TIGIT and Helios Are Highly Expressed on CD4+ T Cells in Sézary Syndrome Patients (2016)

Cutaneous T-cell lymphoma is commonly manifested as skin-restricted mycosis fungoides (MF) or Sézary syndrome (SS), a leukemic variant characterized by erythroderma and circulating malignant CD4+ T cells with features of T helper T(h) type 2 cells (Kim et al., 2005Kim E.J. Hess S. Richardson S.K. Newton S. Showe L.C. Benoit B.M. et al.Immunopathogenesis and therapy of cutaneous T cell lymphoma.J Clin Invest. 2005; 115: 798-812Crossref PubMed Scopus (379) Google Scholar). T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is a recently identified co-inhibitory receptor expressed on the cell surface of activated or regulatory T cells and natural killer cells. Emerging data suggest that TIGIT plays an inhibitory role by down-regulating Th1 and Th17 while enhancing Th2 immune responses (Johnston et al., 2015Johnston R.J. Yu X. Grogan J.L. The checkpoint inhibitor TIGIT limits antitumor and antiviral CD8 T cell responses.Oncoimmunology. 2015; 4: e1036214Crossref PubMed Scopus (32) Google Scholar, Joller et al., 2014Joller N. Lozano E. Burkett P.R. Patel B. Xiao S. Zhu C. et al.Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses.Immunity. 2014; 40: 569-581Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, Kourepini et al., 2016Kourepini E. Paschalidis N. Simoes D.C. Aggelakopoulou M. Grogan J.L. Panoutsakopoulou V TIGIT enhances antigen-specific Th2 recall responses and allergic disease.J Immunol. 2016; 196: 3570-3580Crossref PubMed Scopus (31) Google Scholar, Kurtulus et al., 2015Kurtulus S. Sakuishi K. Ngiow S.F. Joller N. Tan D.J. Teng M.W. et al.TIGIT predominantly regulates the immune response via regulatory T cells.J Clin Invest. 2015; 125: 4053-4062Crossref PubMed Scopus (338) Google Scholar, Zhang et al., 2016Zhang B. Zhao W. Li H. Chen Y. Tian H. Li L. et al.Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155.Cancer Immunol Immunother. 2016; 65: 305-314Crossref PubMed Scopus (44) Google Scholar). Helios is a transcription factor in the Ikaros family present in regulatory T cells. Like TIGIT, Helios has inhibitory activity and plays a role in antitumor immunity (Khaitan et al., 2016Khaitan A. Kravietz A. Mwamzuka M. Marshed F. Ilmet T. Said S. et al.FOXP3+Helios+ regulatory T cells, immune activation and advancing disease in HIV infected children.J Acquir Immune Defic Syndr. 2016; 72: 474-484PubMed Google Scholar, Muto et al., 2015Muto S. Owada Y. Inoue T. Watanabe Y. Yamaura T. Fukuhara M. et al.Clinical significance of expanded Foxp3(+) Helios(-) regulatory T cells in patients with non-small cell lung cancer.Int J Oncol. 2015; 47: 2082-2090PubMed Google Scholar, Sebastian et al., 2016Sebastian M. Lopez-Ocasio M. Metidji A. Rieder S.A. Shevach E.M. Thornton A.M. Helios controls a limited subset of regulatory T cell functions.J Immunol. 2016; 196: 144-155Crossref PubMed Scopus (108) Google Scholar). The expression and role of Helios and TIGIT in SS have not been explored, but given the immunosuppressive nature of the disease, both molecules may potentially contribute to this process. We reported high expression of FCRL3 on CD26– CD4+T cells in SS patients with high blood tumor burden. A microarray analysis of global gene expression that identified FCRL3 also showed significantly increased expression of Helios in CD4+ cells from SS patients with high tumor burden (data not shown) (Wysocka et al., 2014Wysocka M. Kossenkov A.V. Benoit B.M. Troxel A.B. Singer E. Schaffer A. et al.CD164 and FCRL3 are highly expressed on CD4+CD26- T cells in Sezary syndrome patients.J Invest Dermatol. 