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029 CD209+ monocyte-derived myeloid dendritic cells were increased in patients with leukemic cutaneous T-cell lymphoma after extracorporeal photopheresis
Adaptive Immunity & Vaccination | ABSTRACTS 025
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Recognition of mammalian and microbial lipid antigens by human T cells H Jiang3,4, W Zeng1, S Cremers2,4,3 and A de Jong1 1 Dermatology, Columbia University, New York, NY, 2 Pathology and Cell Biology, Columbia University, New York, NY, 3 Medicine, Columbia University, New York, NY and 4 Irving Inst Clin Transl Research, Columbia University, New York, NY The lipids in human stratum corneum and sebum provide a physical barrier that protects against external microbial threats and prevents the loss of water and minerals. We have recently shown that beyond providing a physical barrier, skin lipids also have an immunological role. We identified certain classes of skin lipids, including waxesters, fatty acids, squalene and triglycerides, that can bind to the lipid antigen presenting molecule CD1a and be presented to T cells. CD1a is constitutively expressed at high levels on Langerhans cells, which are located at the interface of the skin lipidome and the skin immune system, supporting an important role for lipid antigen presentation in the skin. Since human skin is colonized by bacteria and fungi that contribute both indirectly (through lipase activity) and directly (through the release of microbial lipids) to the pool of lipids in the skin, an important question is whether CD1a-restricted T cells in the skin can distinguish lipids harboring mammalian fatty acid chains from structurally related lipids that harbor microbe-specific fatty acids chains (e.g methyl branched fatty acids). We are first determining if CD1a preferentially binds microbial fatty acid chains, and secondly, if CD1a-restricted T cell receptors preferentially bind complexes of CD1a with microbial lipids. We have developed and validated a HLPC/MS method to quantify relative amounts of structurally related mammalian and microbial fatty acid structures eluted from CD1a molecules, to determine their lipid binding preference. In addition, we are screening reactivity of T cells isolated from normal human skin against mammalian lipids and microbial analogs loaded onto plate-bound CD1a. Our studies will provide insights in how shifts in the composition of the skin lipidome, which occur in many skin diseases (e.g. atopic dermatitis, psoriasis), affect the activation of lipidspecific T cells in the skin.
Boosting of the delayed-type hypersensitivity response to varicella-zoster virus antigen following zoster vaccination in ageing individuals is associated with a local reduction in regulatory T cells N Patel1,2, D Sandhu1,2, M Vukmanovic-Stejic1, M Rustin2 and A Akbar1 1 Infection & Immunity, University College London, London, United Kingdom and 2 Dermatology, Royal Free London NHS Foundation Trust, London, United Kingdom Ageing is accompanied by an increased risk of varicella-zoster virus (VZV) reactivation and impairment of the delayed-type hypersensitivity (DTH) response to VZV skin test antigen. The aim of this study was to investigate how the live attenuated zoster vaccine alters the kinetics of the DTH response to VZV antigen in old individuals. Healthy volunteers (n¼27, age range 70-93 years) were skin-tested with VZV antigen and those with a robust clinical response (2 individuals) were excluded from further participation, with a further 4 drop-outs. Numbers and proliferation (Ki67+) of CD4+ effector T cells and CD4+ Foxp3+ regulatory T cells at the test site were assessed by immunofluorescence microscopy at Days 1, 3 and 7 after VZV challenge. Volunteers were vaccinated after 4 weeks and repeat skin testing and immunofluorescence microscopy analysis were performed after a further 2-8 months. Overall, zoster vaccination led to a significant boost in clinical responses (p¼0.0002), although in 8 individuals there was no improvement. At Day 7 post-VZV challenge the median number of CD4+ T cells per dermal perivascular infiltrate was significantly increased in vaccinated (40.0) compared with unvaccinated individuals (18.9, p¼0.026). The median percentage of proliferating CD4+ T cells at Day 7 was 9.4% in the vaccinated compared with 4.0% in the unvaccinated (p¼0.162). At Day 7 the median proportion of CD4+ T cells with a regulatory phenotype (Foxp3+) reduced from 19.5% in the unvaccinated to 14.0% in the vaccinated (p¼0.088). This study demonstrates that the zoster vaccine significantly boosts the memory CD4+ T cell response to VZV skin challenge in the old, partially restoring the intensity of the immune response to that seen in the young. Reduced inhibitory signalling as a consequence of fewer local regulatory T cells may be a possible mechanism underlying this vaccineinduced immune enhancement.
