Human Peripheral Blood T-lymphocytes Research Articles

Novel Peptide-Based Plectin-1 CAR T-Cells Target Specifically Pancreatic Cancer Cells

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating cancers, and therapies to treat this malignancy have not evolved rapidly to fulfill the need of affected patients. PDAC, like many solid malignancies, is considered a “cold immunological tumor” where the lack of targetable tumor-associated antigens has hampered the development of successful immunotherapies. Despite newer treatment approaches, PDAC patients with advanced disease have a dismal prognosis. Therefore, there is an urgent need to develop new therapies. Plectin-1 is a high molecular weight protein (∼500 kDa) that participates in cytoskeleton network organization linking intermediate filaments to microtubules and microfilaments. Plectin-1 is aberrantly expressed in the PDAC cellular membrane, contributes to PDAC pathogenesis, and confers a poor prognosis. Plectin-1-targeting peptides (PTP) in mice and humans have been successfully explored for their use in PDAC imaging and targeted payload delivery (small molecules, radioisotopes, and chemotherapy). We sought to take advantage of the specific binding of PTP and develop a peptide-based plectin-1 chimeric antigen receptor (CAR), and test T-cell transduced with this construct in vitro against PDAC cell lines. Methods: Using in silico tools, we optimized different pepto-CAR constructs and selected one that showed the highest levels of cytotoxicity against plectin-1-expressing PDAC cell lines. A myc tag was incorporated in the hinge region for flow cytometry or western blot detection. The intracellular signaling domain of these CAR sequences included 4-1BB and the zeta chain of the CD3 molecule ( Figure 1A). Plasmids containing the CAR sequence were packaged on lentivirus particles using HEK293T cells. Purified plectin-1 CAR lentiviruses were used to transduce T-cells derived from human peripheral blood T-cells obtained and purified from healthy donor whole blood. The expanded CAR T-cells were used for cytotoxic experiments against different cell lines. Results: Expression of plectin-1 CAR was confirmed by flow cytometry using an anti-myc tag antibody ( Figure 1B). Plectin-1 expression was analyzed in different pancreatic cancer cell lines, and the BxPC-3 cells showed the highest expression level ( Figure 1B). Using a luciferase-based assay, we plated 50,000 BxPC-3 cells per well in duplicates with different effector-to-target (E:T) ratios. After 24 hours, luciferin was added, and the plate was read using a luminometer. As a negative control for effector cells, we use T-cells without a CAR ( Figure 1B), and for the target cells, we use the plectin-1 negative lung cancer cell line A549 ( Figure 1B). Plectin-1 CAR T-cells effectively killed BxPC-3 cells at different E:T ratios in a specific manner compared with controls using unmodified T-cells. Furthermore, no cytotoxicity was observed against plectin-1 negative A549 cells ( Figure 1A). Conclusion: Our results show that a novel peptide-based CAR construct design against plectin-1 can induce specific cytotoxicity on plectin-1 expressing PDAC cell line BxPC-3. Furthermore, these data demonstrate the feasibility of using a CAR with a peptide-based antigen binding domain instead of the traditional antibody-derived scFv. This novel peptide-based CAR design offers alternatives for cellular therapy bioengineering. Moreover, our results can significantly impact immune-oncology therapeutic applications, particularly in challenging diseases like pancreatic cancer. Figure 1 . A) In the above section, the Plectin-1 CAR construct is depicted. Cytotoxicity of unmodified T-cells and Plectin-1 CAR T-cells against BxPC-3 and A549 is shown below. B) Expression of Plectin-1 CAR in unmodified T-cells and Plectin-1 CAR T-cells is shown on the left. The expression of plectin-1 antigen on the surface of BxPC-3 cells and A549 cells is shown on the right. PTP: plectin-1 targeting peptide.

Anticancer effects of herbal medicine Emelia-M, Mshikazi and Delosma H against human leukaemia cells.

