Sarah Q. Crome

CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL‐23 receptor (IL‐23R), CCR6, CD161, and the transcription factor retinoic acid‐related orphan receptor C (RORC), and originate from a CD161+CD4+ naïve T‐cell precursor in response to the combined activity of IL‐1β and IL‐23. We show here that not only CD4+TCRαβ+, but also CD8+TCRαβ+, CD4−CD8− TCRαβ+, and CD4−CD8− TCRγδ+ circulating lymphocytes that produce IL‐17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8+ and CD4−CD8− umbilical cord blood T cells that already express RORC and IL‐23R mRNA and that can be induced to differentiate into IL‐17‐producing cells in the presence of IL‐1β and IL‐23. Finally, we provide evidence that umbilical cord blood naïve CD4+CD161− T cells, upon lentivirus‐mediated transduction with RORC2 can acquire the ability to express IL‐23R, IL‐1RI, and CD161, as well as to produce IL‐17. Taken together, these data allow to conclude that T‐cell subsets able to produce IL‐17, as well as precursors of IL‐17‐producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2‐dependent.

Translational Mini-Review Series on Th17 Cells: Function and regulation of human T helper 17 cells in health and disease

OTHER ARTICLES PUBLISHED IN THIS MINI‐REVIEW SERIES ON Th17 CELLS
Induction of interleukin‐17 production by regulatory T cells. Clin Exp Immunol 2009; doi:10.1111/j.1365‐2249.2009.04038.x
Are T helper 17 cells really pathogenic in autoimmunity? Clin Exp Immunol 2009; doi:10.1111/j.1365‐2249.2009.04039.x
CD4+ T helper cells: functional plasticity and differential sensitivity to regulatory T cell‐mediated regulation. Clin Exp Immunol 2009; doi:10.1111/j.1365‐2249.2009.04040.x
Development of mouse and human T helper 17 cells. Clin Exp Immunol 2009; doi:10.1111/j.1365‐2249.2009.04041.x

Cutting Edge: Increased IL-17–Secreting T Cells in Children with New-Onset Type 1 Diabetes

CD4+FOXP3+ regulatory T cells are essential for immune tolerance, and murine studies suggest that their dysfunction can lead to type 1 diabetes (T1D). Human studies assessing regulatory T cell dysfunction in T1D have relied on analysis of FOXP3-expressing cells. Recently, distinct subsets of CD4+FOXP3+ T cells with differing function were identified. Notably, CD45RA−CD25intFOXP3low T cells lack suppressive function and secrete the proinflammatory cytokine IL-17. Therefore, we evaluated whether the relative fractions of CD4+FOXP3+ subsets are altered in new-onset T1D subjects. We report that children with new-onset T1D have an increased proportion of CD45RA−CD25intFOXP3low cells that are not suppressive and secrete significantly more IL-17 than other FOXP3+ subsets. Moreover, these T1D subjects had a higher proportion of both CD4+ and CD8+ T cells that secrete IL-17. The bias toward IL-17–secreting T cells in T1D suggests a role for this proinflammatory cytokine in the pathogenesis of disease.

Human Th1 and Th17 Cells Exhibit Epigenetic Stability at Signature Cytokine and Transcription Factor Loci

The linear model of Th cell lineage commitment is being revised due to reports that mature Th cells can trans-differentiate into alternate lineages. This ability of Th cells to reprogram is thought to be regulated by epigenetic mechanisms that control expression of transcription factors characteristic of opposing lineages. It is unclear, however, to what extent this new model of Th cell plasticity holds true in human Th cell subsets that develop under physiological conditions in vivo. We isolated in vivo-differentiated human Th1 and Th17 cells, as well as intermediate Th1/17 cells, and identified distinct epigenetic signatures at cytokine (IFNG and IL17A) and transcription factor (TBX21, RORC, and RORA) loci. We also examined the phenotypic and epigenetic stability of human Th17 cells exposed to Th1-polarizing conditions and found that although they could upregulate TBX21 and IFN-γ, this occurred without loss of IL-17 or RORC expression, and resulted in cells with a Th1/17 phenotype. Similarly, Th1 cells could upregulate IL-17 upon enforced expression of RORC2, but did not lose expression of IFN-γ or TBX21. Despite alterations in expression of these signature genes, epigenetic modifications were remarkably stable aside from the acquisition of active histone methylation marks at cytokine gene promoters. The limited capacity of human Th17 and Th1 cells to undergo complete lineage conversion suggests that the bipotent Th1/17 cells may arise from Th1 and/or Th17 cells. These data also question the broad applicability of the new model of Th cell lineage plasticity to in vivo-polarized human Th cell subsets.

