The major barrier to effective non-viral T cell genome targeting of large DNA sequences has been the toxicity of the DNA 10 . While the introduction of short singlestranded oligodeoxynucleotide (ssODN) HDR templates does not cause significant T cell death, it has been shown that larger linear double stranded (dsDNA) templates are toxic at high concentrations 11,12 . Contrary to expectations, we found that co-electroporation of human primary T cells with CRISPR-Cas9 ribonucleoprotein (Cas9 RNP 13,14 ) complexes and long (>1kb) linear dsDNA templates reduced the toxicity associated with the dsDNA template (Extended Data Fig 1). Cas9 RNPs were co-electroporated with a dsDNA HDR template designed to introduce an N-terminal GFP-fusion in the housekeeping peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/183418 doi: bioRxiv preprint first posted online Aug. 31, 2017; 3 gene RAB11A (Fig. 1a). Systematic exploration of this approach while optimizing for both viability and efficiency ( Fig. 1b and Extended Data Fig. 2) resulted in GFP expression in ~50% of cells in both primary human CD4+ and CD8+ T cells. The method was reproducibly efficient while maintaining high cell viability and expandability (Fig. 1c, d, e, and Extended Data Fig. 3). The system is also compatible with current manufacturing protocols for cell therapies as it could be applied to fresh or cryopreserved cells, bulk T cells or FACS-sorted sub-populations, and cells from whole blood or leukapheresis (Extended Data Fig. 4).We next confirmed that the system could be applied broadly by targeting sequences in different locations throughout the genome. We efficiently engineered GFP+ primary T cells by generating fusions with different genes (Fig. 2a and Fig. 3a and Extended Data Fig. 14). One mutation, c.530A>G, creates a premature stop codon. With non-viral genome targeting, we were able to correct the mutation and observe IL2RA expression on the surface of corrected T cells from the patient (Fig. 3b). Long dsDNA templates led to efficient correction of the mutations. Because only two base pair changes were necessary (one to correct the mutation and one to silently remove the gRNA's PAM sequence), a short single-stranded DNA (~120 bps) could also be used to make the correction. These single-stranded DNAs were able to correct the mutation at high frequencies, although the efficiency of correction was lower than with the longer dsDNA template (Extended Data Fig. 15, 16).Correction was successful in T cells from all three siblings, but lower rates of IL2RA expression were seen in compound het 3, which could be due to altered cell-state associated with the patient's disease or the fact she was the only sibling treated with immunosuppressive therapy (Extended Data Table 1 and Extended Data Fig. 17). The second mutation identified, c.800delA, causes a frameshift in the reading frame of the final IL2RA exon. This frameshift mutation c...

• Human IL-6 improves T-cell engraftment and serum IgG production in humanized mice.• IgG-switched memory B cells in IL-6 knock-in mice displayed a diverse antibody repertoire and high specificity against immunized antigen.Humanized mice are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, the existing models cannot support robust adaptive immune responses, especially the generation of class-switched, antigen-specific antibody responses. Here we describe a new mouse strain, in which human interleukin 6 (IL-6) gene encoding the cytokine that is important for B-and T-cell differentiation was knocked into its respective mouse locus. The provision of human IL-6 not only enhanced thymopoiesis and periphery T-cell engraftment, but also significantly increased class switched memory B cells and serum immunoglobulin G (IgG). In addition, immunization with ovalbumin (OVA) induced OVA-specific B cells only in human IL-6 knock-in mice. These OVA-specific antibodies displayed the highest frequency of somatic mutation, further suggesting that human IL-6 is important for efficient B-cell activation and selection. We conclude that human IL-6 knock-in mice represent a novel and improved model for human adaptive immunity without relying on complex surgery to transplant human fetal thymus and liver. These mice can therefore be used to exploit or evaluate immunization regimes that would be unethical or untenable in humans. (Blood. 2017;129(8):959-969)

Autoimmune regulator (AIRE) mutations result in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome characterized by defective central T cell tolerance and the production of many autoantibodies targeting tissue-specific antigens and cytokines. By studying CD3- and AIRE-deficient patients, we found that lack of either T cells or AIRE function resulted in the peripheral accumulation of autoreactive mature naïve B cells. Proteomic arrays and Biacore affinity measurements revealed that unmutated antibodies expressed by these autoreactive naïve B cells recognized soluble molecules and cytokines including insulin, IL-17A, and IL-17F, which are AIRE-dependent thymic peripheral tissue antigens targeted by autoimmune responses in APECED. AIRE-deficient patients also displayed decreased frequencies of regulatory T cells (Tregs) that lacked common TCRβ clones found instead in their conventional T cell compartment, thereby suggesting holes in the Treg TCR repertoire of these patients. Hence, AIRE-mediated T cell/Treg selection normally prevents the expansion of autoreactive naïve B cells recognizing peripheral self-antigens.

The human VH4-34 gene segment encodes intrinsically self-reactive antibodies that recognize I/i carbohydrates. Schickel et al. show that these self-reactive clones may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached.

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