scholarly journals Diffuse Large B Cell Pdtx in Humanized Mice Are Valuable Models to Study Host-Lymphoma Interactions and Immune-Modulating Agents

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2406-2406
Author(s):  
Giorgia Zanetti ◽  
Giuseppina Astone ◽  
Luca Cappelli ◽  
William Chiu ◽  
Maria Teresa Cacciapuoti ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Ex Vivo ◽  
Human Immune System ◽  
Human T Cells ◽  
Human Immune

Abstract Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory. Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining. Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells. After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis. Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. Figure 1 Figure 1. Disclosures Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

Transient accumulation of human mature thymocytes and regulatory T cells with CD28 superagonist in “human immune system” Rag2-/-γc-/- mice

Blood ◽  
2006 ◽  
Vol 108 (1) ◽  
pp. 238-245 ◽  
Author(s):  
Nicolas Legrand ◽  
Tom Cupedo ◽  
Anja U. van Lent ◽  
Menno J. Ebeli ◽  
Kees Weijer ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Opportunistic Infections ◽  
Human Immune System ◽  
Independent Manner ◽  
Human T Cell ◽  
Human Immune

Efficient and quick reconstitution of T-cell compartments in lymphopenic patients is of great importance to prevent opportunistic infections, but remains difficult to achieve. Human T-cell proliferation in a T-cell-receptor (TCR)-independent manner is possible in vitro with superagonist anti-CD28 antibodies, and such molecules are therefore promising therapeutic tools. Here, we investigated the in vivo effects of superagonist anti-CD28 treatment on human developing and mature T cells, in the recently developed model of “human immune system” BALB/c Rag2-/-γc-/- mice. Our results show that superagonist anti-CD28 treatment transiently induces a 7-fold increase in thymocyte numbers and up to 18-fold accumulation of mature thymocytes. The increased thymic production lead to transient accumulation of mature T cells in the periphery at the peak of treatment effect (day 6). In addition, long-term peripheral T-cell depletion was induced. Furthermore, the concomitant selective expansion and accumulation of suppressive CD4+CD25+FoxP3+ T cells was induced in a transient manner. Superagonist anti-CD28 therapy could therefore be of clinical interest in humans, both for beneficial effect on thymic T-cell production as well as regulatory T-cell accumulation. (Blood. 2006;108:238-245)

scholarly journals Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Josephine F. Reijneveld ◽  
Mira Holzheimer ◽  
David C. Young ◽  
Kattya Lopez ◽  
Sara Suliman ◽  
...  
Keyword(s):  
T Cells ◽  
Fatty Acid ◽  
Immune System ◽  
T Cell ◽  
Cross Reactivity ◽  
Lipid Binding ◽  
Human Immune System ◽  
Human T Cell ◽  
Human T Cells ◽  
Pure Compounds

AbstractThe cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

721 AT1412, a patient-derived CD9 antibody in preclinical development promoting tumor immune infiltration and inducing tumor rejection

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A763-A763
Author(s):  
Remko Schotte ◽  
Julien Villaudy ◽  
Martijn Kedde ◽  
Wouter Pos ◽  
Daniel Go ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Tumor Cells ◽  
Tumor Rejection ◽  
Human Immune System ◽  
Preclinical Development ◽  
High Affinity ◽  
Human Immune ◽  
A375 Melanoma

BackgroundAdaptive immunity to cancer cells forms a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether tumor-targeting antibodies could be isolated from a patient that cured (now 13 years tumor-free) metastatic melanoma following adoptive transfer of ex vivo expanded autologous T cells.MethodsPatient‘s peripheral blood B cells were isolated and tested for the presence of tumor-reactive B cells using AIMM’s immmortalisation technology. Antibody AT1412 was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 binds the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities in cancer and induces ADCC and ADCP by effector cells.ResultsSpontaneous immune rejection of tumors was observed in human immune system (HIS) mouse models implanted with CD9 genetically-disrupted A375 melanoma (A375-CD9KO) tumor cells, while A375wt cells were not cleared. Most notably, no tumor rejection of A375-CD9KO tumors was observed in NSG mice, indicating that blockade of CD9 makes tumor cells susceptible to immune rejection.CD9 has been described to regulate integrin signaling, e.g. LFA-1, VLA-4, VCAM-1 and ICAM-1. AT1412 was shown to modulate CD9 function by enhancing adhesion and transmigration of T cells to endothelial (HUVEC) cells. AT1412 was most potently enhancing transendothelial T-cell migration, in contrast to a high affinity version of AT1412 or other high affinity anti-CD9 reference antibodies (e.g. ALB6). Enhanced immune cell infiltration is also observed in immunodeficient mice harbouring a human immune system (HIS). AT1412 strongly enhanced CD8 T-cell and macrophage infiltration resulting in tumor rejection (A375 melanoma). PD-1 checkpoint blockade is further sustaining this effect. In a second melanoma model carrying a PD-1 resistant and highly aggressive tumor (SK-MEL5) AT1412 together with nivolumab was inducing full tumor rejection, while either one of the antibodies alone did not.ConclusionsThe safety of AT1412 has been assessed in preclinical development and is well tolerated up to 10 mg/kg (highest dose tested) by non human primates. AT1412 demonstrated a half-life of 8.5 days, supporting 2–3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. First in Human clinical study is planned to start early 2021.Ethics ApprovalStudy protocols were approved by the Medical Ethical Committee of the Leiden University Medical Center (Leiden, Netherlands).ConsentBlood was obtained after written informed consent by the patient.

