scholarly journals PI3K δ /γ Inhibition Enhances the Expansion and Anti-Tumor Cytotoxicity of CART Cells for CLL Patients

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4795-4795
Author(s):  
Shuhua Wang ◽  
Christopher R. Funk ◽  
Sruthi Ravindranathan ◽  
Kevin Chen ◽  
Edmund K. Waller
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
Tumor Cytotoxicity ◽  
Privately Held

Abstract Background: While CD19-targeted chimeric antigen receptor (CAR) based T cell therapy has shown promise in the treatment of chronic lymphocytic leukemia (CLL), overall efficacy is limited due to impaired T-cell fitness. We have previously shown that dual inhibition of PI3Kδ and PI3Kγ enhanced mitochondrial mass and ex vivo expansion of central and stem cell memory T cells from CLL patients(Funk, 2019,Journal for Immunotherapy of Cancer). In this study, we hypothesized that pharmacological inhibition of these pathways during ex vivo culture would increase the expansion and in vivo anti-tumor cytotoxicity. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of CLL patients by ficol hypaque centrifugation. T cells were negatively selected using MACS beads and transduced with CD19 CAR lentivirus (encoding CD28 or 41BB co-stimulatory domains) and stimulated with anti-CD3/CD28 beads in media containing 30 U/mL interleukin-2 with or without the PI3Kδ/γ inhibitor duvelisib (Duv) for 15 days. NOG mice were engrafted with the OSU-CLL cell line for 14 to 18 days with tumor burden measured by flow cytometry of blood samples from the mice, comprising a mean 0.15% of peripheral nucleated cell content. Control-CART or Duv-CART were injected by tail vein injection. Frequencies of CART, OSU-CLL cells and immune checkpoint molecule expression of CART or T cells in blood were measured by serial flow cytometry. Kaplan-Meier survival plots were represented as recipient survival on the indicated days Results: Treatment with either CD28 or 4-1BB Duv-CART cells led to significantly prolonged survival relative to control-CART (Figure 1, P<0.05). Recipients of Duv-CART cleared circulating OSU-CLL faster than control CART (Figure 2. P<0.05 at day26 for CD28 CART) and exhibited greater peak expansion and persistence of total CART and CD8+ CART. Recipients of Duv-CART cells had significantly greater in vivo persistence and expansion of total CART and CD8+ CART (Figure 3, P<0.001, day14 after CART were infused). Consistent with improved survival, both CD28 Duv-CART and 4-BB Duv-CART show reduced expression of LAG3, TIM3 and PD1 in the CD4 (Figure 4) and CD8+ subsets at earlier time point in vivo. (* p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001). Conclusions: Inhibition of PI3Kd/g during CART cell culture decreased the expression of immune checkpoint molecules and enhanced in vivo expansion leading to greater efficacy in eliminating CLL. Figure 1 Figure 1. Disclosures Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

scholarly journals 588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A623-A623
Author(s):  
Yannick Rakké ◽  
Lucia Campos Carrascosa ◽  
Adriaan van Beek ◽  
Valeska de Ruiter ◽  
Michael Doukas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Colorectal Carcinoma ◽  
Liver Metastases ◽  
Mismatch Repair ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Human Tumour ◽  
Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

scholarly journals PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1959-1959
Author(s):  
Jeong A Park ◽  
Hong fen Guo ◽  
Hong Xu ◽  
Nai-Kong V. Cheung
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
Activated T Cells ◽  
The Impact ◽  
Anti Tumor Activity

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

Reconstitution of CD52-Negative CD4 T Cells after Alemtuzumab-Based T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1182-1182
Author(s):  
Eva M Wagner ◽  
Aline N Lay ◽  
Sina Wenzel ◽  
Timo Schmitt ◽  
Julia Hemmerling ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Lymphocytic Leukemia ◽  
Cell Depletion ◽  
Down Regulation ◽  
T Cell Depletion ◽  
Hematopoietic Stem

