High frequency of intragraft IFN-γ producing CD8+ T cells is predictive of lung allograft rejection

Abstract We have recently shown that ex vivo manufactured, rapamycin-resistant donor Th2 cells (Th2.R cells) prevent the rejection of allogeneic bone marrow grafts through a process that involves IL-4-mediated polarization of host T cells towards a Th2 phenotype. Because donor T cell therapy lacks feasibility in the setting of cardiac allograft transplantation using cadaveric donors, we developed an approach to prevent rejection that incorporates host Th2 cell therapy prior to organ transplantation. The aims of the study were to: (1) develop a method for the ex vivo manufacture of rat Th2.R cells and inject such syngeneic Th2.R cells just prior to class I and class II disparate cardiac allografting; (2) determine whether such adoptive host Th2.R cell transfer polarizes post-transplant immunity towards a Th2 phenotype; and (3) evaluate in a preliminary manner whether Th2.R cell transfer reduces graft rejection in a model that uses sub-optimal cyclosporine therapy. Recipient-type (Black Norway, BN) CD4+ T cells were co-stimulated with anti-rat anti-CD3 and –CD28 antibodies in the presence of rapamycin, rrIL4, rhuIL-2, and rhIL7 (3-day culture interval). The Th2.R cell cytokine phenotype was determined by intra-cellular flow cytometry and multi-analyte testing of supernatants obtained after repeat co-stimulation. Th2.R cells were injected (i.v.; 1 × 107 cells) 2–3 hours prior to cardiac allografting (donor strain, Dark Agouti; DA). Cardiac function was evaluated clinically by cardiac palpation through day 28 post-grafting. At day 28, histology studies and immune function studies were performed, including assessment of host-anti-donor alloreactivity. Engraftment controls (cohort #1) and rejection controls (cohort #2) received cyclosporine (CSA) through the full 28 days or only through day 18, respectively; the experimental cohort (#3) received host Th2.R cells followed by the short-course of CSA. Supernatants from the Th2.R cell product contained 1500 pg/ml IL-4 and only 50 pg/ml of IFN-γ; the frequency of IL-4+ cells and IFN-γ+ cells was 10% and 1%, respectively. Clinical rejection was observed in cohort #2 (3/3 subjects) whereas all subjects in cohorts #1 and #3 had full cardiac function through day 28 (cohort #1- 2/3 subjects; cohort #3- 3/3 subjects). Relative to rejection controls, Th2.R cell recipients had a reduced frequency of intra-cardiac CD8+ T cells (%CD8+ T cells, 9.2 ± 6.2 vs. 1.1 ± 0.3; p<0.001) and spleen CD8+ T cells (1.9 ± 0.2 vs. 0.5 ± 0.2; p=0.014). Furthermore, after ex vivo re-stimulation with donor-type dendritic cells, intra-cardiac CD8+ T cells from Th2.R cell recipients tended to produce less IFN-γ relative to rejection controls (pg/ml in supernatant; 15.7 ± 1.4 vs. 44.2 ± 1.4; p=0.07) and tended to produce increased IL-4 relative to rejection controls (1974 ± 140 vs. 260 ± 291; p=0.07). This study demonstrates a feasible and successful methodology for inducing recipient based immunotolerance by generating rapamycin-resistant Th2 cells in the rat species.. Adoptive transfer of host-type Th2.R cells just prior to fully genetically-disparate cardiac allograft transplantation results in a shift towards Th2 cytokines post-transplant, a reduction in the frequency of intra-cardiac T cell infiltration, and shows promise as a novel method to prevent cardiac allograft rejection.

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Phase 1 study of safety, tolerability and immunogenicity of the human telomerase (hTERT)-encoded DNA plasmids INO-1400 and INO-1401 with or without IL-12 DNA plasmid INO-9012 in adult patients with solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-003019 ◽

2021 ◽

Vol 9 (7) ◽

pp. e003019

Author(s):

Robert H Vonderheide ◽

Kimberly A Kraynyak ◽

Anthony F Shields ◽

Autumn J McRee ◽

Jennifer M Johnson ◽

...

