent to produce IL-22, and such cells were found in both wild type and Rag2−/− mice. Our data suggest that during pulmonary infection of mice with K. pneumoniae, conventional NK cells are required for optimal host defense, which includes the production of IL-22.

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Th17 cells, which all express the chemokine receptor CCR6, are implicated in many immune-mediated disorders such as psoriasis and multiple sclerosis. We found that expression levels of CCR6 on human effector/memory CD4+ T cells reflect a continuum of Th17 differentiation. By evaluating the transcriptome in cells with increasing CCR6, we detected progressive up-regulation of ZBTB16, which encodes the BTB-ZF transcription factor PLZF. Using ChIP for modified histones, p300, and PLZF, we identified enhancer-like sites at −9/−10 kb and −13/−14 kb from the upstream transcription start site of CCR6 that bind PLZF in CCR6+ cells. For Th cells from adult blood, both in the CCR6+ memory population and in naïve cells activated ex vivo, knockdown of ZBTB16 down-regulated CCR6 and other Th17-associated genes. ZBTB16 and RORC (which encodes the “master regulator” RORγt) cross-regulate each other, and PLZF binds at the RORC promoter in CCR6+ cells. In naive Th cells from cord blood, ZBTB16 expression was confined to the CD161+ cells, which are the Th17 cell precursors. ZBTB16 was not expressed in mouse Th17 cells and Th17 cells could be made from luxoid mice, which harbor an inactivating mutation in Zbtb16. These studies demonstrate a role for PLZF as an activator of transcription important both for Th17 differentiation and the maintenance of the Th17 phenotype in human cells, expand the role of PLZF as a critical regulator in the human adaptive immune system, and identify a novel, essential element in a regulatory network that is of significant therapeutic interest.

Many mediators and regulators of extravasation by bona fide human memory-phenotype T cells remain undefined. Mucosal-associated invariant T (MAIT) cells are innate-like, antibacterial cells that we found excelled at crossing inflamed endothelium. They displayed abundant selectin ligands, with high expression of FUT7 and ST3GAL4, and expressed CCR6, CCR5, and CCR2, which played non-redundant roles in trafficking on activated endothelial cells. MAIT cells selectively expressed CCAAT/enhancer-binding protein delta (C/EBPδ). Knockdown of C/EBPδ diminished expression of FUT7, ST3GAL4 and CCR6, decreasing MAIT cell rolling and arrest, and consequently the cells’ ability to cross an endothelial monolayer in vitro and extravasate in mice. Nonetheless, knockdown of C/EBPδ did not affect CCR2, which was important for the step of transendothelial migration. Thus, MAIT cells demonstrate a program for extravasastion that includes, in part, C/EBPδ and C/EBPδ-regulated genes, and that could be used to enhance, or targeted to inhibit T cell recruitment into inflamed tissue.