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AbstractBackgroundAdoptive transfer of chimeric antigen receptor (CAR)-expressing natural killer (NK) cells has demonstrated success against hematological malignancies. Efficacy against solid tumors has been limited by poor NK cell survival and function in the suppressive tumor microenvironment (TME). To enhance efficacy against solid tumors, stimulatory cytokines have been incorporated into CAR-NK cell therapeutic approaches. However, current cytokine strategies have limitations, including systemic toxicities, exogenous dependencies, and unwanted TME bystander effects. Here, we aimed to overcome these limitations by modifying CAR-NK cells to express a constitutively active interleukin (IL)-7 receptor, termed C7R, capable of providing intrinsic CAR-NK cell activation that does not rely on or produce exogenous signals nor activate bystander cells.MethodsWe examined persistence, antitumor function, and transcriptional profiles of CAR-NK cells coexpressing C7R in a novel tumor immune microenvironment (TiME) co-culture system and against hematologic and solid tumor xenografts in vivo.ResultsPeripheral blood NK cells expressing a CAR directed against the solid tumor antigen GD2 and modified with C7R demonstrated enhanced tumor killing and persistence in vitro compared with CAR-NK cells without cytokine support and similar functions to CAR-NK cells supplemented with recombinant IL-15. C7R.CAR-NK cells exhibited enhanced survival and proliferation within neuroblastoma TiME xenografts in vivo but produced poor long-term tumor control compared with CAR-NK cells supplemented with IL-15. Similar results were seen using C7R-expressing CD19.CAR-NK cells against CD19+leukemia xenografts. Gene expression analysis revealed that chronic signaling via C7R induced a transcriptional signature consistent with intratumor stressed NK cells with blunted effector function. We identified gene candidates associated with chronic cytokine-stressed NK cells that could be targeted to reduce CAR-NK cell stress within the solid TME.ConclusionC7R promoted CAR-NK cell survival in hostile TMEs independent of exogenous signals but resulted in poor antitumor function in vivo. Our data reveals the detrimental role of continuous IL-7 signaling in CAR-NK cells and provides insights into proper application of cytokine signals when attempting to enhance CAR-NK cell antitumor activity.

AbstractObjectivesTumour necrosis factor inhibitors (TNFi) are widely used and effective as treatment for immune-mediated inflammatory diseases (IMIDs). However, TNFi therapy causes a faster waning of antibody responses following vaccination. The underlying cause by which TNFi affect humoral immunity remains to be elucidated. The formation of long-lasting, high-affinity antibodies after vaccination results from germinal centre (GC)-derived, T cell-dependent B-cell responses. Therefore, this study investigated how TNFi affect the formation and maintenance of antigen-specific B- and CD4+ T-cell responses following SARS-CoV-2 mRNA vaccination.MethodsSARS-CoV-2 spike-specific B-cell responses were characterised using spectral flow cytometry. Spike-specific CD4+ T cells were measured using an activation-induced marker assay. 15 patients with inflammatory bowel disease (IBD) treated with TNFi were compared with 9 IBD patients without systemic immunosuppression and 10 healthy controls.ResultsSpike-specific CD4+T-cell frequency and phenotype, including T follicular helper cells, were not affected by TNFi. Total spike-specific B-cell frequencies were reduced in TNFi-treated patients. Deep phenotyping revealed lower IgG+memory B-cell frequencies in TNFi-treated patients 3¨C6 months after vaccination. These data were confirmed in TNFi-treated rheumatoid arthritis patients. Interestingly, already at day 7 after the second vaccination, TNFi therapy reduced the induction of class-switched CD11c- CD71+activated B cells, which are believed to be GC-derived. Conversely, CD11c+B cells, associated with extrafollicular B-cell responses, were not affected by TNFi therapy.ConclusionsThese data suggest that TNFi therapy affects the differentiation of GC-derived B cells, which may explain its effect on humoral immune responses.

A well-balanced maternal immune system is crucial to maintain fetal tolerance in case of infections during pregnancy. Immune adaptations include an increased secretion of soluble mediators to protect the semi-allogeneic fetus from excessive pro-inflammatory response. B lymphocytes acquire a higher capacity to express CD83 and secrete soluble CD83 (sCD83) upon exposure to bacteria-derived components such as LPS. CD83 possesses immune modulatory functions and shows a promising therapeutic potential against inflammatory conditions. The administration of sCD83 to pregnant mice reduces LPS-induced abortion rates. The increased CD83 expression by endometrial B cells as compared to peripheral blood B cells suggests its modulatory role in the fetal tolerance, especially in the context of infection. We postulate that in pregnancy, CD83 expression and release is controlled by pregnancy-related hormones. The intra- and extracellular expression of CD83 in leukocytes from peripheral blood or decidua basalis and parietalis at term were analyzed by flow cytometry. After treatment with pregnancy-related hormones and LPS, ELISA and qPCR were performed to study sCD83 release and CD83 gene expression, respectively. Cleavage prediction analysis was used to find potential proteases targeting CD83. Expression of selected proteases was analyzed by ELISA. Higher levels of CD83 were found in CD11c+ dendritic cells, CD3+ T cells and CD19+ B cells from decidua basalis and decidua parietalis after LPS-stimulation in vitro. An increase of intracellular expression of CD83 was also detected in CD19+ B cells from both compartments. Stimulated B cells displayed significantly higher percentages of CD83+ cells than dendritic cells and T cells from decidua basalis and peripheral blood. Treatment of B lymphocytes with pregnancy-related molecules (E2, P4, TGF-¦Â1 and hCG) enhanced the LPS-mediated increase of CD83 expression, while dexamethasone led to a reduction. Similarly, the release of sCD83 was increased under TGF-¦Â1 treatment but decreased upon dexamethasone stimulation. Finally, we found that the hormonal regulation of CD83 expression is likely a result from a balance between gene transcription from CD83 and the modulation of the metalloproteinase MMP-7. Thus, data supports and complements our previous murine studies on hormonal regulation of CD83 expression, reinforcing its immunomodulatory relevance in anti-bacterial responses during pregnancy.