2014; 134: 229-236Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). A recent report linked the expression of FCRL3 with the expression of TIGIT and Helios (Bin Dhuban et al., 2015Bin Dhuban K. d’Hennezel E. Nashi E. Bar-Or A. Rieder S. Shevach E.M. et al.Coexpression of TIGIT and FCRL3 identifies Helios+ human memory regulatory T cells.J Immunol. 2015; 194: 3687-3696Crossref PubMed Scopus (78) Google Scholar). Our data show significantly increased percentages of TIGIT+ and Helios+ CD4+ T cells in patients with SS (mean = 33.6%, P = 0.02 and mean = 35.7%, P = 0.0001, respectively) when compared with patients with MF and healthy donors (HDs) (Figure 1a and b). TIGIT and Helios were also highly expressed in the skin of SS and MF patients compared with atopic dermatitis, psoriasis, and control skin (HD) (Figure 1c–e). TIGIT mRNA in skin from SS and MF patients was 327- and 110-fold increased, respectively, compared with levels in atopic dermatitis patients or control skin (Figure 1c). Similarly, Helios mRNA was increased in the skin of SS and MF patients at 77- and 13.6-fold, respectively (Figure 1d), along with FCRL3 at 765- and 68.3-fold for SS and MF, respectively, when compared with HD skin (Figure 1e). The data presented in Figure 1a distinguish patients with a high and low percentages of TIGIT+ CD4+ T cells. A thorough analysis of patients with high TIGIT expression (mean = 79.3%, standard deviation [SD] = 9.7) (Figure 2a) showed that patients’ CD4+ T cells also show a high percentage of CD26– cells (mean = 87.1%, SD = 9.6), express a single TCRVβ (mean = 85.9%, SD = 11.3), highly express Helios (mean = 46.7%, SD = 19.4), FCRL3 (mean = 64.9% SD = 25.7), and CD164 (mean = 56.7%, SD = 26.6), but low PD1 (mean = 22.1%, SD = 15.8). Importantly, TIGIT expression correlated positively and significantly with CD26 negativity and with a single TCRVβ (Figure 2b and c) but not with the expression of Helios, FCRL3, and CD164 because of the variability between patients. Patients with low expression of TIGIT on CD4+ T cells (mean = 7.9%, SD = 5.12) (Figure 2d) also show a low percentage of CD26 negativity (mean = 28.5%, SD = 18.7), no detectable single TCRVβ, low expression of Helios (mean = 29.6%, SD = 26.1), FCRL3 (mean = 11.9%, SD = 13.5), CD164 (mean = 21.5%, SD = 22.7), and PD1 (mean = 10.0%, SD = 12.6). This group of patients lacks a well-defined cell surface malignant phenotype, with the percentages of CD4+ T cells expressing the aforementioned molecules being significantly lower compared with high TIGIT-expressing patients (P < 0.05 for all tested molecules), implying that low TIGIT-expressing patients have a low circulating tumor burden. We analyzed the clinical differences between the high and low TIGIT/Helios groups in terms of tumor burden in the blood, stage, and LDH and found that all patients in the high-TIGIT group (n = 9) had a diagnosis of advanced B2 disease, whereas among 16 patients with low TIGIT expression, 10 (62.5%) patients had diagnoses of B0 and 6 (37.5%) patients of B1 disease. TIGIT is a known activation marker and is expressed on chronically activated, exhausted CD4+ T cells (Chew et al., 2016Chew G.M. Fujita T. Webb G.M. Burwitz B.J. Wu H.L. Reed J.S. et al.TIGIT marks exhausted T cells, correlates with disease progression, and serves as a target for immune restoration in HIV and SIV infection.PLoS Pathog. 2016; 12: e1005349Crossref PubMed Scopus (192) Google Scholar, Le Mercier et al., 2015Le Mercier I. Lines J.L. Noelle R.J. Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators.Front Immunol. 