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.

CagA-specific Gastric CD8+ Tissue-Resident T Cells Control Helicobacter pylori During the Early Infection Phase.

BACKGROUND & AIMS: Infection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. METHODS: We comprehensively characterize gastric H pylori-specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. RESULTS: We define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. CONCLUSIONS: Our results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.

Helicobacter pylori promotes colorectal carcinogenesis by deregulating intestinal immunity and inducing a mucus-degrading microbiota signature.

OBJECTIVE: Helicobacter pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbour a nearly twofold increased risk to develop colorectal cancer (CRC). However, a direct causal and functional connection between H. pylori infection and colon cancer is lacking. DESIGN: We infected two Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive analysis of H. pylori-induced changes in intestinal immune responses and epithelial signatures via flow cytometry, chip cytometry, immunohistochemistry and single cell RNA sequencing. Microbial signatures were characterised and evaluated in germ-free mice and via stool transfer experiments. RESULTS: H. pylori infection accelerated tumour development in Apc-mutant mice. We identified a unique H. pylori-driven immune alteration signature characterised by a reduction in regulatory T cells and pro-inflammatory T cells. Furthermore, in the intestinal and colonic epithelium, H. pylori induced pro-carcinogenic STAT3 signalling and a loss of goblet cells, changes that have been shown to contribute-in combination with pro-inflammatory and mucus degrading microbial signatures-to tumour development. Similar immune and epithelial alterations were found in human colon biopsies from H. pylori-infected patients. Housing of Apc-mutant mice under germ-free conditions ameliorated, and early antibiotic eradication of H. pylori infection normalised the tumour incidence to the level of uninfected controls. CONCLUSIONS: Our studies provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis. Therefore, implementation of H. pylori status into preventive measures of CRC should be considered.

Increased LIGHT expression and activation of non-canonical NF-κB are observed in gastric lesions of MyD88-deficient mice upon Helicobacter felis infection.

Helicobacter pylori infection induces a number of pro-inflammatory signaling pathways contributing to gastric inflammation and carcinogenesis. Among those, NF-κB signaling plays a pivotal role during infection and malignant transformation of the gastric epithelium. However, deficiency of the adaptor molecule myeloid differentiation primary response 88 (MyD88), which signals through NF-κB, led to an accelerated development of gastric pathology upon H. felis infection, but the mechanisms leading to this phenotype remained elusive. Non-canonical NF-κB signaling was shown to aggravate H. pylori-induced gastric inflammation via activation of the lymphotoxin ß receptor (LTßR). In the present study, we explored whether the exacerbated pathology observed in MyD88-deficient (Myd88-/-) mice was associated with aberrant activation of non-canonical NF-κB. Our results indicate that, in the absence of MyD88, H. felis infection enhances the activation of non-canonical NF-κB that is associated with increase in Cxcl9 and Icam1 gene expression and CD3+ lymphocyte recruitment. In addition, activation of signal transducer and activator of transcription 3 (STAT3) signaling was higher in Myd88-/- compared to wild type (WT) mice, indicating a link between MyD88 deficiency and STAT3 activation in response to H. felis infection. Thereby, MyD88 deficiency results in accelerated and aggravated gastric pathology induced by Helicobacter through activation of non-canonical NF-κB.

Helicobacter pylori γ-glutamyl transferase contributes to colonization and differential recruitment of T cells during persistence.

Helicobacter pylori γ-glutamyl transferase (gGT) is a key bacterial virulence factor that is not only important for bacterial gastric colonization but also related to the development of gastric pathology. Despite accumulating evidence for pathogenic and immunologic functions of H. pylori gGT, it is still unclear how it supports gastric colonization and how its specific effects on the host's innate and adaptive immune responses contribute to colonization and pathology. We have compared mice showing similar bacterial load after infection with gGT-proficient or gGT-deficient H. pylori to analyse the specific role of the enzyme during infection. Our data indicate that H. pylori gGT supports initial colonization. Nevertheless, bacteria lacking gGT can still colonize and persist. We observed that the presence of gGT during infection favoured a proinflammatory innate and adaptive immune response. Notably, H. pylori gGT activity was linked to increased levels of IFNγ, which were attributed to a differential recruitment of CD8+ T cells to the stomach. Our data support an essential role for H. pylori gGT in gastric colonization and further suggest that gGT favours infiltration of CD8+ cells to the gastric mucosa, which might play an important and yet overlooked role in the pathogenesis of H. pylori.

Dual-Track Clearance of Circulating Bacteria Balances Rapid Restoration of Blood Sterility with Induction of Adaptive Immunity.

Efficient clearance of bacteremia prevents life-threatening disease. Platelet binding to intravascular bacteria, a process involving platelet glycoprotein GPIb and bacterial opsonization with activated complement C3, influences blood clearance and anti-infective immunity. Using intravital microscopy of the bloodstream of mice infected with Listeria monocytogenes, we show that bacterial clearance is not a uniform process but a "dual-track" mechanism consisting of parallel "fast" and "slow" pathways. "Slow clearance" is regulated by time-dependent bacterial opsonization, stochastic platelet binding, and capture of bacteria-platelet-complexes via the complement receptor of the immunoglobulin superfamily, CRIg. The mechanism spares some bacteria from "fast clearance" and rapid destruction in the liver via Kupffer cell scavenger receptors, keeping them available for adaptive immunity induction by splenic CD8α(+) dendritic cells. We consistently find "fast" and "slow" clearance patterns for a broad panel of other Gram+ and Gram- bacteria. Thus, dual-track clearance balances rapid restoration of blood sterility with induction of specific antibacterial immunity.