CD4(+) type 1 T regulatory (Tr1) cells are induced in the periphery and have a pivotal role in promoting and maintaining tolerance. The absence of surface markers that uniquely identify Tr1 cells has limited their study and clinical applications. By gene expression profiling of human Tr1 cell clones, we identified the surface markers CD49b and lymphocyte activation gene 3 (LAG-3) as being stably and selectively coexpressed on mouse and human Tr1 cells. We showed the specificity of these markers in mouse models of intestinal inflammation and helminth infection and in the peripheral blood of healthy volunteers. The coexpression of CD49b and LAG-3 enables the isolation of highly suppressive human Tr1 cells from in vitro anergized cultures and allows the tracking of Tr1 cells in the peripheral blood of subjects who developed tolerance after allogeneic hematopoietic stem cell transplantation. The use of these markers makes it feasible to track Tr1 cells in vivo and purify Tr1 cells for cell therapy to induce or restore tolerance in subjects with immune-mediated diseases.
Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.
Summary
Current therapeutic regimens for acute myeloid leukaemia (AML) are still associated with high rates of relapse. Immunotherapy with T‐cells genetically modified to express chimeric antigen receptors (CARs) represents an innovative approach. Here we investigated the targeting of the interleukin three receptor alpha (IL3RA; CD123) molecule, which is overexpressed on AML bulk population, CD34+ leukaemia progenitors, and leukaemia stem cells (LSC) compared to normal haematopoietic stem/progenitor cells (HSPCs), and whose overexpression is associated with poor prognosis. Cytokine‐induced killer (CIK) cells were transduced with SFG‐retroviral‐vector encoding an anti‐CD123 CAR. Transduced cells were able to strongly kill CD123+ cell lines, as well as primary AML blasts. Interestingly, secondary colony experiments demonstrated that anti‐CD123.CAR preserved in vitro HSPCs, in contrast to a previously generated anti‐CD33.CAR, while keeping an identical cytotoxicity profile towards AML. Furthermore, limited killing of normal monocytes and CD123‐low‐expressing endothelial cells was noted, thus indicating a low toxicity profile of the anti‐CD123.CAR. Taken together, our results indicate that CD123‐specific CARs strongly enhance anti‐AML CIK functions, while sparing HSPCs and normal low‐expressing antigen cells, paving the way to develop novel immunotherapy approaches for AML treatment.
IL-10-producing CD4+ type 1 regulatory T (Tr1) cells, defined based on their ability to produce high levels of IL-10 in the absence of IL-4, are major players in the induction and maintenance of peripheral tolerance. Tr1 cells inhibit T-cell responses mainly via cytokine-dependent mechanisms. The cellular and molecular mechanisms underlying the suppression of APC by Tr1 cells are still not completely elucidated. Here, we defined that Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)- and perforin (PRF)-dependent mechanism that requires HLA class I recognition, CD54/lymphocyte function-associated antigen (LFA)-1 adhesion, and activation via killer cell Ig-like receptors (KIRs) and CD2. Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1-cell activation, is necessary for defining Tr1-cell target specificity. We also showed that high frequency of GZB-expressing CD4+ T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10-producing CD4+ T cells. In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions that lead to selective killing of myeloid cells and possibly bystander suppression.
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