Keyword(s):

Pancreatic Cancer ◽

T Cells ◽

Cd8 T Cells ◽

Plasmid Dna ◽

Phase 1 ◽

Dna Encoding ◽

Cancer Types ◽

Ifn Γ ◽

Human Telomerase ◽

Disease Free

BackgroundHuman telomerase reverse transcriptase (hTERT) is frequently classified as a ‘universal’ tumor associated antigen due to its expression in a vast number of cancers. We evaluated plasmid DNA-encoded hTERT as an immunotherapy across nine cancer types.MethodsA phase 1 clinical trial was conducted in adult patients with no evidence of disease following definitive surgery and standard therapy, who were at high risk of relapse. Plasmid DNA encoding one of two hTERT variants (INO-1400 or INO-1401) with or without plasmid DNA encoding interleukin 12 (IL-12) (INO-9012) was delivered intramuscularly concurrent with the application of the CELLECTRA constant-current electroporation device 4 times across 12 weeks. Safety assessments and immune monitoring against native (germline, non-mutated, non-plasmid matched) hTERT antigen were performed. The largest cohort of patients enrolled had pancreatic cancer, allowing for additional targeted assessments for this tumor type.ResultsOf the 93 enrolled patients who received at least one dose, 88 had at least one adverse event; the majority were grade 1 or 2, related to injection site. At 18 months, 54.8% (51/93) patients were disease-free, with median disease-free survival (DFS) not reached by end of study. For patients with pancreatic cancer, the median DFS was 9 months, with 41.4% of these patients remaining disease-free at 18 months. hTERT immunotherapy induced a de novo cellular immune response or enhanced pre-existing cellular responses to native hTERT in 96% (88/92) of patients with various cancer types. Treatment with INO-1400/INO-1401±INO-9012 drove hTERT-specific IFN-γ production, generated hTERT-specific CD4+ and CD8+ T cells expressing the activation marker CD38, and induced hTERT-specific activated CD8 +CTLs as defined by cells expressing perforin and granzymes. The addition of plasmid IL-12 adjuvant elicited higher magnitudes of cellular responses including IFN-γ production, activated CD4+ and CD8+ T cells, and activated CD8+CTLs. In a subset analysis of pancreatic cancer patients, the presence of immunotherapy-induced activated CD8+ T cells expressing PD-1, granzymes and perforin correlated with survival.ConclusionsPlasmid DNA-encoded hTERT/IL-12 DNA immunotherapy was well-tolerated, immune responses were noted across all tumor types, and a specific CD8+ phenotype increased by the immunotherapy was significantly correlated with survival in patients with pancreatic cancer.

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CD8 T Cells Use IFN-γ To Protect against the Lethal Effects of a Respiratory Poxvirus Infection

The Journal of Immunology ◽

10.4049/jimmunol.1400256 ◽

2014 ◽

Vol 192 (11) ◽

pp. 5415-5425 ◽

Cited By ~ 21

Author(s):

John Goulding ◽

Georges Abboud ◽

Vikas Tahiliani ◽

Pritesh Desai ◽

Tarun E. Hutchinson ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Lethal Effects ◽

Ifn Γ

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Vaccination with DNA Encoding the Immunodominant LACK Parasite Antigen Confers Protective Immunity to Mice Infected with Leishmania major

Journal of Experimental Medicine ◽

10.1084/jem.186.7.1137 ◽

1997 ◽

Vol 186 (7) ◽

pp. 1137-1147 ◽

Cited By ~ 255

Author(s):

Sanjay Gurunathan ◽

David L. Sacks ◽

Daniel R. Brown ◽

Steven L. Reiner ◽

Hughes Charest ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Protective Immunity ◽

Leishmania Major ◽

Protective Efficacy ◽

Dna Vaccination ◽

Attractive Alternative ◽

Dna Immunization ◽

Dna Encoding ◽

Ifn Γ

To determine whether DNA immunization could elicit protective immunity to Leishmania major in susceptible BALB/c mice, cDNA for the cloned Leishmania antigen LACK was inserted into a euykaryotic expression vector downstream to the cytomegalovirus promoter. Susceptible BALB/c mice were then vaccinated subcutaneously with LACK DNA and challenged with L. major promastigotes. We compared the protective efficacy of LACK DNA vaccination with that of recombinant LACK protein in the presence or absence of recombinant interleukin (rIL)-12 protein. Protection induced by LACK DNA was similar to that achieved by LACK protein and rIL-12, but superior to LACK protein without rIL-12. The immunity conferred by LACK DNA was durable insofar as mice challenged 5 wk after vaccination were still protected, and the infection was controlled for at least 20 wk after challenge. In addition, the ability of mice to control infection at sites distant to the site of vaccination suggests that systemic protection was achieved by LACK DNA vaccination. The control of disease progression and parasitic burden in mice vaccinated with LACK DNA was associated with enhancement of antigen-specific interferon-γ (IFN-γ) production. Moreover, both the enhancement of IFN-γ production and the protective immune response induced by LACK DNA vaccination was IL-12 dependent. Unexpectedly, depletion of CD8+ T cells at the time of vaccination or infection also abolished the protective response induced by LACK DNA vaccination, suggesting a role for CD8+ T cells in DNA vaccine induced protection to L. major. Thus, DNA immunization may offer an attractive alternative vaccination strategy against intracellular pathogens, as compared with conventional vaccination with antigens combined with adjuvants.

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NKG2D+ IFN-γ+ CD8+ T Cells Are Responsible for Palladium Allergy

PLoS ONE ◽

10.1371/journal.pone.0086810 ◽

2014 ◽

Vol 9 (2) ◽

pp. e86810 ◽

Cited By ~ 13

Author(s):

Mitsuko Kawano ◽

Masafumi Nakayama ◽

Yusuke Aoshima ◽

Kyohei Nakamura ◽

Mizuho Ono ◽

...

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Ifn Γ

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Virally Activated CD8 T Cells Home to Mycobacterium bovis BCG-Induced Granulomas but Enhance Antimycobacterial Protection Only in Immunodeficient Mice

Infection and Immunity ◽

10.1128/iai.00943-06 ◽

2006 ◽

Vol 75 (3) ◽

pp. 1154-1166 ◽

Cited By ~ 10

Author(s):

Laura H. Hogan ◽

Dominic O. Co ◽

Jozsef Karman ◽

Erika Heninger ◽

M. Suresh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mycobacterium Bovis ◽

Cd8 T Cells ◽

Bacterial Load ◽

Granulomatous Inflammation ◽

Cd8 T Cell ◽

Activated T Cells ◽

Immunodeficient Mice ◽

Ifn Γ

ABSTRACT The effect of secondary infections on CD4 T-cell-regulated chronic granulomatous inflammation is not well understood. Here, we have investigated the effect of an acute viral infection on the cellular composition and bacterial protection in Mycobacterium bovis strain bacille Calmette-Guérin (BCG)-induced granulomas using an immunocompetent and a partially immunodeficient murine model. Acute lymphocytic choriomeningitis virus (LCMV) coinfection of C57BL/6 mice led to substantial accumulation of gamma interferon (IFN-γ)-producing LCMV-specific T cells in liver granulomas and increased local IFN-γ. Despite traffic of activated T cells that resulted in a CD8 T-cell-dominated granuloma, the BCG liver organ load was unaltered from control levels. In OT-1 T-cell-receptor (TCR) transgenic mice, ovalbumin (OVA) immunization or LCMV coinfection of BCG-infected mice induced CD8 T-cell-dominated granulomas containing large numbers of non-BCG-specific activated T cells. The higher baseline BCG organ load in this CD8 TCR transgenic animal allowed us to demonstrate that OVA immunization and LCMV coinfection increased anti-BCG protection. The bacterial load remained substantially higher than in mice with a more complete TCR repertoire. Overall, the present study suggests that peripherally activated CD8 T cells can be recruited to chronic inflammatory sites, but their contribution to protective immunity is limited to conditions of underlying immunodeficiency.