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Identification of a gain-of-function STAT3 mutation (p.Y640F) in lymphocytic variant hypereosinophilic syndrome S Walker1, C Wang2, T Walradt2, BS Hong2, JR Tanner3, J Levinsohn2, G Goh4, A Subtil2, SR Lessin5, W Heymann9,10, EC Vonderheid6, BA King2, R Lifton7,4 and J Choi3,8 1 Medicine, Dana Farber Cancer Institute, Boston, MA, 2 Dermatology, Yale School of Medicine, New Haven, CT, 3 Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, 4 Genetics, Yale School of Medicine, New Haven, CT, 5 Dermatology, Fox Chase Cancer Center, Philadelphia, PA, 6 Sydney Kimmel Cancer Center, Johns Hopkins Medical Institutes, Baltimore, MD, 7 Howard Hughes Medical Institute, New Haven, CT, 8 Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, 9 Dermatology, Cooper Medical School, Camden, NJ and 10 Dermatology, Perelman School of Medicine, Philadelphia, PA Idiopathic hypereosinophilic syndrome (HES) is a heterogeneous group of disorders characterized by 1) persistent peripheral eosinophilia, 2) target organ pathology mediated by infiltrating eosinophils, and 3) the absence of known infectious or allergic causes of hypereosinophilia. Lymphocytic variant HES (L-HES) is a unique subtype of HES characterized by eosinophilia and eosinophil-infiltrating lesions of the skin, soft tissue, and rarely internal organs. In L-HES, an abnormal T cell clone (most commonly CD3-CD4+) produces pathological amounts of eosinophil-promoting Th2 cytokines, such as IL-4, IL-5, and IL-13. These Th2 cytokines are thought to drive the polyclonal expansion of eosinophils in the skin, the subcutaneous tissue, and the blood. To date, the molecular mechanisms underlying L-HES pathogenesis have been unclear. By exome sequencing, we identified a somatic gain-offunction mutation in STAT3 (p.Y640F) in one patient with L-HES. This mutation renders STAT3 constitutively active, and constitutively active STAT3 has been previously shown sufficient to enforce Th2 differentiation. In two additional cases of L-HES, we did not find activating STAT3 mutations; however, RNA-Seq demonstrated evidence of STAT3 activation in all three cases of L-HES. These findings suggest a novel targetable signaling node that can be used to treat this otherwise recalcitrant disease.
Close similarities in global gene expression profiling between skin and lung homing T cells after skin and lung infection Y Pan, T Tian, S Loftus, S Divito, A De Masson, R Clark, R Fuhlbrigge and TS Kupper Dermatology, BWH/HMS, Boston, MA Compared to the well studied tissue-specific homing of lymphocytes to skin and gut, factors that mediate tissue-specific homing to lung are largely unknown. Previous work has shown that in draining lymph nodes after Vaccinia virus (VACV) infection, by 60 hours after infection antigen specific CD8+ T cells can be identified that have divided from 0-5 times (P0-P5). Imprinting of T cells with tissue-specific homing molecules is initiated during this series of cell divisions. In this study, we employed transcriptional microarrays to reveal the global gene expression profile of CD8+ T cells generated via distinct infection routes. OT-1 T cells loaded with CFSE were injected into syngeneic mice, and 24 hours later mice were immunized by epidermal skin infection (es.i), intra-tracheal (i.tra.) or intra-peritoneal infection (i.p.) with VACV-ova, which expresses the OT-1 peptide under an early gene promoter. At 60 hours after infection, we harvested draining lymph nodes (inguinal, mediastinal, and mesenteric, respectively) and sorted cells that had undergone between 0 and 5 cell divisions, as determined by CFSE. RNA was extracted from these cells, and was analyzed by transcriptional profiling. By contrast to i.p. (74/116 genes, p¼0.51), s.s. generated CD8+ Teff cells exhibiting similar gene transcriptional profile with those generated via i.tra.(92/116 genes, p<0.0004), including chemokine receptors CCR4, CCR8 and CCR10. This was confirmed by principal component analysis. Next we evaluated the capacity of es.i. in generating lung CD8+ tissue resident memory T (TRM) cells. More than half of lung as many CD8+ TRM cells could be generated via es.i., as compared to i.tra., suggesting es.i. as a viable approach for generating lung CD8+ TRM cells. The commonalities between transcriptional profiles of skin homing and lung homing T cells is important not only for vaccine strategies, but also for understanding biological phenomena such as the “atopic march” and the tuberculin skin test reaction.
