The amalgam of naive CD4+ T cell transcriptional states is reconfigured by helminth infection to dampen the amplitude of the immune response.

Naive CD4+ T cells in specific pathogen-free (SPF) mice are characterized by transcriptional heterogeneity and subpopulations distinguished by the expression of quiescence, the extracellular matrix (ECM) and cytoskeleton, type I interferon (IFN-I) response, memory-like, and T cell receptor (TCR) activation genes. We demonstrate that this constitutive heterogeneity, including the presence of the IFN-I response cluster, is commensal independent insofar as being identical in germ-free and SPF mice. By contrast, Nippostrongylus brasiliensis infection altered this constitutive heterogeneity. Naive T cell-intrinsic transcriptional changes acquired during helminth infection correlated with and accounted for decreased immunization response to an unrelated antigen. These compositional and functional changes were dependent variables of helminth infection, as they disappeared at the established time point of its clearance in mice. Collectively, our results indicate that the naive T cell pool is subject to dynamic transcriptional changes in response to certain environmental cues, which in turn permutes the magnitude of the immune response.

Predominance of M2 macrophages in gliomas leads to the suppression of local and systemic immunity.

Glioblastoma is a highly prevalent and aggressive form of primary brain tumor. It represents approximately 56% of all the newly diagnosed gliomas. Macrophages are one of the major constituents of tumor-infiltrating immune cells in the human gliomas. The role of immunosuppressive macrophages is very well documented in correlation with the poor prognosis of patients suffering from breast, prostate, bladder and cervical cancers. The current study highlights the correlation between the tumor-associated macrophage phenotypes and glioma progression. We observed an increase in the pool of M2 macrophages in high-grade gliomas, as confirmed by their CD68 and CD163 double-positive phenotype. In contrast, less M1 macrophages were noticed in high-grade gliomas, as evidenced by the down-regulation in the expression of CCL3 marker. In addition, we observed that higher gene expression ratio of CD163/CCL3 is associated with glioma progression. The Kaplan-Meier survival plots indicate that glioma patients with lower expression of M2c marker (CD163), and higher expression of M1 marker (CCL3) had better survival. Furthermore, we examined the systemic immune response in the peripheral blood and noted a predominance of M2 macrophages, myeloid-derived suppressor cells and PD-1+ CD4 T cells in glioma patients. Thus, the study indicates a high gene expression ratio of CD163/CCL3 in high-grade gliomas as compared to low-grade gliomas and significantly elevated frequency of M2 macrophages and PD-1+ CD4 T cells in the blood of tumor patients. These parameters could be used as an indicator of the early diagnosis and prognosis of the disease.

The role of macrophages in skin homeostasis.

The skin and its appendages comprise the largest and fastest growing organ in the body. It performs multiple tasks and maintains homeostatic control, including the regulation of body temperature and protection from desiccation and from pathogen invasion. The skin can perform its functions with the assistance of different immune cell populations. Monocyte-derived cells are imperative for the completion of these tasks. The comprehensive role of macrophages and Langerhans cells in establishing and maintaining skin homeostasis remains incompletely defined. However, over the past decade, innovations in mouse genetics have allowed for advancements in the field. In this review, we explore different homeostatic roles of macrophages and Langerhans cells, including wound repair, follicle regeneration, salt balance, and cancer regression and progression in the skin. The understanding of the precise functions of myeloid-derived cells in the skin under basal conditions can help develop specific therapies that aid in skin and hair follicle regeneration and cutaneous cancer prevention.

T-cell exhaustion in tuberculosis: pitfalls and prospects.

