Ileana S. Mauldin

The Protein Tyrosine Phosphatase SHP-1 Modulates the Suppressive Activity of Regulatory T Cells

The importance of regulatory T cells (Tregs) for immune tolerance is well recognized, yet the signaling molecules influencing their suppressive activity are relatively poorly understood. In this article, through in vivo studies and complementary ex vivo studies, we make several important observations. First, we identify the cytoplasmic tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) as an endogenous brake and modifier of the suppressive ability of Tregs; consistent with this notion, loss of SHP-1 expression strongly augments the ability of Tregs to suppress inflammation in a mouse model. Second, specific pharmacological inhibition of SHP-1 enzymatic activity via the cancer drug sodium stibogluconate potently augmented Treg suppressor activity both in vivo and ex vivo. Finally, through a quantitative imaging approach, we directly demonstrate that Tregs prevent the activation of conventional T cells and that SHP-1–deficient Tregs are more efficient suppressors. Collectively, our data reveal SHP-1 as a critical modifier of Treg function and a potential therapeutic target for augmenting Treg-mediated suppression in certain disease states.

The tyrosine phosphatase SHP-1 dampens murine Th17 development

Th17 cells represent a subset of CD4+ T helper cells that secrete the proinflammatory cytokine IL-17. Th17 cells have been ascribed both a beneficial role in promoting clearance of pathogenic fungi and bacteria, and a pathogenic role in autoimmune diseases. Here we identify the tyrosine phosphatase SHP-1 as a critical regulator of Th17 development, using 3 complementary approaches. Impaired SHP-1 activity through genetic deletion of SHP-1, transgenic expression of an inducible dominant negative SHP-1, or pharmacologic inhibition of SHP-1 strongly promotes the development of Th17. Ex vivo Th17 skewing assays demonstrate that genetic or pharmacologic disruption of SHP-1 activity in T cells results in a hyper-response to stimulation via IL-6 and IL-21, 2 cytokines that promote Th17 development. Mechanistically, we find that SHP-1 decreases the overall cytokine-induced phosphorylation of STAT3 in primary CD4+ T cells. These data identify SHP-1 as a key modifier of IL-6-and IL-21-driven Th17 development via regulation of STAT3 signaling and suggest SHP-1 as a potential new therapeutic target for manipulating Th17 differentiation in vivo.

Vaccination with Melanoma Helper Peptides Induces Antibody Responses Associated with Improved Overall Survival

Purpose: A melanoma vaccine incorporating six peptides designed to induce helper T-cell responses to melanoma antigens has induced Th1-dominant CD4+ T-cell responses in most patients, and induced durable clinical responses or stable disease in 24% of evaluable patients. The present study tested whether this vaccine also induced antibody (Ab) responses to each peptide, and whether Ab responses were associated with T-cell responses and with clinical outcome. Experimental Design: Serum samples were studied from 35 patients with stage III-IV melanomas vaccinated with 6 melanoma helper peptides (6MHP). IgG Ab responses were measured by ELISA. Associations with immune response and overall survival were assessed by log-rank test and χ2 analysis of Kaplan–Meier data. Results: Ab responses to 6MHP were detected by week 7 in 77% of patients, and increased to peak 6 weeks after the last vaccine and persisted to 6 months. Ab responses were induced most frequently to longer peptides. Of those with T-cell responses, 82% had early Ab responses. Survival was improved for patients with early Ab response (P = 0.0011) or with early T-cell response (P < 0.006), and was best for those with both Ab and T-cell responses (P = 0.0002). Conclusions: Vaccination with helper peptides induced both Ab responses and T-cell responses, associated with favorable clinical outcome. Such immune responses may predict favorable clinical outcome to guide combination immunotherapy. Further studies are warranted to understand mechanisms of interaction of these Abs, T-cell responses, and tumor control. Clin Cancer Res; 21(17); 3879–87. ©2015 AACR.

