Visceral leishmaniasis (VL), a parasitic disease caused by Leishmania donovani, requires a robust CD4+ T-cells response to control parasite replication by interferon-gamma (IFN-γ) production and activation of macrophages. However, in VL patients, the anti-parasitic CD4+ T-cell responses are ineffective for reasons that are still unclear. Our recent study reporting a transcriptional signature of CD4+ T-cell isolated from the peripheral blood of active VL patients showed enhanced expression of genes related to cytotoxicity. This study investigates the cytotoxic potential of CD4+ T-cells in VL patients, focusing on the expression of the key cytotoxic molecules granzyme B (GZMB), granulysin (GNLY), perforin (PRF), natural killer cell granule protein 7 (NKG7) and the degranulation capacity of CD4+ T-cells during infection. We observed significant upregulation of these cytotoxic markers in CD4⁺ T-cells from VL patients, particularly prior to anti-parasitic drug treatment (D0), suggesting activation of these cells. However, the degranulation capacity, as indicated by CD107a expression, was comparable between VL patients and endemic controls (ECs), suggesting potential functional impairment in the cytotoxic response. Yet, antigen-specific GZMB secretion in whole blood cultures and intracellular GZMB production in CD4+ T-cell subsets (notably Th1, Th9 and Th17/22 cells) were enhanced in VL patients, indicating a robust antigen-specific response, though this did not translate into effective parasite control. These findings highlight a paradox whereby CD4+ T-cells from VL patients have heightened production of cytotoxic molecules, but lack translocation of CD107a to the cell surface, which is associated with an inability to eliminate parasites. This study provides new insights into the immune dysfunction in VL, highlighting a potential role for the cytotoxic phenotype that develops in parasite-specific CD4+ T-cells. Targeting these pathways may offer novel therapeutic strategies to enhance immune responses and improve clinical outcomes in VL.

Robust T helper 1 (Th1) cell responses, which activate macrophages to kill intracellular parasites, are required to control Leishmania infection. Yet, visceral leishmaniasis (VL) patients do not control the infection despite the expansion of CD4+ T cells and increased IFN-γ expression in the spleen. Chemokines and/or chemokine receptors are involved in cellular migration and are critical in the inflammatory response. In a recent study that defined a transcriptional signature for CD4+ T cells from active VL patients, we found several differentially expressed chemokine receptor genes in CD4+ T cells compared to healthy endemic controls (EC). Since CD4+ T cells play crucial roles in parasite clearance, a better understanding of the role of altered chemokine receptor expression on CD4+ T cells and their different subsets during VL could inform future treatment strategies. In this study, we examined the gene expression and surface protein expression of differentially expressed chemokine receptors found in human VL subjects, relative to endemic controls (EC) by real-time qPCR and multicolor flow cytometry, respectively. We measured chemokine levels in plasma by enzyme-linked immunosorbent assay (ELISA) and performed transwell migration assays and flow cytometry to measure the migratory potential of CD4+ T cell subsets. We found elevated mRNA and surface protein expression of CCR5, while reduced CCR4 and CCR6 expression in CD4+ T cells from VL patients compared to EC. The frequency of CCR5 expressing Th1 cells was increased in peripheral blood, indicating the expansion of CCR5+ Th1 cells that may be responsible for Th1 cells trafficking towards infected tissues. Lower CCR4 expression was found on regulatory T (Treg) cells and central memory T (Tcm) cells, possibly explaining the reduced frequencies of these cells in peripheral blood during VL. The frequency of CCR6 expressing CD4+ T cells was also found to be lower in VL patients. Our results show that VL patients possess unique chemokine receptor expression patterns on the cell surface of CD4+ T cells, compared to EC. We also found increased levels of CCL3, CCL5 and CCL20 in VL plasma compared to EC. However, no changes were observed for CCL17 levels in VL plasma compared to EC, but their levels increased following treatment. We also observed reduced migration of VL CD4+ T cells, relative to other lymphocytes and mononuclear cells, irrespective of exogenous CCL5 presence. However, the frequency of CD4+ T cells migrating in response to CCL5 in EC individuals was similar. Additionally, we noted the frequency of CCR5+ Th1 cells was increased in VL patients compared to EC. However, no enhancement was seen in the migratory capacity of CCR5+ CD4+ T cells in the presence of recombinant CCL5. This suggests that the CCR5 receptor signalling pathway is less responsive towards exogenous CCL5, potentially due to high levels of CCL3 and CCL5 present in VL plasma, which may saturate surface CCR5. The upregulation of CCR5 expression and downregulation of CCR4 and CCR6 by CD4+ T cells distinguished VL patients from healthy individuals. These findings provide new insights into VL pathogenesis and could direct the development of new and improved disease diagnostics and therapeutics for treatment of VL.

