Human Purified CD8+T Cells: Ex vivo Expansion Model to Generate a Maximum Yield of Functional Cytotoxic Cells

While T cell-based cancer immunotherapies have shown great promise, there remains a need to understand how individual metastatic tumor environments impart local T cell dysfunction. At advanced stages, cancers that metastasize to the pleural space can result in a malignant pleural effusion (MPE) that harbors abundant tumor and immune cells, often exceeding 108 leukocytes per liter. Unlike other metastatic sites, MPEs are readily and repeatedly accessible via indwelling catheters, providing an opportunity to study the interface between tumor dynamics and immunity. In the current study, we examined CD8+ T cells within MPEs collected from patients with heterogeneous primary tumors and at various stages in treatment to determine (1) if these cells possess anti-tumor activity following removal from the MPE, (2) factors in the MPE that may contribute to their dysfunction, and (3) the phenotypic changes in T cell populations that occur following ex vivo expansion. Co-cultures of CD8+ T cells with autologous CD45― tumor containing cells demonstrated cytotoxicity (p = 0.030) and IFNγ production (p = 0.003) that inversely correlated with percent of myeloid derived suppressor cells, lactate, and lactate dehydrogenase (LDH) within the MPE. Ex vivo expansion of CD8+ T cells resulted in progressive differentiation marked by distinct populations expressing decreased CD45RA, CCR7, CD127, and increased inhibitory receptors. These findings suggest that MPEs may be a source of tumor-reactive T cells and that the cellular and acellular components suppress optimal function.

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The Critical Requirement of the NKG2D Receptor On CD8+ T Cells in Killing Myeloma Cells.

Blood ◽

10.1182/blood.v114.22.4733.4733 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4733-4733

Author(s):

Laleh X. Talebian ◽

Dawn A Fischer ◽

Zbigniew M Szczepiorkowski ◽

Charles L Sentman ◽

Kenneth R Meehan

Keyword(s):

T Cells ◽

Mhc Class I ◽

Cd8 T Cells ◽

Research Funding ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Class I ◽

Myeloma Cells ◽

Nkg2d Receptor ◽

Tumor Cell Killing

Abstract Abstract 4733 Autologous stem cell transplant for myeloma improves survival, but the immunologic mechanisms accounting for this improvement are unknown. Our data indicate that NKG2D+CD8+T cells may be one reason for this benefit. NKG2D, one of four NK cell activating receptors, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. We identified methods for ex vivo expansion of cyclophosphamide – mobilized blood progenitor cells (BPCs) from myeloma patients, as part of a clinical trial (Cytotherapy 2008). Mobilized BPC are cultured in serum-free media with IL-2 (50 IU/ml) and OKT3 (50ng/ml). After 7 days, the CD8+ T cells are isolated, evaluated and tested. Myeloma cells from patients' marrow aspirates are used as targets in cytotoxicity assays. Phenotypic analysis of the expanded cells and the patients' primary myeloma cells was performed using flow cytometry. Ex vivo expansion of cyclophosphamide-mobilized BPCs induces CD8+ T cells that acquire the NKG2D receptor during ex vivo expansion (P < 0.03). Three of the 6 known NKG2D ligands are strongly expressed on patients' myeloma cells, including MICA, ULBP1 and ULBP3. Using cytotoxicity assays, autologous effector cells recognize and kill the patient's autologous tumor cells (E:T 50:1, P< 0.001). When using K562 leukemia cells as targets (lack MHC class I), the NKG2D+CD8+ T cells kill K562, supporting an MHC class I-independent mechanism of tumor cell killing. Blocking the NKG2D receptor on CD8+ T cells prevents killing of autologous myeloma cells (P < 0.009). NKG2D-mediated cytotoxicity correlates with the amount of ligand expression on the target. The NKG2D receptor on CD8+T cells recognizes ligands expressed on autologous myeloma cells. The NKG2D+CD8+T cells aggressively kill myeloma cells in a non–MHC restricted and TCR-independent manner. Blocking NKG2D on CD8+ T cells prevents killing of autologous myeloma cells. Since tumor cells often down regulate MHC Class I expression, the ability of NKG2D+CD8+ T cells to recognize and kill autologous myeloma cells in a non-MHC restricted manner may contribute to the beneficial effects in the treatment of myeloma. Ongoing experiments are testing these methods in mouse models and defining the molecular mechanisms involved. Disclosures: Szczepiorkowski: Cersus Corporation: Research Funding; CaridianBCT: Research Funding; BASF: Research Funding; Terumo Corporation: Research Funding; Fenwel, Inc - Scientific Advisory Board: Research Funding. Meehan:Berlex Pharmaceutical: Research Funding.