Leukaemia is one of the three major types of blood cancers that lead to the overproduction of abnormal white blood cells. Emelia M (EMB), Mshikazi and Delosma H are herbal medicines that are being used by traditional healers in KwaZulu-Natal, South Africa to treat leukaemia and other diseases. To gain insight into the safety (non-toxic effect), anti-cancer activity, mechanisms of action and phytochemical profiles of traditional herbal medicines (Emelia M (EMB), Mshikazi and Delosma H) in South Africa. The viability of human peripheral blood mononuclear cells (PBMCs), monocytic (THP-1) and T-lymphocyte (Jurkat) cell lines exposed to varying concentrations of aqueous extracts of the three herbal medicines were assessed using adenosine triphosphate (ATP) assay. All three extracts showed a dose-dependent effect on the viability of PBMCs. Cell viability decreased with increasing concentrations of extracts when compared with the untreated cells at 24 and 48 hours. The inhibitory activities (IC50) of the extract were found in the order of Mshikazi > EMB, > Delosma H. All the extracts induced apoptosis with minimal necrosis. Many bioactive compounds that have been previously reported to have anticancer effects were identified in the extracts. Mshikazi extract significantly inhibited the growth of THP-1 and Jurkat cells and induced cell death through apoptosis than the other two extracts.

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit Tcell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of Tcell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human Tcell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood Tcells and TIL. Tumor-specific Tcells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. CISH expression was associated with Tcell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing Tcell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade invivo. CISH negatively regulates human Tcell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Possible effects of the exposure to ionizing radiation on the patients recovered from COVID-19

The aim. To conduct an analytical literature review on the possible impact of SARS-CoV-2 on the radiosensitivity of the human body and justify the relevance of radiobiological research in this area.
 Materials and methods. Analysis of data from biological dosimetry / indication of radiation lesions of human peripheral blood T-lymphocyte chromosomes under medical irradiation for comparison with radiosensitivity in the patients recovered from COVID-19 (Scopus International Scientific Metric Database, IAEA guidelines, 2011).
 Results. With the ongoing COVID-19 pandemic, forecasting and clarifying of the mechanisms of distant effects resulting from interactions between ionizing radiation and the SARS-CoV-2 virus play an important role. The difficulty in solving this problem is caused by the fact that the global science has no exhaustive information on the possible influence of this virus on radiation-induced effects. The attention of the professional community is drawn to the possible impact of SARS-CoV-2 on the radiosensitivity of the body of patients recovered from COVID-19 and a hypothesis is first proposed regarding the mechanism on how to increase it based on the development of systemic long-term inflammation. Therefore, clinical trials of low-dose radiotherapy for the treatment of COVID-19-related pneumonia involve preliminary radiobiological studies to answer the following question: does the SARS-CoV-2 virus affect the radiosensitivity of the human body? Long-term experience of the author of this paper in biodosimetric (cytogenetic) studies allows her to recommend the peripheral blood lymphocyte test system with chromosome aberration’s analysis as the most radiosensitive cell model.
 Conclusions. Clinical trials of low-dose radiotherapy for the treatment of COVID-19 pneumonia involve a preliminary radiobiological study to answer the following question: does the SARS-CoV-2 virus affect the radiosensitivity of the human body? The most optimal approach for the solution of this problem is the use of test-system of human peripheral blood lymphocytes’ culture with the subsequent cytogenetic analysis. It will allow investigating changes in the “dose-effect” “cell cycle stage-effect” dependencies, as well as changes in individual radiosensitivity under the influence of SARS-CoV-2 virus

124P Effective silencing of PD-1 gene in human peripheral blood T-lymphocytes by self-deliverable siRNA

Like all monoclonal antibodies, inhibitors of immune checkpoints (ICIs) can only interact with cell surface receptors, but not with intracellular proteins, which can be a promising target for cancer immunotherapy. ICIs can only block the receptor (e.g., PD-1), but not decrease its level. In contrast, small interfering RNAs (siRNAs) trigger mRNA degradation resulting in protein synthesis inhibition. It is possible to regulate the levels of both intracellular and extracellular target proteins using siRNAs.

