Paul L. Stein

CD1d-Expressing Breast Cancer Cells Modulate NKT Cell-Mediated Antitumor Immunity in a Murine Model of Breast Cancer Metastasis

Background Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression. Methodology/Principal Findings In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors. Conclusions/Significance The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.

Inhibition of TYK2 and JAK1 Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis by Inhibiting IL-22 and the IL-23/IL-17 Axis

Psoriasis is a chronic autoimmune disease affecting the skin and characterized by aberrant keratinocyte proliferation and function. Immune cells infiltrate the skin and release proinflammatory cytokines that play important roles in psoriasis. The Th17 network, including IL-23 and IL-22, has recently emerged as a critical component in the pathogenesis of psoriasis. IL-22 and IL-23 signaling is dependent on the JAK family of protein tyrosine kinases, making JAK inhibition an appealing strategy for the treatment of psoriasis. In this study, we report the activity of SAR-20347, a small molecule inhibitor with specificity for JAK1 and tyrosine kinase 2 (TYK2) over other JAK family members. In cellular assays, SAR-20347 dose dependently (1 nM–10 μM) inhibited JAK1- and/or TYK2-dependent signaling from the IL-12/IL-23, IL-22, and IFN-α receptors. In vivo, TYK2 mutant mice or treatment of wild-type mice with SAR-20347 significantly reduced IL-12–induced IFN-γ production and IL-22–dependent serum amyloid A to similar extents, indicating that, in these models, SAR-20347 is probably acting through inhibition of TYK2. In an imiquimod-induced psoriasis model, the administration of SAR-20347 led to a striking decrease in disease pathology, including reduced activation of keratinocytes and proinflammatory cytokine levels compared with both TYK2 mutant mice and wild-type controls. Taken together, these data indicate that targeting both JAK1- and TYK2-mediated cytokine signaling is more effective than TYK2 inhibition alone in reducing psoriasis pathogenesis.

PHENOTYPIC DIFFERENCES BETWEEN MICE DEFICIENT IN XIAP AND SAP, TWO FACTORS TARGETED IN X-LINKED LYMPHOPROLIFERATIVE SYNDROME (XLP)

Mutations in the X-linked inhibitor of apoptosis (XIAP) have recently been identified in patients with the rare genetic disease, X-linked lymphoproliferative syndrome (XLP), which was previously thought to be solely attributable to mutations in a distinct gene, SAP. To further understand the roles of these two factors in the pathogenesis of XLP, we have compared mice deficient in Xiap with known phenotypes of Sap-null mice. We show here that in contrast to Sap-deficient mice, animals lacking Xiap have apparently normal NKT cell development and no apparent defect in humoral responses to T cell-dependent antigens. However, Xiap-deficient cells were more susceptible to death upon infection with the murine herpesvirus MHV-68 and gave rise to more infectious virus. These differences could be rescued by restoration of XIAP. These data provide insight into the differing roles of XIAP and SAP in the pathogenesis of XLP.

The Adaptor Molecule Signaling Lymphocytic Activation Molecule-Associated Protein (SAP) Regulates IFN-γ and IL-4 Production in Vα14 Transgenic NKT Cells via Effects on GATA-3 and T-bet Expression

NKT cells comprise a rare regulatory T cell population of limited TCR diversity, with most cells using a Vα14Jα18 TCR. These cells exhibit a critical dependence on the signaling adapter molecule, signaling lymphocytic activation molecule-associated protein (SAP), for their ontogeny, an aspect not seen in conventional αβ T cells. Prior studies demonstrate that SAP enhances TCR-induced activation of NF-κB in CD4+ T cells. Because NF-κB is required for NKT cell development, SAP might promote the ontogeny of this lineage by signaling to NF-κB. In this study, we demonstrate that forced expression of the NF-κB target gene, Bcl-xL, or inhibitory NF-κB kinase β, a catalytic subunit of the IκB kinase complex essential for NF-κB activation, fails to restore NKT cell development in sap−/− mice, suggesting that SAP mediates NKT cell development independently of NF-κB. To examine the role of SAP in NKT cell function, we generated NKT cells in sap−/− mice by expressing a transgene encoding the Vα14Jα18 component of the invariant TCR. These cells bound α-galactosylceramide-loaded CD1d tetramers, but exhibited a very immature CD24+NK1.1− phenotype. Although sap−/− tetramer-reactive cells proliferated in response to TCR activation, they did not produce appreciable levels of IL-4 or IFN-γ. The reduction in cytokine production correlated with the near absence of GATA-3 and T-bet, key transcription factors regulating cytokine expression and maturation of NKT cells. Ectopic expression of GATA-3 partially restored IL-4 production by the NKT cells. Collectively, these data suggest that by promoting GATA-3 and T-bet expression, SAP exerts control over NKT cell development and mature NKT cell cytokine production.