For over 40 years, ivermectin has served as an effective anti-parasitic drug used in human and veterinary medicine. In laboratory animal facilities it is used prophylactically or therapeutically to maintain the health status of the colony or experimentally in studies. Although ivermectin is generally safe to use, there are reports of neurotoxicity associated with ivermectin crossing the blood-brain barrier due to overdosing or blood-brain barrier dysfunction. In mice, P-glycoprotein maintains the blood-brain barrier and mice with a mutation in the P-glycoprotein encoding gene mdr1a are 50-100 times more sensitive to ivermectin. Signs of neurotoxicity include ataxia, bradypnea, recumbency, tremor, and death. We report neurotoxicity after ivermectin administration was used for the purpose of eradicating the murine-specific intestinal nematode Heligmosomoides polygyrus in C57BL/6NTac and C57BL/6NCrl mice. The mice were dewormed by subcutaneous administration of 10 or 20 mg/kg ivermectin to eradicate all stages of Heligmosomoides polygyrus. At 24-48h after deworming, 5% (n = 4) of the mice presented with tremor, ataxia, and/or head tilt. The affected mice were euthanised and gross pathological findings were found in one of the four mice (left-sided hydronephrosis). We assume that the observed neurological effects were due to defects in the blood-brain barrier, overdosing or individual sensitivity. This report provides a reason for caution when deworming laboratory mice subcutaneously with ivermectin at doses of 10 mg/kg or higher. L'ivermectine est un m & eacute;dicament antiparasitaire efficace utilis & eacute; depuis plus de 40 ans en m & eacute;decine humaine et v & eacute;t & eacute;rinaire. Elle est utilis & eacute;e & agrave; titre prophylactique ou th & eacute;rapeutique dans les installations pour animaux de laboratoire afin de maintenir l'& eacute;tat de sant & eacute; de la colonie ou dans le cadre d'& eacute;tudes exp & eacute;rimentales. Bien que l'utilisation de l'ivermectine soit g & eacute;n & eacute;ralement sans danger, il existe des rapports de neurotoxicit & eacute; associ & eacute;e au franchissement de la barri & egrave;re h & eacute;mato-enc & eacute;phalique par l'ivermectine en raison d'un surdosage ou d'un dysfonctionnement de cette barri & egrave;re. Chez la souris, la glycoprot & eacute;ine P maintient la barri & egrave;re h & eacute;mato-enc & eacute;phalique et les souris pr & eacute;sentant une mutation du g & egrave;ne codant pour la glycoprot & eacute;ine P mdr1a sont 50 & agrave; 100 fois plus sensibles & agrave; l'ivermectine. Les signes de neurotoxicit & eacute; comprennent l'ataxie, la bradypn & eacute;e, la position couch & eacute;e, les tremblements et la mort. Nous rapportons une neurotoxicit & eacute; apr & egrave;s administration d'ivermectine dans le but d'& eacute;radiquer le n & eacute;matode intestinal sp & eacute;cifique de la souris Heligmosomoides polygyrus chez les souris C57BL/6NTac et C57BL/6NCrl. Les souris ont fait l'objet d'un traitement vermifuge par administration sous-cutan & eacute;e de 10 ou 20 mg/kg d'ivermectine pour & eacute;radiquer tous les stades d'Heligmosomoides polygyrus. 24 & agrave; 48 heures apr & egrave;s l'administration du vermifuge, 5% (n = 4) des souris pr & eacute;sentaient des tremblements, une ataxie et/ou une inclinaison de la t & ecirc;te. Les souris affect & eacute;es ont & eacute;t & eacute; euthanasi & eacute;es et des signes pathologiques g & eacute;n & eacute;raux ont & eacute;t & eacute; trouv & eacute;s chez l'une des quatre souris (hydron & eacute;phrose gauche). Nous supposons que les effets neurologiques observ & eacute;s & eacute;taient dus & agrave; des d & eacute;fauts de la barri & egrave;re h & eacute;mato-enc & eacute;phalique, & agrave; un surdosage ou & agrave; une sensibilit & eacute; individuelle. Ce rapport justifie la prudence lors du traitement vermifuge des souris de laboratoire par voie sous-cutan & eacute;e avec de l'ivermectine & agrave; des doses de 10 mg/kg ou plus. Seit & uuml;ber 40 Jahren ist Ivermectin ein wirksames Antiparasitikum, das in der Human- und Veterin & auml;rmedizin eingesetzt wird. In der Versuchstierhaltung dient es prophylaktisch oder therapeutisch zur Gew & auml;hrleistung des Gesundheitszustands der Kolonie oder zu Versuchen in Studien. Obgleich Ivermectin im Allgemeinen sicher in der Anwendung ist, gibt es Berichte & uuml;ber Neurotoxizit & auml;t im Zusammenhang mit dem & Uuml;berschreiten der Blut-Hirn-Schranke durch Ivermectin aufgrund einer & Uuml;berdosierung oder einer St & ouml;rung der Blut-Hirn-Schranke. Bei M & auml;usen wird die Blut-Hirn-Schranke durch das P-Glykoprotein aufrechterhalten, und M & auml;use mit einer Mutation im P-Glykoprotein-kodierenden Gen mdr1a sind 50 bis 100 Mal empfindlicher gegen & uuml;ber Ivermectin. Zu den Anzeichen von Neurotoxizit & auml;t geh & ouml;ren Ataxie, Bradypnoe, R & uuml;ckenlage, Tremor und Tod. Wir berichten & uuml;ber Neurotoxizit & auml;t nach Verabreichung von Ivermectin zur Eradikation des m & auml;usespezifischen Darmnematoden Heligmosomoides polygyrus bei C57BL/6NTac- und C57BL/6NCrl-M & auml;usen. Die M & auml;use wurden durch subkutane Verabreichung von 10 bzw. 20 mg/kg Ivermectin entwurmt, um alle Stadien von Heligmosomoides polygyrus zu beseitigen. 24 bis 48 Stunden nach der Entwurmung traten bei 5% (n = 4) der M & auml;use Tremor, Ataxie und/oder Kopfneigung auf. Die betroffenen M & auml;use wurden euthanasiert und bei einer der vier M & auml;use wurde ein schwerwiegender pathologischer Befund festgestellt (linksseitige Hydronephrose). Wir gehen davon aus, dass die beobachteten neurologischen Effekte auf Defekte der Blut-Hirn-Schranke, & Uuml;berdosierung oder individuelle Empfindlichkeit zur & uuml;ckzuf & uuml;hren sind. Dieser Bericht gibt Anlass zur Vorsicht bei der subkutanen Entwurmung von Laborm & auml;usen mit Ivermectin in Dosen von 10 mg/kg oder h & ouml;her. Durante m & aacute;s de 40 a & ntilde;os, la ivermectina ha sido un eficaz f & aacute;rmaco antiparasitario utilizado tanto en la medicina humana como en la veterinaria. En los centros de animales de laboratorio se utiliza profil & aacute;ctica o terap & eacute;uticamente para mantener el estado de salud de la colonia o experimentalmente en estudios. Aunque la ivermectina es generalmente segura de usar, existen informes de neurotoxicidad asociados con el cruce de la barrera hematoencef & aacute;lica por parte de la ivermectina debido a una sobredosis o a una disfunci & oacute;n de la barrera hematoencef & aacute;lica. En los ratones, la glicoprote & iacute;na P mantiene la barrera hematoencef & aacute;lica y los ratones con mutaci & oacute;n en el gen mdr1a que codifica la glicoprote & iacute;na P son entre 50 y 100 veces m & aacute;s sensibles a la ivermectina. Los signos de neurotoxicidad incluyen ataxia, bradipnea, dec & uacute;bito, temblores y la muerte. Informamos de la neurotoxicidad tras la administraci & oacute;n de ivermectina para erradicar el nematodo intestinal Heligmosomoides polygyrus- que afecta espec & iacute;ficamente a murinos- en ratones C57BL/6NTac y C57BL/6NCrl. Los ratones fueron desparasitados mediante la administraci & oacute;n subcut & aacute;nea de 10 o 20 mg/kg de ivermectina para erradicar todas las fases del Heligmosomoides polygyrus. A las 24 - 48 horas despu & eacute;s de la desparasitaci & oacute;n, el 5% (n = 4) de los ratones presentaron temblor, ataxia y/o inclinaci & oacute;n de la cabeza. Los ratones afectados fueron sacrificados y se encontraron hallazgos patol & oacute;gicos macrosc & oacute;picos en uno de los cuatro ratones (hidronefrosis del lado izquierdo). Suponemos que los efectos neurol & oacute;gicos observados se debieron a defectos en la barrera hematoencef & aacute;lica, sobredosis o sensibilidad individual. Este informe advierte sobre la necesidad de precauci & oacute;n al desparasitar ratones de laboratorio por v & iacute;a subcut & aacute;nea con ivermectina con dosis de 10 mg/kg o superiores.