Human melanomas and ovarian cancers overexpressing mechanical barrier molecule genes lack immune signatures and have increased patient mortality risk

ABSTRACT We have identified eight genes whose expression in human melanoma metastases and ovarian cancers is associated with a lack of Th1 immune signatures. They encode molecules with mechanical barrier function in the skin and other normal tissues and include filaggrin (FLG), tumor-associated calcium signal transducer 2 (TACSTD2), and six desmosomal proteins (DST, DSC3, DSP, PPL, PKP3, and JUP). This association has been validated in an independent series of 114 melanoma metastases. In these, DST expression alone is sufficient to identify melanomas without immune signatures, while FLG and the other six putative barrier molecules are overexpressed in a different subset of melanomas lacking immune signatures. Similar associations have been identified in a set of 186 ovarian cancers. RNA-seq data from 471 melanomas and 307 ovarian cancers in the TCGA database further support these findings and also reveal that overexpression of barrier molecules is strongly associated with early patient mortality for melanoma (p = 0.0002) and for ovarian cancer (p < 0.01). Interestingly, this association persists for FLG for melanoma (p = 0.012) and ovarian cancer (p = 0.006), whereas DST overexpression is negatively associated with CD8+ gene expression, but not with patient survival. Thus, overexpression of FLG or DST identifies two distinct patient populations with low immune cell infiltration in these cancers, but with different prognostic implications for each. These data raise the possibility that molecules with mechanical barrier function in skin and other tissues may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction.

Topical treatment of melanoma metastases with imiquimod, plus administration of a cancer vaccine, promotes immune signatures in the metastases

IntroductionInfiltration of cancers by T cells is associated with improved patient survival and response to immune therapies; however, optimal approaches to induce T cell infiltration of tumors are not known. This study was designed to assess whether topical treatment of melanoma metastases with the TLR7 agonist imiquimod plus administration of a multipeptide cancer vaccine will improve immune cell infiltration of melanoma metastases.Patients and methods Eligible patients were immunized with a vaccine comprised of 12 melanoma peptides and a tetanus toxoid-derived helper peptide, and imiquimod was applied topically to metastatic tumors daily. Adverse events were recorded, and effects on the tumor microenvironment were evaluated from sequential tumor biopsies. T cell responses were assessed by IFNγ ELIspot assay and T cell tetramer staining. Patient tumors were evaluated for immune cell infiltration, cytokine and chemokine production, and gene expression.Results and conclusionsFour eligible patients were enrolled, and administration of imiquimod and vaccination were well tolerated. Circulating T cell responses to the vaccine was detected by ex vivo ELIspot assay in 3 of 4 patients. Treatment of metastases with imiquimod induced immune cell infiltration and favorable gene signatures in the patients with circulating T cell responses. This study supports further study of topical imiquimod combined with vaccines or other immune therapies for the treatment of melanoma.

TLR2/6 agonists and interferon-gamma induce human melanoma cells to produce CXCL10

Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor‐reactive T‐cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T‐cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T‐cell infiltration into the TME. Here, we show that toll‐like receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP‐2 or FSL‐1) synergize with interferon‐gamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T‐cell migration into melanoma metastases.

Immune Cell Infiltration and Tertiary Lymphoid Structures as Determinants of Antitumor Immunity

Limited representation of intratumoral immune cells is a major barrier to tumor control. However, simply enhancing immune responses in tumor-draining lymph nodes or through adoptive transfer may not overcome the limited ability of tumor vasculature to support effector infiltration. An alternative is to promote a sustained immune response intratumorally. This idea has gained traction with the observation that many tumors are associated with tertiary lymphoid structures (TLS), which organizationally resemble lymph nodes. These peri- and intratumoral structures are usually, but not always, associated with positive prognoses in patients. Preclinical and clinical data support a role for TLS in modulating immunity in the tumor microenvironment. However, there appear to be varied functions of TLS, potentially based on their structure or location in relation to the tumor or the origin or location of the tumor itself. Understanding more about TLS development, composition, and function may offer new therapeutic opportunities to modulate antitumor immunity.