Fyn Promotes Th17 Differentiation by Regulating the Kinetics of RORγt and Foxp3 Expression

Th17 cells constitute a proinflammatory CD4+ T cell subset that is important for microbial clearance, but also are implicated as propagators of various autoimmune pathologies. Evidence suggests that Th17 cells share common progenitors with immunosuppressive CD4+ inducible regulatory T cells (TREG) and that the developmental pathways of these two subsets are reciprocally regulated. In this study, we show evidence that the Src family tyrosine kinase Fyn helps regulate this Th17/TREG balance. When placed under Th17-skewing conditions, CD4+ T cells from fyn−/− mice had decreased levels of IL-17, but increased expression of the TREG transcription factor Foxp3. The defect in IL-17 expression occurred independently of the ectopic Foxp3 expression and correlated with a delay in retinoic acid-related orphan receptor γt upregulation and an inability to maintain normal STAT3 activation. Fyn-deficient Th17 cells also exhibited delayed upregulation of Il23r, Il21, Rora, and Irf4, as well as aberrant expression of Socs3, suggesting that Fyn may function upstream of a variety of molecular pathways that contribute to Th17 polarization. The fyn−/− mice had fewer IL-17+CD4+ T cells in the large intestinal lamina propria compared with littermate controls. Furthermore, after transfer of either wild-type or fyn−/− naive CD4+ T cells into Rag1−/− hosts, recipients receiving fyn−/− cells had fewer IL-17–producing T cells, indicating that Fyn may also regulate Th17 differentiation in vivo. These results identify Fyn as a possible novel regulator of the developmental balance between the Th17 cell and TREG subsets.

Lck Mediates Th2 Differentiation through Effects on T-bet and GATA-3

The Src family kinase Lck has been shown to be crucial in T cell signaling and development. However, its role in Th effector functions is not well understood. Lck has previously been shown to play a role in the cytokine expression of Th2 cells, but the mechanism by which Lck influences Th2 effector functions is unknown. Using a mouse model, we report that Lck is important in regulating the expression of IL-4 in Th2 skewed cells but is not as necessary for the expression of Th2 cytokines IL-5, IL-10, and IL-13. Furthermore, in the absence of Lck, T-bet and GATA-3 expression is aberrant. Moreover, this atypical expression pattern of T-bet and GATA-3 correlates with increased histone 3 acetylation at the Ifng locus and production of the Th1 cytokine IFN-γ. We find overexpression of GATA-3 restores IL-4 expression in lck−/− Th2 cells; this indicates that the decreased IL-4 expression is due in part to reduced amounts of GATA-3. Taken together, these data imply that Lck mediates Th2 differentiation through effects on T-bet and GATA-3.

Lck regulates IL-10 expression in memory-like Th1 cells

The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck−/− Th1 skewed cells have normal expression of T‐bet and produce IFN‐γ at WT levels. However, there is a 3‐fold increase in IL‐10 producing cells in the mutant cultures. These cells do not have elevated levels of IL‐4, GATA3, IL‐17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3+ T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL‐10 in the lck−/− Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L+ (naïve) cells are similar to WT cells. Furthermore, this IL‐10 expression appears to be dependent on IL‐12 and correlates with elevated c‐Maf. These data highlight a previously unappreciated role for Lck in regulating IL‐10 in Th1 cells.

SAP is required for the development of innate phenotype in H2-M3--restricted Cd8(+) T cells.