Changes in human peripheral blood mononuclear cell (HPBMC) populations and T-cell subsets associated with arsenic and polycyclic aromatic hydrocarbon exposures in a Bangladesh cohort.

Exposures to environmental arsenic (As) and polycyclic aromatic hydrocarbons (PAH) have been shown to independently cause dysregulation of immune function. Little data exists on the associations between combined exposures to As and PAH with immunotoxicity in humans. In this work we examined associations between As and PAH exposures with lymphoid cell populations in human peripheral blood mononuclear cells (PBMC), as well as alterations in differentiation and activation of B and T cells. Two hundred men, participating in the Health Effects of Arsenic Longitudinal Study (HEALS) in Bangladesh, were selected for the present study based on their exposure to As from drinking water and their cigarette smoking status. Blood and urine samples were collected from study participants. We utilized multiparameter flow cytometry in PBMC to identify immune cells (B, T, monocytes, NK) as well as the T-helper (Th) cell subsets (Th1, Th2, Th17, and Tregs) following ex vivo activation. We did not find evidence of interactions between As and PAH exposures. However, individual exposures (As or PAH) were associated with changes to immune cell populations, including Th cell subsets. Arsenic exposure was associated with an increase in the percentage of Th cells, and dose dependent changes in monocytes, NKT cells and a monocyte subset. Within the Th cell subset we found that Arsenic exposure was also associated with a significant increase in the percentage of circulating proinflammatory Th17 cells. PAH exposure was associated with changes in T cells, monocytes and T memory (Tmem) cells and with changes in Th, Th1, Th2 and Th17 subsets all of which were non-monotonic (dose dependent). Alterations of immune cell populations caused by environmental exposures to As and PAH may result in adverse health outcomes, such as changes in systemic inflammation, immune suppression, or autoimmunity.

Hypoxia and TGF-β1 induce tissue resident memory phenotype cells from human peripheral blood T-cells in vitro

Abstract Tissue resident memory cells (TRM) are a recently identified subset of memory T-cells important in frontline defense against viral diseases and recent reports suggest T-cells with this phenotype are important in anti-tumor immunity. However, relatively little is known regarding TRM differentiation and endogenous TRM are notoriously difficult to isolate, impeding their study in basic research and downstream applications. Thus a means to generate TRM in vitro would be desirable. It is known that TGF-β is critical in establishing TRM populations and attempts to identify other factors that can induce differentiation to resident phenotype have focused on cytokines. Oxygen tension is another factor that distinguishes the circulation from peripheral tissues; thus we hypothesized that hypoxia may contribute to a TGF-β1-induced TRM phenotype in human CD8+ T-cells. We demonstrate that differentiation of human peripheral blood CD8+ T-cells in hypoxia and TGF-β1 in vitro led to the development of a TRM phenotype, characterized by a significant increase in CD69+CD103+ cells expressing CD49a, CD101, and PD-1 – hallmarks of human TRM. At the transcriptional level we observed upregulation of genes associated with TRM differentiation and downregulation of genes associated with memory cell recirculation. Hypoxia and TGF-β1 appear to synergize to induce a resident phenotype as their combination produced a significantly larger population of TRM phenotype cells than the additive effects of either condition alone. Our findings identify a previously unreported cue for TRM differentiation and enable a facile means of generating human TRM phenotype cells in vitro for basic studies and translational applications such as adoptive cellular therapies.

Isolation of Human Peripheral Blood T-Lymphocytes.

Peripheral blood is the most common source of T-lymphocytes for in vitro culture. Here, we present a simple and standardized method for small- or large-scale isolation of viable T-lymphocytes and other mononuclear cells from fresh peripheral blood or buffy coat blood samples using the density gradient centrifugation. T-cells obtained using the protocol described here can be used for a variety of downstream analysis, including cellular, molecular, and functional assays.

