Allogeneic Chimeric Antigen Receptor-Invariant Natural Killer T Cells Exert Both Direct and Indirect Antitumor Effects through Host CD8 T Cell Cross-Priming

Chimeric antigen receptor (CAR) T cells have shown impressive results in refractory B-cell malignancies. Unfortunately, to date, commercially available cells, as well as most products tested in clinical trials, are autologous CAR T cells whose widespread use is limited by the logistical and financial burdens related to their ad hoc generation. The development of universal allogeneic CAR T cell products to be used off-the-shelf across MHC-barriers has faced major limitations, namely the risk of Graft-versus-Host-Disease (GvHD) induction and the rejection of the administered cells by the host immune system. Invariant Natural Killer-T (iNKT) cells are innate lymphocytes that are deprived of any GvHD induction potential but that display antitumor effects both directly, through the production of cytotoxic effector molecules, and indirectly, through the enhancement of NK and CD8 T cell-mediated immune responses. Preclinical studies using xenogeneic mouse models have demonstrated the feasibility of using iNKT cells as a platform for CAR-based therapies, and two clinical trials are currently ongoing. In order to study the interaction of CD19-specific iNKT CAR cells with the host immune system, we transduced iNKT cells ex vivo expanded from FVB/N mice with a CAR composed of the variable region cloned from the 1D3 hybridoma recognizing murine CD19 linked to a portion of the murine CD28 molecule and to the cytoplasmic region of the murine CD3-ζ molecule. The cytotoxic potential of CD19-iNKT CAR was confirmed in an in vitro cytotoxic assay against the CD19-expressing A20 lymphoma cell line, revealing a strong, dose dependent cytotoxic effect of CD19-CAR iNKT cells. Accordingly, and similarly to what was previously reported in xenogeneic studies, FVB/N (H-2Kq) derived iNKT CAR (2x10e6 cells iv) significantly improved survival of mice after administration to major histocompatibility complex (MHC)-mismatched, immunodeficient BALB/c (H-2Kd) Rag2-/- gamma-chain-/- mice receiving A20 cells (2x10e4 cells iv; Figure 1A) without inducing any signs of GvHD. To test the efficacy of iNKT CAR cells in the presence of host immune cells, we tested the antitumor activity mediated by iNKT CAR against A20 cells in BALB/c mice receiving sublethal irradiation (4.4 Gy), resulting in only a partial and transient lymphopenia. In this model, the antitumor effect of iNKT CAR cells was greatly enhanced, leading to long-term survival of the great majority of treated mice (Figure 1B). Such a difference in iNKT CAR effect between mice that genetically lacked lymphocytes and mice with only partial lymphopenia suggested the participation of host derived lymphocytes in the antitumor effect. To test the hypothesis that host CD8 T cell activation via cross-priming could at least partially mediate the indirect antitumor effect of iNKT CAR cells, we repeated the experiment, employing as recipients BALB/c BATF3-/- mice, in which CD8 T cell cross-priming is impaired as a result of the absence of BATF3-dependent CD103+ CD8a+ dendritic cells that are thought to present antigens through CD1d to the invariant T cell receptor expressed by iNKT cells. Interestingly, the iNKT CAR effect was partially abrogated in A20-receiving BATF3-/- mice as compared to WT mice (Figure 1C), supporting the hypothesis that the impact of iNKT CAR cells is mediated at least partially by the activation of host CD8 T cells via their cross-priming. To formally demonstrate the synergistic effect between allogeneic iNKT CAR and autologous CD8 T cells, we employed an autologous bone marrow transplantation model, co-administering allogeneic iNKT CAR with autologous CD8 T cells at the time of transfer of T-cell-depleted autologous bone marrow cells and A20 lymphoma cells into lethally irradiated (8.8 Gy) BALB/c recipients. Co-administration of allogeneic iNKT CAR and autologous CD8 T cells resulted in a synergistic effect, significantly extending animal survival (Figure 1D) compared to mice receiving no treatment, as well as to mice receiving either allogeneic iNKT or autologous CD8 T cells alone. Collectively, these results represent the first demonstration of an immune adjuvant effect exerted by an allogeneic CAR cell product toward the autologous immune system of the host, suggesting that the effect of the administered cells would last longer than the physical persistence of the allogeneic cells after they will be rejected by the host immune system. Figure 1 Disclosures Mackall: Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Identification of Dual Positive CD19+/CD3+ T Cells in an Apheresis Product Undergoing Chimeric Antigen Receptor (CAR) Transduction