Abstract The human CD52 molecule is the target of the monoclonal antibody Alemtuzumab, which is used for treating patients with chemo-refractory chronic lymphocytic leukemia as well as for T cell depletion (TCD) in the context of allogeneic hematopoietic stem cell transplantation (HSCT). The molecule is expressed on the surface of lymphocytes, dendritic cells and to a lesser extent on blood-derived monocytes. Previously, investigators have demonstrated that the surface expression of CD52 on T cells is down-regulated after in vitro incubation with Alemtuzumab. By treating purified human CD4 T cells over 4 hours with 10 μg/mL Alemtuzumab in medium supplemented with 10% human AB serum in vitro, we observed a strong decrease of CD52 expression by flow cytometry with a maximum 3–7 days after incubation. The CD52 down-regulation was also found at weaker intensity on CD8 T cells. From previous studies in chronic lymphocytic leukemia patients, it is known that Alemtuzumab treatment also leads to a down-regulation of CD52 on T cells in vivo. However, similar experiments have not been performed in allogeneic HSCT patients receiving Alemtuzumab in vivo for T cell depletion. We therefore analyzed the expression of CD52 on human peripheral blood mononuclear cells isolated at repeated time points from 22 allogeneic HSCT patients after reduced-intensity conditioning with fludarabine and melphalan and in vivo T cell depletion with Alemtuzumab (100 mg). Half of the patients received prophylactic CD8-depleted donor lymphocyte infusions (DLI) to promote immune reconstitution. By flow cytometry, we observed that the CD52 expression on monocytes, B cells, and natural killer cells remained unaltered after transplantation and was not influenced by the application of DLI. In contrast, the majority of CD4 T cells were CD52-negative (median, 72%) after transplantation and they remained CD52-negative in patients who did not receive DLI throughout the first year after HSCT. The permanent lack of CD52 expression could not be explained by a continuous effect of Alemtuzumab, because earlier studies have shown that the antibody is not present in active plasma concentrations beyond day +60 after HSCT. In contrast, patients receiving CD8-depleted DLI demonstrated a significant increase in the proportion of CD52-positive CD4 T cells. In three of our patients (DLI: n=2, non-DLI: n=1) we analyzed the donor chimerism of CD52-positive and CD52-negative CD4 T cells sorted with high purity by flow cytometry. Three months after HSCT (before DLI), the proportion of donor T cells was clearly higher among the CD52-negative compared to the small proportion of CD52-positive cells in all patients (44% vs. 10%, 83% vs. 0%, and 100% vs. 40%). In the patient who did not receive DLI, the donor T cell chimerism remained mixed in the CD52-negative and CD52-positive fractions on days 200 (CD52-negative: 95%; CD52-positive: 15%) and 350 (CD52-negative: 92%; CD52-positive: 65%). In contrast, the two patients receiving CD8-depleted DLI showed a strong increase in the proportion of CD52-positive CD4 T cells that were of complete donor origin. Altogether, CD52 is permanently down-regulated in reconstituting CD4 T cells following HSCT with an Alemtuzumab-based TCD regimen unless DLI are applied. Our data support the idea of an active mechanism for CD52 down-regulation in CD4 T cells that is not related to B cells and natural killer cells and that appears to differently affect donor and host T cells, respectively.

scholarly journals The Injury Response to DNA Damage Promotes Anti-Tumor Immunity

2020 ◽  
Author(s):  
Ganapathy Sriram ◽  
Lauren Milling ◽  
Jung-Kuei Chen ◽  
Wuhbet Abraham ◽  
Erika D. Handly ◽  
...  
Keyword(s):  
Dna Damage ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Injured Cell ◽  
Ifn Γ

ABSTRACTInhibition of immune checkpoints has shown promising results in the treatment of certain tumor types. However, the majority of cancers do not respond to immune checkpoint inhibition (ICI) treatment, indicating the need to identify additional modalities that enhance the response to immune checkpoint blockade. In this study, we identified a tumor-tailored approach using ex-vivo DNA damaging chemotherapy-treated tumor cells as a live injured cell adjuvant. Using an optimized ex vivo system for dendritic cell-mediated T-cell IFN-γ induction in response to DNA-damaged tumor cells, we identified specific dose-dependent treatments with etoposide and mitoxantrone that markedly enhance IFN-γ production by T-cells. Unexpectedly, the immune-enhancing effects of DNA damage failed to correlate with known markers of immunogenic cell death or with the extent of apoptosis or necroptosis. Furthermore, dead tumor cells alone were not sufficient to promote DC cross-presentation and induce IFN-γ in T-cells. Instead, the enhanced immunogenicity resided in the fraction of injured cells that remained alive, and required signaling through the RIPK1, NF-kB and p38MAPK pathways. Direct in vivo translation of these findings was accomplished by intra-tumoral injection of ex vivo etoposide-treated tumor cells as an injured cell adjuvant, in combination with systemic anti-PD1/CTLA4 antibodies. This resulted in increased intra-tumoral CD103+ dendritic cells and circulating tumor antigen-specific CD8+ T-cells, leading to enhanced anti-tumor immune responses and improved survival. The effect was abrogated in BATF3-deficient mice indicating that BATF3+ DCs are required for appropriate T-cell stimulation by live but injured DNA-damaged tumor cells. Notably, injection of the free DNA-damaging drug directly into the tumor failed to elicit such an enhanced anti-tumor response as a consequence of simultaneous damage to dendritic cells and T-cells. Finally, the DNA damage induced injured cell adjuvant and systemic ICI combination, but not ICI alone, induced complete tumor regression in a subset of mice who were then able to reject tumor re-challenge, indicating induction of a long-lasting anti-tumor immunological memory by the injured cell adjuvant treatment in vivo.