A Gut Microbial Mimic that Hijacks Diabetogenic Autoreactivity to Suppress Colitis

The gut microbiota contributes to the development of normal immunity but, when dysregulated, can promote autoimmunity through various non-antigen-specific effects on pathogenic and regulatory lymphocytes. Here, we show that an integrase expressed by several species of the gut microbial genus Bacteroides encodes a low-avidity mimotope of the pancreatic β cell autoantigen islet-specific glucose-6-phosphatase-catalytic-subunit-related protein (IGRP206-214). Studies in germ-free mice monocolonized with integrase-competent, integrase-deficient, and integrase-transgenic Bacteroides demonstrate that the microbial epitope promotes the recruitment of diabetogenic CD8+ Tcells to the gut. There, these effectors suppress colitis by targeting microbial antigen-loaded, antigen-presenting cells in an integrin β7-, perforin-, and majorhistocompatibility complex class I-dependent manner. Like their murine counterparts, human peripheral blood Tcells also recognize Bacteroides integrase. These data suggest that gut microbial antigen-specific cytotoxic Tcells may have therapeutic value in inflammatory bowel disease and unearth molecular mimicry as a novel mechanism by which the gut microbiota can regulate normal immune homeostasis. PAPERCLIP.

Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.

Effective adoptive immunotherapy has proved elusive for many types of human cancer, often due to difficulties achieving robust expansion of natural tumor-specific T-cells from peripheral blood. We hypothesized that antigen-driven T-cell expansion might best be triggered in vitro by acute activation of innate immunity to mimic a life-threatening infection. Unfractionated peripheral blood mononuclear cells (PBMC) were subjected to a two-step culture, first synchronizing their exposure to exogenous antigens with aggressive surrogate activation of innate immunity, followed by ?-chain cytokine-modulated T-cell hyperexpansion. Step 1 exposure to GM-CSF plus paired Toll-like receptor agonists (resiquimod and LPS), stimulated abundant IL-12 and IL-23 secretion, as well as upregulated co-stimulatory molecules and CD11c expression within the myeloid (CD33+) subpopulation. Added synthetic long peptides (>20aa) derived from widely expressed oncoproteins (MUC1, HER2/neu and CMVpp65), were reliably presented to CD4+ T-cells and cross-presented to CD8+ T-cells. Both presentation and cross-presentation demonstrated proteasomal and Sec61 dependence that could bypass the endoplasmic reticulum. Step 2 exposure to exogenous IL-7 or IL-7+IL-2 produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific T-cells with a predominant interferon-?-producing T1-type, as well as the antigen-specific ability to lyse tumor targets. Other ?-chain cytokines and/or combinations were initially proliferogenic, but followed by a contractile phase not observed with IL-7 or IL-7+IL-2. Regulatory T-cells were minimally propagated under these culture conditions. This mechanistically rational culture sequence, effective even for unvaccinated donors, enables rapid preparation of T-cells recognizing tumor-associated antigens expressed by the majority of human cancers, including pancreatic cancers, breast cancers and glioblastomas.

Bacterial Heat Shock Protein GroEL (Hsp64) Exerts Immunoregulatory Effects on T Cells by Utilizing Apoptosis.

Aggregatibacter actinomycetemcomitans (Aa) expresses a 64-kDa GroEL protein belonging to the heat shock family of proteins. This protein has been shown to influence human host cells, but the apoptotic capacity of the GroEL protein regarding T cells is not yet known. The purpose of this study was to investigate the ability of A. actinomycetemcomitans GroEL (AaGroEL) protein to induce human peripheral blood T-cell apoptosis. Endogenous, purified AaGroEL protein was used as an antigen. In AaGroEL-treated T cells, the data indicated that phosphatidylserine exposure, an early apoptotic event, was dose- and time-dependent. The AaGroEL-treated T cells were also positive for active caspase-3 in a dose-dependent manner. The rate of AaGroEL-induced apoptosis was suppressed by the addition of the general caspase inhibitor Z-VAD-FMK. Furthermore, cleaved caspase-8 bands (40/36 kDa and 23 kDa) were identified in cells responding to AaGroEL. DNA fragmentation was also detected in the AaGroEL-treated T cells. Overall, we demonstrated that the endogenous GroEL from A. actinomycetemcomitans has the capacity to induce T-cell apoptosis.