Naive and Ex Vivo Activated Human T Cells Generate Consistent Engraftment and Lethal Graft-Versus-Host Disease (GvHD) in NOD SCID β 2M Null Mice: A New Xenogeneic Model for GvHD.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3106-3106
Author(s):  
Bruno Nervi ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
Gerhard Bauer ◽  
Jon Walker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Activation Markers ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Human T Cells ◽  
The Impact

Abstract GvHD remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion. The human GvHD pathophysiology includes recipient tissue destruction and proinflammatory cytokine production associated with the conditioning regimen; donor T cells become allo-activated, proliferate, and mediate tissue injury in various organs, including the liver, skin, and gut. Modern therapeutic strategies to control GvHD while maintaining the beneficial graft-versus-leukemia effects require ex vivo T cell stimulation and expansion. Multiple studies have demonstrated that these ex vivo expanded T cells exhibit decreased survival and function in vivo, including reduced alloreactivity and GvHD potential. Unfortunately no in vivo models exist to consistently examine the impact of ex vivo manipulation of human T cells (HuT) on T cell function. Naive HuT were compared to HuT activated using CD3/28 beads (XcyteTMDynabeads) with 50 U/ml IL-2 for 4 days (Act). We initially evaluated the HuT engraftment and GvHD potential of naive and Act in RAG2γ null mice (n=22) conditioned with clodronate liposomes on day −1 and 350cGy on day 0, as previously described by others. We injected 107 and 1.5x107 naive or Act HuT intravenously (iv). All mice exhibited low HuT engraftment and no lethal GvHD. NOD SCIDβ 2M null mice (β 2M) were next conditioned with 250cGy on day −1 (n=34), or 300cGy on day 0 (n=21). 107 naive vs Act HuT were injected retroorbitaly (ro). Lower HuT doses or iv injection resulted in no expansion or GvHD. Engraftment of HuT in peripheral blood of recipient mice was evaluated weekly by FACS and euthanasia was performed if mice lost > 20% body weight. 60% of the mice conditioned with 250cGy that received naive HuT developed lethal GvHD, in comparison to 75% of mice that received 300cGy and nave HuT, and 100% of mice that received 300cGy and Act HuT. Table 1 250cGy 300cGy Naive (n=34) Naive (n=8) Activated (n=13) *p<0.02 PB engraftment (%HuT) 20%±15 33%±21 59%±19 Lethal GvHD 60% 75% 100% All mice receiving 300cGy had well preserved CD4/CD8 ratios (1–1.5). Tissue infiltration was greatest in mice that had received 300cGy and Act HuT (spleen, liver, lung, kidney: 50–70%). Of interest, serum levels of hu IFNγ dramatically increased over time in all mice who went on to develop lethal GvHD (day 3=270 ug/ml and day 15=36,000 ug/ml) compared to mice that did not develop lethal GvHD (day 10=40 ug/ml and day 17=1,020 ug/ml)(p<0.05). Interestingly, the up-regulation of the activation markers CD25 and CD30 in HuT, and IFNγ production predicted lethal GvHD in β 2M null mice. In summary, we developed a xenogeneic model of lethal GvHD where naive or ex vivo Act HuT injected ro in sublethaly irradiated β 2M not only engraft, expand in vivo, but also infiltrate and damage different mouse target organs. HuT are allo-activated against mouse antigens and damage the target tissues, sharing the major characteristics of human GvHD and causing the death of mice. This model will allow us to study the effects of specific ex vivo T cell manipulation including transduction, selection, expansion, and the depletion or addition of various T cells and other cellular subsets on the outcome of GvHD, to determine improved therapeutic interventions.

