The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

SummaryCD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and longterm replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to cc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16 INK4A transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16 INK4A or p19 ARF expression (as shown using T cells from CDKN2A À/À mice) but was associated with expression of transcripts encoding p15 INK4B , another protein involved in senescence induction. We conclude that T-cell-receptor-and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16 INK4A and p19 ARF .

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“…c). Our results are in line with the observed hypo‐responsiveness of late‐stage TCR‐stimulated human T cells with a KLRG1 hi phenotype …”

Section: Discussionsupporting

confidence: 89%

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B

et al. 2015

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“…For instance, Rappl et al elucidated one possible mechanism of terminally-differentiated, late stage T cell hypofunction as being due to impaired TCR synapse formation from immobility of TCR membrane surface components, which can be bypassed by introducing a 1 st generation CAR construct that confers tumor reactivity by signaling through normal T cell signaling components. 46 The mechanism of the hypofunction described here seems to be different as demonstrated by dysfunctional proximal T cell signaling. The described in vivo model is inadequate to assess TCR function as it lacks human antigen-presenting cells.…”

Section: Discussionmentioning

confidence: 81%

Multifactorial T-cell Hypofunction That Is Reversible Can Limit the Efficacy of Chimeric Antigen Receptor–Transduced Human T cells in Solid Tumors

Moon

Wang

Dolfi

et al. 2014

Clinical Cancer Research

345

293

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Purpose Immunotherapy using vaccines or adoptively transferred tumor infiltrating lymphocytes (TILs) is limited by T cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine if a similar tumor-induced inhibition occurred with genetically-modified cytotoxic T cells expressing chimeric antibody receptors (CARs) targeting tumor-associated-antigens. Methods Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4-1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin- expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways. Results CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction appeared to be multifactorial and was associated with upregulation of intrinsic T cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD-1, LAG3, TIM3, 2B4). Conclusions Advanced generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell-based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD-1 pathway antagonism may augment human CAR T cell function.

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“…The combined transfection of specific TCR and TCRz genes in T cells may be an ideal method to manufacture redirected T cells for immunotherapy. For example, the transgenic expression of the CD3z signaling molecule chimeric antigen receptor, which is specific for CMV, recovered hyporesponsive T cells to full effector functions, released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells (Rappl et al, 2012). Clonally expanded T cells from patients with leukemia possess a leukemia-associated, antigen-specific TCR , and these T cells have a specific cytotoxic ability against antileukemia cells; however, their activation was poor due to deficiencies in TCR signaling, including decreased TCRz expression (Chen et al, 2000;Li, 2008;Chen et al, 2009).…”

mentioning

confidence: 99%

Upregulated TCRζ Enhances Interleukin-2 Production in T-Cells from Patients with CML

Zha

Chen

Yang

et al. 2012

DNA and Cell Biology

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T-cell immunodeficiency is a common feature in patients with chronic myeloid leukemia (CML), and deficiency in CD3 levels was detected in T cells from these patients, which may represent a characteristic that is related to a lower T cell activation. In this study, we explored the possibility that forced TCRζ gene expression may upreg-u-late T cell receptor (TCR) signaling activation and reverse interleukin-2 (IL-2) production in T cells from patients with CML. A recombinant eukaryotic vector expressing TCRζ was transfected into T cells by nucleofection. Phosphorylated TCRζ, phosphorylated NF-κB, and the IL-2 level in TCRζ-transfected CD3+T cells that were activated with anti-CD3 and anti-CD28 antibodies were measured by Western blot and enzyme-linked immunosorbent assay (ELISA). Significantly increased TCRζ levels were found in TCRζ-transfected CD3+T cells. After CD3 and CD28 antibody stimulation, a significantly higher phosphorylated TCRζ chain level was demonstrated, and an increased IL-2 production in TCRζ-upregulated T cells was associated with the increased expression of the phosphorylated NF-κB. In conclusion, TCRζ gene transfection could restore TCRζ chain deficiency and enhance IL-2 production in T cells from patients with CML. It is possible that TCRζ chain reconstitution in leukemia-specific, clonally expanded T cells will effectively increase their activation of antileukemia cytotoxicity.

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The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

Cited by 12 publications

References 34 publications

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B

Control of CD8 T cell proliferation and terminal differentiation by active STAT5 and CDKN2A/CDKN2B

Multifactorial T-cell Hypofunction That Is Reversible Can Limit the Efficacy of Chimeric Antigen Receptor–Transduced Human T cells in Solid Tumors

Upregulated TCRζ Enhances Interleukin-2 Production in T-Cells from Patients with CML

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