scholarly journals Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 679-679
Author(s):  
Michelle J. Cox ◽  
Fabrice Lucien-Matteoni ◽  
Reona Sakemura ◽  
Justin C. Boysen ◽  
Yohan Kim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Ex Vivo ◽  
Data Safety Monitoring Board ◽  
Lymphocytic Leukemia ◽  
Cell Dysfunction

Treatment with CD19-directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes and was FDA-approved in acute lymphoblastic leukemia and non-Hodgkin B-cell lymphoma. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and in part may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. In this study, we aimed to investigate the interaction between circulating EVs isolated from CLL patient plasma (designated as CLL-derived EVs) and CART19 cells. We enumerated and immunophenotyped circulating EVs from platelet free plasma in untreated patients with CLL. We determined their interaction with CART19 cells using second generation, 41BB co-stimulated, lentiviral transduced CART19 cells generated in the laboratory from normal donors (FMC63-41BBζ CART cells). Our findings indicate that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation against the CD19+ mantle cell lymphoma cell line Jeko-1 (Figure 1A). Next, we characterized CLL-derived EVs using nanoscale flow cytometric analysis of surface proteins and compared to healthy controls. Although the total EV particle count was not different between CLL and healthy controls (Figure 1B), there were significantly higher PD-L1+ EVs in patients with CLL (Figure 1C). Based on these results, we sought to assess the physical interaction between CLL-derived EVs and CART cells from normal individuals. When CLL-derived EVs were co-cultured with CART19 and CLL B cells and imaged with super-resolution microscopy, EVs were localized at the T cell-tumor junction (Figure 1D). Furthermore, CLL-derived EVs are captured by T cells as indicated by a significant reduction in the absolute count of EVs when co-cultured with resting T cells (Figure 1E). Having demonstrated that 1) there is an excess of PD-L1+ EVs in patients with CLL (Figure 1C) and 2) CLL-derived EVs physically interact with CART cells (Figures 1D-E), we sought to establish their functional impact on CART19 cells. Here, CART19 cells were stimulated with irradiated CD19+ JeKo-1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL-derived EVs. There was a significant upregulation of inhibitory receptors such as PD-1 and CTLA-4 on the T cells (Figure 1F). This is associated with a reduction in CART effector cytokines (i.e., TNFβ) at higher concentrations of EVs (Figure 1G), suggesting a state of exhaustion in activated CART19 cells in the presence of CLL-derived EVs. This was further supported by transcriptome interrogation of CART19 cells. Here, CART19 cells were stimulated via 24-hour co-culture with the irradiated CD19+ cell line JeKo-1, in the presence of CLL-derived EVs at ratios of 10:1 and 1:1 EV:CART19 and then isolated by magnetic sorting. RNA sequencing of these activated CART19 cells indicated a significant upregulation of AP-1 (FOS-JUN) and YY1 (Figures 1H), known critical pathways in inducing T cell exhaustion. Finally, to confirm the impact of CLL-derived EVs on CART19 functions in vivo, we used our xenograft model for relapsed mantle cell lymphoma. Here, immunocompromised NOD-SCID-ɣ-/- mice were engrafted with the CD19+ luciferase+ cell line JeKo-1 (1x106 cells I.V. via tail vein injection). Engraftment was confirmed through bioluminescent imaging and mice were randomized to treatment with 1) untreated, 2) CART19 cells, or 3) CART19 cells co-cultured ex vivo with CLL-derived EVs for six hours prior to injection. A single low dose of CAR19 (2.5x105) was injected, to induce relapse. Treatment with CART19 cells that were co-cultured ex vivo with CLL-derived EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Figure 1I). Our results indicate that CLL-derived EVs induce significant CART19 cell dysfunction in vitro and in vivo, through a direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way through which circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients. Disclosures Cox: Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. Kenderian:Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

scholarly journals Attenuation of graft-versus-host disease and graft rejection by ex vivo immunotoxin elimination of alloreactive T cells in an H-2 haplotype disparate mouse combination