H2-M3–restricted T cells have a preactivated surface phenotype, rapidly expand, and produce cytokines upon stimulation, and, as such, are classified as innate T cells. Unlike most innate T cells, M3-restricted T cells also express CD8αβ coreceptors and a diverse TCR repertoire: hallmarks of conventional MHC Ia-restricted CD8+ T cells. Although invariant NKT cells are also innate T cells, they are selected exclusively on hematopoietic cells (HC), whereas M3-restricted T cells can be selected on either hematopoietic or thymic epithelial cells. Moreover, their phenotypes differ depending on what cells mediate their selection. Although there is a clear correlation between selection on HC and development of innate phenotype, the underlying mechanism remains unclear. Signaling lymphocyte activation molecule-associated protein (SAP) is required for the development of invariant NKT cells and mediates signals from signaling lymphocyte activation molecule receptors that are exclusively expressed on HC. Based on their dual selection pathway, M3-restricted T cells present a unique model for studying the development of innate T cell phenotype. Using both polyclonal and transgenic mouse models, we demonstrate that although M3-restricted T cells are capable of developing in the absence of SAP, SAP is required for HC-mediated selection, development of preactivated phenotype, and heightened effector functions of M3-restricted T cells. These findings are significant because they directly demonstrate the need for SAP in HC-mediated acquisition of innate T cell phenotype and suggest that, due to their SAP-dependent HC-mediated selection, M3-restricted T cells develop a preactivated phenotype and an intrinsic ability to proliferate faster upon stimulation, allowing for an important role in the early response to infection.

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma

CD1d‐restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT‐cell TCR transgenic mouse model, we demonstrated that CD1d‐expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT‐cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP‐deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine‐producing capacity and diminished cytotoxicity to CD1d‐expressing lymphoma cells. The impaired IL‐4 production by SAP‐deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA‐3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X‐linked lymphoproliferative disease caused by mutations in SAP.

Abstract A041: Preclinical development of a preventive vaccine against ovarian cancer

Survival of cancer patients with high-grade serous carcinoma of the ovary (HGSCs) remains less than 30% five years after diagnosis, despite initial chemosensitivity of most HGSCs. We hypothesized that a tumor antigen-targeted vaccine capable of eliciting a robust cell-mediated immune response may help prevent ovarian cancer. Mesothelin (meso) is overexpressed by ovarian, lung and pancreatic cancers and is 57% conserved at the protein level between mouse and human. To elicit cell-mediated immune responses directed against mesothelin, we combined a human mesothelin recombinant protein with various adjuvants including: aluminum hydroxide gel (alum, for Th2 immune response); TLR4 ligand (synthetic lipid MPL, for Th1 response); squalene-oil-in-water (AddaVaxTM, for both Th1 and Th2 responses); and STING (stimulator of interferon genes) ligand (CDN) that activates innate immunity and triggers type 1 IFN response and NF-κB-dependent cytokine secretion. We detected robust anti-meso antibody (Ab) responses after 1 prime and 2 boosts with meso/alum/MPL or meso/CDN/AddaVax. The highest and most stable Ab titers were obtained with CDN/AddaVax-based adjuvants. Four groups of female C57BL/6 mice were immunized with alum/MPL or CDN/AddaVax with or without meso (n = 12 per group). Six weeks after the prime immunization, cells from a luciferase-transduced syngeneic mouse ovarian cancer cell line, ID8 (Luc-ID8) were injected orthotopically in the left ovary of the mice. In vivo bioluminescent imaging (BLI) suggested a 92% take rate of tumors 3 weeks after ID8-Luc injections in mice immunized with meso/alum/MPL or adjuvants only. However, in the group immunized with meso/CDN/Addavax only 30% of the mice showed a detectable signal 3 weeks after ID8-Luc implantation. After 10 weeks the average BLI signal was still significantly lower than in all the other groups. Furthermore, the majority of mice immunized with alum/MPL +/- meso developed ascites by 12 weeks; no significant difference of peritoneal leukocyte numbers or phenotype was observed between the groups immunized with alum/MPL vs. meso/alum/MPL. In contrast, 7 out 11 mice immunized with meso/CDN/Addavax did not develop ascites through 14 weeks post tumor challenge; most of the tumors were unilateral, smaller, with more T and B cells present in the peritoneal lavages compared to the other groups. Immunizations with CDN/AddaVax or meso/CDN/AddaVax strongly increased the frequency of CD62L-CD44+ effector memory T cells, stimulated innate immunity with M1 polarization, profoundly decreased MDSC levels in peritoneal lavages, and correlated with a less aggressive tumor phenotype (EpCAM+ PD-L1-). HE 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A041.