T-cells play an important role in immunity but when these cells are overexposed to specific antigens, their function may decline. This state is usually referred to as exhaustion and the T-cells show reduced proliferation and functions such as cytokine release. T-cell exhaustion has been observed in several cancers as well as in chronic infections such as tuberculosis (TB). In chronic Mycobacterium tuberculosis (Mtb) infection, T-cells may express the exhaustion phenotype and show a progressive loss of secretion of IL-2, IFN-γ and TNF-α. In some cancers and chronic infection models, blocking the exhaustion phenotype can be achieved with the so-called checkpoint inhibitors. This results in tumor control and more effective immunity. However, in the case of TB, the T-cell exhaustion results are quite ambiguous. Hence, there is a need to investigate and explain the contribution of checkpoint at a molecular level to the outcome of events in chronic TB. Such information could help to guide the success of new therapies against chronic TB. This review highlights the mechanism through which T-cells undergo exhaustion and the approaches that can avert such events. This will help to design immunotherapies that can reinvigorate T-cell potency to protect patients from TB.

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.

CBLs downregulation foretells T cell ubiquitination leading to autoimmunity.

In a study published in the July issue of Immunity, Li et al.1 demonstrate that expression of the E3 ubiquitin ligases CBL and CBL-B is downregulated in Tfh cells in SLE with Tfh cell expansion and autoimmunity. This leads to reduced ubiquitination of the T cell costimulator ICOS which regulates proteostasis of the Tfh cell transcription factor BCL6 via chaperone-mediated autophagy.

AP-1-independent NFAT signaling maintains follicular T cell function in infection and autoimmunity.

Coordinated gene expression programs enable development and function of T cell subsets. Follicular helper T (Tfh) cells coordinate humoral immune responses by providing selective and instructive cues to germinal center B cells. Here, we show that AP-1-independent NFAT gene expression, a program associated with hyporesponsive T cell states like anergy or exhaustion, is also a distinguishing feature of Tfh cells. NFAT signaling in Tfh cells, maintained by NFAT2 autoamplification, is required for their survival. ICOS signaling upregulates Bcl6 and induces an AP-1-independent NFAT program in primary T cells. Using lupus-prone mice, we demonstrate that genetic disruption or pharmacologic inhibition of NFAT signaling specifically impacts Tfh cell maintenance and leads to amelioration of autoantibody production and renal injury. Our data provide important conceptual and therapeutic insights into the signaling mechanisms that regulate Tfh cell development and function.

Functionally and metabolically divergent melanoma-associated macrophages originate from common bone-marrow precursors.

Melanomas display high numbers of tumor-associated macrophages (TAMs), which correlate with worse prognosis. Harnessing macrophages for therapeutic purposes has been particularly challenging due to their heterogeneity, based on their ontogeny and function and driven by the tissue-specific niche. In the present study, we used the YUMM1.7 model to better understand melanoma TAM origin and dynamics during tumor progression, with potential therapeutic implications. We identified distinct TAM subsets based on F4/80 expression, with the F4/80 high fraction increasing over time and displaying tissue-resident-like phenotype. While skin-resident macrophages showed mixed on-togeny, F4/80 + TAM subsets in i.d. YUMM1.7 tumors originated almost exclusively from bone-marrow precursors. Mul-tiparametric analysis of macrophage phenotype showed a temporal divergence of F4/80 + TAM subpopulations, which also differed from skin-resident subsets, and from their monocytic precursors. Overall, F4/80 + TAMs displayed co-ex-pression of M1- and M2-like canonical markers, while RNA-seq and pathway analysis showed differential immunosup-pressive and metabolic profiles. GSEA showed F4/80 high TAMs to rely on oxidative phosphorylation, with increased proliferation and protein secretion while F4/80 low cells had high pro-inflammatory and intracellular signaling pathways, with lipid and polyamine metabolism. Overall, the present in-depth characterization provides further evidence of the ontogeny of the evolving melanoma TAMs, whose gene expression profiles matched recently-identified TAM clusters in other tumor models and human cancers. These findings provide evidence for potentially targeting specific immunosup-pressive TAMs in advanced tumor stages.

Functionally and Metabolically Divergent Melanoma-Associated Macrophages Originate from Common Bone-Marrow Precursors.