2015; 6: 418Crossref PubMed Scopus (132) Google Scholar, Pauken and Wherry, 2014Pauken K.E. Wherry E.J. TIGIT and CD226: tipping the balance between costimulatory and coinhibitory molecules to augment the cancer immunotherapy toolkit.Cancer Cell. 2014; 26: 785-787Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Furthermore, as we show, it is highly expressed on CD26− TCRVβ+ CD4+ T cells, defined as the malignant population in SS. We observed significantly decreased production of IFN-γ and IL-2 by TIGIT+ compared with TIGIT– CD4+ T cells from patients with high TIGIT expression and advanced B2 disease (Figure 2e and f). This is consistent with the reported function of TIGIT-positive, exhausted T cells and is strongly suggestive that TIGIT is associated with an immunosuppressive phenotype typically observed in advanced SS by being expressed on both exhausted and malignant CD4+ T cells (Joller et al., 2014Joller N. Lozano E. Burkett P.R. Patel B. Xiao S. Zhu C. et al.Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses.Immunity. 2014; 40: 569-581Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, Kourepini et al., 2016Kourepini E. Paschalidis N. Simoes D.C. Aggelakopoulou M. Grogan J.L. Panoutsakopoulou V TIGIT enhances antigen-specific Th2 recall responses and allergic disease.J Immunol. 2016; 196: 3570-3580Crossref PubMed Scopus (31) Google Scholar, Kurtulus et al., 2015Kurtulus S. Sakuishi K. Ngiow S.F. Joller N. Tan D.J. Teng M.W. et al.TIGIT predominantly regulates the immune response via regulatory T cells.J Clin Invest. 2015; 125: 4053-4062Crossref PubMed Scopus (338) Google Scholar). In contrast to patients with high TIGIT expression, TIGIT+ CD4+ T cells from patients with low TIGIT expression produce IFN-γ and IL-2 at levels comparable to their TIGIT–cells (Figure 2g and h). These results suggest that low TIGIT expression in these patients may be linked to activation status of CD4 T cells rather than to an exhausted phenotype. The precise pathogenic nature of cells expressing TIGIT and Helios molecules in cutaneous T-cell lymphoma currently remains unknown. TIGIT+ and/or Helios+ CD4+ T cells may represent malignant cells, exhausted cells, or nonconventional CD4+ T regulatory cells, lacking Foxp3 and CD25 (data not shown). Malignant Foxp3+CD25– CD4+ T cells with suppressive activity have been identified in SS patients, further suggesting that the typical phenotype of T regulatory cells may be challenged in the context of cutaneous T-cell lymphoma (Heid et al., 2009Heid J.B. Schmidt A. Oberle N. Goerdt S. Krammer P.H. Suri-Payer E. et al.FOXP3+CD25- tumor cells with regulatory function in Sezary syndrome.J Invest Dermatol. 2009; 129: 2875-2885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). High expression of TIGIT and Helios identifies CD4+ T cells with impaired immunological functions, primarily among patients with an advanced stage of SS, suggesting that high expression of these molecules may correlate with a poor prognosis. However, further studies are needed to define in detail the functional and biological significance of TIGIT- and Helios-expressing CD4+ T cells in SS and the potential use of these markers as therapeutic targets. Donation of blood and skin samples by patients and healthy donors, with written informed consent, conformed to the protocols approved by institutional review boards of the University of Pennsylvania and Washington University. The authors state no conflict of interest. This work was supported by a translational research grant from the Leukemia and Lymphoma Society (AHR); a developmental project on grant 5P50CA174523-02 from the National Cancer Institute (AHR); National Institute of Arthritis and Musculoskeletal and Skin Diseases, K23-AR68433 to JT and K08-AR065577-03 to BSK; American Skin Association Research Grant to BSK; an R01 CA 132098 (LCS); and by the Penn Cutaneous Lymphoma Charitable Research Fund.