Reduced DNA double-strand break repair capacity and risk of squamous cell carcinoma of the head and neck--A case-control study.

Tobacco smoke and alcohol use play important roles in the etiology of squamous cell carcinoma of the head and neck (SCCHN). Smoking causes DNA damage, including double-strand DNA breaks (DSBs), that leads to carcinogenesis. To test the hypothesis that suboptimal DSB repair capacity is associated with risk of SCCHN, we applied a flow cytometry-based method to detect the DSB repair phenotype first in four EBV-immortalized human lymphoblastoid cell lines and then in human peripheral blood T-lymphocytes (PBTLs). With this blood-based laboratory assay, we conducted a pilot case-control study of 100 patients with newly diagnosed, previously untreated SCCHN and 124 cancer-free controls of non-Hispanic whites. We found that the mean DSB repair capacity level was significantly lower in cases (42.1%) than that in controls (54.4%) (P<0.001). When we used the median DSB repair capacity level in the controls as the cutoff value for calculating the odds ratios (ORs) with adjustment for age, sex, smoking and drinking status, the cases were more likely than the controls to have a reduced DSB repair capacity (adjusted OR=1.93; 95% confidence interval, CI=1.04-3.56, P=0.037), especially for those subjects who were ever drinkers (adjusted OR=2.73; 95% CI=1.17-6.35, P=0.020) and had oropharyngeal tumors (adjusted OR=2.17; 95% CI=1.06-4.45, P=0.035). In conclusion, these findings suggest that individuals with a reduced DSB repair capacity may be at an increased risk of developing SCCHN. Larger studies are warranted to confirm these preliminary findings.

The modulating effect of carumopticum on human peripheral blood T-cells radiation toxicity

The modulating effect of carumopticum on human peripheral blood T-cells radiation toxicity

Abstract P6-08-34: p16INK4a expression and chemotherapy toxicity in women with early stage breast cancer

Abstract Background: Increased expression of p16INK4a, a molecular marker of aging, is a hallmark of increased cellular senescence in most mammalian tissues. In human peripheral blood T-lymphocytes, expression of p16INK4a increases 10-fold between 20 and 80 years of age (Liu et al, Aging Cell, 2009). We hypothesized that higher "molecular age," as evidenced by increased T-cell expression of p16INK4a at the time of initiation of breast cancer (BC) chemotherapy (CRx), predicts increased treatment-related toxicity. Methods: Patients (pts) with early breast cancer scheduled to receive neoadjuvant (NA) or adjuvant (Adj) chemotherapy (CRx) had p16INK4a evaluation performed prior to treatment. Expression of p16INK4a mRNA in CD3+ T-lymphocytes was determined using TaqMan real time quantitative reverse transcription polymerase chain reaction. Grade 3 and 4 (G3/4) hematologic (H) and non-hematologic (NH) toxicities (NCI CTCAE version 4) were assessed during and within 4 weeks of completion of CRx. Wilcoxon Rank Sum tests compared p16INK4a between groups. Results: 93 pts with Stage I-III BC and complete toxicity data during and within 4 weeks after completion of CRx have been accrued. Median age (range) was 52 (25-76). 48 (52%) of pts were hormone receptor (HR) positive, 21 (23%) HER-2 positive, and 24 (26%) triple-negative. 39 (42%) received NA and 54 (58%) received Adj CRx. 57 pts (61%) received an anthracycline containing combination and 21 (23%) cyclophosphamide/docetaxel (TC). 85 pts (91%) received pegfilgrastim. Overall, 69 pts (74%) had a G3/4 toxicity during or ≤4 wks of CRx, 54% NH and 40% H. 22 pts (24%) were hospitalized for CRx related toxicity and 11 (12%) of these admissions were for neutropenic fever (NF). 23 (25%) reported G3/4 fatigue during CRx and had significantly higher p16INK4a values at baseline than those without fatigue (p=0.03). There was no significant association of baseline p16INK4a and other NH or H toxicity or hospitalization in this cohort. Conclusion: In this small sample of pts treated with anthracycline and non-anthracycline containing NA and Adj CRx regimens, there was a significant association of baseline p16INK4a and G3/4 fatigue. The heterogeneity of treatment and use of pegfilgrastim in almost all pts limits the power of this study to find significant relationships between p16INK4a and other toxicities. The cohort for this study is being expanded to further explore p16INK4a as a predictor for G3/4 toxicity. Support: Breast Cancer Treatment Foundation, New York, NY; University Cancer Research Fund, University of North Carolina, Chapel Hill. Citation Format: Hyman Muss, Allison Deal, Arti Hurria, Natalia Mitin, Chad Torrice, Krishnamurthy Janakiraman, Trevor Jolly, Grant Williams, Shani Alston, Jerard West, Laura Zavala, Vani Katheria, Norman Sharpless. p16INK4a expression and chemotherapy toxicity in women with early stage breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-34.