Tumor-associated macrophages (TAMs) can be widely heterogeneous, based on their ontogeny and function, and driven by the tissue-specific niche. TAMs are highly abundant in the melanoma tumor microenvironment (TME), usually correlating with worse prognoses. However, the understanding of their diversity may be harnessed for therapeutic purposes. Here, we used the clinically relevant YUMM1.7 model to study melanoma TAM origin and dynamics during tumor progression. In i.d. YUMM1.7 tumors, we identified distinct TAM subsets based on F4/80 expression, with the F4/80high fraction increasing over time and displaying a tissue-resident-like phenotype. While skin-resident macrophages showed mixed ontogeny, F4/80+ TAM subsets in the melanoma TME originated almost exclusively from bone-marrow precursors. A multiparametric analysis of the macrophage phenotype showed a temporal divergence of the F4/80+ TAM subpopulations, which also differed from the skin-resident subsets and their monocytic precursors. Overall, the F4/80+ TAMs displayed co-expressions of M1- and M2-like canonical markers, while RNA sequencing showed differential immunosuppressive and metabolic profiles. Gene-set enrichment analysis (GSEA) revealed F4/80high TAMs to rely on oxidative phosphorylation, with increased proliferation and protein secretion, while F4/80low cells had high pro-inflammatory and intracellular signaling pathways, with lipid and polyamine metabolism. Overall, we provide an in-depth characterization of and compelling evidence for the BM-dependency of melanoma TAMs. Interestingly, the transcriptomic analysis of these BM-derived TAMs matched macrophage subsets with mixed ontogeny, which have been observed in other tumor models. Our findings may serve as a guide for identifying potential ways of targeting specific immunosuppressive TAMs in melanoma.

Topical arginase inhibition decreases growth of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinomas (cSCC) are among the most commonly diagnosed malignancies, causing significant morbidity and mortality. Tumor-associated macrophage (TAM) expression of arginase is implicated in tumor progression, and therapeutic use of arginase inhibitors has been studied in various cancers. However, investigating potential cSCC immunotherapies including arginase inhibition in pre-clinical models is hampered by the lack of appropriate tumor models in immunocompetent mice. PDV is a cSCC cell line derived from chemical carcinogenesis of mouse keratinocytes. PDVC57 cells were derived from a PDV tumor in C57BL/6 (B6) mice. Unlike PDV, PDVC57 tumors grow consistently in B6 mice, and have increased TAMs, decreased dendritic and T cell intra-tumor infiltration. Arginase inhibition in cSCC tumors using Nω-hydroxy-nor-arginine (nor-NOHA) reduced tumor growth in B6 mice but not immunodeficient Rag1-deficient mice. nor-NOHA administration increased dendritic and T cell tumor-infiltration and PD-1 expression. The combination of nor-NOHA and anti-PD-1 therapy with nivolumab enhanced anti-PD-1 therapeutic efficacy. This study demonstrates the therapeutic potential of transcutaneous arginase inhibition in cSCC. A competent immune microenvironment is required for tumor growth inhibition using this arginase inhibitor. Synergistic co-inhibition of tumor growth in these results, supports further examination of transcutaneous arginase inhibition as a therapeutic modality for cSCC.

Repeat tick exposure elicits distinct immune responses in guinea pigs and mice.

Ticks deposit salivary proteins into the skin during a bite to mediate acquisition of a blood meal. Acquired resistance to tick bites has been demonstrated to prevent Borrelia burgdorferi sensu lato (s.l.) transmission. However, the mechanism of resistance, as well as the protective antigens, have remained elusive. To address these unknowns, we utilized a guinea pig model of tick resistance and a mouse model of permissiveness. Guinea pigs developed immunity after multiple Ixodes scapularis tick infestations, characterized by rapid tick detachment and impaired feeding. In comparison, mice tolerated at least 6 infestations with no significant impact on feeding. We analyzed the bite sites by RNA-sequencing and histology, identifying several inflammatory pathways in tick immune animals, such as FcεRI signaling and complement activation, and activation of coagulation pathways that could impair local blood flow. Together, these results identify important pathways altered during tick rejection and potential tick proteins that could serve as vaccine candidates.

TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-αß Signaling and Restricts Tumor Progression.

During tumor progression, macrophages shift their protective M1-phenotype to pro-tumorigenic M2-subtype. Therefore, conversion of M2 to M1 phenotype may be a potential therapeutic intervention. TLRs are important pathogen recognition receptors expressed by cells of the immune system. Recently, a crucial role of TLR-3 has been suggested in cancer. Consequently, in the current study, we defined the role of TLR-3 in the reversion of M2-macrophages to M1. We analyzed the role of TLR-3 stimulation for skewing M2-macrophages to M1 at mRNA and protein level through qRT-PCR, flow cytometry, western blotting, and ELISA. The effectiveness of TLR-3L stimulation to revert M2-macrophages to M1 was evaluated in the murine tumor model. To determine the role of IFN-αß signaling in vitro and in vivo, we used Ifnar1-/- macrophages and anti-IFN-αß antibodies, respectively. We observed upregulation of M1-specific markers MHC-II and costimulatory molecules like CD86, CD80, and CD40 on M2-macrophages upon TLR-3 stimulation. In contrast, reduced expression of M2-indicators CD206, Tim-3, and pro-inflammatory cytokines was noticed. The administration of TLR-3L in the murine tumor reverted the M2-macrophages to M1-phenotype and regressed the tumor growth. The mechanism deciphered for macrophage reversion and controlling the tumor growth is dependent on IFN-αß signaling pathway. The results indicate that the signaling through TLR-3 is important in protection against tumors by skewing M2-macrophages to protective M1-subtype.

Antibody response against PhoP efficiently discriminates among healthy individuals, tuberculosis patients and their contacts.

Tuberculosis continues to be one of the most devastating global health problem. Its diagnosis will benefit in timely initiation of the treatment, cure and therefore reduction in the transmission of the disease. Tests are available, but none can be comprehensively relied on for its diagnosis; especially in TB-endemic zones. PhoP is a key player in Mycobacterium tuberculosis virulence but nothing has been known about its role in the diagnosis of TB. We monitored the presence of anti-PhoP antibodies in the healthy, patients and their contacts. In addition, we also measured antibodies against early secretory antigens ESAT-6 and CFP-10, and latency associated antigen Acr-1 to include proteins that are associated with the different stages of disease progression. Healthy subjects showed high antibody titer against PhoP than patients and their contacts. In addition, a distinct pattern in the ratio of Acr-1/PhoP was observed among all cohorts. This study for the first time demonstrates a novel role of anti-PhoP antibodies, as a possible marker for the diagnosis of TB and therefore will contribute in the appropriate action and management of the disease.

Distinct Strategies Employed by Dendritic Cells and Macrophages in Restricting Mycobacterium tuberculosis Infection: Different Philosophies but Same Desire.

Dendritic cells (DCs) and macrophages (Mϕs) are professional antigen-presenting cells (APCs) that can efficiently phagocytose Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). It is quite interesting to mention here that DCs and Mϕs use distinct strategies to combat and eliminate Mtb. Similarly, Mtb employs different mechanisms to counteract the action of DCs and Mϕs. Mϕs are evolved with specialized, innate, defensive machinery to restrict growth of Mtb at the initial phase of infection. However, DCs are more endowed toward initiating adaptive immunity by activating naïve T cells. During encounter with Mtb, DCs and Mϕs deliver discrete functions via triggering through different pattern recognition receptors (PRRs) expressed by these APCs. Mtb-infected DCs and Mϕs show differential expression of genes encoding cytokines, chemokines, costimulatory molecules, and adhesion molecules. Interestingly, Mtb impairs the immune defensive machinery by exploiting various PRRs. Remarkably, selective signaling through PRRs by Mtb abrogates the bactericidal activity of Mϕs, but subverts differentiation of monocytes to DCs. In this article, we highlight the role of PRRs in inducing distinct immune response by DCs and Mϕs against Mtb. Concurrently, we also discuss smart strategies exploited by Mtb to impair the function of host DCs and Mϕs.

