Conserved stromal-immune cell circuits secure B cell homeostasis and function

B cell zone reticular cells (BRCs) form stable microenvironments that direct efficient humoral immunity with B cell priming and memory maintenance being orchestrated across lymphoid organs. However, a comprehensive understanding of systemic humoral immunity is hampered by the lack of knowledge of global BRC sustenance, function and major pathways controlling BRC-immune cell interactions. Here we dissected the BRC landscape and immune cell interactome in human and murine lymphoid organs. In addition to the major BRC subsets underpinning the follicle, including follicular dendritic cells, PI16$^{+}$ RCs were present across organs and species. As well as BRC-produced niche factors, immune cell-driven BRC differentiation and activation programs governed the convergence of shared BRC subsets, overwriting tissue-specific gene signatures. Our data reveal that a canonical set of immune cell-provided cues enforce bidirectional signaling programs that sustain functional BRC niches across lymphoid organs and species, thereby securing efficient humoral immunity

Viral vector-mediated reprogramming of the fibroblastic tumor stroma sustains curative melanoma treatment.

The tumor microenvironment (TME) is a complex amalgam of tumor cells, immune cells, endothelial cells and fibroblastic stromal cells (FSC). Cancer-associated fibroblasts are generally seen as tumor-promoting entity. However, it is conceivable that particular FSC populations within the TME contribute to immune-mediated tumor control. Here, we show that intratumoral treatment of mice with a recombinant lymphocytic choriomeningitis virus-based vaccine vector expressing a melanocyte differentiation antigen resulted in T cell-dependent long-term control of melanomas. Using single-cell RNA-seq analysis, we demonstrate that viral vector-mediated transduction reprogrammed and activated a Cxcl13-expressing FSC subset that show a pronounced immunostimulatory signature and increased expression of the inflammatory cytokine IL-33. Ablation of Il33 gene expression in Cxcl13-Cre-positive FSCs reduces the functionality of intratumoral T cells and unleashes tumor growth. Thus, reprogramming of FSCs by a self-antigen-expressing viral vector in the TME is critical for curative melanoma treatment by locally sustaining the activity of tumor-specific T cells

Bone morphogenic protein-4 availability in the cardiac microenvironment controls inflammation and fibrosis in autoimmune myocarditis

Myocarditis is an inflammatory heart disease that leads to loss of cardiomyocytes and frequently precipitates fibrotic remodeling of the myocardium, culminating in heart failure. However, the molecular mechanisms underlying immune cell control and maintenance of tissue integrity in the inflamed cardiac microenvironment remain elusive. In this study, we found that bone morphogenic protein-4 (BMP4) gradients maintain cardiac tissue homeostasis by single-cell transcriptomics analyses of inflamed murine and human myocardial tissues. Cardiac BMP pathway dysregulation was reflected by reduced BMP4 serum concentration in patients with myocarditis. Restoration of BMP signaling by antibody-mediated neutralization of the BMP inhibitors gremlin-1 and gremlin-2 ameliorated T cell-induced myocardial inflammation in mice. Moreover, progression to inflammatory cardiomyopathy was blocked through the reduction of fibrotic remodeling and preservation of cardiomyocyte integrity. These results unveil the BMP4–gremlin axis as a druggable pathway for the treatment of myocardial inflammation, limiting the severe sequelae of cardiac fibrosis and heart failure

Salmonella Typhi Porins OmpC and OmpF Are Potent Adjuvants for T-Dependent and T-Independent Antigens

Several microbial components, such as bacterial DNA and flagellin, have been used as experimental vaccine adjuvants because of their inherent capacity to efficiently activate innate immune responses. Likewise, our previous work has shown that the major Salmonella Typhi (S. Typhi) outer membrane proteins OmpC and OmpF (porins) are highly immunogenic protective antigens that efficiently stimulate innate and adaptive immune responses in the absence of exogenous adjuvants. Moreover, S. Typhi porins induce the expression of costimulatory molecules on antigen-presenting cells through toll-like receptor canonical signaling pathways. However, the potential of major S. Typhi porins to be used as vaccine adjuvants remains unknown. Here, we evaluated the adjuvant properties of S. Typhi porins against a range of experimental and clinically relevant antigens. Co-immunization of S. Typhi porins with ovalbumin (OVA), an otherwise poorly immunogenic antigen, enhanced anti-OVA IgG titers, antibody class switching, and affinity maturation. This adjuvant effect was dependent on CD4+ T-cell cooperation and was associated with an increase in IFN-γ, IL-17A, and IL-2 production by OVA-specific CD4+ T cells. Furthermore, co-immunization of S. Typhi porins with an inactivated H1N1 2009 pandemic influenza virus experimental vaccine elicited higher hemagglutinating anti-influenza IgG titers, antibody class switching, and affinity maturation. Unexpectedly, co-administration of S. Typhi porins with purified, unconjugated Vi capsular polysaccharide vaccine (Vi CPS)—a T-independent antigen—induced higher IgG antibody titers and class switching. Together, our results suggest that S. Typhi porins OmpC and OmpF are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens

Dendritic cell-specific delivery of Flt3L by coronavirus vectors secures induction of therapeutic antitumor immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8(+) T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8(+) T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity

Evolution of <i>Salmonella Typhi</i> outer membrane protein-specific T and B cell responses in humans following oral Ty21a vaccination: A randomized clinical trial

