Genome-wide off-target analyses of CRISPR/Cas9-mediated T-cell receptor engineering in primary human T cells.

OBJECTIVES: Exploiting the forces of human T cells for treatment has led to the current paradigm of emerging immunotherapy strategies. Genetic engineering of the T-cell receptor (TCR) redirects specificity, ablates alloreactivity and brings significant progress and off-the-shelf options to emerging adoptive T-cell transfer (ACT) approaches. Targeted CRISPR/Cas9-mediated double-strand breaks in the DNA enable knockout or knock-in engineering. METHODS: Here, we perform CRISPR/Cas9-mediated TCR knockout using a therapeutically relevant ribonucleoprotein (RNP) delivery method to assess the safety of genetically engineered T-cell products. Whole-genome sequencing was performed to analyse whether CRISPR/Cas9-mediated DNA double-strand break at the TCR locus is associated with off-target events in human primary T cells. RESULTS: TCRα chain and TCRß chain knockout leads to high on-target InDel frequency and functional knockout. None of the predicted off-target sites could be confirmed experimentally, whereas whole-genome sequencing and manual Integrative Genomics Viewer (IGV) review revealed 9 potential low-frequency off-target events genome-wide. Subsequent amplification and targeted deep sequencing in 7 of 7 evaluable loci did not confirm these low-frequency InDels. Therefore, off-target events are unlikely to be caused by the CRISPR/Cas9 engineering. CONCLUSION: The combinatorial approach of whole-genome sequencing and targeted deep sequencing confirmed highly specific genetic engineering using CRISPR/Cas9-mediated TCR knockout without potentially harmful exonic off-target effects.

A multifunctional mouse model to study the role of Samd3.

The capacity to develop immunological memory is a hallmark of the adaptive immune system. To investigate the role of Samd3 for cellular immune responses and memory development, we generated a conditional knock-out mouse including a fluorescent reporter and a huDTR cassette for conditional depletion of Samd3-expressing cells. Samd3 expression was observed in NK cells and CD8 T cells, which are known for their specific function against intracellular pathogens like viruses. After acute viral infections, Samd3 expression was enriched within memory precursor cells and the frequency of Samd3-expressing cells increased during the progression into the memory phase. Similarly, during chronic viral infections, Samd3 expression was predominantly detected within precursors of exhausted CD8 T cells that are critical for viral control. At the functional level however, Samd3-deficient CD8 T cells were not compromised in the context of acute infection with Vaccinia virus or chronic infection with Lymphocytic choriomeningitis virus. Taken together, we describe a novel multifunctional mouse model to study the role of Samd3 and Samd3-expressing cells. We found that Samd3 is specifically expressed in NK cells, memory CD8 T cells, and precursor exhausted T cells during viral infections, while the molecular function of this enigmatic gene remains further unresolved.

BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.

Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

TALEN-mediated genome engineering to generate targeted mice.

Genetic mouse models are critical for biomedical research to understand gene function and pathophysiology. In the last years, the generation of genetic mouse models has been revolutionized by the emergence of transcription activator-like effector nucleases (TALENs). TALENs are programmable, sequence-specific DNA-binding proteins fused to a non-specific endonuclease domain used as powerful tools for site-specific induction of DNA double-strand breaks. These result in disruption of the gene product of the targeted locus by mutations induced during repair by error-prone non-homologous end-joining. Alternatively, these DNA double-strand breaks can be exploited to integrate a user-defined sequence by homologous recombination if an appropriate repair plasmid is provided. In this review, we highlight the major technological improvements for genome editing in murine oocytes which have been achieved using TALENs, discuss current limitations of the technology, suggest strategies to broadly apply TALENs, and describe possible future directions to facilitate gene editing in murine oocytes.