Blood ◽

10.1182/blood-2019-129436 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4471-4471

Author(s):

Liora M Schultz ◽

Shabnum Patel ◽

Sneha Ramakrishna ◽

Alice Bertaina ◽

Neehar Bhatia ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cell Subset ◽

Advisory Board ◽

Advisory Committees ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory ◽

Equity Ownership

Following CAR T cell therapy, many patients receive consolidative hematopoietic stem cell transplantation (HSCT), with the available donor pool recently expanding to include genetically engineered cell products. One such example with striking early promise is the α/Β T-cell depleted haploidentical HSCT (α/Β haplo-HSCT) given with bystander α/Β genetically modified T cells, termed BPX-501 cells. These cells are engineered to express an inducible caspase 9 (iCas9) suicide vector that can be activated by the AP1903 (Rimiducid) dimerizing agent in the event of graft-versus-host disease (GVHD) (US NCT03301168). Here, we report the identification of an unexpected dual expressing CD19+/CD3+ T cell subset in a 10 year old patient who underwent apheresis collection for CAR T cell manufacturing upon relapse six months after α/Β haplo-HSCT and BPX-501 cell addback. A leukapheresis product was collected and CD4 and CD8 T cells were selected from this product for CAR transduction using the Miltenyi CliniMACS Prodigy. T cell purity was assessed using flow cytometry. Characterization of his T cell product at this stage revealed an aberrant cell population expressing both surface CD3 and CD19 (Fig 1a) and lacking additional B cell surface markers, excluding leukemic origin of these cells. This patient was the recipient of the described BPX-501 cell product. BPX-501 cells are also engineered to express a truncated version of CD19 to permit tracking of these modified T cells in patients, post-infusion. We hypothesized that these dual positive CD19+/CD3+ cells were the BPX-501 cells derived from his paternal haploidentical donor and still circulating despite leukemia relapse. We utilized the patient's leukapheresis sample to better characterize these cells. Flow cytometry confirmed the presence of this dual positive CD19+/CD3+ population in the apheresis product. We subsequently treated the apheresis product with Rimiducid in vitro and observed elimination of the CD19+/CD3+ cell subset in a dose dependent manner, thus confirming that these cells were BPX-501 cells (Fig 1b). We additionally investigated the fate of the BPX-501 cells following CAR transduction and observed an absence of this subset post-CAR transduction (Fig 1a). Cells co-expressing CD19-targeting CARs and surface CD19 were not observed in the final manufactured product. The most likely explanation for this phenomenon is in vitro fratricide of the CD19+ T cell population by the CD19-specific CAR+ T cells in culture. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR T therapy and engineered α/Β haplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant add-back products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy. Disclosures Majzner: Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy; Xyphos Inc.: Consultancy. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Feldman:Octane Biotech, Inc.: Employment; Personalized Medicine Initiative Science: Membership on an entity's Board of Directors or advisory committees.

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Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽

10.1182/blood-2019-130815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 779-779 ◽

Cited By ~ 2

Author(s):

Zinaida Good ◽

Jay Y. Spiegel ◽

Bita Sahaf ◽

Meena B. Malipatlolla ◽

Matthew J. Frank ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

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Disruption of T Cell Homeostasis in Mice Expressing a T Cell–Specific Dominant Negative Transforming Growth Factor β II Receptor

Journal of Experimental Medicine ◽

10.1084/jem.191.7.1187 ◽

2000 ◽

Vol 191 (7) ◽

pp. 1187-1196 ◽

Cited By ~ 190

Author(s):