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 288-298 ◽  
Author(s):  
M Cavazzana-Calvo ◽  
JL Stephan ◽  
S Sarnacki ◽  
S Chevret ◽  
C Fromont ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Graft Rejection ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
In Vivo Model ◽  
Host Disease ◽  
Graft Versus Host

A mouse anti-interleukin-2 receptor A-chain-specific PC61-immunotoxin (PC61-IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61-IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodepletion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61-IT-treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allo-depletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 x 10(6) T-cell-depleted BM cells and 0.5 x 10(6) PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

Vaccination Strategies for Patients with B-CLL.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2106-2106
Author(s):  
Fatma V Okur ◽  
Eric Yvon ◽  
Gianpietro Dotti ◽  
George Carrum ◽  
Helen E. Heslop ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Th2 Cells ◽  
Ifn Γ ◽  
Adenoviral Vaccine

Abstract B-chronic lymphocytic leukemia (B-CLL) cells express tumor associated antigens that may generate a T cell mediated immune response, but present these antigens poorly. Moreover, patients with B-CLL often have poor immune function due to the disease or its treatment. We have shown that expression of transgenic CD40L increases the immunogenecity of human B-CLL cells ex vivo and in vivo, and that this effect can be potentiated by co-expression of transgenic IL2. Previous studies described outcomes when adenoviral vectors were used to obtain gene transfer, but because of the complexities and expense of manufacture of viral vectors, and their lingering safety concerns, we determined whether it was possible to use electroporation (with the MaxCyte device) as a physical means of transferring CD40L and IL2 plasmids to produce vaccines with similar biological properties in vitro and in vivo. Table 1 compares the phenotype of the vaccines using each vector. Table 1. Comparision of immunogenic characteristics and viability of the adenoviral and plasmid vaccines Type of Vaccine CD40L (%) CD80 (%) CD86 (%) IL-2 (pg/ml/10e6 cells) Viability (%) IL2 CD40L All the values are given as mean ± SE. * P&lt; 0.01, Paired Student’s t test. Adenoviral Pre 0.2 ± 0.01 2.6 ± 2.4 7.5 ± 3.9 Post 66.1 ± 5.5* 50.2 ± 7.8* 69.5 ±11* 253.5 ± 82.6 93.6 94.2 Plasmid Pre 1.3 ± 0.85 11.5 ± 6.2 19.7 ± 6.8 Post 55.5 ± 5.1* 19.2 ± 9.3 26.4 ± 9.7 4806.6 ±1398.9 84.4 88.4 Vaccines made by both approaches met the release criteria for CD40L and IL2 expression (CD40L ≥20% and IL-2 ≥ 150 pg/ml/1x10e6 cells ), but expression of IL2 was higher in the plasmid vaccines, expression of CD40L was equivalent in each and expression of the additional co-stimulatory molecules CD80 and CD86 (induced after CD40 activation by transgenic CD40L) was higher in the adenoviral vaccines. Fourteen patients were given adenoviral-vaccines and nine the plasmid transduced cells. Each of these patients received up to 18 s.c. injections of IL-2 secreting and CD40L expressing tumor cells. Both types of vaccine were well tolerated. Table 2 shows the results of culturing patient T cells with autologous B-CLL tumor cells. Table 2. Comparision of anti-B-CLL T cell responses induced by adenoviral and plasmid vaccines Type of Vaccine Pre-vaccine After 3rd vaccine After 6th vaccine All the values were are given as mean ± SE. *P&lt;0.05, Wilcoxon Signed Ranks test Adenoviral 307.3 ± 293.9 375 ± 306.8 656.8 ± 373.8 IFN-γ spots/10e6 T cells&#x2028; IL-5 spots/10e6 T cells 0 12.8 ± 7.9 5.8 ± 2.3 Plasmid 31.1 ± 14.8 38 ± 17.8 32.9 ± 19.5 IFN-γ spots/10e6 T cells&#x2028; IL-5 spots/10e6 T cells 4 ± 2.7 14 ± 10.2 203.9 ± 156.3* After 3 and 6 injections, both the adenoviral and plasmid vaccines had induced a rise in spot forming cells (SFC) for IL5, a cytokine associated with Th2 cells, but the rise was greatest in the recipients of the electroporated plasmid vaccine. By contrast, only the adenoviral vaccine induced a rise in SFC that produced IFN-γ, a cytokine associated with Th1 cells. Studies using MHC class I and II blocking antibodies showed that the IL5 and IFN-γ responses to both types of vaccine were mediated by HLA restricted T lymphocytes. The 1-year progression-free survival rates (PFS) for adenoviral vaccine group and plasmid vector group were 43% and 22% respectively. Figure 1 shows 1-year PFS rates for each group. Hence electroporation provides a more rapid and simpler means of preparing IL2/CD40L expressing B-CLL vaccines, but the cells express higher levels of IL2 and lower levels of “secondary” co-stimulator molecules than adenoviral vaccines, and produce an anti-tumor immune response of different polarity. Currently, we are evaluating electroporation of mRNA encoded CD40L which appears to augment upregulation of additional costimulatory molecules. Figure Figure

