B cells have been long studied for their role and function in the humoral immune system. Apart from generating antibodies and an antibody-mediated memory response against pathogens, B cells are also capable of generating cell-mediated immunity. It has been demonstrated by several groups that B cells can activate antigen-specific CD4 and CD8 T cells, and can have regulatory and cytotoxic effects. The function of B cells as professional antigen presenting cells (APCs) to activate T cells has been largely understudied. This, however, requires attention as several recent reports have demonstrated the importance of B cells within the tumor microenvironment, and B cells are increasingly being evaluated as cellular therapies. Antigen presentation through B cells can be through antigen-specific (B cell receptor (BCR) dependent) or antigen non-specific (BCR independent) mechanisms and can be modulated by a variety of intrinsic and external factors. This review will discuss the pathways and mechanisms by which B cells present antigens, and how B cells differ from other professional APCs.

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFβ. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Transforming growth factor-beta (TGFβ) is a potent immunosuppressive cytokine that inhibits the anti-tumor responses of NK cells and T cells. However, the stimulation of natural killer (NK) cells with pro-inflammatory cytokines decreases NK cell sensitivity to TGFβ. Herein, we sought to determine if TGFβ imprinting (TGFβi) during NK cell activation and expansion would decrease NK cell sensitivity to TGFβ suppression. To this end, we demonstrate that the activation of NK cells during chronic IL-2 stimulation and TGFβi potently induces NK cell hypersecretion of interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) in response to tumor targets which persists for at least one month in vitro after the removal of TGFβ. TGFβi NK cell cytokine hypersecretion is induced following both cytokine and tumor activation. Further, TGFβi NK cells have a marked suppression of SMAD3 and T-bet which is associated with altered chromatin accessibility. In contrast to their heightened cytokine secretion, TGFβi NK cells downregulate several activating receptors, granzyme and perforin, and upregulate TRAIL, leading to cell-line-specific alterations in cytotoxicity. These findings may impact our understanding of how TGFβ affects NK cell development and anti-tumor function.

Concurrent blockade of different checkpoint receptors, notably PD-1 and CTLA-4, elicits greater antitumor activity for some tumor types, and the combination of different checkpoint receptor inhibitors is an active area of clinical research. We have previously demonstrated that anti-tumor vaccination, by activating CD8 + T cells, increases the expression of PD-1, CTLA-4, LAG-3 and other inhibitory receptors, and the anti-tumor efficacy of vaccination can be increased with checkpoint blockade. In the current study, we sought to determine whether anti-tumor vaccination might be further improved with combined checkpoint blockade. Using an OVA-expressing mouse tumor model, we found that CD8 + T cells activated in the presence of professional antigen presenting cells (APC) expressed multiple checkpoint receptors; however, T cells activated without APCs expressed LAG-3 alone, suggesting that LAG-3 might be a preferred target in combination with vaccination. Using three different murine tumor models, and peptide or DNA vaccines targeting three tumor antigens, we assessed the effects of vaccines with blockade of PD-1 and/or LAG-3 on tumor growth. We report that, in each model, the anti-tumor efficacy of vaccination was increased with PD-1 and/or LAG-3 blockade. However, combined PD-1 and LAG-3 blockade elicited the greatest anti-tumor effect when combined with vaccination in a MycCaP prostate cancer model in which PD-1 blockade alone with vaccination targeting a "self" tumor antigen had less efficacy. These results suggest anti-tumor vaccination might best be combined with concurrent blockade of both PD-1 and LAG-3, and potentially other checkpoint receptors whose expression is increased on CD8 + T cells following vaccine-mediated activation.