Philip J. Lucas ◽

Seong-Jin Kim ◽

Spencer J. Melby ◽

Ronald E. Gress

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Growth Factor ◽

Cd8 T Cells ◽

Transforming Growth Factor ◽

Cd8 T Cell ◽

Dominant Negative ◽

T Cell Homeostasis ◽

Lymphocyte Homeostasis

The immune system, despite its complexity, is maintained at a relative steady state. Mechanisms involved in maintaining lymphocyte homeostasis are poorly understood; however, recent availability of transgenic (Tg) and knockout mouse models with altered balance of lymphocyte cell populations suggest that cytokines play a major role in maintaining lymphocyte homeostasis. We show here that transforming growth factor (TGF)-β plays a critical role in maintaining CD8+ T cell homeostasis in a Tg mouse model that specifically overexpresses a dominant negative TGF-β II receptor (DNRII) on T cells. DNRII T cell Tg mice develop a CD8+ T cell lymphoproliferative disorder resulting in the massive expansion of the lymphoid organs. These CD8+ T cells are phenotypically “naive” except for the upregulation of the cell surface molecule CD44, a molecule usually associated with memory T cells. Despite their dominance in the peripheral lymphoid organs, CD8+ T cells appear to develop normally in the thymus, suggesting that TGF-β exerts its homeostatic control in the peripheral immune system.

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An exploratory clinical trial on the safety and efficacy of iNKT cell and PD-1+CD8+ T cell-based innovative immunotherapy in patients with lung adenocarcinoma

10.21203/rs.3.rs-70176/v1 ◽

2020 ◽

Author(s):

Xiaobo Cheng ◽

Jing Wang ◽

Chenli Qiu ◽

Yanling Jin ◽

Bili Xia ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

T Cell ◽

Lung Adenocarcinoma ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Inkt Cell ◽

Tolerability Profile ◽

Inkt Cells ◽

Ct Examination

Abstract Background and purposeThe antitumor activities of invariant natural killer T (iNKT) cells and CD8+ T cells have been confirmed. As tumor-specific CD8+ T cells, PD-1+CD8+ T cells kill tumor cells relatively effectively and specifically. We aimed to assess the feasibility, toxicity, antitumor efficacy, and biological correlates of response in patients with advanced lung adenocarcinoma treated with iNKT cells and PD-1+CD8+ T cells for the first time. MethodsWe performed a single-arm, prospective, single-center exploratory clinical trial that is ongoing and registered at ClinicalTrials.gov (NCT03093688). We expanded autologous iNKT cells and PD-1+CD8+ T cells in vitro using our patented technology. Patients were infused with autologous iNKT cells (1×10 8 ~1×10 10 ), PD-1+CD8+ T cells (1×10 7 ~1×10 9 ) and dendritic cells (DCs) (5×10 5 ) every 3-5 weeks, which was considered 1 cycle. The study was conducted from March 1, 2017. The primary endpoint were safety and objective tumor response. ResultsAn analysis of the completed first stage is reported here. A total of 3 consecutive patients were enrolled and received autologous iNKT cell and PD-1+CD8+ T cell-based immunotherapy (≥4 cycles). The first patient received 16 cycles. Computed tomography (CT) examination revealed a stable disease (SD) response after 4 cycles and a progressive disease (PD) response after 11 cycles. The overall survival (OS) time was 20 months. The second patient received 10 cycles. CT examinaton revealed an SD response after 4 cycles and a PD response after 9 cycles. The OS time was 10 months. The third patient received 6 cycles. CT examination revealed an SD response. The OS time was 5.5 months. The patients suffered from only fever, chills, soreness, headache, dizziness, fatigue, sweating, a sick feeling, nausea, vomiting and shortness of breath and received symptomatic treatment. ConclusionsiNKT cell and PD-1+CD8+ T cell-based immunotherapy showed a manageable tolerability proﬁle and effective antitumor activity and was selected as a prospective antitumor therapy.Trial registrationThe protocol had retrospectively registered at clinical trial.gov, Unique identifer: NCT03093688, date of registration: March 01, 2017;https://www.clinicaltrials.gov/ct2/show/NCT03093688?term=NCT03093688&rank=1

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Peptide-Induced Antigen-Specific CD4+CD25+FoxP3+ T Cells Suppress Cytotoxicity T Cell Responses Directed Against the AAV Capsid.