Innate Immunity Holding the Flanks until Reinforced by Adaptive Immunity against Mycobacterium tuberculosis Infection.

T cells play a cardinal role in imparting protection against Mycobacterium tuberculosis (Mtb). However, ample time is required before T-cells are able to evoke efficient effector responses in the lung, where the mycobacterium inflicts disease. This delay in T cells priming, which is termed as lag phase, provides sufficient time for Mtb to replicate and establish itself within the host. In contrast, innate immunity efficiently curb the growth of Mtb during initial phase of infection through several mechanisms. Pathogen recognition by innate cells rapidly triggers a cascade of events, such as apoptosis, autophagy, inflammasome formation and nitric oxide production to kill intracellular pathogens. Furthermore, bactericidal mechanisms such as autophagy and apoptosis, augment the antigen processing and presentation, thereby contributing substantially to the induction of adaptive immunity. This manuscript highlights the role of innate immune mechanisms in restricting the survival of Mtb during lag phase. Finally, this article provides new insight for designing immuno-therapies by targeting innate immune mechanisms to achieve optimum immune response to cure TB.

NOD-2 and TLR-4 Signaling Reinforces the Efficacy of Dendritic Cells and Reduces the Dose of TB Drugs against Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the leading killer infectious diseases. TB patients are inflicted with devastating side effects and the toxicity of a lengthy drug regime, accentuating an urgent need to explore newer and safer treatment methods. Recently, an improved understanding of host-pathogen interaction has opened new avenues for TB treatment, including immunotherapy. This has emboldened us to devise a novel strategy to restrict Mycobacterium tuberculosis(Mtb) growth by activating dendritic cells (DCs) through the NOD-2 and TLR-4 molecules of innate immunity. Triggered DCs show a robust release of cytokines and nitric oxide, autophagy and improved migration towards the lymph nodes, and consequently impede the intracellular survival of Mtb. Of note, this approach enhanced the efficacy of TB drugs by reducing their dose to a 5-fold lesser concentration than recommended. In vivo administration of ligands of NOD-2 (NOD-2L) and TLR-4 (TLR-4L) substantially increased the pool of effector memory CD4 and CD8 T cells. Additionally, NOD-2L and TLR-4L, in conjunction with the reduced dose of isoniazid, substantially declined the Mtb burden in the lungs. In the future, adjunct therapy involving NOD-2L, TLR-4L and TB drugs may have enough potential to reduce the dose and duration of treatment of TB patients.

Stimulation through CD40 and TLR-4 Is an Effective Host Directed Therapy against Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading cause of morbidity and mortality among all infectious diseases. Failure of Bacillus Calmette Guerin as a vaccine and serious side-effects and toxicity due to long-term TB drug regime are the major hurdles associated with TB control. The problem is further compounded by the emergence of drug-resistance strains of Mycobacterium tuberculosis (Mtb). Consequently, it demands a serious attempt to explore safer and superior treatment approaches. Recently, an improved understanding of host-pathogen interaction has opened up new avenues for immunotherapy for treating TB. Although, dendritic cells (DCs) show a profound role in generating immunity against Mtb, their immunotherapeutic potential needs to be precisely investigated in controlling TB. Here, we have devised an approach of bolstering DCs efficacy against Mtb by delivering signals through CD40 and TLR-4 molecules. We found that DCs triggered through CD40 and TLR-4 showed increased secretion of IL-12, IL-6, and TNF-α. It also augmented autophagy. Interestingly, CD40 and TLR-4 stimulation along with the suboptimal dose of anti-TB drugs significantly fortified their efficacy to kill Mtb. Importantly, animals treated with the agonists of CD40 and TLR-4 boosted Th1 and Th17 immunity. Furthermore, it amplified the pool of memory CD4 T cells as well as CD8 T cells. Furthermore, substantial reduction in the bacterial burden in the lungs was observed. Notably, this adjunct therapy employing immunomodulators and chemotherapy can reinvigorate host immunity suppressed due to drugs and Mtb. Moreover, it would strengthen the potency of drugs in curing TB.