Perivascular Adipose Tissue Harbors Atheroprotective IgM-Producing B Cells

Adipose tissue surrounding major arteries (Perivascular adipose tissue or PVAT) has long been thought to exist to provide vessel support and insulation. Emerging evidence suggests that PVAT regulates artery physiology and pathology, such as, promoting atherosclerosis development through local production of inflammatory cytokines. Yet the immune subtypes in PVAT that regulate inflammation are poorly characterized. B cells have emerged as important immune cells in the regulation of visceral adipose tissue inflammation and atherosclerosis. B cell-mediated effects on atherosclerosis are subset-dependent with B-1 cells attenuating and B-2 cells aggravating atherosclerosis. While mechanisms whereby B-2 cells aggravate atherosclerosis are less clear, production of immunoglobulin type M (IgM) antibodies is thought to be a major mechanism whereby B-1 cells limit atherosclerosis development. B-1 cell-derived IgM to oxidation specific epitopes (OSE) on low density lipoproteins (LDL) blocks oxidized LDL-induced inflammatory cytokine production and foam cell formation. However, whether PVAT contains B-1 cells and whether atheroprotective IgM is produced in PVAT is unknown. Results of the present study provide clear evidence that the majority of B cells in and around the aorta are derived from PVAT. Interestingly, a large proportion of these B cells belong to the B-1 subset with the B-1/B-2 ratio being 10-fold higher in PVAT relative to spleen and bone marrow. Moreover, PVAT contains significantly greater numbers of IgM secreting cells than the aorta. ApoE−/− mice with B cell-specific knockout of the gene encoding the helix-loop-helix factor Id3, known to have attenuated diet-induced atherosclerosis, have increased numbers of B-1b cells and increased IgM secreting cells in PVAT relative to littermate controls. Immunostaining of PVAT on human coronary arteries identified fat associated lymphoid clusters (FALCs) harboring high numbers of B cells, and flow cytometry demonstrated the presence of T cells and B cells including B-1 cells. Taken together, these results provide evidence that murine and human PVAT harbor B-1 cells and suggest that local IgM production may serve to provide atheroprotection.

Intratumoral interferon-gamma increases chemokine production but fails to increase T cell infiltration of human melanoma metastases

IntroductionOptimal approaches to induce T cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T cell recruitment and may be induced by IFN. This study tests the hypothesis that intratumoral administration of IFNγ will induce CXCL9–11 and will induce T cell recruitment and anti-tumor immune signatures in melanoma metastases.Patients and methodsNine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides and received IFNγ intratumorally. Effects on the tumor microenvironment were evaluated in sequential tumor biopsies. Adverse events (AEs) were recorded. T cell responses to vaccination were assessed in PBMC by IFNγ ELISPOT assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression, and gene expression.ResultsVaccination and intratumoral administration of IFNγ were well tolerated. Circulating T cell responses to vaccine were detected in six of nine patients. IFNγ increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone, nor the addition of IFNγ promoted immune cell infiltration or induced anti-tumor immune gene signatures.ConclusionThe melanoma vaccine induced circulating T cell responses, but it failed to infiltrate metastases, thus highlighting the need for combination strategies to support T cell infiltration. A single intratumoral injection of IFNγ induced T cell-attracting chemokines; however, it also induced secondary immune regulation that may paradoxically limit immune infiltration and effector functions. Alternate dosing strategies or additional combinatorial treatments may be needed to promote trafficking and retention of tumor-reactive T cells in melanoma metastases.

Formation and phenotypic characterization of CD49a, CD49b and CD103 expressing CD8 T cell populations in human metastatic melanoma

ABSTRACT Integrins α1β1 (CD49a), α2β1 (CD49b) and αEβ7 (CD103) mediate retention of lymphocytes in peripheral tissues, and their expression is upregulated on tumor infiltrating lymphocytes (TIL) compared to circulating lymphocytes. Little is known about what induces expression of these retention integrins (RI) nor whether RI define subsets in the tumor microenvironment (TME) with a specific phenotype. Human metastatic melanoma-derived CD8 TIL could be grouped into five subpopulations based on RI expression patterns: RIneg, CD49a+ only, CD49a+CD49b+, CD49a+CD103+, or positive for all three RI. A significantly larger fraction of the CD49a+ only subpopulation expressed multiple effector cytokines, whereas CD49a+CD103+ and CD49a+CD49b+ cells expressed IFNγ only. RIneg and CD49a+CD49b+CD103+ CD8 TIL subsets expressed significantly less effector cytokines overall. Interestingly, however, CD49a+CD49b+CD103+ CD8 expressed lowest CD127, and highest levels of perforin and exhaustion markers PD-1 and Tim3, suggesting selective exhaustion rather than conversion to memory. To gain insight into RI expression induction, normal donor PBMC were cultured with T cell receptor (TCR) stimulation and/or cytokines. TCR stimulation alone induced two RI+ cell populations: CD49a single positive and CD49a+CD49b+ cells. TNFα and IL-2 each were capable of inducing these populations. Addition of TGFβ to TCR stimulation generated two additional populations; CD49a+CD49bnegCD103+ and CD49a+CD49b+CD103+. Taken together, our findings identify opportunities to modulate RI expression in the TME by cytokine therapies and to generate subsets with a specific RI repertoire in the interest of augmenting immune therapies for cancer or for modulating other immune-related diseases such as autoimmune diseases.