scholarly journals Altered Ex Vivo Expression of Caspase 8, Caspase 9, and Bcl-2 Is Associated with T-Cell Hyporeactivity in Patients with Paracoccidioidomycosis

2009 ◽  
Vol 16 (6) ◽  
pp. 953-955 ◽  
Author(s):  
Camila R. Cacere ◽  
Maria J. S. Mendes-Giannini ◽  
Antonio Carlos F. do Valle ◽  
Alberto J. S. Duarte ◽  
Gil Benard
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Tolerant State

ABSTRACT To better understand the T-cell hyporesponsiveness of patients with paracoccidioidomycosis, we tested the hypothesis that the T cells were committed to apoptosis. We show here that T cells of patients with paracoccidioidomycosis overexpress caspase 9 and caspase 8 but express low Bcl-2 levels and that interleukin-2 was unable to revert the hyporesponsiveness. These data suggest that the T cells would in vivo be driven to a tolerant state and apoptosis.

scholarly journals Duvelisib Promotes Mitochondrial Fusion and Epigenetic Reprogramming to Drive Therapeutic T Cell Persistence and Function

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1714-1714
Author(s):  
Kevin Z. Chen ◽  
Christopher R. Funk ◽  
Shuhua Wang ◽  
Aditi Sharma ◽  
Edmund K. Waller ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Cultures ◽  
Ex Vivo ◽  
Mitochondrial Dynamics ◽  
Epigenetic Reprogramming ◽  
Lymphocytic Leukemia ◽  
Mitochondrial Fusion ◽  
Memory T Cell