Blood ◽

10.1182/blood.v116.21.3769.3769 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3769-3769 ◽

Cited By ~ 1

Author(s):

Daniel J. Hui ◽

Etiena Basner-Tschakarjan ◽

Gary C. Pien ◽

William D. Martin ◽

Annie S. DeGroot ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

T Cell Responses ◽

Cd8 T Cell ◽

Cell Responses ◽

Equity Ownership

Abstract Abstract 3769 Recent advances in adeno-associated viral (AAV) vector-mediated gene transfer continue to offer hope for the correction of monogenic disorders such as hemophilia B. However, unanticipated T cell responses directed against viral capsid epitopes may limit the efficacy of AAV gene transfer. A phase I clinical study in which an AAV2 vector expressing human factor IX (FIX) was delivered systemically provided the first evidence that AAV vector administration at high doses may trigger the expansion of memory CD8+ T cells directed against AAV capsid epitopes. This response was associated with the loss of FIX transgene expression and a transient increase in liver enzymes. Additional studies in human subjects undergoing AAV gene transfer suggest that the capsid antigen load is an important determinant of capsid-specific T cell activation. Thus, strategies for the modulation of capsid T cell responses could contribute to achieving sustained transgene expression following high dose delivery of AAV. MHC class II peptide ligands identified within the human IgG Fc fragment (Tregitopes, Blood 2008;112:3303) have been shown to expand regulatory T cells (Tregs). Restimulation of human peripheral blood mononuclear cells (PBMC) in vitro with AAV capsid antigen in the presence of Tregitopes resulted in the suppression of capsid-specific CD8+ T cells and in the expansion of CD4+CD25+FoxP3+ Tregs. To better define the nature of Tregitope-induced Tregs, we depleted CD25+ cells from PBMC prior to in vitro restimulation. This completely prevented capsid-specific CTL suppression and the expansion of Tregs, suggesting that Tregitopes act by expanding natural Tregs. Cytokine ELISA on conditioned media from PBMC co-cultured with AAV antigen and Tregitopes showed a 50% decrease in IL-2 levels and a >500-fold increase in IL-10 levels. These results suggest that the effect of Tregitopes may be cytokine mediated. To test this hypothesis, we used a transwell system in which the CD4+ T cell fraction of Tregitope-restimulated PBMC was co-cultured with the capsid-specific CD8+ T cells. Without cell contact, a nearly 50% suppression of anti-capsid CD8+ T cell responses was observed. Further evidence supporting the role of cytokine-mediated suppression came from the observation that Tregitope-treated capsid-specific CD8+ T cells appeared to be anergic, and depletion of CD4+ T cells (Tregs) followed by a 24-hour incubation of CD8+CD4− T cells with IL-2 restored >80% of CTL activity. Finally, antigen specificity of Tregitope-induced Tregs was tested by expanding PBMC in vitro with HLA-B*0702-restricted epitopes from either the AAV capsid or the Epstein-Barr Virus (EBV). After in vitro restimulation, negatively-isolated CD4+ T cells expanded in the presence of EBV antigen and Tregitopes were co-incubated with either CD8+ T cells expanded against the AAV capsid or against EBV. Suppression of CTL activity was observed only when EBV Tregs were co-incubated with EBV CD8+ T cells. Similarly, Tregs isolated from AAV and Tregitope cultures suppressed AAV-specific CD8+ T cells but not EBV-specific CD8+T cells. These results suggest that inhibition of CD8+ T cell responses is antigen-specific. We conclude that Tregitopes induce the expansion of Tregs, which can mediate a potent antigen-specific inhibition of CD8+ T cell responses directed to the AAV capsid. Disclosures: Hui: Children's Hospital of Philadelphia: Patents & Royalties. Martin:EpiVax: Employment, Equity Ownership, Patents & Royalties. DeGroot:EpiVax: Employment, Equity Ownership. High:Children's Hospital of Philadelphia: Patents & Royalties. Mingozzi:Children's Hospital of Philadelphia: Patents & Royalties.

