Wojciech Szeliga

Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments

Th17 cells play an active role in autoimmune diseases. However, the nature of Th17 cells is poorly understood in cancer patients. We studied Th17 cells, the associated mechanisms, and clinical significance in 201 ovarian cancer patients. Tumor-infiltrating Th17 cells exhibit a polyfunctional effector T-cell phenotype, are positively associated with effector cells, and are negatively associated with tumor-infiltrating regulatory T cells. Tumor-associated macrophages promote Th17 cells through interleukin-1beta (IL-1beta), whereas tumor-infiltrating regulatory T cells inhibit Th17 cells through an adenosinergic pathway. Furthermore, through synergistic action between IL-17 and interferon-gamma, Th17 cells stimulate CXCL9 and CXCL10 production to recruit effector T cells to the tumor microenvironment. The levels of CXCL9 and CXCL10 are associated with tumor-infiltrating effector T cells. The levels of tumor-infiltrating Th17 cells and the levels of ascites IL-17 are reduced in more advanced diseases and positively predict patient outcome. Altogether, Th17 cells may contribute to protective human tumor immunity through inducing Th1-type chemokines and recruiting effector cells to the tumor microenvironment. Inhibition of Th17 cells represents a novel immune evasion mechanism. This study thus provides scientific and clinical rationale for developing novel immune-boosting strategies based on promoting the Th17 cell population in cancer patients.

Induction of IL-17+ T Cell Trafficking and Development by IFN-γ: Mechanism and Pathological Relevance in Psoriasis

Th1 and Th17 T cells are often colocalized in pathological environments, yet Th1-derived IFN-γ inhibits Th17 cell development in vitro. We explored the physiologic basis of this paradox in humans. In this study, we demonstrate increased the number of CD4+ and CD8+ IL-17+ T cells in skin lesions of psoriasis. Furthermore, we show that myeloid APCs potently support induction of IL-17+ T cells, and that this activity is greatly increased in psoriasis. We tested stimuli that might account for this activity. Th1 cells and IFN-γ are increased in psoriatic blood and lesional skin. We show that IFN-γ programs myeloid APCs to induce human IL-17+ T cells via IL-1 and IL-23. IFN-γ also stimulates APC production of CCL20, supporting migration of IL-17+ T cells, and synergizes with IL-17 in the production of human β-defensin 2, an antimicrobial and chemotactic protein highly overexpressed by psoriatic keratinocytes. This study reveals a novel mechanistic interaction between Th1 and IL-17+ T cells, challenges the view that Th1 cells suppress Th17 development through IFN-γ, and suggests that Th1 and IL-17+ T cells may collaboratively contribute to human autoimmune diseases.

ENDOGENOUS IL-17 CONTRIBUTES TO REDUCED TUMOR GROWTH AND METASTASIS

It has been reported that ectopically expressed interleukin-17 (IL-17) in tumor cells suppresses tumor progression through enhanced antitumor immunity in immune competent mice or promote tumor progression through an increase in inflammatory angiogenesis in immune-deficient mice. The role of endogenous IL-17 in tumor immunity remains undefined. Here we showed that tumor growth and lung metastasis were enhanced in IL-17-deficient mice, associated with decreased interferon-gamma(+) natural killer cells and tumor specific interferon-gamma(+) T cells in the tumor draining lymph nodes and tumors. Together with the published data showing that in vitro transforming growth factor-beta and IL-6-polarized Th17 cells induce tumor regression, our work supports the notion that endogenous IL-17 or/and Th17 cells may play a protective role in tumor immunity.

IL-17+ Regulatory T Cells in the Microenvironments of Chronic Inflammation and Cancer

Foxp3+CD4+ regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17+CD4+ T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17+Foxp3+CD4+ T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17+Foxp3+CD4+ T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17+Foxp3+CD4+ T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17+Foxp3+CD4+ T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6+ T cells or Treg cells. IL-17+Foxp3+CD4+ T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17+Foxp3+ cells may be “inflammatory” Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.

Human TH17 cells are long-lived effector memory cells.