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CD209+ monocyte-derived myeloid dendritic cells were increased in patients with leukemic cutaneous T-cell lymphoma after extracorporeal photopheresis T Langridge, M Austin, X Zhang, L Shiue, M Duvic and X Ni Department of Dermatology, The University of Texas MD Anderson Cancer Center, Houston, TX Our previous study found that numbers of myeloid dendritic cells (mDC) and their HLA-DR expression were increased after extracorporeal photopheresis (ECP) in patients with leukemic cutaneous T-cell lymphoma (L-CTCL). To further define the subset of ECP-affected mDCs, we assessed the expression of CD209 on mDCs in L-CTCL patients over a 6-month ECP treatment course. It is known that CD209 or DC-SIGN, a transmembrane receptor, is dominantly expressed on monocyte-derived mDCs (Mo-mDC), and can be used as a biomarker for MomDCs. Nineteen L-CTCL patients who started ECP therapy with the CELLEX photopheresis system were enrolled in the study. The peripheral blood was collected at baseline, at Day 2, 1month, 3-months, and 6-months after initial ECP treatment, and mDC populations as well as CD209+ mDC subsets were assessed by multi-color flow cytometry. At baseline, about one third of patients showed a low number of mDCs, but all patients had lower than normal CD209+ mDC counts. The average number of CD209+ mDCs was 0.064% out of peripheral blood mononuclear cells (PBMC) in normal donors (n¼3), but only 0.006% out of PBMCs in patients (n¼19, p<0.05). The CD209+ mDC subset accounts for about 66.2% of total mDCs in normal donors compared to only 13.9% in patients (p<0.05). Eight patients finished 3 and/ or 6-month treatment, with 5 clinical responders and 3 non-responders. The average absolute CD209+ mDC counts were increased by about 3-fold at 3-months and 2-fold at 6-months. The average percentages of CD209+ mDCs of PBMCs went up from 0.008% at baseline to 0.015% at 3 months and remained the same at 6 months. Our results suggest that patients with L-CTCL have a deficiency in monocyte-derived myeloid dendritic cells which may be attributed to the suppressed immunity. ECP may exert a positive effect on monocyte-derived myeloid dendritic cells, which might enhance antigen processing and improve anti-tumor immunity in patients with L-CTCL.
Blockade of multiple immune checkpoints cooperatively enhanced the antitumor effect of melanoma-specific CTLs T Inozume2, T Yaguchi1, T Kawamura2, Y Kawakami1 and S Shimada2 1 Division of Cellular Signaling, Keio University, University of Yamanashi, Chuo, Japan and 2 Dermatology, University of Yamanashi, Chuo, Japan Anti-PD-1 and anti-CTLA-4 antibody dramatically improved the overall survival of melanoma patients. However, further developments are still required, particularly, in the enhancement of the anti-tumor effect and the prevention of autoimmunity. Our previous study suggested that a blockade of multiple immune checkpoint molecules that are selectively and highly expressed on tumor-infiltrating CTLs (for example, TIGIT, TIM-3, and LAG-3) may enhance the antitumor effect without increasing the risk of autoimmunity. However, the synergistic effect of these molecules in human melanoma-specific human CTLs remains unclear. In this study, we examined the synergistic effects of the multiple immune checkpoint molecules in human melanoma-specific CTLs. We compared the impacts of overexpression of CD155 (a ligand for TIGIT), Galectin 9 (a ligand for TIM-3), and MHC class II (a ligand for LAG-3) on melanoma cells in the effector phase in vitro. These molecules were overexpressed on a melanoma cell line alone or in combination with other molecules by retroviral transduction, and the transfectants were cocultured with melanoma-specific CTLs that highly upregulate TIGIT, TIM-3, and LAG-3 by antigen stimulation. In addition, the effect of coblockade of these molecules by the antibodies was assessed in the coculture assay. The activation status of CTL was assessed by cytokine production. The suppressive effect of CD155 was the highest in the ligand molecules overexpressed on melanoma cells. In addition, the suppressive effect of coexpression of CD155 and PD-L1 was the highest compared with those of other combinations. In the blocking assay, the best CTL activation was obtained by coblockade of TIGIT and PD-1 signals. In our experiments, coblockade of TIGIT and PD-1 signals were the most effective in activating melanoma-specific CTLs in the effector phase, compared with those of other combinations.