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Allotransplantation Is Associated With Exacerbation of CD8 T-Cell Senescence: The Particular Place of the Innate CD8 T-Cell Component

Frontiers in Immunology ◽

10.3389/fimmu.2021.674016 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lauren Daniel ◽

Marion Tassery ◽

Clara Lateur ◽

Antoine Thierry ◽

André Herbelin ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Cd8 T Cells ◽

Immune Cell ◽

Cell Subset ◽

Cd8 T Cell ◽

Cmv Infection ◽

Senescent Phenotype ◽

Ifn Γ

Immunosenescence is a physiological process that is associated with changes in the immune system, particularly among CD8 T-cells. Recent studies have hypothesized that senescent CD8 T-cells are produced with chronologic age by chronic stimulation, leading to the acquisition of hallmarks of innate-like T-cells. While conventional CD8 T-cells are quite well characterized, CD8 T-cells sharing features of NK cells and memory CD8 T-cells, are a newly described immune cell population. They can be distinguished from conventional CD8 T-cells by their combined expression of panKIR/NKG2A and Eomesodermin (E), a unique phenotype closely associated with IFN-γ production in response to innate stimulation. Here, we first provided new evidence in favor of the innate character of panKIR/NKG2A(+) E(+) CD8 T-cells in normal subjects, documenting their position at an intermediate level in the innateness gradient in terms of both innate IFN-γ production and diminished mitochondrial mass. We also revealed that CD8 E(+) panKIR/NKG2A(+) T-cells, hereafter referred to as Innate E(+) CD8 T-cells, exhibit increased senescent (CD27(-) CD28(-)) phenotype, compared to their conventional memory counterparts. Surprisingly, this phenomenon was not dependent on age. Given that inflammation related to chronic viral infection is known to induce NK-like marker expression and a senescence phenotype among CD8 T-cells, we hypothesized that innate E(+) CD8 T-cells will be preferentially associated with exacerbated cellular senescence in response to chronic alloantigen exposure or CMV infection. Accordingly, in a pilot cohort of stable kidney allotransplant recipients, we observed an increased frequency of the Innate E(+) CD8 T-cell subset, together with an exacerbated senescent phenotype. Importantly, this phenotype cannot be explained by age alone, in clear contrast to their conventional memory counterparts. The senescent phenotype in CD8 T-cells was further increased in cytomegalovirus (CMV) positive serology transplant recipients, suggesting that transplantation and CMV, rather than aging by itself, may promote an exacerbated senescent phenotype of innate CD8 T-cells. In conclusion, we proposed that kidney transplantation, via the setting of inflammatory stimuli of alloantigen exposure and CMV infection, may exogenously age the CD8 T-cell compartment, especially its innate component. The physiopathological consequences of this change in the immune system remain to be elucidated.

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Circulating DNA for Molecular Response Prediction, Characterization of Resistance Mechanisms and Quantification of CAR T-Cells during Axicabtagene Ciloleucel Therapy

Blood ◽

10.1182/blood-2019-129015 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 550-550 ◽

Cited By ~ 5

Author(s):

Brian Sworder ◽

David M. Kurtz ◽

Charles Macaulay ◽

Matthew J. Frank ◽

Stefan Alig ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Resistance Mechanisms ◽