Uncontrolled regeneration leads to neoplastic transformation1-3. The intestinal epithelium requires precise regulation during continuous homeostatic and damage-induced tissue renewal to prevent neoplastic transformation, suggesting that pathways unlinking tumour growth from regenerative processes must exist. Here, by mining RNA-sequencing datasets from two intestinal damage models4,5 and using pharmacological, transcriptomics and genetic tools, we identified liver X receptor (LXR) pathway activation as a tissue adaptation to damage that reciprocally regulates intestinal regeneration and tumorigenesis. Using single-cell RNA sequencing, intestinal organoids, and gain- and loss-of-function experiments, we demonstrate that LXR activation in intestinal epithelial cells induces amphiregulin (Areg), enhancing regenerative responses. This response is coordinated by the LXR-ligand-producing enzyme CYP27A1, which was upregulated in damaged intestinal crypt niches. Deletion of Cyp27a1 impaired intestinal regeneration, which was rescued by exogenous LXR agonists. Notably, in tumour models, Cyp27a1 deficiency led to increased tumour growth, whereas LXR activation elicited anti-tumour responses dependent on adaptive immunity. Consistently, human colorectal cancer specimens exhibited reduced levels of CYP27A1, LXR target genes, and B and CD8 T cell gene signatures. We therefore identify an epithelial adaptation mechanism to damage, whereby LXR functions as a rheostat, promoting tissue repair while limiting tumorigenesis.

