The Impact of Ex Vivo Clinical Grade Activation Protocols on Human T-cell Phenotype and Function for the Generation of Genetically Modified Cells for Adoptive Cell Transfer Therapy

BackgroundAlthough the presence and activity of tumor infiltrating lymphocytes (TILs) have been shown to be important factors for survival and response to immunotherapy for multiple cancer types, the benefits of immunotherapy in soft tissue sarcomas (STS) have been limited, and novel approaches are needed. In this study, we sought to characterize the phenotype and function of tumor infiltrating natural killer (NK) and T cells in STS patients and to evaluate clinically relevant strategies to augment TIL function.MethodsUsing both prospectively collected blood and tumor tissue from STS patients undergoing surgical resection (n = 21) and archived specimens (n = 45), we performed flow cytometry and immunohistochemistry to evaluate the extent of peripheral and intratumoral CD3-CD56+ NK and CD8+ T cell phenotype and function as predictors of outcome. We also analyzed TCGA data and the peripheral blood of dogs with spontaneous osteosarcoma receiving inhaled IL-15 on a clinical trial to evaluate the association of CD3-NKp46+ NK and CD8+ T cell activation as well as TIGIT upregulation with outcome. Finally, we stimulated patient PBMCs and TILs ex vivo with IL-15 and a novel human anti-TIGIT antibody to assess the impact of combination therapy on NK and T cell phenotype and function. Parametric and non-parametric statistical tests were used where appropriate. Univariate and multivariate survival analyses were performed by Cox proportional hazards models.ResultsCompared to peripheral expression, intratumoral NK and T cells showed an activated and exhausted phenotype by CD69 and TIGIT, respectively. Ex vivo TIL stimulation with IL-15 further increased markers of activation and function including CD69, Ki67, IFNg, and granzyme B, while increasing expression of exhaustion marker TIGIT. Analysis of a retrospective STS cohort and TCGA STS gene expression confirmed the association of TILs with improved prognosis. Dogs with metastatic osteosarcoma receiving inhaled IL-15 exhibited upregulation of activation markers and TIGIT. In vitro, IL-15 and TIGIT blockade of both peripheral and intratumoral NK cells increased cytotoxicity against sarcoma cell lines and increased expression of degranulation marker CD107a compared to IL-15 alone.ConclusionsTILs are associated with improved survival in STS, and tumor infiltrating NK and T cells show features of both increased activation and increased exhaustion. Tumor-infiltrating NK and T cells respond to IL-15 stimulation, but simultaneously further upregulate TIGIT with the combination of IL-15 and TIGIT blockade showing greatest cytotoxic effects. Overall, our data suggest that the combination of IL-15 and TIGIT blockade is a promising clinical strategy in STS.Ethics ApprovalAll experiments involving human and canine patients were approved by the respective Institutional Review Boards at the University of California, Davis, Schools of Medicine (Protocol #218204-9) and Veterinary Medicine (IACUC #20179).

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Mass Cytometry Analysis of Human T Cell Phenotype and Function

Methods in Molecular Biology - T-Helper Cells ◽

10.1007/978-1-4939-1212-4_7 ◽

2014 ◽

pp. 55-68 ◽

Cited By ~ 2

Author(s):

Evan W. Newell ◽

Lai Li Yun

Keyword(s):

T Cell ◽

Cell Phenotype ◽

Mass Cytometry ◽

Human T Cell ◽

And Function

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Retinoic acid-responsive CD8 effector T-cells are selectively increased in IL-23-rich tissue in gastrointestinal GvHD

Blood ◽

10.1182/blood.2020005170 ◽

2020 ◽

Author(s):

Jennifer A Ball ◽

Andrew James Clear ◽

James Aries ◽

Sarah Charrot ◽

Caroline Besley ◽

...

Keyword(s):