Advisory Board ◽

Advisory Committees ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory ◽

Equity Ownership

Background: Anti-CD19 chimeric antigen receptor (CAR19) T-cells have significant activity in patients with relapsed/refractory DLBCL (rrDLBCL). While the majority of rrDLBCL patients receiving axicabtagene ciloleucel (Axi-cel)achieve complete responses, a significant subset of patients experience disease progression (Locke FL, et al. Lancet Oncol. 2019). Circulating tumor DNA (ctDNA) analysis has demonstrated utility for predicting therapeutic benefit in DLBCL, as well as for detecting emergent resistance mechanisms to targeted therapies. Here we apply cell-free DNA (cfDNA) analysis to patients receiving Axi-cel, to characterize molecular responses, resistance mechanisms, and to track CAR19 cells. Methods: We performed Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) on DNA from germline and plasma samples collected prior to CAR T-cell infusion, multiple time-points post infusion, and, where available, at the time of relapse from 30 patients receiving Axi-cel for rrDLBCL at Stanford University. We designed a novel hybrid-capture panel and analysis pipeline designed to detect both tumor variants, as well as Axi-cel specific recombinant retroviral sequences to quantify CAR19 levels in cfDNA. Tumor variants were identified prior to and following Axi-cel therapy to assess for emergent variants, and Axi-cel specific sequences were quantified. Results: The median follow-up for the 30 patients after Axi-cel infusion was 10 months, with 47% (14/30) of patients experiencing disease progression after Axi-cel therapy. We identified an average of 164.3 SNVs per case (range:1-685) before Axi-cel therapy; the most common coding variants identified at baseline were in MLL2 (29.2%), BCL2 (22.5%), and TP53 (19.3%). When treated as a continuous variable, pretreatment ctDNA levels were prognostic of PFS (HR 2.16, 95% CI 1.11-4.21, P=0.02). Using a previously established ctDNA threshold to stratify disease burden (2.5 log10(hGE/mL); Kurtz et al. JCO 2018), we observed significantly superior PFS in patients with low pretreatment ctDNA levels treated with Axi-cel (Fig. 1A). In the majority of Axi-cel treated patients (62.9%), ctDNA was detectable at day 28, and PFS was significantly longer in patients with undetectable ctDNA at this time-point (Fig. 1B). Multiple putative resistance mechanisms were identified at relapse after Axi-cel, including emergent variants in CD19, HVEM, and TP53, as well as copy number gains in PD-L1 (Fig. 1C). For example, in one patient, a CD19 stop-gain mutation, which was not detected prior to treatment or at the time of the first interim PET scan, emerged at the time of relapse (Fig. 1D). Finally, we found cfDNA evidence for Axi-cel DNA in 74% of patients 28 days after therapy, including in patients without evidence of circulating CAR T-cells in PBMCs. Axi-cel levels in cfDNA as measured by CAPP-Seq were significantly correlated with CAR19 flow cytometry (Pearson r=0.55, P=.015; Fig. 1E). Conclusions: Baseline and interim ctDNA measurements have prognostic significance in DLBCL patients being treated with CAR19 T-cells, and potential emergent resistance mutations, including in CD19, can be identified in patients via cfDNA analysis. Quantification of CAR19 T-cells using cfDNA is significantly correlated with flow cytometric quantification, indicating that these cells can be quantified via cfDNA. Taken together, these data indicate that cfDNA analysis is a powerful tool for predicting response to CAR19 therapy, identifying genomic determinants of resistance and quantifying CAR19 cells, which may in turn inform the next therapeutic steps. Figure 1: A) Kaplan Meier analysis of PFS, with patients stratified based on pre-Axi-cel therapy ctDNA level, above and below a previously established threshold (2.5 log10[haploid Genome Equivalents/mL]). B) A Kaplan Meier plot depicting PFS stratification for patients with detectable versus undetectable ctDNA at day 28 after Axi-cel infusion. C) Oncoprint depicting selected emergent and baseline tumor variants in progressors and non-progressors after Axi-cel therapy. D) Change in mean ctDNA variant allele frequency (VAF) and emergence of a CD19 stop-gain mutation (CD19 pTrpX) at the time of relapse in a patient who initially achieved a CR at day 28 after CAR19 infusion. E) Relationship between CAR19 T-cell quantification by cfDNA and flow cytometry. (ND: Not detected) Disclosures Kurtz: Roche: Consultancy. Chabon:Lexent Bio Inc: Consultancy. Khodadoust:Corvus Pharmaceuticals: Research Funding. Majzner:Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Diehn:Roche: Consultancy; Quanticell: Consultancy; Novartis: Consultancy; AstraZeneca: Consultancy; BioNTech: Consultancy. Miklos:Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees. Alizadeh:Genentech: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

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Recombinant CD19 Glycomutant Accurately and Reproducibly Detects CD19-Directed CAR-T Cells By Flow Cytometry

