Adele Y. Wang

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

Leishmania Exosomes Modulate Innate and Adaptive Immune Responses through Effects on Monocytes and Dendritic Cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ–producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100−/− L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100−/− leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100−/− exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

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.

A Regulatory T-Cell Gene Signature Is a Specific and Sensitive Biomarker to Identify Children With New-Onset Type 1 Diabetes

Type 1 diabetes (T1D) is caused by immune-mediated destruction of insulin-producing β-cells. Insufficient control of autoreactive T cells by regulatory T cells (Tregs) is believed to contribute to disease pathogenesis, but changes in Treg function are difficult to quantify because of the lack of Treg-exclusive markers in humans and the complexity of functional experiments. We established a new way to track Tregs by using a gene signature that discriminates between Tregs and conventional T cells regardless of their activation states. The resulting 31-gene panel was validated with the NanoString nCounter platform and then measured in sorted CD4+CD25hiCD127lo Tregs from children with T1D and age-matched control subjects. By using biomarker discovery analysis, we found that expression of a combination of six genes, including TNFRSF1B (CD120b) and FOXP3, was significantly different between Tregs from subjects with new-onset T1D and control subjects, resulting in a sensitive (mean ± SD 0.86 ± 0.14) and specific (0.78 ± 0.18) biomarker algorithm. Thus, although the proportion of Tregs in peripheral blood is similar between children with T1D and control subjects, significant changes in gene expression can be detected early in disease process. These findings provide new insight into the mechanisms underlying the failure to control autoimmunity in T1D and might lead to a biomarker test to monitor Tregs throughout disease progression.

Cellular magnetic resonance imaging of monocyte-derived dendritic cell migration from healthy donors and cancer patients as assessed in a scid mouse model

BACKGROUND AIMS. The use of dendritic cells (DC) as an adjuvant in cell-based immunotherapeutic cancer vaccines is a growing field of interest. A reliable and non-invasive method to track the fate of autologous DC following their administration to patients is required in order to confirm that clinically sufficient numbers are reaching the lymph node (LN). We demonstrate that an immunocompromised mouse model can be used to conduct translational studies employing cellular magnetic resonance imaging (MRI). Such studies can provide clinically relevant information regarding the migration potential of clinical-grade DC used in cancer immunotherapies. METHODS. Human monocyte-derived dendritic cells (mo-DC) were generated from negatively selected monocytes obtained from either healthy donors or cancer patients. DC were labeled with superparamagnetic iron oxide (SPIO) nanoparticles in order to track them in vivo in a CB17scid mouse model using cellular MRI. SPIO did not have any adverse effects on DC phenotype or function, independent of donor type. Cellular MRI readily detected migration of SPIO-loaded DC in CB17scid mice. No differences in migration were observed between DC obtained from healthy donors and those obtained from donors undergoing autologous stem cell transplant for cancer therapy. CONCLUSIONS. Cellular MRI provided semi-quantitative image data that corresponded with data obtained by digital morphometry, validating cellular MRIs potential to assess DC migration in DC-based cancer immunotherapy clinical trials.

Natural killer T cells constitutively expressing the interleukin-2 receptor α chain early in life are primed to respond to lower antigenic stimulation.

Invariant natural killer T (iNKT) cells are known to constitutively express the high affinity interlukin‐2 receptor α chain (CD25) in neonates, but the functional consequence of this phenotype is unknown. Here, we show that high numbers of CD25‐expressing iNKT cells are present early in gestation and represent a significant proportion of the developing immune system. Despite their activated phenotype, neonatal iNKT cells express high levels of the Krüppel‐like factor‐2, a transcription factor associated with quiescent T cells, and require de novo T‐cell receptor and CD28 co‐stimulation to proliferate. In contrast to bona fide CD4/CD25‐expressing regulatory T cells, neonatal iNKT cells do not suppress T‐cell responses, indicating that they do not represent an immunosuppressive cell subset. Evidence that neonatal iNKT cells respond to dramatically reduced amounts of CD1d‐restricted antigen compared with adult iNKT cells or T cells, and that their proliferation can be induced in the absence of early interleukin‐2 suggest that constitutive expression of CD25 ‘primes’ neonatal iNKT cells to respond rapidly to low amounts of antigen. This unique phenotype, which is distinct from adult iNKT cells, as well as other CD25‐expressing activated T or regulatory T cells, may be important to ensure stability of a structurally limited peripheral iNKT‐cell repertoire early in life.

T regulatory cell chemokine production mediates pathogenic T cell attraction and suppression

T regulatory cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and nonharmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require these cells to be near their targets of suppression; however, it is not known how Treg-to-target proximity is controlled. Here, we found that Tregs attract CD4+ and CD8+ T cells by producing chemokines. Specifically, Tregs produced both CCL3 and CCL4 in response to stimulation, and production of these chemokines was critical for migration of target T cells, as Tregs from Ccl3-/- mice, which are also deficient for CCL4 production, did not promote migration. Moreover, CCR5 expression by target T cells was required for migration of these cells to supernatants conditioned by Tregs. Tregs deficient for expression of CCL3 and CCL4 were impaired in their ability to suppress experimental autoimmune encephalomyelitis or islet allograft rejection in murine models. Moreover, Tregs from subjects with established type 1 diabetes were impaired in their ability to produce CCL3 and CCL4. Together, these results demonstrate a previously unappreciated facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their therapeutic effect in autoimmunity and transplantation.

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.

T Regulatory Cells and Cancer Immunotherapy

The immune system is comprised of a network of regulatory and effector mechanisms that is balanced to eliminate threats to the body, while preventing uncontrolled inflammatory and auto-immune responses that would otherwise harm self. T regulatory (Treg) cells are central to this process and are required to maintain self-tolerance by actively inhibiting self-reactive T cells by a variety of direct and indirect mechanisms. Numerous studies have shown that Treg cells also exist in tumors, promote a suppressive environment that interferes with antitumor immunity, and are a major barrier to successful immunotherapy. In this review we will discuss evidence for how different types of CD4+ Treg cells are involved in antitumor immunity in both mice and humans. We also highlight strategies that have successfully inhibited Treg cells in animal models of cancer and in human clinical trials. In addition, the known effects of Treg cells on current immunotherapy approaches will be discussed. A better understanding of how Treg cells hinder antitumor immunity is required to optimize current therapeutic regimens and to make effective cancer immunotherapy a reality for an increased number of patients.