Tumor-Targeted Human CD4+ CD25hi Regulatory T Cells Effectively Inhibit T Cell –mediated Rejection of CD19+ Tumors in An in Vivo xenotransplant Tumor Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3901-3901
Author(s):  
James Lee ◽  
Michel Sadelain ◽  
Renier J. Brentjens
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
B Cell ◽  
Clinical Setting ◽  
Effector T Cells ◽  
Effector T Cell ◽  
Human T Cells ◽  
Anti Tumor Activity

Abstract The genetic targeting of human T cells to selected tumor antigens offers a novel means to investigate human immunobiology and treat cancer. T cells may be genetically modified to target specific antigens through the introduction of genes encoding chimeric antigen receptors (CARs). We have previously demonstrated that human T cells targeted in this manner to the CD19 antigen, expressed on normal B cells as well as most B cell tumors, eradicate systemic human CD19+ B cell malignancies in SCID-Beige mice. However, in the clinical setting, the anti-tumor efficacy of these T cells may be impaired by endogenous suppressive elements of the host immune system, including CD4+ CD25hi Foxp3+ regulatory T cells (Tregs). Significantly, Tregs are often increased in the blood and infiltrate the tumor of cancer patients which has been correlated with poor patient outcome and ineffective anti-tumor immunity. In order to study the in vivo impact of Tregs on adoptive therapy with CD19 targeted effector T cells, we developed a murine model wherein human Tregs, similarly targeted to the tumor, are infused prior to adoptive transfer of targeted cytotoxic T cells. To do so, we initially isolated natural Tregs from healthy donor peripheral blood mononuclear cells. Isolated Tregs were subsequently modified to express CARs through retroviral gene transfer. Subsequently, CAR+ Tregs were rapidly expanded either by activation on NIH-3T3 fibroblasts modified to express CD19 and the CD80 costimulatory ligand (3T3(CD19/CD80)), or non-specifically using CD3/CD28 antibodycoated magnetic beads. Expanded CAR+ Tregs exhibited potent suppressive function in vitro inhibiting both effector T cell proliferation as well as cytotoxicity. In vivo, CAR+ Tregs specifically traffic to established tumor in SCID-Beige mice. Significantly, injection of CD19-targeted Tregs into SCID-Beige mice bearing established human CD19+ tumors at 24 hours prior to infusion with CD19-targeted effector T cells, completely abrogated effector T cell function even at Treg:Teff ratios as low as 1:8. We further found that full suppression was dependant both on Treg localization to the tumor site as well as in vivo activation through the CAR. Finally, we show that a pre-conditioning regimen with low-dose cyclophosphamide, which failed to eradicate tumor, was able to reverse the CAR+ Treg mediated inhibition and restore the anti-tumor activity by the targeted effector T cells. In conclusion, we have developed a robust model ideally suited to the study of in vivo Treg-Teff interactions. Furthermore, the data generated from this model to date have significant implications with respect to the application of adoptive T cell therapies in the clinical setting. Namely, the presence of endogenous Tregs at the site of tumor is likely to significantly compromise the anti-tumor activity of adoptively transferred tumor targeted T cells. This inhibition may be reversed by preconditioning regimens designed to eradicate endogenous Tregs. The findings presented here should be considered in the design of future clinical trials utilizing T cell-based adoptive therapies of cancer.

T Cells Engineered with a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Have Long Term Persistence and Induce Durable Remissions in Children with Relapsed, Refractory ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 380-380 ◽  
Author(s):  
Stephan A. Grupp ◽  
Shannon L Maude ◽  
Pamela Shaw ◽  
Richard Aplenc ◽  
David M. Barrett ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Dose Range ◽  
Single Chain

Abstract BACKGROUND CARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors. RESULTS 30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. Figure 1 Figure 1. All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONS: CTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. Disclosures Grupp: Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.

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 p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Valérie Janelle ◽  
Mathieu Neault ◽  
Marie-Ève Lebel ◽  
Dave Maurice De Sousa ◽  
Salix Boulet ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cellular Senescence ◽  
Ex Vivo ◽  
Cell Dysfunction ◽  
Human T Cells ◽  
Car T ◽  
P38mapk Signaling ◽  
T Cell Dysfunction

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

scholarly journals Efficient Acquisition of Fully Human Antibody Genes against Self-Proteins by Sorting Single B Cells Stimulated with Vaccines Based on Nitrated T Helper Cell Epitopes

2019 ◽  
Vol 2019 ◽  
pp. 1-16
Author(s):  
Liangliang Jiang ◽  
Tao Jiang ◽  
Jianhua Luo ◽  
Yanliang Kang ◽  
Yue Tong ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
B Cell ◽  
Immune Tolerance ◽  
Human Antibody ◽  
T Helper ◽  
Human Immune System ◽  
Helper Epitope ◽  
Human Immune