Understanding the development and maintenance of immunological memory is important for efforts to eliminate parasitic diseases like leishmaniasis. Leishmaniasis encompasses a range of pathologies, resulting from infection with protozoan parasites belonging to the subgenera Leishmania and Viannia of the genus Leishmania. A striking feature of these infections is that natural or drug-mediated cure of infection generally confers life-long protection against disease. The generation of protective T cell responses are necessary to control Leishmania infections. CD4+ T helper (Th) cells orchestrate immune responses in leishmaniasis and IFNγ+ Tbet+ CD4+ T (Th1) cells are required for the activation of phagocytes to kill captured or resident parasites, while other Th cell subset, including FoxP3+ natural regulatory T cells and Th2 cells can promote disease progression by suppressing the activities of Th1 cells. Upon resolution of a primary Leishmania infection, different subsets of CD4+ T cells, including tissue-resident memory T cells, effector memory T cells, central memory T cells, and short-lived effector T cells, help to confer resistance against reinfection. To maintain long-term protective Leishmania-specific CD4+ T cells responses, it is believed that persistent parasites or re-exposure to parasites at regular intervals is required (concomitant immunity). Despite the advances in our understanding about the immune responses during leishmaniasis, the generation of long-lasting protective immunity via vaccination has yet to be achieved. In this review, we summarize our current understanding about the formation and maintenance of immunological memory and control of leishmaniasis at the individual and population level. We will focus on Indian visceral leishmaniasis and discuss T cell responses that contribute to susceptibility to leishmaniasis, parasite persistence in populations and the environment, as well as describing advances in the development of leishmaniasis vaccines aimed at inducing protective CD4+ T cell responses.

Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.

Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1β and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses.

Influenza virus contributes substantially to the global human and animal disease burden. To protect individuals against disease, strategies are needed to minimize the time an individual is at risk of developing disease symptoms. Passive immunization using avian IgY antibodies can protect individuals against a variety of pathogens, including influenza virus. Yet the effect of IgY administration on generation of protective immunity is largely unknown. To address the effect of passive immunization on the host immune response development, adult or aged, male and female C57BL/6NCrl mice received chicken IgY anti-H5N1, normal IgY or PBS intranasally four hours before, and 20 hours after intranasal infection with H1N1 influenza A virus (PR8). The mice receiving cross-reactive IgY anti-H5N1 were protected from disease and developed influenza virus-specific memory T cells similar to control-treated mice. When re-challenged with PR8 35 days post primary infection IgY anti-H5N1-treated mice were fully protected. Moreover, when challenged with heterologous H3N2 influenza A virus (X-31) or with PR8 three months post infection the mice were protected against severe disease and death, albeit a slight transient weight loss was noted. The results show that passive immunization with IgY anti-H5N1 is safe and protects mice against disease induced by influenza virus without inhibiting development of protective immunity after virus exposure. This indicate that passive immunization can be used as prophylactic therapy in combination with immunization to prevent disease.