Inflammatory Effects of Ex Vivo Human Th17 Cells Are Suppressed by Regulatory T Cells

Th17 cells are proinflammatory cells associated with many immune-mediated diseases. Major factors limiting the study of human Th17 cells are the lack of an accepted method for their in vitro differentiation or for isolation of a homogenous population of Th17 cells that do not cosecrete IFN-γ. To overcome these hurdles, we established a novel method to isolate in vivo differentiated Th17 cells from peripheral blood by sorting CD161+CCR4+CCR6+CXCR3−CD4+ T cells. The resulting cells produce high levels of IL-17 but not IFN-γ, express high levels of retinoic acid-related orphan receptor variant 2, and maintain this phenotype upon expansion. Ex vivo Th17 cells exhibit a low cytotoxic potential and are hyporesponsive to polyclonal anti-CD3/anti-CD28 stimulation. Importantly, ex vivo Th17 cells were susceptible to suppression by both naive and memory regulatory T cells (Tregs), which inhibited production of IL-17, IL-22, and CXCL8. Moreover, Tregs suppressed the antifibrotic effects of Th17 cells in a wound-healing model. These findings provide new tools for the study of normal and pathological functions of bona fide Th17 cells in humans. They also provide new insight into the cross-talk between Th17 cells and immune and nonimmune cells, and they establish the paradigm that adoptive Treg-based therapies may effectively limit Th17-mediated inflammation.

The role of retinoic acid-related orphan receptor variant 2 and IL-17 in the development and function of human CD4+ T cells.

Th17 cells are defined by their capacity to produce IL‐17, and are important mediators of inflammation and autoimmunity. Human Th17 cells express high levels of the retinoic acid‐related orphan receptor variant 2 (RORC2), but it is currently unclear whether expression of this transcription factor alone is sufficient to recapitulate all the known properties of Th17 cells. We used lentivirus‐mediated transduction to investigate the role of RORC2 in defining aspects of the human Th17 cell lineage. Expression of RORC2 induced production of IL‐17A, IL‐22, IL‐6 and TNF‐α, a Th17‐cell‐associated chemokine receptor profile and upregulation of CD161. RORC2‐transduced T cells were hypo‐responsive to TCR‐mediated stimulation, a property shared with ex vivo Th17 cells and overcome by addition of exogenous IL‐2 or IL‐15. Co‐culture experiments revealed that RORC2‐expressing cells were partially resistant to Treg cells since production of IL‐17 and proliferation were not suppressed. Evidence that IL‐17 stimulates CD4+ T cells to produce IL‐2 and proliferate suggested that the resistance of Th17 cells to Treg‐mediated suppression may be partly attributed to IL‐17 itself. These findings demonstrate that expression of RORC2 in T cells has functional consequences beyond altering cytokine production and provides insight into the factors regulating the development of human Th17 cells.

Human CD4+FOXP3+ regulatory T cells produce CXCL8 and recruit neutrophils

One of the defining features of the majority of FOXP3+ Tregs is their inability to produce typical T‐cell‐derived cytokines. Little is known, however, about their capacity to produce chemokines. As Tregs are constitutively present in, and rapidly traffic to, non‐lympoid tissues, we hypothesized that they may produce chemokines to direct the composition of cells that infiltrate inflamed tissues. Surprisingly, we found that Tregs produce high amounts of CXCL8 (IL‐8), a potent neutrophil chemoattractant. Tregs also produced other CC and CXC family chemokines, including CCL2‐5, CCL7, and CXCL10. Whereas ectopic expression of FOXP3 suppressed cytokine production, it significantly induced CXCL8. Moreover, supernatants from Tregs attracted neutrophils via a CXCL8‐dependent mechanism. These data provide the first evidence that although classical Tregs are defined by their lack of proinflammatory cytokine production, they secrete significant quantities of chemokines and thus may have an unappreciated role in directing the recruitment of immune cells.

Adenoviral-transduced dendritic cells are susceptible to suppression by T regulatory cells and promote interleukin 17 production

Dendritic cell (DC) vaccines offer a robust platform for the development of cancer vaccines, but their effectiveness is thought to be limited by T regulatory cells (Tregs). Recombinant adenoviruses (RAdV) have been used successfully to engineer tumor antigen expression in DCs, but the impact of virus transduction on susceptibility to suppression by Tregs is unknown. We investigated the functional consequences of exposure to adenovirus on interactions between human monocyte-derived DCs and Tregs. Since the development of Tregs is linked to that of pro-inflammatory Th17 cells, the role of Th17 cells and IL-17-producing Tregs in the context of DC-based immunotherapies was also investigated. We found that Tregs potently suppressed the co-stimulatory capacity of RAdV-transduced DCs, regardless of whether the DCs were maturated by inflammatory cytokines or by exposure to Th1 or Th17 cells. Furthermore, exposure of Tregs to RAdV-exposed DCs increased IL-17 production and suppressive capacity, and correlated with enhanced secretion of IL-1β and IL-6 by DCs. The findings that DCs exposed to RAdV are suppressed by Tregs, promote Treg plasticity, and enhance Treg suppression indicates that strategies to limit Tregs will be required to enhance the efficacy of such DC-based immunotherapies.