Single B cell antibody technology is a method for isolating antigen-specific B cells from human peripheral blood and obtaining antibody genes in developing antibody drugs. However, owing to immune tolerance to autoantigen, human autoantigen-specific B cells are difficult to acquire by conventional single B cell technology. In this study, we constructed a nitrated T-cell epitope named NitraTh by incorporating p-nitrophenylalanine into a universal T helper epitope. NitraTh had enhanced ability to activate CD4+ T cells and can be recognized by CD4+ T cells with different HLA class II haplotypes. This NitraTh can also break immune tolerance to autoantigens, such as human epidermal growth factor receptor 2 (HER2) and cannabinoid receptor 1, and induce strong specific IgM+ B cell responses in vitro. HER2-NitraTh vaccine can also stimulate the generation of HER2-specific IgG+ B cells in human immune system mice, which was established by cotransplanting lymphocytes and autologous dendritic cells in immunodeficient mice. We obtained 30 fully human IgG antibody genes by sorting single B cells from the human immune system mice immunized with HER2-NitraTh vaccine. The analysis of antibody genes showed that sorted B cells underwent the extensive somatic mutation of the antibody genes. We randomly selected eight genes for cloning, six of which expressed antibodies that can bind to HER2. Hence, we provided a convenient and effective method in acquiring fully human antibody genes against self-proteins, which can be used in developing therapeutic antibody drugs.

Quantitative Assessment of the Impact of Ex Vivo Manipulation on the In Vivo Functionality of Human T Cells in RAG2−/− γc−/− mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 450-450
Author(s):  
Rozemarijn S. van Rijn ◽  
Elles R. Simonetti ◽  
Gert Storm ◽  
Mark Bonyhadi ◽  
Anton Hagenbeek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Critical Parameters ◽  
In Vivo Evaluation ◽  
Human T Cells ◽  
The Impact

Abstract T cells retrovirally modified to express therapeutic genes encoding cytokines, exogenous TCRs or suicide molecules represent a novel class of immune therapeutics of great potency. However, recent clinical trials using retrovirally-modified T cells have indicated that T cells exhibit a diminished reactivity upon ex vivo manipulation. In addition, virus-specific memory T cells seem to be lost during gene transfer. In a BNML rat model we have shown that the culture procedure is one of the critical parameters. To preserve T cell reactivity, reliable models are required which permit readout of human T cell activity. We recently developed a huPBMC-RAG2−/−γc−/− mouse model for xenogeneic graft-versus-host disease (xGVHD), in which iv injection of 15 x 106 human T cells into RAG2−/−γc−/− mice consistently leads to high level engraftment and lethal xGVHD within 3 weeks in 80% of mice (van Rijn et al, Blood 2003). We have now used this model to analyze in vivo functionality of human T cells following different ex vivo culture procedures. For this, we cultured human T cells for 7 days with either of the two currently available clinically applicable stimulation conditions: 1) via CD3 and 2) via CD3/CD28. In addition, we included CD3/CD28/4-1BB stimulation to explore the effect of extensive costimulation. Mice were injected with escalating doses T cells. HuCD45+ cells in peripheral blood were measured by FACS. Lethal xGVHD occurred at only 6 times (90.106) the dose of fresh cells for CD3-stimulated T cells and 3 times for CD3/28- or CD3/28/4-1BB-stimulated cells. About 20% of surviving mice developed chronic xGVHD, independent of culture method. While lethal xGVHD was always associated with very high levels of engraftment (up to 95%) engraftment levels in chronic mice ranged from 1–75%. To compare the impact of the different culture conditions on in vivo T cell function, we analyzed engraftment potential. The fraction of huCD45+ cells was plotted against the time and the areas under the curves were compared. Based on a total of 68 mice, statistical analysis showed a 2-fold improvement of engraftment potential for C28-costimulated human T cells compared to CD3-stimulated cells (P<0.0001). Additional ligation of 4-1BB did not increase engraftment potential. In addition, different T cell subsets (naïve, memory, effector) were monitored based on the combined expression of CD45RA, CD27 and CCR7. For all primary T cells and variably cultured T cells, a strikingly similar pattern was observed in vivo. After 3 weeks mainly effector and memory effector T cells (both CD4+ and CD8+) could be detected, suggesting a (xeno-)antigen-driven survival and expansion. This was a very consistent observation independent of donor, culture condition, engraftment level or severity of disease. In conclusion, in vitro costimulation preserves in vivo functionality of human T cells and should therefore be included in future clinical protocols for ex vivo manipulation of T cells. These data show the feasibility to use the huPBMC-RAG2−/−γc−/− model for in vivo evaluation of in vitro effects on human T cells. This model is the most sensitive to date for in vivo evaluation of human T cells and will be a promising new tool for the study of human T cells in, for instance, autoimmune disease, cancer and infectious diseases like AIDS.

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