Viral infections, such as with cytomegalovirus (CMV), remain a major risk factor for mortality and morbidity of transplant recipients because of their requirement for lifelong immunosuppression (IS). Antiviral drugs often cause toxicity and sometimes fail to control disease. Thus, regeneration of the antiviral immune response by adoptive antiviral T cell therapy is an attractive alternative. Our recent data, however, show only short-term efficacy in some solid organ recipients, possibly because of malfunction in transferred T cells caused by ongoing IS. We developed a vector-free clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based good manufacturing practice (GMP)-compliant protocol that efficiently targets and knocks out the gene for the adaptor protein FK506-binding protein 12 (FKBP12), required for the immunosuppressive function of tacrolimus. This was achieved by transient delivery of ribonucleoprotein complexes into CMV-specific T cells by electroporation. We confirmed the tacrolimus resistance of our gene-edited T cell products in vitro and demonstrated performance comparable with non-tacrolimus-treated unmodified T cells. The alternative calcineurin inhibitor cyclosporine A can be administered as a safety switch to shut down tacrolimus-resistant T cell activity in case of adverse effects. Furthermore, we performed safety assessments as a prerequisite for translation to first-in-human applications.

Activity of the CXCR3alt-CXCL11 chemokine system in CD8 ⁺ stem-like T cells predicts chemotherapy response in patients with bladder cancer.

Regulatory T cells (T _reg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of T _reg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited T _reg cell products. Here, we examined different T _reg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each T _reg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory T _reg cell populations, including a hitherto unidentified naïve-like memory T _reg cell subset, outperformed late-differentiated effector memory–like T _reg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional T _reg cell products could be achieved by depleting the small effector memory–like T _reg cell subset before manufacturing. Considering T _reg cell subset composition appears critical to maintain lineage stability in the final cell product.

Viral infections have a major impact on morbidity and mortality of immunosuppressed solid organ transplant (SOT) patients because of missing or failure of adequate pharmacologic antiviral treatment. Adoptive antiviral T-cell therapy (AVTT), regenerating disturbed endogenous T-cell immunity, emerged as an attractive alternative approach to combat severe viral complications in immunocompromised patients. AVTT is successful in patients after hematopoietic stem cell transplantation where T-cell products (TCPs) are manufactured from healthy donors. In contrast, in the SOT setting TCPs are derived from/applied back to immunosuppressed patients. We and others demonstrated feasibility of TCP generation from SOT patients and first clinical proof-of-concept trials revealing promising data. However, the initial efficacy is frequently lost long-term, because of limited survival of transferred short-lived T-cells indicating a need for next-generation TCPs. Our recent data suggest that Rapamycin treatment during TCP manufacture, conferring partial inhibition of mTOR, might improve its composition. The aim of this study was to confirm these promising observations in a setting closer to clinical challenges and to deeply characterize the next-generation TCPs. Using cytomegalovirus (CMV) as model, our next-generation Rapamycin-treated (Rapa-)TCP showed consistently increased proportions of CD4⁺ T-cells as well as CD4⁺ and CD8⁺ central-memory T-cells (T_CM). In addition, Rapamycin sustained T-cell function despite withdrawal of Rapamycin, showed superior T-cell viability and resistance to apoptosis, stable metabolism upon activation, preferential expansion of T_CM, partial conversion of other memory T-cell subsets to T_CM and increased clonal diversity. On transcriptome level, we observed a gene expression profile denoting long-lived early memory T-cells with potent effector functions. Furthermore, we successfully applied the novel protocol for the generation of Rapa-TCPs to 19/19 SOT patients in a comparative study, irrespective of their history of CMV reactivation. Moreover, comparison of paired TCPs generated before/after transplantation did not reveal inferiority of the latter despite exposition to maintenance immunosuppression post-SOT. Our data imply that the Rapa-TCPs, exhibiting longevity and sustained T-cell memory, are a reasonable treatment option for SOT patients. Based on our success to manufacture Rapa-TCPs from SOT patients under maintenance immunosuppression, now, we seek ultimate clinical proof of efficacy in a clinical study.

Identifying the key toxic players within an in-vivo toxic syndrome is crucial to develop targeted therapies. Here, we established a novel method that characterizes the effect of single substances by means of an ex-vivo incubation set-up. We found that primary human spermatozoa elicit a distinct motile response on a (uremic) toxic milieu. Specifically, this approach describes the influence of a bulk toxic environment (uremia) as well as single substances (uremic toxins) by real-time analyzing motile cellular behavior. We established the human spermatozoa-based toxicity testing (HSTT) for detecting single substance-induced toxicity to be used as a screening tool to identify in-vivo toxins. Further, we propose an application of the HSTT as a method of clinical use to evaluate toxin-removing interventions (hemodialysis).

The significance of the tumor microenvironment (TME) in predicting immunotherapy efficacy is increasingly acknowledged. However, the complexity of the TME necessitates novel technological approaches for the precise characterization of individual cell types, functional phenotypes, and heterocellular spatial interactions. This study utilizes a streamlined multiplex cyclic immunohistochemistry (cycIHC) protocol for detailed TME annotation. Unlike proprietary methods, cycIHC relies on iterative cycles of conventional immunohistochemistry, using off-the-shelf antibodies and reagents, followed by digitalization, chromogen removal, and antibody stripping. The method was combined with open-source tools for the coregistration of individual staining cycles. Using clear cell renal cell carcinoma (ccRCC) as a model, the protocol was applied for granular annotation of cellular and acellular structures in the tumor boundary zone. Our results demonstrate that the tumor periphery, particularly the pseudocapsule of ccRCC, is homogeneously organized across the 3D scale, yet exhibits distinct cellular distribution gradients of T and B cells. These patterns correspond to deposited extracellular matrix proteins, especially collagen types I and VI. Our findings indicate an instructive impact of extracellular matrix proteins on defining the spatial organization of immune cells in the TME of ccRCC. The developed cycIHC method facilitates detailed characterization of the TME and may enhance the understanding of tumor-immune cell interactions.