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Recognition of mammalian and microbial lipid antigens by human T cells H Jiang3,4, W Zeng1, S Cremers2,4,3 and A de Jong1 1 Dermatology, Columbia University, New York, NY, 2 Pathology and Cell Biology, Columbia University, New York, NY, 3 Medicine, Columbia University, New York, NY and 4 Irving Inst Clin Transl Research, Columbia University, New York, NY The lipids in human stratum corneum and sebum provide a physical barrier that protects against external microbial threats and prevents the loss of water and minerals. We have recently shown that beyond providing a physical barrier, skin lipids also have an immunological role. We identified certain classes of skin lipids, including waxesters, fatty acids, squalene and triglycerides, that can bind to the lipid antigen presenting molecule CD1a and be presented to T cells. CD1a is constitutively expressed at high levels on Langerhans cells, which are located at the interface of the skin lipidome and the skin immune system, supporting an important role for lipid antigen presentation in the skin. Since human skin is colonized by bacteria and fungi that contribute both indirectly (through lipase activity) and directly (through the release of microbial lipids) to the pool of lipids in the skin, an important question is whether CD1a-restricted T cells in the skin can distinguish lipids harboring mammalian fatty acid chains from structurally related lipids that harbor microbe-specific fatty acids chains (e.g methyl branched fatty acids). We are first determining if CD1a preferentially binds microbial fatty acid chains, and secondly, if CD1a-restricted T cell receptors preferentially bind complexes of CD1a with microbial lipids. We have developed and validated a HLPC/MS method to quantify relative amounts of structurally related mammalian and microbial fatty acid structures eluted from CD1a molecules, to determine their lipid binding preference. In addition, we are screening reactivity of T cells isolated from normal human skin against mammalian lipids and microbial analogs loaded onto plate-bound CD1a. Our studies will provide insights in how shifts in the composition of the skin lipidome, which occur in many skin diseases (e.g. atopic dermatitis, psoriasis), affect the activation of lipidspecific T cells in the skin.
Boosting of the delayed-type hypersensitivity response to varicella-zoster virus antigen following zoster vaccination in ageing individuals is associated with a local reduction in regulatory T cells N Patel1,2, D Sandhu1,2, M Vukmanovic-Stejic1, M Rustin2 and A Akbar1 1 Infection & Immunity, University College London, London, United Kingdom and 2 Dermatology, Royal Free London NHS Foundation Trust, London, United Kingdom Ageing is accompanied by an increased risk of varicella-zoster virus (VZV) reactivation and impairment of the delayed-type hypersensitivity (DTH) response to VZV skin test antigen. The aim of this study was to investigate how the live attenuated zoster vaccine alters the kinetics of the DTH response to VZV antigen in old individuals. Healthy volunteers (n¼27, age range 70-93 years) were skin-tested with VZV antigen and those with a robust clinical response (2 individuals) were excluded from further participation, with a further 4 drop-outs. Numbers and proliferation (Ki67+) of CD4+ effector T cells and CD4+ Foxp3+ regulatory T cells at the test site were assessed by immunofluorescence microscopy at Days 1, 3 and 7 after VZV challenge. Volunteers were vaccinated after 4 weeks and repeat skin testing and immunofluorescence microscopy analysis were performed after a further 2-8 months. Overall, zoster vaccination led to a significant boost in clinical responses (p¼0.0002), although in 8 individuals there was no improvement. At Day 7 post-VZV challenge the median number of CD4+ T cells per dermal perivascular infiltrate was significantly increased in vaccinated (40.0) compared with unvaccinated individuals (18.9, p¼0.026). The median percentage of proliferating CD4+ T cells at Day 7 was 9.4% in the vaccinated compared with 4.0% in the unvaccinated (p¼0.162). At Day 7 the median proportion of CD4+ T cells with a regulatory phenotype (Foxp3+) reduced from 19.5% in the unvaccinated to 14.0% in the vaccinated (p¼0.088). This study demonstrates that the zoster vaccine significantly boosts the memory CD4+ T cell response to VZV skin challenge in the old, partially restoring the intensity of the immune response to that seen in the young. Reduced inhibitory signalling as a consequence of fewer local regulatory T cells may be a possible mechanism underlying this vaccineinduced immune enhancement.