Identifying the Factors Modulating the Efficacy of CAR-T Cell Therapy

T cells modified to express tumour-directed chimeric antigen receptors (CARs) have shown anti-tumor efficacy in early phase clinical trials. There is no consensus yet about the optimal CAR T- cell dose needed for achieve optimal anti-leukemic activity. Early phase clinical trials have shown that the level of in vivo expansion of CAR T-cells rather than CAR T-cell dose determines the efficacy of CAR-T cell therapy. Here we identify factors that modify CAR-T cell expansion and anti-tumour activity in vitro and in vivo in a ‘humanised’ mouse model using CARs targeting CD19+ B-cell leukaemia (CAR19 T-cells). Human peripheral and cord blood T-cells were genetically modified using second generation of CAR19-T cells engineered to express CD3zeta and co-stimulatory CD28 domains cloned into PiggyBac-transposon vector. Antigen-specific stimulation using autologous mononuclear cells was used to enrich and expand CAR19 T-cells. CAR19 T-cells induced cytolysis of leukaemia cells in vitro and exhibited anti-tumour activity in vivo in xenograft mouse model of leukaemia. Antigen-specific stimulation induced rapid activation and proliferation of CAR19 T-cells, however, terminal effector differentiation and activated T cell death limited CAR19-T cell expansion. We have analysed CAR19 T-cell expansion using the panel of primary CD19+leukaemia cells expressing different levels of CD19 antigen. CAR19 T-cells expansion and cytolytic function correlated with the level of CD19 expression on target cells. In addition, increasing CD19 expression in leukaemia cells using hypomethylating agent 5-aza-2′-deoxycytidine (5-AZA) promoted antigen-specific recognition of target cells by CAR19-T cells and increased their expansion. Treatment with 5-AZA also increased CAR expression on the CAR19 T-cells, however, cytotoxicity towards CAR19 T cells mediated by the agent delayed CAR19T-cell expansion. Interestingly, allogeneic stimulation of CAR19 T-cells using CD3/CD28 activation of TCR signalling or co-culture with allogeneic bone marrow stroma cells promoted CAR19 T-cell expansion, however, addition of antigen-specific target cells (CD19+leukaemia cells) attenuated allogeneic expansion indicating the antagonism between antigen-specific and allogeneic CAR19 T- cell activation.Effector to target (E:T) cell ratio appears to be a strong determinate of CAR19 T-cell expansion and cytolytic activity towards cancer cells. CAR19 T-cells used at high E:T ratios exhibit higher cytolytic activity compared to effector cells used at a low E:T. Cell division analysis demonstrats lower proliferation of CAR19 T-cells when used at a high E:T compared to those used at low E:T ratio. The repetitive antigen-specific activation of CAR19 T-cells occurring at a high density of target cells (low E:T ratio) promotes proliferation, however, also acts to induce terminal effector differentiation resulting in rapid T cell extinction due to activated T cell death. Thus the excessive proliferation and rapid extinction of CAR19 T-cells may account for the low efficacy of CAR19 T- cell therapy when given at low E:T ratios. Similar effects were observed in a stem cell reconstituted mouse model where stem cell-derived CD19+ B cells were targeted by CAR19 T-cells. A single infusion of CAR19-T cells infused at a high E:T ratio in mice with low B cell engraftment resulted in efficient B cell depletion while the same dose of CAR19 T-cells infused to mice with high B cell engraftment level(low E:T ratio) was less efficient. These findings show that rapid exhaustion of effector cells used at low E:T ratio prevents long lasting anti-tumor effects justifying the need of pre-conditioning in patients with advanced tumors.. DisclosuresNo relevant conflicts of interest to declare.

