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Cerebral (Aβ) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aβ/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aβ/pTau, however, appears to vary depending on the animal model. Our prior work suggested that antigen-specific memory CD8 T ("hiT") cells act upstream of Aβ/pTau after brain injury. Here, we examine whether hiT cells influence sporadic AD-like pathophysiology upstream of Aβ/pTau. Examining neuropathology, gene expression, and behavior in our hiT mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. We identify an age-related factor acting upstream of Aβ/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD. [ABSTRACT FROM AUTHOR]

Background: Chimeric antigen receptor (CAR) T-cell therapy has proven to be a valuable new treatment option for patients with B-cell malignancies. However, by applying selective pressure, outgrowth of antigen-negative tumor cells can occur, eventually resulting in relapse. Subsequent rescue by administration of CAR-T cells with different antigen-specificity indicates that those tumor cells are still sensitive to CAR-T treatment and points towards a multi-target strategy. Due to their natural tumor sensitivity and highly cytotoxic nature, natural killer (NK) cells are a compelling alternative to T cells, especially considering the availability of an off-the-shelf unlimited supply in the form of the clinically validated NK-92 cell line.Methods: Given our goal to develop a flexible system whereby the CAR expression repertoire of the effector cells can be rapidly adapted to the changing antigen expression profile of the target cells, electrotransfection with CD19-/BCMA-CAR mRNA was chosen as CAR loading method in this study. We evaluated the functionality of mRNA-engineered dual-CAR NK-92 against tumor B-cell lines and primary patient samples. In order to test the clinical applicability of the proposed cell therapy product, the effect of irradiation on the proliferative rate and functionality of dual-CAR NK-92 cells was investigated.Results: Co-electroporation of CD19 and BMCA CAR mRNA was highly efficient, resulting in 88.1% dual-CAR NK-92 cells. In terms of CD107a degranulation, and secretion of interferon (IFN)-γ and granzyme B, dual-CAR NK-92 significantly outperformed single-CAR NK-92. More importantly, the killing capacity of dual-CAR NK-92 exceeded 60% of single and dual antigen-expressing cell lines, as well as primary tumor cells, in a 4h co-culture assay at low effector to target ratios, matching that of single-CAR counterparts. Furthermore, our results confirm that dual-CAR NK-92 irradiated with 10 Gy cease to proliferate and are gradually cleared while maintaining their killing capacity.Conclusions: Here, using the clinically validated NK-92 cell line as a therapeutic cell source, we established a readily accessible and flexible platform for the generation of highly functional dual-targeted CAR-NK cells. [ABSTRACT FROM AUTHOR]

The Wilms’ tumor protein 1 (WT1) is a well-known and prioritized tumor-associated antigen expressed in numerous solid and blood tumors. Its abundance and immunogenicity have led to the development of different WT1-specific immune therapies. The driving player in these therapies, the WT1-specific T-cell receptor (TCR) repertoire, has received much less attention. Importantly, T cells with high affinity against the WT1 self-antigen are normally eliminated after negative selection in the thymus and are thus rare in peripheral blood. Here, we developed computational models for the robust and fast identification of WT1-specific TCRs from TCR repertoire data. To this end, WT137-45 (WT1-37) and WT1126-134 (WT1-126)-specific T cells were isolated from WT1 peptide-stimulated blood of healthy individuals. The TCR repertoire from these WT1-specific T cells was sequenced and used to train a pattern recognition model for the identification of WT1-specific TCR patterns for the WT1-37 or WT1-126 epitopes. The resulting computational models were applied on an independent published dataset from acute myeloid leukemia (AML) patients, treated with hematopoietic stem cell transplantation, to track WT1-specific TCRs in silico. Several WT1-specific TCRs were found in AML patients. Subsequent clustering analysis of all repertoires indicated the presence of more diverse TCR patterns within the WT1-specific TCR repertoires of AML patients in complete remission in contrast to relapsing patients. We demonstrate the possibility of tracking WT1-37 and WT1-126-specific TCRs directly from TCR repertoire data using computational methods, eliminating the need for additional blood samples and experiments for the two studied WT1 epitopes. [ABSTRACT FROM AUTHOR]

• High number of stable, pure and functional Tregs upon GMP-compliant cell culture • Tregs can be engineered by TCR-encoding mRNA electroporation • mRNA-engineered Tregs remain functional and phenotypically and epigenetically stable Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance. This unique capacity of Tregs, in combination with proof-of-principle in preclinical studies, highlights the potential clinical use of Tregs for the treatment of autoimmunity and transplant rejection. Although proven to be safe and well tolerated in the first clinical trials, only modest clinical results were observed. In this regard, it has been hypothesized that current challenges lie in the development of antigen-specific Tregs. Here, we present an innovative, good manufacturing practices (GMP)-compliant manufacturing protocol for Tregs applicable in a clinical-grade setting, allowing efficient and safe redirection of Treg specificity. First, a soluble polymer conjugated with antibodies to CD3 and CD28 and high amounts of exogenous IL-2 for in vitro Treg expansion resulted in a >70-fold and 185-fold increase of a pure population of CD4+CD127−CD25hi Tregs and CD4+CD127−CD25+CD45RA+ Tregs, respectively. Next, as a proof-of-principle, expanded Tregs were engineered by means of TCR-encoding mRNA electroporation to generate antigen-specific Tregs. This resulted in an expression of the newly introduced TCR in up to 85% of Tregs. Moreover, we did not observe a negative effect on the phenotype of Tregs, as demonstrated by the expression of FOXP3, Helios, CTLA-4 and CCR4, nor on the TSDR methylation status. Importantly, mRNA-engineered Tregs were still able to induce in vitro suppression of effector T cells and produced anti-inflammatory, but not pro-inflammatory, cytokines when activated. In conclusion, our findings demonstrate that high numbers of stable and functional Tregs can be obtained with high purity and successfully engineered for gain of function, in a GMP-compliant manner. We envisage that this clinical-grade protocol will provide solid basis for future clinical application of mRNA-engineered Tregs. [ABSTRACT FROM AUTHOR]

Antigen recognition through the T cell receptor (TCR) aβ heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naïve T cells with high specificity to expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naïve hosts. In this study, we use high-throughput sequencing of memory CD4 TCRβ repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCRβ sequence patterns into a hepatitis B epitope-specific annotation model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T cell clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination. [ABSTRACT FROM AUTHOR]