The Probiotic Compound VSL#3 Modulates Mucosal, Peripheral, and Systemic Immunity Following Murine Broad-Spectrum Antibiotic Treatment

There is compelling evidence linking the commensal intestinal microbiota with host health and, in turn, antibiotic induced perturbations of microbiota composition with distinct pathologies. Despite the attractiveness of probiotic therapy as a tool to beneficially alter the intestinal microbiota, its immunological effects are still incompletely understood. The aim of the present study was to assess the efficacy of the probiotic formulation VSL#3 consisting of eight distinct bacterial species (including Streptococcus thermophilus, Bifidobacterium breve, B. longum, B. infantis, Lactobacillus acidophilus, L. plantarum, L. paracasei, and L. delbrueckii subsp. Bulgaricus) in reversing immunological effects of microbiota depletion as compared to reassociation with a complex murine microbiota. To address this, conventional mice were subjected to broad-spectrum antibiotic therapy for 8 weeks and perorally reassociated with either VSL#3 bacteria or a complex murine microbiota. VSL#3 recolonization resulted in restored CD4+ and CD8+ cell numbers in the small and large intestinal lamina propria as well as in B220+ cell numbers in the former, whereas probiotic intervention was not sufficient to reverse the antibiotic induced changes of respective cell populations in the spleen. However, VSL#3 application was as efficient as complex microbiota reassociation to attenuate the frequencies of regulatory T cells, activated dendritic cells and memory/effector T cells in the small intestine, colon, mesenteric lymph nodes, and spleen. Whereas broad-spectrum antibiotic treatment resulted in decreased production of cytokines such as IFN-γ, IL-17, IL-22, and IL-10 by CD4+ cells in respective immunological compartments, VSL#3 recolonization was sufficient to completely recover the expression of the anti-inflammatory cytokine IL-10 without affecting pro-inflammatory mediators. In summary, the probiotic compound VSL#3 has an extensive impact on mucosal, peripheral, and systemic innate as well as adaptive immunity, exerting beneficial anti-inflammatory effects in intestinal as well as systemic compartments. Hence, VSL#3 might be considered a therapeutic immunomodulatory tool following antibiotic therapy

Schlussbericht

Neuromyelitis optica spectrum disorders (NMOSD) form a rare group of severe antibody-mediated autoimmune diseases (AID) of the central nervous system, requiring lifelong therapy with potentially severe side effects. Aquaporin-4 (AQP4) has been identified as a specific target of autoantibodies in most NMOSD patients. AQP4-specific T cells are centrally involved in disease pathogenesis and chronification. They support the formation of anti-AQP-4-antibody-producing plasma cells and are required for disease transfer in animal models. Dynamic changes in autoreactive CD4 T cell activity in NMOSD may therefore explain why autoantibody levels do not correlate with clinical disease activity. Thus, autoreactive T cell characteristics should provide fundamental insight into disease etiology and allow for refined diagnosis, prognosis as well as personalized therapy of NMOSD. However, technical limitations so far prevented the characterization of AQP4-specific T cells and their NMOSD-associated quantitative, phenotypic or functional alterations. We have developed powerful technologies for the characterization of human effector- and regulatory autoantigen-specific CD4 T cells. In a proof of principle experiment we identified a unique and discriminative phenotype of AQP4-specific T cells in AQP4 antibody-positive but not -negative NMOSD patients. Our consortium also provides unique expertise to study T:B cell interaction, to determine the full spectrum of peptides recognized by selected TCR and to characterize patients microbiota. We propose to molecularly and functionally characterize autoreactive T cells and their interaction with B cells, as well as to define their self and foreign, e.g. microbiota-associated, peptide/MHC antigens as potential trigger of the pathogenic response. Integration of a biotech company will allow developing optimized analysis tools for their rapid application in basic research and clinical routine. Our project on autoreactive lymphocyte characteristics will thus make a pioneer contribution to the understanding of human AIDs, which will provide the basis for the future development of refined diagnostics, beyond mere antibody analysis, and for disease prognosis, monitoring as well as autoantigen-specific therapeutics. We anticipate that the results on disease-relevant T cell characteristics, which will be obtained using this human AID with well-characterized target autoantigens, will also reach out to other more frequent autoimmune diseases with less well-defined autoantigen targets, such as multiple sclerosis, type 1 diabetes or rheumatoid arthritis

