ResearchPad - 2436 https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Immunosuppressant indulges EBV reactivation and related lymphoproliferative disease by inhibiting Vδ2<sup>+</sup> T cells activities after hematopoietic transplantation for blood malignancies]]> https://www.researchpad.co/article/Ne3aeecc1-2420-4966-aac4-128b28ea58b7 Following the extensive use of immunosuppressive drugs in the clinic, immunosuppression-associated side effects have received increasing attention. Epstein-Barr virus (EBV) reactivation and related lymphoproliferative diseases (LPD) are the lethal complications observed after allogeneic hematopoietic cell transplantation (alloHCT). While studies generally suggest an association between immunosuppressants and EBV reactivation, the effects of specific immunosuppressive drugs and which T-cell subsets mediate these correlations are unclear. Vδ2+ T cells are correlated with EBV reactivation after alloHCT. Researchers have not determined whether Vδ2+ T-cell activities are affected by immunosuppressants and thereby facilitate EBV reactivation and related LPD.MethodsA clinical cohort study of 170 patients with hematopoietic malignancies who received haploidentical hematopoietic cell transplantation (haploHCT) was performed to investigate whether the early cessation of mycophenolate mofetil (MMF) decreases EBV reactivation and related LPD and to determine whether this change is associated with the recovery of Vδ2 + T cells after transplantation. The effects of MMF on the expansion and anti-EBV capacity of Vδ2+ T cells were detected in vitro and in an immunodeficient mouse model.ResultsA reduction in the course of MMF significantly improved the recovery of Vδ2+ T cells from 30 to 90 days after haploHCT (p=0.002, p=0.042 and p=0.035, respectively), accompanied by a significant decrease in EBV reactivation (from 26% to 13%, p=0.033) and EBV-LPD (from 10.6% to 2.4%, p=0.029). The day-30 Vδ2+ T level remained an independent factor for EBV reactivation in patients with different MMF durations (p=0.007). In the in-vitro experiments, MMF inhibited Vδ2+ T-cell expansion and its cytotoxicity on EBV-transformed malignant cells. Furthermore, the therapeutic and prophylactic effects of adoptively transferred human Vδ2+ T cells were attenuated by the MMF treatment in immunodeficient mice with EBV-LPD.ConclusionsThese results elucidated a negative effect of immunosuppressants on the anti-EBV capacity of Vδ2+ T cells. Strategies that appropriately relieve the immunosuppression may improve anti-EBV immunity by increasing the activity of Vδ2+ T cells after alloHCT. ]]> <![CDATA[Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia]]> https://www.researchpad.co/article/N5c7fbe25-a7eb-4795-b424-89c4f2555a9a While achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy.MethodsFirst, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by ‘2S stimulation’ through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM-knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM-knockout or TP53-knockout cells was also performed.ResultsPrimary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. ‘2S’ stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells’ in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells’ survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012).ConclusionsWhile in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells. ]]> <![CDATA[Regulatory T-cell depletion in the setting of autologous stem cell transplantation for multiple myeloma: pilot study]]> https://www.researchpad.co/article/N9c447f12-cac5-4cb3-a83e-dafdcd4ed33c

Background

Progression after high-dose melphalan with autologous stem cell transplantation (ASCT) in multiple myeloma (MM) may be due in part to immune dysfunction. Regulatory T (Treg) cells reconstitute rapidly after ASCT and inhibit immune responses against myeloma cells.

Methods

We performed a randomized study to evaluate two methods of Treg depletion in patients with MM undergoing ASCT. No Treg depletion was performed in the control ASCT arm. An anti-CD25 monoclonal antibody (basiliximab 20 mg IV) was administered on day +1 post-ASCT in the in vivo Treg depletion (IVTRD) arm. Tregs were depleted from autologous stem cell (ASC) grafts with anti-CD25 microbeads and the CliniMACS device in the ex vivo Treg depletion (EVTRD) arm.

Results

Fifteen patients were enrolled, five in each arm. The conditioning regimen was melphalan 200 mg/m2. Primary objectives included assessments of efficiency of IVTRD/EVTRD, kinetics of Treg depletion and recovery following ASCT, and safety. EVTRD removed 90% of CD4+CD25+ cells from ASC grafts. IVTRD and EVTRD led to reductions in Treg frequency between days +7 and +90 post-transplant compared with the control (p=0.007 and p<0.001, respectively).

Conclusions

IVTRD and EVTRD are feasible and significantly reduce and delay Treg recovery post-ASCT for MM, and serve as a platform for using post-transplant immunotherapies to improve post-ASCT outcomes.

Trial registration number

NCT01526096.

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<![CDATA[Increased antitumor efficacy of PD-1-deficient melanoma-specific human lymphocytes]]> https://www.researchpad.co/article/N6da0b129-47d3-4c0f-8573-ac212b407402

Background

Genome editing offers unique perspectives for optimizing the functional properties of T cells for adoptive cell transfer purposes. So far, PDCD1 editing has been successfully tested mainly in chimeric antigen receptor T (CAR-T) cells and human primary T cells. Nonetheless, for patients with solid tumors, the adoptive transfer of effector memory T cells specific for tumor antigens remains a relevant option, and the use of high avidity T cells deficient for programmed cell death-1 (PD-1) expression is susceptible to improve the therapeutic benefit of these treatments.

Methods

Here we used the transfection of CAS9/sgRNA ribonucleoproteic complexes to edit PDCD1 gene in human effector memory CD8+ T cells specific for the melanoma antigen Melan-A. We cloned edited T cell populations and validated PDCD1 editing through sequencing and cytometry in each T cell clone, together with T-cell receptor (TCR) chain’s sequencing. We also performed whole transcriptomic analyses on wild-type (WT) and edited T cell clones. Finally, we documented in vitro and in vivo through adoptive transfer in NOD scid gamma (NSG) mice, the antitumor properties of WT and PD-1KO T cell clones, expressing the same TCR.

Results

Here we demonstrated the feasibility to edit PDCD1 gene in human effector memory melanoma-specific T lymphocytes. We showed that PD-1 expression was dramatically reduced or totally absent on PDCD1-edited T cell clones. Extensive characterization of a panel of T cell clones expressing the same TCR and exhibiting similar functional avidity demonstrated superior antitumor reactivity against a PD-L1 expressing melanoma cell line. Transcriptomic analysis revealed a downregulation of genes involved in proliferation and DNA replication in PD-1-deficient T cell clones, whereas genes involved in metabolism and cell signaling were upregulated. Finally, we documented the superior ability of PD-1-deficient T cells to significantly delay the growth of a PD-L1 expressing human melanoma tumor in an NSG mouse model.

Conclusion

The use of such lymphocytes for adoptive cell transfer purposes, associated with other approaches modulating the tumor microenvironment, would be a promising alternative to improve immunotherapy efficacy in solid tumors.

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<![CDATA[Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia]]> https://www.researchpad.co/article/N3af984d3-f741-4e12-8d7b-de82d60bdd17

Background

While achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy.

Methods

First, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by ‘2S stimulation’ through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM-knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM-knockout or TP53-knockout cells was also performed.

Results

Primary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. ‘2S’ stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells’ in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells’ survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012).

Conclusions

While in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells.

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