Helen Linda Warmuth

Adoptive cellular therapies have shown enormous potential, but are complicated by personalization. Because of HLA mismatch, rejection of transferred T cells frequently occurs, compromising the T-cell graft's functionality. This obstacle has led to the development of human leukocyte antigen (HLA) knock-out (KO) T cells as universal donor cells. Whether such editing directly affects T-cell functionality remains poorly understood. In addition, HLA KO T cells are susceptible to missing-self recognition through NK cells and lack of canonical HLA class I expression may represent a safety hazard. Engineering of non-canonical HLA molecules could counteract NK cell recognition, but further complicates the generation of cell products. We here show that HLA KO does not alter T-cell functionality in vitro and in vivo. While HLA KO abrogates allogeneic T-cell responses, it elicits NK-cell recognition. To circumvent this problem, we demonstrate that selective editing of individual HLA class I molecules in primary human T cells is possible. Such "HLA reduction" not only inhibits T-cell alloreactivity and NK-cell recognition simultaneously, but also preserves the T-cell graft's canonical HLA class I expression. In the presence of allogeneic T cells and NK cells, T cells with remaining expression of a single, matched HLA class I allele show improved functionality in vivo in comparison to conventional allogeneic T cells. Since reduction to only a few, most frequent HLA haplotypes would already be compatible with large shares of patient populations, this approach significantly extends the toolbox to generate broadly applicable cellular products.

Engagement of the inhibitory T cell receptor programmed cell death protein 1 (PD-1) associates with dysfunctional states of pathogen- or tumor-specific T cells. Accordingly, systemic antibody-mediated blockade of PD-1 has become a central target for immunotherapies but is also associated with severe toxicities due to loss of peripheral tolerance. Therefore, selective ablation of PD-1 expression on adoptively transferred T cells through direct genetic knockout (KO) is currently being explored as an alternative therapeutic approach. However, since PD-1 might also be required for the regulation of physiological T cell function and differentiation, the suitability of PD-1 as an engineering target is controversial. In this study, we systematically investigated the maintenance of T cell functionality after CRISPR/Cas9-mediated PD-1 KO in vivo during and after acute and chronic antigen encounter. Under all tested conditions, PD-1 ablation preserved the persistence, differentiation, and memory formation of adoptively transferred receptor transgenic T cells. Functional PD-1 KO T cells expressing chimeric antigen receptors (CARs) targeting CD19 could be robustly detected for over 390 d in a syngeneic immunocompetent mouse model, in which constant antigen exposure was provided by continuous B cell renewal, representing the longest in vivo follow-up of CAR-T cells described to date. PD-1 KO CAR-T cells showed no evidence for malignant transformation during the entire observation period. Our data demonstrate that genetic ablation of PD-1 does not impair functionality and longevity of adoptively transferred T cells per se and therefore may be pursued more generally in engineered T cell-based immunotherapy to overcome a central immunosuppressive axis.