Blood ◽

10.1182/blood-2021-148229 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1724-1724

Author(s):

Delaney R. Kirchmeier ◽

Alyssa Sheih ◽

Salvatore Fiorenza ◽

Alexandre V. Hirayama ◽

Cassie Chou ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Stock Options ◽

Advisory Board ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T Cell ◽

Car T ◽

Scientific Advisory ◽

Privately Held

Abstract Background CD19-directed chimeric antigen receptor (CAR)-modified T cell therapy expansion and persistence in peripheral blood has been shown to correlate with responses against B cell malignances (Hay, Blood, 2018; Ayuk, Blood Adv, 2021). CAR-T cell enumeration is typically achieved through quantitative PCR (qPCR) or flow cytometry (FC). qPCR, however, requires techniques that may not be locally validated and, unlike FC, cannot simultaneously address T cell immunophenotype. Existing FC reagents to detect CD19 CAR-T cells are mostly specific for a given CAR sequence or bind non-specifically to the introduced protein (e.g. Protein A). Utilizing glycosylation data from the structure of CD19(Teplykov, Proteins, 2018), we created a stable, biotinylated CD19 ectodomain glycomutant reagent (gmCD19) that binds to the CD19-directed scFv in CAR constructs and can be detected by FC. Methods The gmCD19 ectodomain (amino acids 21-227, N138Q) was expressed in 293 Freestyle™ cells with a C-terminal 6x Histidine-AviTag™ to facilitate expression, purification, FC detection (with fluorophore-bound streptavidin) and enable tetramer formation. We used FC to determine limit of detection (LoD), defined as the percent of detectable CAR-T cells when spiked into peripheral blood mononuclear cells (PBMC) at known concentrations. We used a CD19-directed CAR-T cell that co-expresses truncated EGFR (EGFRt) to benchmark gmCD19 and compare our reagent with commercially available detection reagents. This was quantitated by Pearson correlation coefficient and Bland-Altman measure of bias - defined as the mean of differences between tests on the same sample. We also tested reagent stability by assessing for decrease in mean fluorescence intensity (MFI) and the percent of CAR-T cells detected after multiple freeze-thaw cycles of the gmCD19 reagent. We compared CAR-T cell detection by gmCD19 with qPCR from actual patient samples treated with axicabtagene ciloleucel (axi-cel) and collected into three different anticoagulants. Results gmCD19 detected as few as 0.25% CAR-T cells by FC and was highly correlated with EGFRt expression (r=0.9993, p<0.0001). When benchmarking gmCD19 with EGFRt expression and comparing gmCD19 quantitation with other commercially available CD19 scFv detection reagents (Acro and BioSwan), gmCD19 showed the least bias (0.04 vs -1.225 and 61.66, respectively), and was the only method that demonstrated statistically significant agreement with EGFRt. Following each freeze-thaw cycle of gmCD19, there was no statistically significant decrease in percent of CAR-T cells detected with only a slight decrease in MFI (~2% decrease per cycle). Detection of axi-cel from patient samples was highly correlated with CAR copy number determined by qPCR, regardless of the anticoagulant in which the patient sample was collected (r=0.9387, 0.9849 and 0.9373 for sodium heparin, sodium citrate and EDTA, respectively) with equivalent coefficients of variation (11.0% vs 11.4% for gmCD19 and qPCR, respectively). Conclusion The availability of multiple CD19-directed CAR-T cell products and the importance of monitoring CAR-T cell expansion and persistence in patients undergoing CAR-T cell therapy creates the necessity for an easily applied, stable, and reliable quantitation FC method. We show that our gmCD19 accurately measures CD19-directed CAR-T cells across a variety of CAR-T cell constructs, including commercially available products. Disclosures Sheih: Umoja Biopharma: Current Employment. Fiorenza: Bristol Myers Squibb: Research Funding; Link Immunotherapeutics: Consultancy. Hirayama: Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Honoraria. Chou: Genentech: Current Employment. Gauthier: Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Multerra Bio: Consultancy; Larvol: Consultancy; JMP: Consultancy; Eusapharma: Consultancy. Correnti: Link Immunotherapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Turtle: PACT Pharma: Consultancy; Amgen: Consultancy; Asher Bio: Consultancy; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; T-CURX: Other: Scientific Advisory Board; Century Therapeutics: Consultancy, Other: Scientific Advisory Board; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Nektar Therapeutics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; TCR2 Therapeutics: Research Funding; Allogene: Consultancy.