BackgroundCD4+ T cells play a central role in control of L. donovani infection, through IFN-γ production required for activation of macrophages and killing of intracellular parasites. Impaired control of parasites can in part be explained by hampered CD4+ T cells effector functions in visceral leishmaniasis (VL) patients. In a recent studies that defined transcriptional signatures for CD4+ T cells from active VL patients, we found that expression of the IL-7 receptor alpha chain (IL-7Rα; CD127) was downregulated, compared to CD4+ T cells from endemic controls (ECs). Since IL-7 signaling is critical for the survival and homeostatic maintenance of CD4+ T cells, we investigated this signaling pathway in VL patients, relative to ECs.MethodsCD4+ T cells were enriched from peripheral blood collected from VL patients and EC subjects and expression of IL7 and IL7RA mRNA was measured by real time qPCR. IL-7 signaling potential and surface expression of CD127 and CD132 on CD4+ T cell was analyzed by multicolor flow cytometry. Plasma levels of soluble IL-7 and sIL-7Rα were measured by ELISA.ResultTranscriptional profiling data sets generated previously from our group showed lower IL7RA mRNA expression in VL CD4+ T cells as compared to EC. A significant reduction was, however not seen when assessing IL7RA mRNA by RT-qPCR. Yet, the levels of soluble IL-7Rα (sIL-7Rα) were reduced in plasma of VL patients compared to ECs. Furthermore, the levels of soluble IL-7 were higher in plasma from VL patients compared to ECs. Interestingly, expression of the IL-7Rα protein was higher on VL patient CD4+ T cells as compared to EC, with activated CD38+ CD4+ T cells showing higher surface expression of IL-7Rα compared to CD38- CD4+ T cells in VL patients. CD4+ T cells from VL patients had higher signaling potential baseline and after stimulation with recombinant human IL-7 (rhIL-7) compared to EC, as measured by phosphorylation of STAT5 (pSTAT5). Interestingly, it was the CD38 negative cells that had the highest level of pSTAT5 in VL patient CD4+ T cells after IL-7 stimulation. Thus, despite unaltered or potentially lowered IL7RA mRNA expression by CD4+ T cells from VL patients, the surface expression of the IL-7Rα was higher compared to EC and increased pSTAT5 was seen following exposure to rhIL-7. Accordingly, IL-7 signaling appears to be functional and even enhanced in VL CD4+ T cells and cannot explain the impaired effector function of VL CD4+ T cells. The enhanced plasma IL-7 may serve as part of homeostatic feedback mechanism regulating IL7RA expression in CD4+ T cells.

Background Dendritic cell (DC) vaccines for cancer therapy offer the possibility to let the patient’s own immune system kill cancer cells. However, DC vaccines have shown less efficacy than expected due to failure to induce cancer cell killing and by activating T regulatory cells. Methods We tested if inhibition of signalling via WASp and Arp2/3 using the small molecule CK666 would enhance DC-mediated killing of tumour cells in vitro and in vivo. Results Using CK666 during the ex vivo phase of antigen processing of ovalbumin (OVA), murine and human DCs showed decreased phagosomal acidification, indicating activation of the cross-presentation pathway. When compared to untreated DCs, DCs treated with CK666 during uptake and processing of OVA-induced increased proliferation of OVA-specific CD8 + OT-I T cells in vitro and in vivo. Using the aggressive B16-mOVA melanoma tumour model, we show that mice injected with CK666-treated DCs and OVA-specific CD8 + OT-I T cells showed higher rejection of B16 melanoma cells when compared to mice receiving non-treated DCs. This resulted in the prolonged survival of tumour-bearing mice receiving CK666-treated DCs. Moreover, combining CK666-treated DCs with the checkpoint inhibitor anti-PD1 further prolonged survival. Conclusion Our data suggest that the small molecule inhibitor CK666 is a good candidate to enhance DC cross-presentation for cancer therapy.