T Cells ◽

Retinoic Acid ◽

T Cell ◽

Mononuclear Cells ◽

Effector T Cells ◽

Cell Phenotype ◽

Major Barrier ◽

Hematopoietic Stem ◽

Human T Cell ◽

The Impact

Gastrointestinal (GI) graft-versus-host disease (GvHD) is a major barrier in allogeneic hematopoietic stem-cell transplantation (AHST). The metabolite retinoic acid (RA) potentiates GI-GvHD in mice via alloreactive T-cells expressing the RA-receptor-alpha (RARα), but the role of RA-responsive cells in human GI-GvHD remains undefined. We therefore used conventional and novel sequential immunostaining and flow cytometry to scrutinize RA-responsive T-cells in tissues and blood of AHST patients and characterize the impact of RA on human T-cell alloresponses. Expression of RARα by human mononuclear cells was increased after RA exposure. RARαhi mononuclear cells were increased in GI-GvHD tissue, contained more cellular RA-binding proteins, localized with tissue damage and correlated with GvHD severity and mortality. Using a targeted candidate protein approach we predicted the phenotype of RA-responsive T-cells in the context of increased microenvironmental IL-23. Sequential immunostaining confirmed the presence of a population of RARahi CD8 T-cells with the predicted phenotype, co-expressing the effector T-cell transcription factor T-bet and the IL-23-specific receptor. These cells were increased in GI- but not skin-GvHD tissues and were also selectively expanded in GI-GvHD patient blood. Finally, functional approaches demonstrated RA predominantly increased alloreactive GI-tropic RARahi CD8 effector T-cells, including cells with the phenotype identified in vivo. IL-23-rich conditions potentiated this effect by selectively increasing b7 integrin expression on CD8 effector T-cells and reducing CD4 T-cells with a regulatory cell phenotype. In conclusion we have identified a population of RA-responsive effector T-cells with a distinctive phenotype which are selectively expanded in human GI-GvHD and represent a potential new therapeutic target.

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Epigenetic Suppression of Transgenic T-cell Receptor Expression via Gamma-Retroviral Vector Methylation in Adoptive Cell Transfer Therapy

Cancer Discovery ◽

10.1158/2159-8290.cd-20-0300 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1645-1653

Author(s):

Theodore S. Nowicki ◽

Colin Farrell ◽

Marco Morselli ◽

Liudmilla Rubbi ◽

Katie M. Campbell ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Retroviral Vector ◽

Adoptive Cell Transfer ◽

Receptor Expression ◽

Cell Transfer ◽

Adoptive Cell Transfer Therapy ◽

Epigenetic Suppression

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Ex vivo enrichment of circulating anti-tumor T cells from both cutaneous and ocular melanoma patients: clinical implications for adoptive cell transfer therapy

Cancer Immunology Immunotherapy ◽

10.1007/s00262-011-1179-z ◽

2011 ◽

Vol 61 (8) ◽

pp. 1169-1182

Author(s):

Tonia Mazzarella ◽

Valeria Cambiaghi ◽

Nathalie Rizzo ◽

Lorenzo Pilla ◽

Danilo Parolini ◽

...

Keyword(s):

T Cells ◽

Ex Vivo ◽

Ocular Melanoma ◽

Adoptive Cell Transfer ◽

Clinical Implications ◽

Cell Transfer ◽

Melanoma Patients ◽

Adoptive Cell Transfer Therapy

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Strong induction of 4-1BB, a growth and survival promoting costimulatory receptor, in HTLV-1–infected cultured and patients' T cells by the viral Tax oncoprotein

Blood ◽

10.1182/blood-2007-10-115220 ◽

2008 ◽

Vol 111 (9) ◽

pp. 4741-4751 ◽

Cited By ~ 21

Author(s):

Klemens Pichler ◽

Tarek Kattan ◽

Juliane Gentzsch ◽

Andrea K. Kress ◽

Graham P. Taylor ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Ex Vivo ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Viral Oncoprotein ◽

Cell Leukemia Virus Type ◽

Human T Cell ◽

The Impact

AbstractHuman T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia, stimulates the growth of infected T cells in cultures and in nonleukemic patients. In the latter, HTLV-1 is found in long-term persisting T-cell clones. The persistence of normal T cells is controlled by the growth-stimulating and antiapoptotic functions of costimulatory receptors, while the growth-stimulating HTLV-1 functions are mediated by the viral oncoprotein Tax. Here we analyzed the impact of Tax on costimulatory receptors in T cells with repressible Tax and found that among these receptors 4-1BB (TNFRSF9/CD137/ILA) was induced most strongly. Up-regulated 4-1BB expression was a consistent feature of all HTLV-1–infected cell lines, whether patient-derived or in vitro transformed. Tax was sufficient to induce the expression of the endogenous 4-1BB gene in uninfected T cells, and it strongly activated (45-fold) the 4-1BB promoter via a single NF-κB site. The ligand of 4-1BB was also found on transformed T-cell lines, opening up the possibility of autostimulation. Moreover, 4-1BB expression in patients' lymphocytes ex vivo correlated with Tax expression, strongly suggesting Tax-mediated 4-1BB activation in vivo. Thus, 4-1BB up-regulation by Tax could contribute to growth, survival, and clonal expansion of the infected cells during persistence and disease.

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