Abstract Chronic lymphocytic leukemia (CLL), a cancer of B-lymphocytes, is the most common leukemia in adults. While current frontline therapies for CLL, such as ibrutinib or combination venetoclax and obintuzumab, have significantly improved clinical outcome for patients with treatment naïve CLL and relapsed and refractory CLL (RR-CLL), complete response (CR) rates for RR-CLL patients on ibrutinib remain between 5-14% and 42% for patients on venetoclax and obintuzumab (1, 2). With the advent of chimeric antigen receptor T cells (CART), CR for RR-CLL have increased to around 26-29%, yet this is in sharp contrast to the 70-93% CR achieved in acute lymphocytic leukemia (2). This discrepancy in response is in part due to the inherently immunosuppressive nature of CLL, such that CLL patients are significantly deficient in CD8 co-receptor expressing (CD8+) T cells, including stem cell-like memory T (Tscm) and central memory T (Tcm) cells (2). As the prevalence of Tscm and Tcm cell populations is directly correlated to the in vivo persistence and efficacy of CART, elucidating translatable mechanisms to selectively expand Tscm and Tcm from CLL patients is key to improving the efficacy CART cell therapy for CLL patients. Memory T cell activation, differentiation, and maintenance are processes that are tightly regulated by mitochondrial fusion, fatty acid oxidation, and oxidative phosphorylation (OXPHOS) (3). Moreover, enforcing T cell mitochondrial fusion improves CART cell efficacy against solid tumors (4). As metabolism plays an important role in memory T cell biology, identifying key metabolic pathways that can be targeted ex vivo during CART expansion is of particular interest. To that end, we have shown that dual inhibition of Phosphoinositide 3-Kinase (PI3K) δ/γ isoforms with IPI-145 (duvelisib) during ex vivo T cell manufacturing, preferentially expands CD8+ T cells, including Tscm and Tcm, as well as improves the in vivo persistence (Figure 1A) and cytotoxicity (Figure 1B) of CD19-targeted CART (CD19-CART) (5). To investigate the role of mitochondrial dynamics during ex vivo expansion of duvelisib treated T cell cultures, we stimulated CLL patient-derived T cells with anti-CD3/CD28 beads, re-stimulated T cells on day 9, and harvested T cell cultures on day 15. Immunoblot analysis of day 15 samples indicates that ex vivo duvelisib treatment of CLL patient T cells increases expression of key mitochondrial fusion proteins, mitofusins 1 and 2 (MFN1/2), and decreases serine 637 phosphorylation of mitochondrial fission protein, DRP1, without coincident upregulation of the master regulator of mitochondrial biogenesis, PPARG coactivator 1 alpha (Figure 2A). In addition, duvelisib increased the expression of sirtuins 1 and 3 (SIRT1/3), which have known roles in the post-translational activation of MFN1/2, as well as other epigenetic regulators of memory T cell development and persistence, including FOXO1, TCF1/7, and ID3 (Figure 2B). Taken together, these data suggest that duvelisib promotes mitochondrial fusion and epigenetic reprogramming of T cells during ex vivo expansion. To further interrogate the role of PI3K δ/γ inhibition in mitochondrial dynamics and metabolism, we analyzed T cell cultures following 15 days of duvelisib treatment using a series of extracellular flux and transmission electron microscopy (TEM) experiments. Duvelisib promotes an increase in the total mitochondrial cross-sectional area of both un-transduced and CD19-CAR transduced T cells (Figure 3) and maintains basal, coupled, and spare respiratory capacity of un-transduced T cells on day 15 of expansion (Figure 4). In summary, our data suggest that mitochondrial fusion through MFN1/2 and epigenetic reprogramming facilitate PI3K δ/γ inhibition-mediated ex vivo T cell expansion, where the SIRT1/3-MFN1/2 axis serves as a potential intersection between mitochondrial fusion and epigenetic reprogramming. Figure 1 Figure 1. Disclosures Waller: Verastem Oncology: Consultancy, Research Funding; Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

scholarly journals Regulatory T Cells Selectively Express Toll-like Receptors and Are Activated by Lipopolysaccharide

2003 ◽  
Vol 197 (4) ◽  
pp. 403-411 ◽  
Author(s):  
Iris Caramalho ◽  
Thiago Lopes-Carvalho ◽  
Dominique Ostler ◽  
Santiago Zelenay ◽  
Matthias Haury ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Toll Like Receptors ◽  
Activation Markers ◽  
Inflammatory Reactions ◽  
Bacterial Products

Regulatory CD4 T cells (Treg) control inflammatory reactions to commensal bacteria and opportunist pathogens. Activation of Treg functions during these processes might be mediated by host-derived proinflammatory molecules or directly by bacterial products. We tested the hypothesis that engagement of germline-encoded receptors expressed by Treg participate in the triggering of their function. We report that the subset of CD4 cells known to exert regulatory functions in vivo (CD45RBlow CD25+) selectively express Toll-like receptors (TLR)-4, -5, -7, and -8. Exposure of CD4+ CD25+ cells to the TLR-4 ligand lipopolysaccharide (LPS) induces up-regulation of several activation markers and enhances their survival/proliferation. This proliferative response does not require antigen-presenting cells and is augmented by T cell receptor triggering and interleukin 2 stimulation. Most importantly, LPS treatment increases CD4+ CD25+ cell suppressor efficiency by 10-fold and reveals suppressive activity in the CD4+ CD45RBlow CD25− subset that when tested ex-vivo, scores negative. Moreover, LPS-activated Treg efficiently control naive CD4 T cell–dependent wasting disease. These findings provide the first evidence that Treg respond directly to proinflammatory bacterial products, a mechanism that likely contributes to the control of inflammatory responses.

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