Background Co-endemicity of neglected tropical diseases (NTDs) necessitates that these diseases should be considered concomitantly to understand the relationship between pathology and to support disease management and control programs. The aims of the study were to assess the prevalence of filarial infection in asymptomatic Leishmania donovani infected individuals and the correlation of Wuchereria bancrofti infection with progression to clinical visceral leishmaniasis (VL) in Bihar, India. Methodology/Principal findings Within the Muzaffarpur-TMRC Health and Demographic Surveillance System (HDSS) area, a cohort of Leishmania seropositive (n = 476) or seronegative individuals (n = 1130) were sampled annually for three years for filarial infection and followed for progression to clinical VL. To corroborate the results from the cohort study, we also used a retrospective case-control study of 36 VL cases and 71 controls selected from a subset of the HDSS population to investigate the relationship between progression to clinical VL and the prevalence of filarial infection at baseline. Our findings suggest a higher probability of progression to clinical VL in individuals with a history of filarial infection: in both the cohort and case-control studies, progression to clinical VL was higher among filaria infected individuals (RR = 2.57, p = 0.056, and OR = 2.52, p = 0.046 respectively). Conclusion This study describes that progression to clinical VL disease is associated with serological evidence of prior infection with W. bancrofti. The integration of disease programs for Leishmania and lymphatic filariasis extend beyond the relationship of sequential or co-infection with disease burden. To ensure elimination targets can be reached and sustained, we suggest areas of co-endemicity would benefit from overlapping vector control activities, health system networks and surveillance infrastructure.

Sex is an important biological variable in human biology with consequences for manifestations of various inflammatory diseases. Sex-associated difference is found in circulating T cell immunity as females are more prone to have autoimmune disease, but it is not clear whether tissue residing T cells are specific to sex. To investigate the effects of sex on the pool of resident T cells in human skin, the sex difference on skin T cell profiles were assessed. The effects of testosterone/estradiol on human CD8 T cell differentiation were also addressed. Both bulk and single RNA sequencing data from human skin from public repositories exhibited that female and male have distinct transcriptional signatures. Protein analyses revealed that female skin harbors around twice more cytotoxic and IFNγ producing CD8 resident memory T cells (Trm) in epidermis compared to that in males or testosterone-treated females. Furthermore, in vitro analyses displayed that testosterone hampers development of cytotoxic CD8 Trm from circulating T cells. These differences were not prominent in dermal skin compartment. Collectively, these results lead to regard sex as one of the variables to consider in the composition of epidermal skin T cells.

In contrast to delayed-type hypersensitivity (DTH) and other hallmark reactions of cell-mediated immunity that correlate with vaccine-mediated protection against Mycobacterium tuberculosis, the contribution of vaccine dose on responses that emerge early after infection in the skin with Bacille Calmette-Guérin (BCG) is not well understood. We used a mouse model of BCG skin infection to study the effect of BCG dose on the relocation of skin Dendritic cells (DCs) to draining lymph node (DLN). Mycobacterium antigen 85B-specific CD4+ P25 T cell-receptor transgenic (P25 TCRTg) cells were used to probe priming to BCG in DLN. DC migration and T cell priming were studied across BCG inocula that varied up to 100-fold (104 to 106 Colony-forming units-CFUs). In line with earlier results in guinea pigs, DTH reaction in our model correlated with BCG dose. Importantly, priming of P25 TCRTg cells in DLN also escalated in a dose-dependent manner, peaking at day 6 after infection. Similar dose-escalation effects were seen for DC migration from infected skin and the accompanying transport of BCG to the DLN. BCG-triggered upregulation of co-stimulatory molecules on migratory DCs was restricted to the first 24 hour after infection and was independent of BCG dose over a 10-fold range (105 to 106 CFUs). The dose seemed to be a determinant of the number of total skin DCs that move to the DLN. In summary, our results support the use of higher BCG doses to detect robust DC migration and T cell priming.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

