The TCR has been identified in several species as a heterodimer of two variable chains (reviewed in reference 1) that is associated with a set of invariant polypeptides collectively referred to as CD3 . MHC-restricted antigen recognition by both cytotoxic (mostly CD8') and helper (mostly CD4') T cells is mediated by clonotypic heterodimers of the a/ß type, while a second type of CD3-associated TCR, termed y/S, has recently been discovered on a small subpopulation of human (2) and mouse (3) T cells . mAbs to the TCR and CD3 molecules have been instrumental to the discovery and analysis of the TCR complex . Recent work in the mouse system has especially profited from the generation of mAbs to Vß segments expressed at a frequency detectable in unimmunized T cell populations (4-7), and to the invariant CD3E chain (8) . No mAb to . a constant determinant of the mouse TCR-a/ß is available, however, that could be used to discriminate TCR-a/ß and TCR-,y/8 expressing T cells and thymocytes. In the rat system, analysis of T cell maturation and activation has been hampered by a complete lack of TCR-and CD3-specific monoclonal reagents, despite an otherwise excellent collection of mAbs to cell surface molecules .Here, we describe a new mAb, termed R73, that detects a rat pan T cell surface antigen with the predicted properties of the TCR-a/ß on mature and immature cells of the T cell lineage and reports its functional effects on resting T lymphocytes . Materials and MethodsAnimals. Young adult Wistar and Lewis rats ofboth sexes were obtained from the animal quarters of the Max Planck Institute for Biochemistry, Martinsried, FRG, or from the Zentralinstitut für Versuchstierzucht, Medizinische Hochschule Hannover, FRG. Results obtained did not vary significantly between both strains .Immunization and Cell Fusion . Spleen cells from a BALB/c mouse alternately immunized intraperitoneally with rat T blasts and rat erythrocytes (it was also intended to generate an mAb to rat LFA-3) were fused 3 d after an intravenous injection of 10' rat erythrocytes with This work was funded by a grant from the Bundesministerium für Forschung und Technologie . Generation of the R73 cell line was funded by Genzentrum e.V. J . Exp. MED.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Since there is no common genetic alteration causing resistance to venetoclax in CLL and B cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole exome sequencing, methylated DNA immunoprecipitation sequencing and genome wide CRISPR/Cas9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter which is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity towards venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher OXPHOS and ATP production, resembling the metabolic phenotype that is seen upon venetoclax resistance. While PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity towards both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive DLBCL in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Adoptive cell therapy with chimeric antigen receptor ( CAR )‐engineered T cells produced lasting remissions in the treatment of advanced, so far refractory B‐cell malignancies; however, the elimination of solid tumors remains so far elusive. The low efficacy of CAR T cells is thought to be due to the immune‐repressive milieu within the tumor lesion, predominantly mediated by transforming growth factor‐β ( TGF ‐β) that represses effector T‐cell activities and drives differentiation towards regulatory T cells (Tregs). Seeking to boost antitumor immunity, TGF ‐β is currently targeted by different means in pre‐clinical studies. While a recent clinical trial showed the utility of shielding CAR T cells from TGF ‐β repression, further strategies in counteracting TGF ‐β in the adoptive cell therapy warrant exploration. We here discuss the most recent advances in the field and draw future developments to make CAR T‐cell therapy more potent in the treatment of solid cancer.

The advent of chimeric antigen receptor (CAR) T cells expedited the field of cancer immunotherapy enabling durable remissions in patients with refractory hematological malignancies. T cells redirected for universal cytokine-mediated killing (TRUCKs), commonly referred to as “fourth generation” CAR T-cells, are designed to release engineered payloads upon CAR-induced T-cell activation. Building on the TRUCK technology, we aimed to generate CAR T-cells with a CAR-inducible artificial, self-limiting autocrine loop. To this end, we engineered CAR T-cells with CAR triggered secretion of type-1 interferons (IFNs). At baseline, IFNα and IFNβ CAR T-cells showed similar capacities in cytotoxicity and cytokine secretion compared to conventional CAR T-cells. However, under “stress” conditions of repetitive rounds of antigen stimulation using BxPC-3 pancreas carcinoma cells as targets, anti-tumor activity faded in later rounds while being fully active in destructing carcinoma cells during first rounds of stimulation. Mechanistically, the decline in activity was primarily based on type-1 IFN augmented CAR T-cell apoptosis, which was far less the case for CAR T-cells without IFN release. Such autocrine self-limiting loops can be used for applications where transient CAR T-cell activity and persistence upon target recognition is desired to avoid lasting toxicities.

Chimeric antigen receptor (CAR) modified T cells can induce complete remissions in patients with advanced hematological malignancies. Nevertheless, the efficacy is mostly transient and remains so far poor in the treatment of solid tumors. Crucial barriers to long-term CAR T cell success encompass loss of functional capacities known as “exhaustion”, among others. To extend CAR T cell functionality, we reduced interferon regulatory factor 4 (IRF4) levels in CAR T cells using a one-vector system encoding a specific short-hairpin (sh) RNA along with constitutive CAR expression. At baseline, CAR T cells with downregulated IRF4 showed equal cytotoxicity and cytokine release compared to conventional CAR T cells. However, under conditions of repetitive antigen encounter, IRF4low CAR T cells displayed enhanced functionality with superior cancer cell control in the long-term compared with conventional CAR T cells. Mechanistically, the downregulation of IRF4 in CAR T cells resulted in prolonged functional capacities and upregulation of CD27. Moreover, IRF4low CAR T cells were more sensitive to cancer cells with low levels of target antigen. Overall, IRF4 downregulation capacitates CAR T cells to recognize and respond to target cells with improved sensitivity and endurance.