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Identification of a gain-of-function STAT3 mutation (p.Y640F) in lymphocytic variant hypereosinophilic syndrome S Walker1, C Wang2, T Walradt2, BS Hong2, JR Tanner3, J Levinsohn2, G Goh4, A Subtil2, SR Lessin5, W Heymann9,10, EC Vonderheid6, BA King2, R Lifton7,4 and J Choi3,8 1 Medicine, Dana Farber Cancer Institute, Boston, MA, 2 Dermatology, Yale School of Medicine, New Haven, CT, 3 Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL, 4 Genetics, Yale School of Medicine, New Haven, CT, 5 Dermatology, Fox Chase Cancer Center, Philadelphia, PA, 6 Sydney Kimmel Cancer Center, Johns Hopkins Medical Institutes, Baltimore, MD, 7 Howard Hughes Medical Institute, New Haven, CT, 8 Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL, 9 Dermatology, Cooper Medical School, Camden, NJ and 10 Dermatology, Perelman School of Medicine, Philadelphia, PA Idiopathic hypereosinophilic syndrome (HES) is a heterogeneous group of disorders characterized by 1) persistent peripheral eosinophilia, 2) target organ pathology mediated by infiltrating eosinophils, and 3) the absence of known infectious or allergic causes of hypereosinophilia. Lymphocytic variant HES (L-HES) is a unique subtype of HES characterized by eosinophilia and eosinophil-infiltrating lesions of the skin, soft tissue, and rarely internal organs. In L-HES, an abnormal T cell clone (most commonly CD3-CD4+) produces pathological amounts of eosinophil-promoting Th2 cytokines, such as IL-4, IL-5, and IL-13. These Th2 cytokines are thought to drive the polyclonal expansion of eosinophils in the skin, the subcutaneous tissue, and the blood. To date, the molecular mechanisms underlying L-HES pathogenesis have been unclear. By exome sequencing, we identified a somatic gain-offunction mutation in STAT3 (p.Y640F) in one patient with L-HES. This mutation renders STAT3 constitutively active, and constitutively active STAT3 has been previously shown sufficient to enforce Th2 differentiation. In two additional cases of L-HES, we did not find activating STAT3 mutations; however, RNA-Seq demonstrated evidence of STAT3 activation in all three cases of L-HES. These findings suggest a novel targetable signaling node that can be used to treat this otherwise recalcitrant disease.
Close similarities in global gene expression profiling between skin and lung homing T cells after skin and lung infection Y Pan, T Tian, S Loftus, S Divito, A De Masson, R Clark, R Fuhlbrigge and TS Kupper Dermatology, BWH/HMS, Boston, MA Compared to the well studied tissue-specific homing of lymphocytes to skin and gut, factors that mediate tissue-specific homing to lung are largely unknown. Previous work has shown that in draining lymph nodes after Vaccinia virus (VACV) infection, by 60 hours after infection antigen specific CD8+ T cells can be identified that have divided from 0-5 times (P0-P5). Imprinting of T cells with tissue-specific homing molecules is initiated during this series of cell divisions. In this study, we employed transcriptional microarrays to reveal the global gene expression profile of CD8+ T cells generated via distinct infection routes. OT-1 T cells loaded with CFSE were injected into syngeneic mice, and 24 hours later mice were immunized by epidermal skin infection (es.i), intra-tracheal (i.tra.) or intra-peritoneal infection (i.p.) with VACV-ova, which expresses the OT-1 peptide under an early gene promoter. At 60 hours after infection, we harvested draining lymph nodes (inguinal, mediastinal, and mesenteric, respectively) and sorted cells that had undergone between 0 and 5 cell divisions, as determined by CFSE. RNA was extracted from these cells, and was analyzed by transcriptional profiling. By contrast to i.p. (74/116 genes, p¼0.51), s.s. generated CD8+ Teff cells exhibiting similar gene transcriptional profile with those generated via i.tra.(92/116 genes, p<0.0004), including chemokine receptors CCR4, CCR8 and CCR10. This was confirmed by principal component analysis. Next we evaluated the capacity of es.i. in generating lung CD8+ tissue resident memory T (TRM) cells. More than half of lung as many CD8+ TRM cells could be generated via es.i., as compared to i.tra., suggesting es.i. as a viable approach for generating lung CD8+ TRM cells. The commonalities between transcriptional profiles of skin homing and lung homing T cells is important not only for vaccine strategies, but also for understanding biological phenomena such as the “atopic march” and the tuberculin skin test reaction.
