Long-term persistence and functionality of adoptively transferred antigen-specific T cells with genetically ablated PD-1 expression.

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.

Orthotopic replacement of T-cell receptor α- and ß-chains with preservation of near-physiological T-cell function.

Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and ß-chains combined with orthotopic TCR placement leads to accurate αß-pairing and results in TCR regulation similar to that of physiological T cells.

miRNA92a targets KLF2 and the phosphatase PTEN signaling to promote human T follicular helper precursors in T1D islet autoimmunity.

Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)+CD4+ TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity.

Type 1 diabetes vaccine candidates promote human Foxp3(+)Treg induction in humanized mice.

Immune tolerance is executed partly by Foxp3(+)regulatory T (Treg) cells, which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing ß-cells. The development of autoantigen-specific vaccination strategies for Foxp3(+)Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here, using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice, we provide direct evidence for human autoantigen-specific Foxp3(+)Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4(+)T cells and demonstrate efficient human insulin-specific Foxp3(+)Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable, show increased expression of Treg signature genes such as Foxp3, CTLA4, IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3(+)Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3(+)Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D.

Canine-DCs using different serum-free methods as an approach to provide an animal-model for immunotherapeutic strategies.

Animal-models are the basis of DC-based human immunotherapies. We describe the standardization of a canine-DC-generation protocol using different cytokines and characterize the quality and functional repertoire of the obtained canine-DCs. DCs were generated from healthy dog-PBMCs under serum-free and serum-containing conditions. DC-quality and -quantity was determined by FACS studying the expression-profiles of DC-/costimulatory- and maturation-antigens before/after culture with canine and human monoclonal-antibodies (cmabs/hmabs). Individual DCAgs-(DC-antigens)-expression-profiles were found before and after culture depending on the agents' mode-of-action. With at least one of three serum-free methods (Ca-Ionophore, Picibanil, Cytokines) sufficient DC-amounts were generated. So, canine-DCs can be regularly generated under serum-free conditions and hmabs additionally to cmabs qualify for staining/quantification of canine-cells/DCs. The canine-DCs were functional, shown by T-cell-activation, -proliferation and antigen-specific CTL-responses. In summary, successful, quantitative DC-generation is possible with serum-free methods. DC-based T-cell-vaccination-strategies evaluated for e.g. AML-patients can be tested in the dog and estimated in clinical studies for DC-vaccination-strategies.

In vivo confocal microscopy through-focusing to measure corneal flap thickness after laser in situ keratomileusis.

PURPOSE: To measure flap thickness in laser in situ keratomileusis (LASIK) patients using in vivo confocal microscopy through-focusing (CMTF) and compare measured versus intended flap thickness achieved by 2 microkeratomes, the Automated Corneal Shaper(R) (ACS) (Chiron Bausch & Lomb) and the Hansatome (Bausch & Lomb). SETTING: Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA. METHODS: Twenty-seven eyes of 27 patients were examined by in vivo CMTF 3 to 12 months after LASIK was performed with the ACS (12 patients) or Hansatome (15 patients) microkeratome. The central cornea was scanned, and the epithelium, flap, stroma, and total corneal thickness were measured. Normalized flap thickness (NFT) was also calculated to account for changes in epithelial thickness that may have occurred postoperatively. RESULTS: The mean posterior stromal thickness was 341.1 microm +/- 53.9 (SD) (range 233 to 431 microm) in the ACS group and 320.3 +/- 42.3 microm (range 258 to 382 microm) in the Hansatome group. The mean nonnormalized flap thickness was 132.7 +/- 12.5 microm (range 11 to 151 microm) in the ACS group and 167.4 +/- 21.4 microm (range 141 to 209 microm) in the Hansatome group. The NFT was 129.6 +/- 9.5 microm and 158.4 +/- 22.1 microm, respectively. Both microkeratomes cut significantly less than intended (P <.05); however, the ACS cut a thinner-than-intended thickness in all cases, and the Hansatome cut thicker than intended in 13% of cases. The Hansatome also showed significantly greater variability in flap thickness than the ACS (P <.05). CONCLUSIONS: A significant difference in precision was noted between the 2 microkeratomes. The findings emphasize the importance of performing thickness measurements and the usefulness of in vivo CMTF in making these determinations to ensure the safety and effectiveness of LASIK.