T-cell subsets in allergy and tolerance induction

Antigen-specific T lymphocytes are the central regulators of tolerance versus immune pathology against otherwise innocuous antigens and key targets of antigen-specific immune therapy. Recent advances in the understanding of T cells in tolerance and allergy resulted from improved technologies to directly characterize allergen-specific T cells by multiparameter flow cytometry or single-cell sequencing. This unravelled phenotypically and functionally distinct populations, such as Type 2a T helper cells (Th2a), follicular Th cells (Tfh), regulatory T cells (Treg), Type 1 regulatory T cells (Tr1), and follicular T regulatory cells. Here we will discuss the role of the different Th-cell subsets in the healthy state, during sensitization and development of allergy, and in tolerance induction by allergen immunotherapy (AIT). To date, the mechanisms of AIT as the only causal treatment of allergy are not completely understood. The analyses of allergen-specific T cells directly ex vivo during AIT support the concept of specific-Th2(a) cell deletion rather than an expansion of allergen-specific Tr1 or Treg cells as underlying mechanism

Proliferative activity of antigen-specific CD154+ T cells against bacterial and fungal respiratory pathogens in cystic fibrosis decreases after initiation of highly effective CFTR modulator therapy

Background: Together with impaired mucociliary clearance, lung disease in cystic fibrosis (CF) is driven by dysregulation of innate and adaptive immunity caused by dysfunctional CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), leading to airway infection and hyperinflamma-tion. The highly effective CFTR modulator therapy (HEMT) elexacaftor/tezacaftor/ivacaftor (ETI) generates substantial improvements in clinical outcomes of people with CF (pwCF) by restoration of CFTR activity. Aberrant immune responses of lymphocytes due to CFTR dysfunction has been described in the past, but not the effects of CFTR restoration by HEMT on these cells. We aimed to examine the effect of ETI on the proliferative activity of antigen-specific CD154 (+) T cells against bacterial and fungal species relevant in CF and on total IgG and IgE as markers of B cell adaptive immunity. Results: Mean Ki-67 expression in antigen-specific CD154 (+) T cells against P. aeruginosa, A. fumigatus, S. apiospermum and C. albicans, but not S. aureus, mean total serum IgG and mean total serum IgE decreased significantly after initiation of ETI. No correlation was found to change in sputum microbiology of the examined pathogens. Mean BMI and FEV1 increased significantly. Conclusion: HEMT is associated with decreased antigen-specific CD154 (+) T cell proliferation activity in our cohort, independent of findings in sputum microbiology of the examined pathogens. Together with the observed clinical improvement and the decrease in total IgE and IgG, this indicates effects due to CFTR restoration on CD154 (+) T cells by ETI and a reduction of B cell activation with subsequent lower immunoglobulin synthesis under HEMT therapy. These results endorse earlier evidence of CFTR dysfunction in T and B cells leading directly to aberrant immune responses with hyperinflammation

Decreased inflammatory cytokine production of antigen-specific CD4+ T cells in NMDA receptor encephalitis