<div><p>Vaccination against complex pathogens such as typhoidal and non-typhoidal <i>Salmonella</i> requires the concerted action of different immune effector mechanisms. Outer membrane proteins (Omps) of <i>Salmonella</i> Typhi are potent immunogens, which elicit long-lasting and protective immunity. Here, we followed the evolution of <i>S</i>. Typhi OmpC and F-specific T and B cell responses in healthy volunteers after vaccination with the vaccine strain Ty21a. To follow humoral and cellular immune responses, pre- and post-vaccination samples (PBMC, serum and stool) collected from 15 vaccinated and 5 non-vaccinated individuals. Immunoglobulin levels were assessed in peripheral blood by enzyme-linked immunosorbent assay. B cell and T cell activation were analyzed by flow cytometry. We observed a significant increase of circulating antibody-secreting cells and maximal Omp-specific serum IgG titers at day 25 post vaccination, while IgA titers in stool peaked at day 60. Likewise, Omp-specific CD4⁺ T cells in peripheral blood showed the highest expansion at day 60 post vaccination, concomitant with a significant increase in IFN-γ and TNFα production. These results indicate that <i>S</i>. Typhi Omp-specific B cell responses and polyfunctional CD4⁺ T cell responses evolve over a period of at least two months after application of the live attenuated vaccine. Moreover, these findings underscore the potential of <i>S</i>. Typhi Omps as subunit vaccine components.</p><p><b>Trial registration:</b><a href="https://clinicaltrials.gov/ct2/show/ISRCTN18360696" target="_blank">ISRCTN18360696</a></p></div

Intestinal fibroblastic reticular cell niches control innate lymphoid cell homeostasis and function

Innate lymphoid cells (ILCs) govern immune cell homeostasis in the intestine and protect the host against microbial pathogens. Various cell-intrinsic pathways have been identified that determine ILC development and differentiation. However, the cellular components that regulate ILC sustenance and function in the intestinal lamina propria are less known. Using single-cell transcriptomic analysis of lamina propria fibroblasts, we identify fibroblastic reticular cells (FRCs) that underpin cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Genetic ablation of lymphotoxin-beta receptor expression in Ccl19-expressing FRCs blocks the maturation of CPs into mature ILFs. Interactome analysis shows the major niche factors and processes underlying FRC-ILC crosstalk. In vivo validation confirms that a sustained lymphotoxin-driven feedforward loop of FRC activation including IL-7 generation is critical for the maintenance of functional ILC populations. In sum, our study indicates critical fibroblastic niches within the intestinal lamina propria that control ILC homeostasis and functionality and thereby secure protective gut immunity. Fibroblastic reticular cells (FRCs) support localisation of immune cells in secondary lymphoid tissues but less is known about the lamina propria. Here the authors use scRNA-seq and intestinal infection to characterise FRCs in the intestinal lamina propria and show specialised niches that foster innate lymphoid cells during homeostasis and infection

Generation and in vitro characterization of cytokine-encoding murine coronavirus vectors.

<p>(A) Schematic representation of MHV A59 genome and construction of cytokine-encoding vectors. (B, C) Growth kinetics of the indicated MHV vectors in 17ECl20 packaging cells (B) and macrophages (Mph) (C). Cells were infected at an MOI of 1 and titres in supernatants were determined at the indicated time points. (D) Transduction in bone marrow-derived DCs with murine coronavirus vectors. DCs from B6 mice were transduced with the indicated vectors at a multiplicity of infection (MOI) of 1. Cells were harvested 24 h later and EGFP expression on CD11c⁺ cells was assessed. Pooled data from three independent experiments with values indicating mean percentage ±SEM of EGFP⁺CD11c⁺ cells. (E) Cytokine production induced by Flt3L and IL-2 encoding vectors. DCs or macrophages were transduced at a MOI of 1 and concentration of cytokines in the supernatants was determined by ELISA. Representative data from one out of three independent experiments.</p

Prophylactic and therapeutic tumor immunity against a peripheral solid tumor.

<p>(A) B6 mice were i.v. immunized with 10⁵ pfu of the different vectors or received PBS as control. Seven days later, 5×10⁵ gp-recombinant Lewis lung carcinoma cells were injected s.c. on the left flank. (B) Assessment of therapeutic tumor immunity induced by 10⁵ pfu of the different vectors applied on day 4 post s.c. inoculation with 5×10⁵ gp-recombinant Lewis lung carcinoma cells. Tumor growth was monitored on the indicated days. Values indicate mean tumor volume ±SEM (n=9 mice). Values in parentheses indicate number of growing tumors of inoculated tumors. Statistical analysis in (B) was performed using one way ANOVA with Bonferroni multiple comparison test (**, p< 0.01; ***, p< 0.001; ns, non-significant).</p

In vivo maturation of DCs following vaccination with coronavirus vectors.

<p>B6 mice were i.v. immunized with 10⁶ pfu of the indicated viral vectors or left untreated (mock). (A, B) Transduction of DCs as assessed by EGFP expression. Spleens were collected after 24 h, digested with collagenase and low-density cells were analyzed by flow cytometry. Values in dot plots (A) and bar graph (B) show the mean percentage ± SEM of EGFP⁺IA^bhigh cells gated on CD11c⁺ cells. Data from two independent experiments with three mice per group (n=6). (C, D) Cytokine concentration in serum and spleen homogenates at 24 h post immunization with Flt3L (C) and IL-2 (D) vectors. Pooled data from three independent experiments with three mice per group (mean ±SEM, n=9). (E) Representative histograms showing expression of the DC maturation markers CD40 and CD86 on EGFP⁺CD11c⁺ cells transduced with the indicated vectors. (F) Mean fluorescence values ±SEM (n=9) of CD40 and CD86 expression in transduced EGFP⁺CD11c⁺ cells (+) and non-transduced EGFP⁺CD11c^- cells (-) cells (*, p< 0.05; **, p< 0.01).</p