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CD209+ monocyte-derived myeloid dendritic cells were increased in patients with leukemic cutaneous T-cell lymphoma after extracorporeal photopheresis T Langridge, M Austin, X Zhang, L Shiue, M Duvic and X Ni Department of Dermatology, The University of Texas MD Anderson Cancer Center, Houston, TX Our previous study found that numbers of myeloid dendritic cells (mDC) and their HLA-DR expression were increased after extracorporeal photopheresis (ECP) in patients with leukemic cutaneous T-cell lymphoma (L-CTCL). To further define the subset of ECP-affected mDCs, we assessed the expression of CD209 on mDCs in L-CTCL patients over a 6-month ECP treatment course. It is known that CD209 or DC-SIGN, a transmembrane receptor, is dominantly expressed on monocyte-derived mDCs (Mo-mDC), and can be used as a biomarker for MomDCs. Nineteen L-CTCL patients who started ECP therapy with the CELLEX photopheresis system were enrolled in the study. The peripheral blood was collected at baseline, at Day 2, 1month, 3-months, and 6-months after initial ECP treatment, and mDC populations as well as CD209+ mDC subsets were assessed by multi-color flow cytometry. At baseline, about one third of patients showed a low number of mDCs, but all patients had lower than normal CD209+ mDC counts. The average number of CD209+ mDCs was 0.064% out of peripheral blood mononuclear cells (PBMC) in normal donors (n¼3), but only 0.006% out of PBMCs in patients (n¼19, p<0.05). The CD209+ mDC subset accounts for about 66.2% of total mDCs in normal donors compared to only 13.9% in patients (p<0.05). Eight patients finished 3 and/ or 6-month treatment, with 5 clinical responders and 3 non-responders. The average absolute CD209+ mDC counts were increased by about 3-fold at 3-months and 2-fold at 6-months. The average percentages of CD209+ mDCs of PBMCs went up from 0.008% at baseline to 0.015% at 3 months and remained the same at 6 months. Our results suggest that patients with L-CTCL have a deficiency in monocyte-derived myeloid dendritic cells which may be attributed to the suppressed immunity. ECP may exert a positive effect on monocyte-derived myeloid dendritic cells, which might enhance antigen processing and improve anti-tumor immunity in patients with L-CTCL.
Blockade of multiple immune checkpoints cooperatively enhanced the antitumor effect of melanoma-specific CTLs T Inozume2, T Yaguchi1, T Kawamura2, Y Kawakami1 and S Shimada2 1 Division of Cellular Signaling, Keio University, University of Yamanashi, Chuo, Japan and 2 Dermatology, University of Yamanashi, Chuo, Japan Anti-PD-1 and anti-CTLA-4 antibody dramatically improved the overall survival of melanoma patients. However, further developments are still required, particularly, in the enhancement of the anti-tumor effect and the prevention of autoimmunity. Our previous study suggested that a blockade of multiple immune checkpoint molecules that are selectively and highly expressed on tumor-infiltrating CTLs (for example, TIGIT, TIM-3, and LAG-3) may enhance the antitumor effect without increasing the risk of autoimmunity. However, the synergistic effect of these molecules in human melanoma-specific human CTLs remains unclear. In this study, we examined the synergistic effects of the multiple immune checkpoint molecules in human melanoma-specific CTLs. We compared the impacts of overexpression of CD155 (a ligand for TIGIT), Galectin 9 (a ligand for TIM-3), and MHC class II (a ligand for LAG-3) on melanoma cells in the effector phase in vitro. These molecules were overexpressed on a melanoma cell line alone or in combination with other molecules by retroviral transduction, and the transfectants were cocultured with melanoma-specific CTLs that highly upregulate TIGIT, TIM-3, and LAG-3 by antigen stimulation. In addition, the effect of coblockade of these molecules by the antibodies was assessed in the coculture assay. The activation status of CTL was assessed by cytokine production. The suppressive effect of CD155 was the highest in the ligand molecules overexpressed on melanoma cells. In addition, the suppressive effect of coexpression of CD155 and PD-L1 was the highest compared with those of other combinations. In the blocking assay, the best CTL activation was obtained by coblockade of TIGIT and PD-1 signals. In our experiments, coblockade of TIGIT and PD-1 signals were the most effective in activating melanoma-specific CTLs in the effector phase, compared with those of other combinations.
www.jidonline.org S5