Signaling through NOD-2 and TLR-4 Bolsters the T cell Priming Capability of Dendritic cells by Inducing Autophagy.

T cells play a cardinal role in mediating protection against intracellular pathogens like Mycobacterium tuberculosis (Mtb). It is important to understand the factors that govern the T cell response; thereby can modulate its activity. Dendritic cells (DCs) are the major player in initiation and augmentation of T cell response. Targeting DCs to induce their optimum maturation and activation can lead to a better T cell response. Interestingly, we observed that combinatorial signaling of DCs through NOD-2 and TLR-4 fortified better yield of IL-12p40/70, IL-6 and IFN-γ and upregulated the expression of CD40, CD80 and CD86 costimulatory molecules. Further, we noticed improved phagocytic capabilities of DCs. Furthermore, NOD-2 and TLR-4 induced autophagy in DCs, which enhanced the activation of T cells. This study signifies that NOD-2 and TLR-4 exhibit synergism in invigorating the activity of DCs. Consequently, this strategy may have significant immunotherapeutic potential in bolstering the function of DCs and thus improving the immunity against pathogens.

Triggering through NOD-2 Differentiates Bone Marrow Precursors to Dendritic Cells with Potent Bactericidal activity.

Dendritic cells (DCs) play a crucial role in bridging innate and adaptive immunity by activating naïve T cells. The role of pattern recognition receptors like Toll-Like Receptors and Nod-Like Receptors expressed on DCs is well-defined in the recognition of the pathogens. However, nothing is precisely studied regarding the impact of NOD-2 signaling during the differentiation of DCs. Consequently, we explored the role of NOD-2 signaling in the differentiation of DCs and therefore their capability to activate innate and adaptive immunity. Intriguingly, we observed that NOD-2 stimulated DCs (nDCs) acquired highly activated and matured phenotype and exhibited substantially greater bactericidal activity by robust production of nitric oxide. The mechanism involved in improving the functionality of nDCs was dependent on IFN-αß signaling, leading to the activation of STAT pathways. Furthermore, we also observed that STAT-1 and STAT-4 dependent maturation and activation of DCs was under the feedback mechanism of SOCS-1 and SOCS-3 proteins. nDCs acquired enhanced potential to activate chiefly Th1 and Th17 immunity. Taken together, these results suggest that nDCs can be exploited as an immunotherapeutic agent in bolstering host immunity and imparting protection against the pathogens.

Alteration in the Gut Microbiota Provokes Susceptibility to Tuberculosis.

The microbiota that resides in the gastrointestinal tract provides essential health benefits to the host. In particular, they regulate immune homeostasis. Recently, several evidences indicate that alteration in the gut microbial community can cause infectious and non-infectious diseases. Tuberculosis (TB) is the most devastating disease, inflicting mortality and morbidity. It remains unexplored, whether changes in the gut microbiota can provoke or prevent TB. In the current study, we have demonstrated the antibiotics driven changes in the gut microbial composition and their impact on the survival of Mycobacterium tuberculosis (Mtb) in the lungs, liver, and spleen of infected mice, compared to those with intact microbiota. Interestingly, dysbiosis of microbes showed significant increase in the bacterial burden in lungs and dissemination of Mtb to spleen and liver. Furthermore, elevation in the number of Tregs and decline in the pool of IFN-γ- and TNF-α-releasing CD4 T cells was noticed. Interestingly, fecal transplantation in the gut microbiota disrupted animals exhibited improved Th1 immunity and lesser Tregs population. Importantly, these animals displayed reduced severity to Mtb infection. This study for the first time demonstrated the novel role of gut microbes in the susceptibility to TB and its prevention by microbial implants. In future, microbial therapies may help in treating patients suffering from TB.