Allergic disorders are caused by a combination of hereditary and environmental factors. The hygiene hypothesis postulates that early-life microbial exposures impede the development of subsequent allergic disease. Recently developed “wildling” mice are genetically identical to standard laboratory specific pathogen–free (SPF) mice but are housed under seminatural conditions and have rich microbial exposures from birth. Thus, by comparing conventional SPF mice with wildlings, we can uncouple the impact of lifelong microbial exposures from genetic factors on the allergic immune response. We found that wildlings developed larger populations of antigen-experienced T cells than conventional SPF mice, which included interleukin-10–producing CD4 T cells specific for commensal Lactobacilli strains and allergy-promoting T helper 2 (T H 2) cells. In models of airway exposure to house dust mite (HDM), recombinant interleukin-33, or Alternaria alternata , wildlings developed strong allergic inflammation, characterized by eosinophil recruitment, goblet cell metaplasia, and antigen-specific immunoglobulin G1 (IgG1) and IgE responses. Wildlings developed robust de novo T H 2 cell responses to incoming allergens, whereas preexisting T H 2 cells could also be recruited into the allergic immune response in a cytokine-driven and TCR-independent fashion. Thus, wildling mice, which experience diverse and lifelong microbial exposures, were not protected from developing pathological allergic immune responses. Instead, wildlings mounted robust allergic responses to incoming allergens, shedding new light on the hygiene hypothesis.

To the Editor, Tissue resident memory T cells contribute to localized immunity in barrier tissues such as the skin1 but are also implicated in local relapse of focal inflammatory skin diseases, including allergic contact dermatitis (ACD) and psoriasis.2– 4 To investigate how activation of skin T cells initiates relapsing contact dermatitis, global tissue responses to panTcell activation in skin biopsies from methylisothiazolinone (MI)induced ACD were mapped. Skin biopsies were collected during inflammation, 2 months, and 2 years after resolution of disease (Appendix S1). Despite macroscopic and microscopic diseaseresolution (Figure 1A), genes involved in keratinocyte differentiation and antimicrobial responses remained overexpressed for up to 2 years (Figure 1B,C, Figure S1) and resolved epidermis contained a twofold increase of T cells compared to nonlesional sites (Figure 1D). To model allergen challenge, skin biopsies from patients with lesional or resolved ACD were stimulated with the allergen MI, the panTcell agonist OKT3 or isotype control for 18 h ex vivo (Figure 1E). Epidermal gene expression analysis highlighted the upregulation of genes involved in ACD pathology in resolved biopsies (Figure 1F). Gene ontology pathway analysis of the epidermal transcriptome identified the involvement of several pathways related to Tcell activation in response to MI and/or OKT3 (Figure 1G). Inflammatory transcripts that were highly expressed in lesional ACD (Figure 1B), including CXCL10, IL22, and MMP12, were induced by both MI and OKT3 in biopsies up to 2 years after resolution of ACD (Figure 1F,G). In line with the transcriptome data, MI triggered release of proinflammatory cytokines and chemokines in resolved samples (Figure 1H). Following elicitation of ACD, the development of spongiotic pathology and macroscopic dermatitis requires influx of newly recruited immune cells to the epidermis. In addition to chemotactic signals, tissue remodeling and collagen degradation are crucial to facilitate cellular movements through the skin (Figure 2A). The transcript of the metalloproteinase (MMP)12 is overexpressed in de novo– induced ACD lesions in sensitized skin5 (Figure 2B). In a murine model of ACD, knocking out mmp12 led to reduced skin inflammation.6,7 We found that MMP12 was upregulated in resolved skin following both MI and OKT3 stimulation (Figures 1B,F,G and 2C). MMP12 cleaves collagen I and collagen IV (Figure 2D), one major component of the basement membrane that separates epidermis from dermis. In response to the exposure to ACDrelated cytokines: IFNγ, TNF, and IL17, MMP12 were induced, and MMP12 was released by primary fibroblasts and keratinocytes (Figure 2E– H), indicating that Tcell activation initiate downstream function of stroma cells. Considering the collagen IVdegrading capacity of MMP12 (Figure 2D), and the loss of basement integrity in lesional but not resolved ACD (Figure 2A), we directly tested the impact of MMP12 on basement membrane integrity and Tcell transmigration between the skin compartments. Explants of full thickness healthy human skin were injected intradermally with MMP12, TNF, IL17A, and IFNγ, or control (Figure 2I). MMP12 alone and in combination with cytokines induced local mobilization and accumulation of T cells in dermis (Figure 2J). Proliferating T cells could not be detected by Ki67staining (not shown). Lastly, intradermal injections of MMP12 and ACDassociated cytokines in human skin xenotransplanted onto SCIDbeige mice in vivo8 resulted in degradation of the basement membrane separating the epidermis and dermis, induced local accumulation of T cells and initiated spongiotic pathology (Figure 2K– N). Our data highlight that allergendriven contact dermatitis has a longterm impact on the transcriptome and local pool of T cells in resolved lesions. Both panTcell activation and allergen challenge induced molecular signs of ACDlike inflammation 2 months and 2 years after disease resolution. Activation of resident T cells in resolved ACD induced signals from cytokines capable of activating the stroma to produce MMP12, thereby driving tissue remodeling and pathology (Figure 2O). Pathogenic T cells in ACD thus ignites recurrent and diseasespecific pathology, and limiting their local persistence is a potential aim of future immunomodulatory treatments.