Human TH17 cells function as long-lived effector memory cells in the context of chronic disease. Forever Seventeen “It” is that quality possessed by some which draws all others with its magnetic force. Novelist Elinor Glyn In teen television drama parlance, T helper 17 (TH17) cells have been immunology’s “it girl” for the past few years. Discovered in 2007, this beguiling subset of TH cells is associated with autoimmune disease and long-term antitumor immunity, and the presence of these cells in tumors positively correlates with patient survival. Although mouse TH17 cells are believed to be short-lived, studies of advanced human cancers suggest that human TH17 cells persist, but little is known about the nature of these cells in the context of human disease. Now, Kryczek et al. define the detailed phenotype and functional aspects of human TH17 cells in diverse diseased human tissues and find that these cells resemble terminally differentiated, long-lived memory T cells with a twist. The authors used a well-defined human system to study the defining features and behavior of TH17 cells in the pathological microenvironments of human graft-versus-host disease, ulcerative colitis, and cancer. The fraction of TH17 cells was increased in these tissues during the chronic phases of the diseases. Furthermore, the TH17 cells not only displayed characteristics in common with terminally differentiated memory T cells, but also sported some genetic and functional signatures of their own. For example, disease-associated human TH17 cells wore the standard phenotypic markers of terminal differentiation and, in adoptive transfer experiments, drove persistent antitumor immunity. However, these versatile human T cells also displayed a high capacity for proliferative self-renewal, cell-type plasticity, and enriched expression of antiapoptotic genes. The cell membrane signaling protein Notch and the transcriptional regulatory protein hypoxia-inducible factor–1α (HIF-1α) were shown to jointly regulate the expression and function of the Bcl-2 family of antiapoptosis proteins. An intricate understanding of human TH17 cell biology in disease contexts should reveal new “it” molecules—therapeutic targets that permit TH17-directed treatment in patients with autoimmune diseases and advanced tumors. T helper 17 (TH17) cells have been shown to contribute to multiple disease systems. However, the functional phenotype and survival pattern of TH17 cells as well as the underlying mechanisms that control TH17 cells have been poorly investigated in humans, significantly hampering the clinical targeting of these cells. Here, we studied human TH17 cells in the pathological microenvironments of graft-versus-host disease, ulcerative colitis, and cancer; TH17 cell numbers were increased in the chronic phase of these diseases. Human TH17 cells phenotypically resembled terminally differentiated memory T cells but were distinct from central memory, exhausted, and senescent T cells. Despite their phenotypic markers of terminal differentiation, TH17 cells mediated and promoted long-term antitumor immunity in in vivo adoptive transfer experiments. Furthermore, TH17 cells had a high capacity for proliferative self-renewal, potent persistence, and apoptotic resistance in vivo, as well as plasticity—converting into other types of TH cells. These cells expressed a relatively specific gene signature including abundant antiapoptotic genes. We found that hypoxia-inducible factor–1α and Notch collaboratively controlled key antiapoptosis Bcl-2 family gene expression and function in TH17 cells. Together, these data indicate that human TH17 cells may be a long-lived proliferating effector memory T cell population with unique genetic and functional characteristics. Targeting TH17-associated signaling pathway would be therapeutically meaningful for treating patients with autoimmune disease and advanced tumor.

Cutting Edge: Opposite Effects of IL-1 and IL-2 on the Regulation of IL-17+ T Cell Pool IL-1 Subverts IL-2-Mediated Suppression

In this report, we show that IL-17+CD4+ and IL-17+CD8+ T cells are largely found in lung and digestive mucosa compartments in normal mice. Endogenous and exogenous IL-1 dramatically contribute to IL-17+ T cell differentiation mediated by TGFβ and IL-6. IL-1 is capable of stimulating IL-17+ T cell differentiation in the absence of IL-6. Furthermore, although IL-2 reduces IL-17+ T cell differentiation, IL-1 completely disables this effect. Mechanistically, IL-1 and IL-2 play opposite roles in regulating the expression of several molecules regulating Th17 cell differentiation, including the orphan nuclear receptor RORγt, the IL-1 receptor, and the IL-23 receptor. IL-1 subverts the effects of IL-2 on the expression of these gene transcripts. Altogether, our work demonstrates that IL-6 is important but not indispensable for IL-17+ T cell differentiation and that IL-1plays a predominant role in promoting IL-17+ T cell induction. Thus, the IL-17+ T cell pool may be controlled by the local cytokine profile in the microenvironment.

Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells

Identification of cancer stem cells is crucial for advancing cancer biology and therapy. Several markers including CD24, CD44, CD117, CD133, the G subfamily of ATP‐binding cassette transporters (ABCG), epithelial specific antigen (ESA) and aldehyde dehydrogenase (ALDH) are used to identify and investigate human epithelial cancer stem cells in the literature. We have now systemically analyzed and compared the expression of these markers in fresh ovarian epithelial carcinomas. Although the expression levels of these markers were unexpectedly variable and partially overlapping in fresh ovarian cancer cells from different donors, we reliably detected important levels of CD133 and ALDH in the majority of fresh ovarian cancer. Furthermore, most of these stem cell markers including CD133 and ALDH were gradually lost following in vitro passage of primary tumor cells. However, the expression of ALDH and CD133, but not CD24, CD44 and CD117, could be partially rescued by the in vitro serum‐free and sphere cultures and by the in vivo passage in the immune‐deficient xenografts. ALDH+ and CD133+ cells formed three‐dimensional spheres more efficiently than their negative counterparts. These sphere‐forming cells expressed high levels of stem cell core gene transcripts and could be expanded and form additional spheres in long‐term culture. ALDH+, CD133+ and ALDH+CD133+ cells from fresh tumors developed larger tumors more rapidly than their negative counterparts. This property was preserved in the xenografted tumors. Altogether, the data suggest that ALDH+ and CD133+ cells are enriched with ovarian cancer‐initiating (stem) cells and that ALDH and CD133 may be widely used as reliable markers to investigate ovarian cancer stem cell biology.

FOXP3 Defines Regulatory T Cells in Human Tumor and Autoimmune Disease

Activated T cells may express FOXP3. It is thought that FOXP3 is not a specific marker to determine regulatory T cells (Treg) in humans. Here, we examined the functional phenotype and cytokine profile of the in vitro induced FOXP3(+) T cells, primary FOXP3(+) and FOXP3(-) T cells in patients with ulcerative colitis and tumors including colon carcinoma, melanoma, hepatic carcinoma, ovarian carcinoma, pancreatic cancer, and renal cell carcinoma. We observed similar levels of suppressive capacity of primary FOXP3(+) T cells in blood, tumors, and colitic tissues. Compared with primary FOXP3(-) T cells in the same microenvironment, these primary FOXP3(+) T cells expressed minimal levels of effector cytokines, negligible amount of cytotoxic molecule granzyme B, and levels of suppressive molecules interleukin-10 and PD-1. Although the in vitro activated T cells expressed FOXP3, these induced FOXP3(+) T cells expressed high levels of multiple effector cytokines and were not functionally suppressive. The data reinforce the fact that FOXP3 remains an accurate marker to define primary Tregs in patients with cancer and autoimmune disease. We suggest that the combination of FOXP3 and cytokine profile is useful for further functionally distinguishing primary Tregs from activated conventional T cells.

IL-22(+)CD4(+) T cells promote colorectal cancer stemness via STAT3 transcription factor activation and induction of the methyltransferase DOT1L.

Little is known about how the immune system impacts human colorectal cancer invasiveness and stemness. Here we detected interleukin-22 (IL-22) in patient colorectal cancer tissues that was produced predominantly by CD4(+) T cells. In a mouse model, migration of these cells into the colon cancer microenvironment required the chemokine receptor CCR6 and its ligand CCL20. IL-22 acted on cancer cells to promote activation of the transcription factor STAT3 and expression of the histone 3 lysine 79 (H3K79) methytransferase DOT1L. The DOT1L complex induced the core stem cell genes NANOG, SOX2, and Pou5F1, resulting in increased cancer stemness and tumorigenic potential. Furthermore, high DOT1L expression and H3K79me2 in colorectal cancer tissues was a predictor of poor patient survival. Thus, IL-22(+) cells promote colon cancer stemness via regulation of stemness genes that negatively affects patient outcome. Efforts to target this network might be a strategy in treating colorectal cancer patients.

Interleukin-2 administration alters the CD4+FOXP3+ T-cell pool and tumor trafficking in patients with ovarian carcinoma.

Interleukin (IL)-2 is used in the immunotherapy of patients with certain cancer and HIV infection. IL-2 treatment reliably results in 16% to 20% objective clinical response rate in cancer patients, with significant durability of responses in selected patients. However, the mechanisms of therapeutic activity in responding versus nonresponding patients remain poorly understood. CD4(+)CD25(+)FOXP3(+) regulatory T (Treg) cells contribute to immunosuppressive networks in human tumors. We treated 31 ovarian cancer patients with IL-2. We show that administration of IL-2 induces the proliferation of existent Treg cells in patients with ovarian cancer. The potency of Treg cell proliferation is negatively determined by the initial prevalence of Treg cells, suggesting that Treg cells are a factor for self-controlling Treg cell proliferation. After IL-2 cessation, the number of Treg cells more efficiently dropped in clinical responders than nonresponders. Furthermore, IL-2 treatment stimulates chemokine receptor CXCR4 expression on Treg cells, enables Treg cell migration toward chemokine CXCL12 in the tumor microenvironment, and may enforce Treg cell tumor accumulation. Our findings support the concept that administration of IL-2 numerically and functionally affects the Treg cell compartment. These data provide an important insight in evaluating the clinical benefit and therapeutic prediction of IL-2 treatment in patients with cancer.