Anti-N-methyl-D-aspartate-receptor (NMDAR) encephalitis is the most common autoimmune encephalitis with psychosis, amnesia, seizures and dyskinesias. The disease is mediated by pathogenic autoantibodies against the NR1 subunit that disrupt NMDAR function. Antibody infusion into mouse brains can recapitulate encephalitis symptoms, while active immunization resulted also in strong T cell infiltration into the hippocampus. However, whether T cells react against NMDAR and their specific contribution to disease development are poorly understood. Here we characterized the ex vivo frequency and phenotype of circulating CD4(+) T helper (T-H) cells reactive to NR1 protein using antigen-reactive T cell enrichment (ARTE) in 24 patients with NMDAR encephalitis, 13 patients with LGI1 encephalitis and 51 matched controls. Unexpectedly, patients with NMDAR encephalitis had lower frequencies of CD154-expressing NR1-reactive T-H cells than healthy controls and produced significantly less inflammatory cytokines. No difference was seen in T cells reactive to the synaptic target LGI1 (Leucine-rich glioma-inactivated 1), ubiquitous Candida antigens or neoantigens, suggesting that the findings are disease-specific and not related to therapeutic immunosuppression. Also, patients with LGI1 encephalitis showed unaltered numbers of LGI1 antigen-reactive T cells. The data reveal disease-specific functional alterations of circulating NMDAR-reactive T-H cells in patients with NMDAR encephalitis and challenge the idea that increased pro-inflammatory NMDAR-reactive T cells contribute to disease pathogenesis

The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4(+)CD25(+ )T cells

It is now generally accepted that CD4(+)CD25(+ )T(reg )cells play a major role in the prevention of autoimmunity and pathological immune responses. Their involvement in the pathogenesis of chronic arthritis is controversial, however, and so we examined their role in experimental antigen-induced arthritis in mice. Depletion of CD25-expressing cells in immunized animals before arthritis induction led to increased cellular and humoral immune responses to the inducing antigen (methylated bovine serum albumin; mBSA) and autoantigens, and to an exacerbation of arthritis, as indicated by clinical (knee joint swelling) and histological scores. Transfer of CD4(+)CD25(+ )cells into immunized mice at the time of induction of antigen-induced arthritis decreased the severity of disease but was not able to cure established arthritis. No significant changes in mBSA-specific immune responses were detected. In vivo migration studies showed a preferential accumulation of CD4(+)CD25(+ )cells in the inflamed joint as compared with CD4(+)CD25(- )cells. These data imply a significant role for CD4(+)CD25(+ )T(reg )cells in the control of chronic arthritis. However, transferred T(reg )cells appear to be unable to counteract established acute or chronic inflammation. This is of considerable importance for the timing of T(reg )cell transfer in potential therapeutic applications

Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4+ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4+ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin- coated vesicles to CD4+ T cells leading to enhanced transmigration of CXCR4+ total CD4+ T cells and CXCR3+ effector/memory CD4+ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4+ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4+ T-cell transmigration in vitro as well as migration of CD4+ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3+ CD4+ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4+ T-cell populations during immune surveillance and liver inflammation

Candida-Reactive T Cells for the Diagnosis of Invasive Candida Infection—A Prospective Pilot Study

Background: Blood or tissue culture or histology prove invasive Candida infection, but long time to result, limited feasibility and sensitivity call for new approaches. In this pilot project, we describe the diagnostic potential of quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes in blood of patients with invasive Candida infection.Methods: We used flow cytometry quantitating Candida-reactive, CD4/CD69/CD154 positive lymphocytes from peripheral blood of patients with invasive Candida infection, from patients at risk and healthy volunteers as controls.Results: Elevated levels of Candida-reactive lymphocytes were measured in 13 patients with proven invasive Candida infection and in one patient with probable hepatosplenic candidiasis. Results of three candidemia patients were uninterpretable due to autofluorescence of samples. Twelve of 13 patients had Candida identified to species level by conventional methods, and T cell reactivity correctly identified Candida species in 10 of 12 patients. Nine hematological high-risk patients and 14 healthy donors had no elevated Candida-reactive T cell counts.Conclusions: This Candida-reactive lymphocyte assay correctly identified the majority of patients with invasive Candida infection and the respective species. Our assay has the potential to support diagnosis of invasive Candida infection to species level and to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative

Developmental Stage, Phenotype, and Migration Distinguish Naive- and Effector/Memory-like CD4+ Regulatory T Cells

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin αEβ7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. αE−CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, αE-positive subsets (CD25+ and CD25−) displayed an effector/memory phenotype expressing high levels of E/P-selectin–binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, αE-expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis