IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Oral anaphylaxis to peanut in a mouse model is associated with gut permeability but not with Tlr4 or Dock8 mutations.

BACKGROUND: The etiology of food allergy is poorly understood; mouse models are powerful systems to discover immunologic pathways driving allergic disease. C3H/HeJ mice are a widely used model for the study of peanut allergy because, unlike C57BL/6 or BALB/c mice, they are highly susceptible to oral anaphylaxis. However, the immunologic mechanism of this strain's susceptibility is not known. OBJECTIVE: We aimed to determine the mechanism underlying the unique susceptibility to anaphylaxis in C3H/HeJ mice. We tested the role of deleterious Toll-like receptor 4 (Tlr4) or dedicator of cytokinesis 8 (Dock8) mutations in this strain because both genes have been associated with food allergy. METHODS: We generated C3H/HeJ mice with corrected Dock8 or Tlr4 alleles and sensitized and challenged them with peanut. We then characterized the antibody response to sensitization, anaphylaxis response to both oral and systemic peanut challenge, gut microbiome, and biomarkers of gut permeability. RESULTS: In contrast to C3H/HeJ mice, C57BL/6 mice were resistant to anaphylaxis after oral peanut challenge; however, both strains undergo anaphylaxis with intraperitoneal challenge. Restoring Tlr4 or Dock8 function in C3H/HeJ mice did not protect from anaphylaxis. Instead, we discovered enhanced gut permeability resulting in ingested allergens in the bloodstream in C3H/HeJ mice compared to C57BL/6 mice, which correlated with an increased number of goblet cells in the small intestine. CONCLUSIONS: Our work highlights the potential importance of gut permeability in driving anaphylaxis to ingested food allergens; it also indicates that genetic loci outside of Tlr4 and Dock8 are responsible for the oral anaphylactic susceptibility of C3H/HeJ mice.

Flow cytometric identification of Tfh13 cells in mouse and human.

Anaphylaxis is a life-threatening allergic reaction caused by cross-linking of high-affinity IgE antibodies on the surface of mast cells and basophils. Understanding the cellular mechanisms that lead to high-affinity IgE production is required to develop better therapeutics for preventing this severe reaction. A recently discovered population of T follicular helper Tfh13 cells regulates the production of high-affinity IgE in mouse models of allergy and can also be found in patients with allergies with IgE antibodies against food or aeroallergens. Here we describe optimized protocols for identifying Tfh13 cells in both mice and humans.

Coincidental loss of DOCK8 function in NLRP10-deficient and C3H/HeJ mice results in defective dendritic cell migration.

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.

Triggering through Toll-like receptor 2 limits chronically stimulated T-helper type 1 cells from undergoing exhaustion.

Chronic infections result in T-cell exhaustion, a state of functional unresponsiveness. To control the infection, it is important to salvage the exhausted T cells. In this study, we delivered signals through Toll-like receptor 2 (TLR-2) to reinvigorate functionality in chronically activated T-helper type 1 (Th1) cells. This process significantly augmented the expression of T-bet, interferon γ, interleukin 2, and the antiapoptotic molecule Bcl-2, whereas it dampened the display of the exhaustion markers programmed death receptor 1 (PD-1) and lymphocyte activation gene 3 (Lag-3). Additionally, TLR-2 signaling bolstered the ability of chronically stimulated Th1 cells to activate B cells. Finally, the results were substantiated by observing reduced lung pathology upon administration of TLR-2 agonist in the chronic infection model of tuberculosis. These data demonstrated the importance of TLR-2 in rescuing chronically activated Th1 cells from undergoing exhaustion. This study will pave a way for targeting TLR-2 in developing therapeutic strategies to treat chronic diseases involving loss of Th1 cell function.

Challenges and solutions for a rational vaccine design for TB-endemic regions.

Vaccines have been successful for global eradication or control of dreaded diseases such as smallpox, diphtheria, tetanus, yellow fever, whooping cough, polio, and measles. Unfortunately, this success has not been achieved for controlling tuberculosis (TB) worldwide. Bacillus Calmette Guérin (BCG) is the only available vaccine against TB. Paradoxically, BCG has deciphered success in the Western world but has failed in TB-endemic areas. In this article, we highlight and discuss the aspects of immunity responsible for controlling Mycobacterium tuberculosis infection and factors responsible for the failure of BCG in TB-endemic countries. In addition, we also suggest strategies that contribute toward the development of successful vaccine in protecting populations where BCG has failed.

Manipulation of costimulatory molecules by intracellular pathogens: veni, vidi, vici!!

Some of the most successful pathogens of human, such as Mycobacterium tuberculosis (Mtb), HIV, and Leishmania donovani not only establish chronic infections but also remain a grave global threat. These pathogens have developed innovative strategies to evade immune responses such as antigenic shift and drift, interference with antigen processing/presentation, subversion of phagocytosis, induction of immune regulatory pathways, and manipulation of the costimulatory molecules. Costimulatory molecules expressed on the surface of various cells play a decisive role in the initiation and sustenance of immunity. Exploitation of the "code of conduct" of costimulation pathways provides evolutionary incentive to the pathogens and thereby abates the functioning of the immune system. Here we review how Mtb, HIV, Leishmania sp., and other pathogens manipulate costimulatory molecules to establish chronic infection. Impairment by pathogens in the signaling events delivered by costimulatory molecules may be responsible for defective T-cell responses; consequently organisms grow unhindered in the host cells. This review summarizes the convergent devices that pathogens employ to tune and tame the immune system using costimulatory molecules. Studying host-pathogen interaction in context with costimulatory signals may unveil the molecular mechanism that will help in understanding the survival/death of the pathogens. We emphasize that the very same pathways can potentially be exploited to develop immunotherapeutic strategies to eliminate intracellular pathogens.

Lipidated promiscuous peptide augments the expression of MHC-II molecules on dendritic cells and activates T cells.

BACKGROUND & OBJECTIVES: In spite of the fact that BCG is the most widely used vaccine, tuberculosis (TB) continues to be a major killer disease in TB-endemic regions. Recently, many emerging evidences from the published literature indicate the role of environmental mycobacteria in blocking the processing and presentation of BCG antigens and thereby impairing with suboptimal generation of protective T cells. To surmount this problem associated with BCG, we constructed a novel lipopeptide (L91) by conjugating a promiscuous peptide consisting of CD4 + T-helper epitope of sequence of 91-110 of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys, an agonist of Toll-like receptor-2. METHODS: Mice were immunized subcutaneously with 20 nmol of L91, followed by a booster with 10 nmol, after an interval of 21 days of primary immunization. Animals were sacrificed after seven days of post-booster immunization. L91 induced immune response was characterized by the expression of MHC-II and CD74 on the surface of dendritic cells (DCs) by flowcytometry. Cytokines (IL-4, IL-10, IFN-γ) secretion and anti-peptide antibodies were measured by ELISA. RESULTS: Self-adjuvanting lipopeptide vaccine (L91) was directly bound to MHC-II molecules and without requiring extensive processing for its presentation to T cells. It stimulated and activated dendritic cells and augmented the expression of MHC-II molecules. Further, it activated effector CD4 T cells to mainly secrete interferon (IFN)-γ but not interleukin (IL)-4 and IL-10. L91 did not elicit anti-peptide antibodies. INTERPRETATION & CONCLUSIONS: The findings suggest that L91 evokes maturation and upregulation of MHC class II molecules and promotes better antigen presentation and, therefore, optimum activation of T cells. L91 mainly induces effector Th1 cells, as evidenced by predominant release of IFN-γ, consequently can mount favourable immune response against M. tuberculosis . As L91 does not provoke the generation of anti-peptide antibodies, there is no fear of the efficacy of the vaccine being neutralized by pre-existing anti-mycobacterial antibodies in TB-endemic population. In conclusion, L91 may be considered as a future potential candidate vaccine against TB.

Potential T cell epitopes of Mycobacterium tuberculosis that can instigate molecular mimicry against host: implications in autoimmune pathogenesis.

BACKGROUND: Molecular mimicry between microbial antigens and host-proteins is one of the etiological enigmas for the occurrence of autoimmune diseases. T cells that recognize cross-reactive epitopes may trigger autoimmune reactions. Intriguingly, autoimmune diseases have been reported to be prevalent in tuberculosis endemic populations. Further, association of Mycobacterium tuberculosis (M. tuberculosis) has been implicated in different autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Although, in silico analyses have identified a number of M. tuberculosis specific vaccine candidates, the analysis on prospective cross-reactive epitopes, that may elicit autoimmune response, has not been yet attempted. Here, we have employed bioinformatics tools to determine T cell epitopes of homologous antigenic regions between M. tuberculosis and human proteomes. RESULTS: Employing bioinformatics tools, we have identified potentially cross-reactive T cell epitopes restricted to predominant class I and II alleles of human leukocyte antigens (HLA). These are similar to peptides of mycobacterial proteins and considerable numbers of them are promiscuous. Some of the identified antigens corroborated with established autoimmune diseases linked with mycobacterial infection. CONCLUSIONS: The present study reveals many target proteins and their putative T cell epitopes that might have significant application in understanding the molecular basis of possible T cell autoimmune reactions during M. tuberculosis infections.

Promiscuous peptide of 16 kDa antigen linked to Pam2Cys protects against Mycobacterium tuberculosis by evoking enduring memory T-cell response.

One of the main reasons considered for BCG failure in tuberculosis-endemic areas is impediment by environmental mycobacteria in its processing and generation of memory T-cell response. To overcome this problem, we developed a unique lipopeptide (L91) by linking the promiscuous peptide (sequence 91-110) of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys. L91 does not require extensive antigen processing and generates enduring Th1 memory response. This is evidenced by the fact that L91 significantly improved the activation, proliferation, and generation of protective T cells. Furthermore, L91 surmounts the barrier of major histocompatibility complex polymorphism and induces better protection than BCG. This peptide has self-adjuvanting properties and activates dendritic cells. Importantly, L91 activates T cells isolated from purified protein derivative-positive healthy volunteers that responded weakly to free peptide (F91). In essence, L91 can be a potent future vaccine candidate against tuberculosis.

T follicular helper cells in IgE-mediated pathologies.

T follicular helper (Tfh) cells help orchestrate optimal humoral immunity by helping B cells to produce affinity-matured, class-switched antibodies in germinal centers. Recent studies have unveiled the complexity and heterogeneity in Tfh cell populations, particularly with respect to their cytokine production. These distinct Tfh cell subsets help tune the class, magnitude and quality of the immunoglobulins produced by B cells, thus shaping the course of humoral responses. The Tfh cell-B cell axis-dependent antibody production is mostly beneficial, but at times can go awry and result in the generation of pathologic antibodies that can harm the host. While IgE class of antibodies are infamous for their detrimental role in allergic diseases, emerging evidence is indicative of their pathologic roles in other dysregulated immune states. Here, we discuss the role of Tfh cell subsets in the regulation of pathologic IgE production in allergies and other immunopathologic conditions.

The curious case of a cryptic Cryptosporidium and a missing dendritic cell subset.

Animal models for studying immune responses to Cryptosporidium, a parasite that causes gastrointestinal disease, have been a challenge due to the parasite's poor infectivity in mice. Russler-Germain et al. discovered a 'commensal' strain of Cryptosporidium, capable of stable infection and vertical transmission, that elicits a T helper type 1 (Th1) response to promote intestinal homeostasis.

High-affinity, neutralizing antibodies to SARS-CoV-2 can be made without T follicular helper cells.

T follicular helper (TFH) cells are the conventional drivers of protective, germinal center (GC)­based antiviral antibody responses. However, loss of TFH cells and GCs has been observed in patients with severe COVID-19. As T cell­B cell interactions and immunoglobulin class switching still occur in these patients, noncanonical pathways of antibody production may be operative during SARS-CoV-2 infection. We found that both TFH-dependent and -independent antibodies were induced against SARS-CoV-2 infection, SARS-CoV-2 vaccination, and influenza A virus infection. Although TFH-independent antibodies to SARS-CoV-2 had evidence of reduced somatic hypermutation, they were still high affinity, durable, and reactive against diverse spike-derived epitopes and were capable of neutralizing both homologous SARS-CoV-2 and the B.1.351 (beta) variant of concern. We found by epitope mapping and B cell receptor sequencing that TFH cells focused the B cell response, and therefore, in the absence of TFH cells, a more diverse clonal repertoire was maintained. These data support an alternative pathway for the induction of B cell responses during viral infection that enables effective, neutralizing antibody production to complement traditional GC-derived antibodies that might compensate for GCs damaged by viral inflammation.

Coadministration of interleukins 7 and 15 with bacille Calmette-Guérin mounts enduring T cell memory response against Mycobacterium tuberculosis.

BACKGROUND: The bacille Calmette-Guérin (BCG) vaccine renders protection against tuberculosis in childhood but not in adulthood. This may be due to its failure to induce long-lasting memory T cells. T cell memory is dependent on crucial cytokine signals during the priming phases. Therefore, coadministering the BCG vaccine with cytokines may improve its efficacy. METHODS: A combination of the cytokines interleukin 7 (IL-7) and interleukin 15 (IL-15) or a combination of the cytokines interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha), which are known to influence memory T cell generation, were administered along with BCG to mice. The animals were rested for a period of 240 d before they were challenged with Mycobacterium tuberculosis. Five weeks later, they were killed to study the T cell memory response. RESULTS: Administration of IL-7 and IL-15, but not IL-1, IL-6, and TNF-alpha, with BCG resulted in an improved CD4 and CD8 T cell memory response. Mice injected with BCG supplemented with IL-7 and IL-15 showed enhanced T cell proliferation, T helper 1-type cytokine production, and an increased pool of multifunctional M. tuberculosis-specific memory T cells. Furthermore, there was a statistically significant reduction in the mycobacterial burden in the lungs. CONCLUSION: Our results indicate that supplementation of the BCG vaccine with IL-7 and IL-15 would substantially improve its efficacy by enhancing the T cell memory response.

Evaluation of different generic in silico methods for predicting HLA class I binding peptide vaccine candidates using a reverse approach.

Since CD8+ T cell response is crucial to combat intracellular infections and cancer, identification of class I HLA binding peptides is of immense clinical value. The experimental identification of such peptides is protracted and laborious. Exploiting in silico tools to discover such peptides is an attractive alternative. However, this approach needs a thorough assessment before its elaborate application. We have adopted a reverse approach to evaluate the reliability of eight different servers (inclusive of 55 predictors) by exploiting experimentally proven data. A comprehensive data set of more than 960 peptides was employed to test the efficacy of the programs. We have validated commonly used strategies to predict peptides that bind to seven most prevalent HLA class I alleles. We conclude that four of the eight servers are more adept in predictions. Although the overall predictions for class I MHC binders were superior to class II MHC binders, individual predictors for different alleles belonging to the same program were highly variable in their efficiencies. We have also addressed whether a consensus approach can yield better prediction efficiency. We observed that combining the results from different in silico programs could not increase the efficiency significantly.

Regulation of IgE by T follicular helper cells.

Allergies to food and environmental antigens have steeply grown to epidemic proportions. IgE antibodies are key mediators of allergic disease, including life-threatening anaphylaxis. There is now compelling evidence that one of the hallmarks of anaphylaxis-inducing IgE molecules is their high affinity for allergen, and the cellular pathway to high-affinity IgE is typically through sequential switching of IgG B cells. Further, in contrast to the previously held paradigm that a subset of CD4+ T cells called Th2 cells promotes IgE responses, recent studies suggest that T follicular helper cells are crucial for inducing anaphylactic IgE. Here we discuss recent studies that have enabled us to understand the nature, induction, and regulation of this enigmatic antibody isotype in allergic sensitization.

Divergent T follicular helper cell requirement for IgA and IgE production to peanut during allergic sensitization.

Immunoglobulin A (IgA) is the dominant antibody isotype in the gut and has been shown to regulate microbiota. Mucosal IgA is also widely believed to prevent food allergens from penetrating the gut lining. Even though recent work has elucidated how bacteria-reactive IgA is induced, little is known about how IgA to food antigens is regulated. Although IgA is presumed to be induced in a healthy gut at steady state via dietary exposure, our data do not support this premise. We found that daily food exposure only induced low-level, cross-reactive IgA in a minority of mice. In contrast, induction of significant levels of peanut-specific IgA strictly required a mucosal adjuvant. Although induction of peanut-specific IgA required T cells and CD40L, it was T follicular helper (TFH) cell, germinal center, and T follicular regulatory (TFR) cell-independent. In contrast, IgG1 and IgE production to peanut required TFH cells. These data suggest an alternative paradigm in which the cellular mechanism of IgA production to food antigens is distinct from IgE and IgG1. We developed an equivalent assay to study this process in stool samples from healthy, nonallergic humans, which revealed substantial levels of peanut-specific IgA that were stable over time. Similar to mice, patients with loss of CD40L function had impaired titers of gut peanut-specific IgA. This work challenges two widely believed but untested paradigms about antibody production to dietary antigens: (i) the steady state/tolerogenic response to food antigens includes IgA production and (ii) TFH cells drive food-specific gut IgA.

In silico methods for predicting T-cell epitopes: Dr Jekyll or Mr Hyde?

In silico tools offer an attractive alternative strategy to the cumbersome experimental approaches to identify T-cell epitopes. These computational tools have metamorphosed over the years into complex algorithms that attempt to efficiently predict the binding of a plethora of peptides to HLA alleles. In recent years, the scientific community has embraced these techniques to reduce the burden of wet-laboratory experimentation. Although there are some splendid examples of the utility of these methods, there are also evidences where they fall short and remain inconsistent. Hence, are these computational tools 'Dr Jekyll' or 'Mr Hyde' to the researcher, who wishes to utilize them intrepidly? This article reviews the progress and pitfalls of the in silico tools that identify T-cell epitopes.

Identification of a T follicular helper cell subset that drives anaphylactic IgE.

Cross-linking of high-affinity immunoglobulin E (IgE) results in the life-threatening allergic reaction anaphylaxis. Yet the cellular mechanisms that induce B cells to produce IgE in response to allergens remain poorly understood. T follicular helper (TFH) cells direct the affinity and isotype of antibodies produced by B cells. Although TFH cell-derived interleukin-4 (IL-4) is necessary for IgE production, it is not sufficient. We report a rare population of IL-13-producing TFH cells present in mice and humans with IgE to allergens, but not when allergen-specific IgE was absent or only low-affinity. These "TFH13" cells have an unusual cytokine profile (IL-13hiIL-4hiIL-5hiIL-21lo) and coexpress the transcription factors BCL6 and GATA3. TFH13 cells are required for production of high- but not low-affinity IgE and subsequent allergen-induced anaphylaxis. Blocking TFH13 cells may represent an alternative therapeutic target to ameliorate anaphylaxis.