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Longitudinal Changes in Cd4+, Cd8+ T Cell Phenotype and Activation Marker Expression Following Antiretroviral Therapy Initiation among Patients with Cryptococcal Meningitis

Journal of Fungi ◽

10.3390/jof5030063 ◽

2019 ◽

Vol 5 (3) ◽

pp. 63

Author(s):

Alice Bayiyana ◽

Samuel Okurut ◽

Rose Nabatanzi ◽

Godfrey Zziwa ◽

David R. Boulware ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antiretroviral Therapy ◽

Cell Surface ◽

Cd8 T Cells ◽

Cryptococcal Meningitis ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Effector Memory ◽

Memory Subset

Despite improvement in the prognosis of HIV/AIDS (human immunodeficiency virus/acquired immune deficiency syndrome) patients on antiretroviral therapy (ART), cryptococcal meningitis (CM) still causes 10–15% mortality among HIV-infected patients. The immunological impact of ART on the CD4+ and CD8+ T cell repertoire during cryptococcal co-infection is unclear. We determined longitudinal phenotypic changes in T cell subsets among patients with CM after they initiated ART. We hypothesized that ART alters the clonotypic phenotype and structural composition of CD4+ and CD8+ T cells during CM co-infection. For this substudy, peripheral blood mononuclear cells (PBMC) were isolated at four time points from CM patients following ART initiation during the parent study (ClinicalTrials.gov number, NCT01075152). Phenotypic characterization of CD4+ and CD8+ T cells was done using T cell surface marker monoclonal antibodies by flow cytometry. There was variation in the expression of immunophenotypic markers defining central memory (CD27+CD45R0+), effector memory (CD45R0+CD27–), immune activation (CD38+ and Human Leucocyte Antigen DR (HLA-DR+), and exhaustion (Programmed cell death protein one (PD-1) in the CD4+ T cell subset. In comparison to the CD4+ T cell population, the CD8+ central memory subset declined gradually with minimal increase in the effector memory subset. Both CD4+ and CD8+ T cell immune exhaustion and activation markers remained elevated over 12 weeks. The relative surge and decline in the expression of T cell surface markers outlines a variation in the differentiation of CD4+ T cells during ART treatment during CM co-infection.

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Class I–unrestricted noncytotoxic anti–HTLV-I activity of CD8+ T cells

Blood ◽

10.1182/blood.v96.5.1994 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1994-1995 ◽

Cited By ~ 5

Author(s):

Masako Moriuchi ◽

Hiroyuki Moriuchi

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Membrane Filter ◽

Cd8 T Cell ◽

Class I ◽

Type I ◽

Peripheral Blood Mononuclear ◽

Permeable Membrane

Abstract Although it is widely believed that viral clearance is mediated principally by the destruction of infected cells by cytotoxic T cells, noncytolytic antiviral activity of CD8+ T cells may play a role in preventing the progression to disease in infections with immunodeficiency viruses and hepatitis B virus. We demonstrate here that (1) replication of human T-lymphotropic virus type I (HTLV-I) is more readily detected from CD8+ T-cell–depleted (CD8−) peripheral blood mononuclear cells (PBMCs) of healthy HTLV-I carriers than from unfractionated PBMCs, (2) cocultures of CD8− PBMCs with autologous or allogeneic CD8+ T cells suppressed HTLV-I replication, and (3) CD8+ T-cell anti-HTLV-I activity is not abrogated intrans-well cultures in which CD8+ cells are separated from CD8− PBMCs by a permeable membrane filter. These results suggest that class I-unrestricted noncytolytic anti–HTLV-I activity is mediated, at least in part by a soluble factor(s), and may play a role in the pathogenesis of HTLV-I infection.

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