People living with diabetes who are also infected with M. tuberculosis are more likely to develop tuberculosis disease (TB). Why diabetic patients have an increased risk for developing TB is not well understood.

A series of 1-aryl-4-(phthalimidoalkyl) piperazines and 1-aryl-4-(naphthalimidoalkyl) piperazines were retrieved from a proprietary library based on their high structural similarity to haloperidol, an antipsychotic with antiparasitic activity, and assessed as potential antileishmanial scaffolds. Selected compounds were tested for antileishmanial activity against promastigotes of Leishmania major and Leishmania mexicana in dose-response assays. Two of the 1-aryl-4-(naphthalimidoalkyl) piperazines (compounds 10 and 11) were active against promastigotes of both Leishmania species without being toxic to human fibroblasts. Their activity was found to correlate with the length of their alkyl chains. Further analyses showed that compound 11 was also active against intracellular amastigotes of both Leishmania species. In promastigotes of both Leishmania species, compound 11 induced collapse of the mitochondrial electrochemical potential and increased the intracellular Ca2+ concentration. Therefore, it may serve as a promising lead compound for the development of novel antiparasitic drugs.

AbstractObjectivesThere is an urgent need to be able to identify individuals with asymptomatic Leishmania donovani infection, so their risk of progressing to VL and transmitting parasites can be managed. This study examined transcriptional markers expressed by CD4+ T cells that could distinguish asymptomatic individuals from endemic controls and visceral leishmaniasis (VL) patients.MethodsCD4+ T cells were isolated from individuals with asymptomatic L. donovani infection, endemic controls and VL patients. RNA was extracted and RNAseq employed to identify differentially expressed genes. The expression of one gene and its protein product during asymptomatic infection were evaluated.ResultsAmphiregulin (AREG) was identified as a distinguishing gene product in CD4+ T cells from individuals with asymptomatic L. donovani infection, compared to VL patients and healthy endemic control individuals. AREG levels in plasma and antigen‐stimulated whole‐blood assay cell culture supernatants were significantly elevated in asymptomatic individuals, compared to endemic controls and VL patients. Regulatory T (Treg) cells were identified as an important source of AREG amongst CD4+ T‐cell subsets in asymptomatic individuals.ConclusionIncreased Treg cell AREG expression was identified in individuals with asymptomatic L. donovani infection, suggesting the presence of an ongoing inflammatory response in these individuals required for controlling infection and that AREG may play an important role in preventing inflammation‐induced tissue damage and subsequent disease in asymptomatic individuals.

Influenza virus infection is restricted to airway-associated tissues and elicits both cellular and humoral responses ultimately resulting in generation of memory cells able to initiate a rapid immune response against re-infections. Resident memory T cells confer protection at the site of infection where lung-resident memory T cells are important for protecting the host against homologous and heterologous influenza virus infections. Mapping kinetics of local and systemic T cell memory formation is needed to better understand the role different T cells have in viral control and protection. After infecting BALB/c mice with influenza virus strain A/Puerto Rico/8/1934 H1N1 the main proportion of activated T cells and B cells expressing the early activation marker CD69 was detected in lungs and lung-draining mediastinal lymph nodes. Increased frequencies of activated cells were also observed in the peripheral lymphoid organs spleen, inguinal lymph nodes and mesenteric lymph nodes. Likewise, antigen-specific T cells were most abundant in lungs and mediastinal lymph nodes but present in all organs studied. CD8+CD103-CD49a+ lung-resident T cells expanded simultaneously with timing of viral clearance whereas CD8+CD103+CD49a+ lung-resident T cells was the most abundant subset after resolution of infection and antigen-specific, lung-resident T cells were detected up to seven months after infection. In conclusion, the results in this detailed kinetic study demonstrate that influenza virus infection elicits adaptive immune responses mainly in respiratory tract-associated tissues and that distinct subsets of lung-resident T cells expand at different time points during infection. These findings contribute to the understanding of the adaptive immune response locally and systemically following influenza virus infection and call for further studies on the roles of the lung-resident T cell subsets.