Protein kinase C-δ and -β coordinate flow-induced directionality and deformation of migratory human blood T-lymphocytes.

T-lymphocyte migration under flow is critical for immune responses, but the mechanisms by which flow modulates the migratory behaviors of T-lymphocytes remain unclear. Human peripheral blood T-lymphocytes (PBTLs), when stimulated with phorbol 12-myristate 13-acetate (PMA), stretched their cell bodies dramatically and moved along the flow direction. In contrast, stromal cell-derived factor-1α-stimulated PBTLs deformed and migrated in a random manner. Here we elucidated the molecular mechanisms underlying flow-induced directionality and deformation of PMA-stimulated PBTLs. PMA primed PBTLs for polarization under flow, with protein kinase C (PKC)-δ enriched in the leading edge, PKC-βI in the microtubule organizing center, and PKC-βII in the uropod and peripheral region. PKC-δ regulated cell protrusions in the leading edge through Tiam1/Rac1/calmodulin, whereas PKC-β regulated RhoA/Rho-associated kinase activity and microtubule stability to modulate uropod contractility and detachment. Our findings indicate that PKC-δ and -β coordinate in the cell leading edge and uropod, respectively, to modulate the directionality and deformability of migratory T-lymphocytes under flow.

Manufacture of gene-modified human T-cells with a memory stem/central memory phenotype.

Advances in genetic engineering have made it possible to generate human T-cell products that carry desired functionalities, such as the ability to recognize cancer cells. The currently used strategies for the generation of gene-modified T-cell products lead to highly differentiated cells within the infusion product, and on the basis of data obtained in preclinical models, this is likely to impact the efficacy of these products. We set out to develop a good manufacturing practice (GMP) protocol that yields T-cell receptor (TCR) gene-modified T-cells with more favorable properties for clinical application. Here, we show the robust clinical-scale production of human peripheral blood T-cells with an early memory phenotype that express a MART-1-specific TCR. By combining selection and stimulation using anti-CD3/CD28 beads for retroviral transduction, followed by expansion in the presence of IL-7 and IL-15, production of a well-defined clinical-scale TCR gene-modified T-cell product could be achieved. A major fraction of the T-cells generated in this fashion were shown to coexpress CD62L and CD45RA, and express CD27 and CD28, indicating a central memory or memory stemlike phenotype. Furthermore, these cells produced IFNγ, TNFα, and IL-2 and displayed cytolytic activity against target cells expressing the relevant antigen. The T-cell products manufactured by this robust and validated GMP production process are now undergoing testing in a phase I/IIa clinical trial in HLA-A*02:01 MART-1-positive advanced stage melanoma patients. To our knowledge, this is the first clinical trial protocol in which the combination of IL-7 and IL-15 has been applied for the generation of gene-modified T-cell products.

T-cell co-stimulation through the CD2 and CD28 co-receptors induces distinct signalling responses

Full T-cell activation critically depends on the engagement of the TCR (T-cell receptor) in conjunction with a second signal by co-stimulatory receptors that boost the immune response. In the present study we have compared signalling patterns induced by the two co-receptors CD2 and CD28 in human peripheral blood T-cells. These co-receptors were previously suggested to be redundant in function. By a combination of multi-parameter phosphoflow cytometry, phosphokinase arrays and Western blot analyses, we demonstrate that CD2 co-stimulation induces phosphorylation of the TCR-proximal signalling complex, whereas CD28 activates distal signalling molecules, including the transcription factors NF-κB (nuclear factor κB), ATF (activating transcription factor)-2, STAT3/5 (signal transducer and activator of transcription 3/5), p53 and c-Jun. These signalling patterns were conserved in both naïve and effector/memory T-cell subsets. We show that free intracellular Ca(2+) and signalling through the PI3K (phosphoinositide 3-kinase)/Akt pathway are required for proper CD28-induced NF-κB activation. The signalling patterns induced by CD2 and CD28 co-stimulation lead to distinct functional immune responses in T-cell proliferation and cytokine production. In conclusion, CD2 and CD28 co-stimulation induces distinct signalling responses and functional outcomes in T-cells.

Dynamic reorganization of flotillins in chemokine-stimulated human T-lymphocytes

BackgroundDifferent types of membrane microdomains (rafts) have been postulated to be present in the rear and front of polarized migrating T-lymphocytes. Disruption of rafts by cholesterol sequestration prevents T-cell polarization and migration. Reggie/flotillin-1 and -2 are two highly homologous proteins that are thought to shape membrane microdomains. We have previously demonstrated the enrichment of flotillins in the uropod of human neutrophils. We have now investigated mechanisms involved in chemokine-induced flotillin reorganization in human T-lymphocytes, and possible roles of flotillins in lymphocyte polarization.ResultsWe studied flotillin reorganization and lateral mobility at the plasma membrane using immunofluorescence staining and FRAP (fluorescence recovery after photobleaching). We show that flotillins redistribute early upon chemokine stimulation, and form very stable caps in the uropods of human peripheral blood T-lymphocytes, colocalizing with the adhesion molecule PSGL-1 and activated ezrin/radixin/moesin (ERM) proteins. Chemokine-induced formation of stable flotillin caps requires integrity and dynamics of the actin cytoskeleton, but is not abolished by inhibitors suppressing Rho-kinase or myosin II activity. Tagged flotillin-2 and flotillin-1 coexpressed in T-lymphocytes, but not singly expressed proteins, colocalize in stable caps at the tips of uropods. Lateral mobility of coexpressed flotillins at the plasma membrane is already partially restricted in the absence of chemokine. Incubation with chemokine results in almost complete immobilization of flotillins. Capping is abolished when wild-type flotillin-1 is coexpressed with a mutant of flotillin-2 (G2A) that is unable to interact with the plasma membrane, or with a deletion mutant of flotillin-2 that lacks a putative actin-binding domain. Wild-type flotillin-2 in contrast forms caps when coexpressed with a mutant of flotillin-1 unable to interact with membranes. Transfection of T-lymphocytes with flotillin-2-G2A reduces cell polarization and uropod recruitment of endogenous flotillin-1 and PSGL-1.ConclusionsOur data suggest that stable flotillin cap formation in the rear of polarized T-lymphocytes requires flotillin heterooligomer formation, as well as direct F-actin interactions of flotillin-2 and raft/membrane association of flotillin-2, but not -1. Our data also implicate flotillin-rich actin-dependent membrane microdomains in T-lymphocyte uropod formation.

Integration profile of retroviral vector in gene therapy treated patients is cell‐specific according to gene expression and chromatin conformation of target cell

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.