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The Wild-Type Sequence (wt) p5325–35 Peptide Induces HLA-DR7 and HLA-DR11-Restricted CD4+ Th Cells Capable of Enhancing the Ex Vivo Expansion and Function of Anti-wt p53264–272 Peptide CD8+ T Cells

The Journal of Immunology ◽

10.4049/jimmunol.177.10.6795 ◽

2006 ◽

Vol 177 (10) ◽

pp. 6795-6803 ◽

Cited By ~ 16

Author(s):

Daisuke Ito ◽

Andreas Albers ◽

Yong Xiang Zhao ◽

Carmen Visus ◽

Ettore Appella ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Wild Type ◽

Th Cells ◽

Wild Type Sequence ◽

And Function ◽

Type Sequence

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Faculty Opinions recommendation of CD8 T-cells from most HIV-infected patients lack ex vivo HIV-suppressive capacity during acute and early infection.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717996871.793475361 ◽

2013 ◽

Author(s):

Susana Asin ◽

Christiane Rollenhagen

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Ex Vivo ◽

Early Infection ◽

Suppressive Capacity

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Ex Vivo Expansion of HER2-Specific T Cells for the Treatment of HER2-Overexpressing Breast Cancer

10.21236/ada416629 ◽

2003 ◽

Author(s):

Keith L. Knutson ◽

Mary Disis

Keyword(s):

Breast Cancer ◽

T Cells ◽

Ex Vivo ◽

Ex Vivo Expansion

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Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

Blood Advances ◽

10.1182/bloodadvances.2019001091 ◽

2020 ◽

Vol 4 (10) ◽

pp. 2143-2157 ◽

Cited By ~ 1

Author(s):

Alak Manna ◽

Timothy Kellett ◽

Sonikpreet Aulakh ◽

Laura J. Lewis-Tuffin ◽

Navnita Dutta ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Xenograft Model ◽

Lymphocytic Leukemia ◽

Dependent Manner

Abstract Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

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602 STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0602 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A637-A637

Author(s):

Manoj Chelvanambi ◽

Ronald Fecek ◽

Jennifer Taylor ◽

Walter Storkus

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Immune Cell ◽

Ex Vivo ◽

In Vitro Assays ◽

Type I ◽

Vascular Normalization ◽

S 100 ◽

Transcriptional Changes

BackgroundThe degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Hence, enhancement of TIL prevalence is a preferred clinical endpoint, one that may be achieved via administration of agents that normalize the tumor vasculature (VN) leading to improved immune cell recruitment and/or that induce the development of local tertiary lymphoid structures (TLS) within the tumor microenvironment (TME).MethodsLow-dose STING agonist ADU S-100 (5 μg/mouse) was delivered intratumorally to established s.c. B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation under an IACUC-approved protocol. Treated and control, untreated tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via qPCR, with corollary immune cell composition changes determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 μg/mL ADU S-100 (vs PBS control) and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For TCRβ-CDR3 analyses, CDR3 was sequenced from gDNA isolated from enzymatically digested tumors and splenocytes.ResultsWe report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of angiostatic factors including Tnfsf15 (Vegi), Cxcl10 and Angpt1, and TLS inducing factors including Ccl19, Ccl21, Lta, Ltb and Tnfsf14 (Light). Therapeutic responses from intratumoral STING activation were characterized by increased vascular normalization (VN), enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neo-genesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ex vivo ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), IL-36, inflammatory chemokines and type I interferons. TLS formation was associated with the development of a therapeutic TIL TCR repertoire enriched in T cell clonotypes uniquely detected within the tumor but not the peripheral circulation in support or local T cell cross-priming within the TME.ConclusionsOur data support the premise that i.t. delivery of STING agonist promotes a pro-inflammatory TME in support of VN and TLS formation, leading to the local expansion of unique TIL repertoire in association with superior anti-melanoma efficacy.

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