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mTOR signaling promotes a robust and continuous production of IFN-γ by human memory CD8+T cells and their proliferation

European Journal of Immunology ◽

10.1002/eji.201445086 ◽

2015 ◽

Vol 45 (3) ◽

pp. 893-902 ◽

Cited By ~ 9

Author(s):

Ruka Setoguchi ◽

Yui Matsui ◽

Kousuke Mouri

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Continuous Production ◽

Human Memory ◽

Mtor Signaling ◽

Memory Cd8 T Cells ◽

Ifn Γ

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CD8+ T Cells in the Lesional Skin of Atopic Dermatitis and Psoriasis Patients Are an Important Source of IFN-γ, IL-13, IL-17, and IL-22

Journal of Investigative Dermatology ◽

10.1038/jid.2012.456 ◽

2013 ◽

Vol 133 (4) ◽

pp. 973-979 ◽

Cited By ~ 128

Author(s):

DirkJan Hijnen ◽

Edward F. Knol ◽

Yoony Y. Gent ◽

Barbara Giovannone ◽

Scott J.P. Beijn ◽

...

Keyword(s):

T Cells ◽

Atopic Dermatitis ◽

Cd8 T Cells ◽

Lesional Skin ◽

Ifn Γ

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148 Identification of prostate-restricted epithelial antigens for transgenic T cell adoptive therapy against prostate cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0148 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A161-A161

Author(s):

Diana DeLucia ◽

Tiffany Pariva ◽

Roland Strong ◽

Owen Witte ◽

John Lee

Keyword(s):

Prostate Cancer ◽

T Cells ◽

T Cell ◽

Single Cell ◽

Cd8 T Cells ◽

Adoptive Therapy ◽

Mhc I ◽

Epithelial Antigens ◽

Ifn Γ ◽

Stimulation Of

BackgroundIn advanced prostate cancer (PCa), progression to castration-resistant PCa (CRPC) is inevitable and novel therapies for CRPC are needed. Adoptive transfer of T cells targeting tumor antigens is a promising approach in the cancer field. Unfortunately, identifying antigens expressed exclusively in prostate tumor cells has been challenging. Since the prostate is not an essential organ, we alternatively selected prostate-restricted epithelial antigens (PREAs) expressed in both malignant and normal prostate tissue for transgenic T cell studies.MethodsRNA-seq data sets identifying genes enriched in PCa were cross-referenced with the NIH Genotype-Expression database to identify PREAs. Using a novel molecular immunology approach, select PREAs and major histocompatibility complex class I (MHC-I) molecules were co-expressed in HEK293F cells, from which MHC–peptide complexes were efficiently isolated. Peptides were eluted and sequenced by mass spectrometry. Peptide–MHC binding was validated with a T2 stabilization assay and peptide immunodominance was determined using an interferon-γ (IFN-γ) ELISpot assay following stimulation of healthy HLA-A2+ peripheral blood mononuclear cells (PBMC) with peptide pools. Following peptide stimulation, CD8+ T cells with peptide-specific T cell receptors (TCR) were enriched by peptide–MHC-I dextramer labeling and fluorescence activated cell sorting for single cell TCR α/β chain sequencing.ResultsWe identified 11 A2+ peptides (8 previously unpublished) from prostatic acid phosphatase (ACPP), solute carrier family 45 member 3 (SLC45A3), and NK3 homeobox 1 (NKX3.1) that bound to HLA-A2 with varying affinities. Extended culture stimulation of PBMC with peptide pools from each PREA, compared to the standard overnight culture, revealed a greater number of IFN-γ producing cells overall and a greater breadth of response across all the peptides. Antigen specific CD8+ T cells were detectable at low frequencies in both male and female healthy PBMC for 7 of the 11 peptides. Dextramer-sorted antigen-specific cells were used for single-cell paired TCR αβ sequencing and transgenic T cell development.ConclusionsThrough this work we identified HLA-A2-presented antigenic peptides from the PREAs ACPP, SLC45A3, and NKX3.1 that can induce the expansion of IFN-γ producing CD8+ T cells. Through peptide–MHC-I dextramer labeling, we isolated PREA-specific CD8+ T cells and characterized TCR αβ sequences with potential anti-tumor functionality. Our results highlight a rapid and directed platform for the development of MHC-I-restricted transgenic CD8+ T cells targeting lineage-specific proteins expressed in prostate epithelia for adoptive therapy of advanced PCa.

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