Assessment of serum granulysin and cathepsin-L levels in vitiligo patients.

OBJECTIVE: Cellular and humoral immunity plays a role in the pathogenesis of vitiligo. T lymphocytes and natural killer cells involved in cellular immunity carry out their cytotoxic activities through perforin/granzyme-dependent granule exocytosis, in which granulysin and cathepsin-L are also involved. The aim of this study was to investigate the possible role of serum granulysin and cathepsin-L in the etiopathogenesis of vitiligo and their association with disease activity and severity. METHODS: This randomized, prospective case-control study was conducted with 46 vitiligo patients admitted to the hospital for vitiligo between January and November 2021 and 46 healthy volunteers of similar age and gender. Serum levels of granulysin and cathepsin-L were measured by the enzyme-linked immunosorbent assay method. RESULTS: The mean serum levels of granulysin and cathepsin-L were statistically significantly higher in vitiligo patients compared with the control group (p=0.048 and p=0.024, respectively). There was no statistically significant correlation between serum granulysin and serum cathepsin-L levels and disease severity in the patient group (r=0.30, p=0.062 and r=0.268, p=0.071, respectively). Disease activity also showed no significant association with serum granulysin and cathepsin-L levels (p=0.986 and p=0.962, respectively). CONCLUSION: Although granulysin and cathepsin-L are molecules involved in the pathogenesis of vitiligo, the use of these molecules may not be helpful in assessing disease activity and severity. It may be helpful to conduct comprehensive and prospective studies to find new molecules to fill the gap in this area.

Outlining the skin-homing and circulating CLA+NK cells in patients with severe atopic dermatitis.

Atopic dermatitis (AD) is a complex, multifactorial skin disease, characterized by pruritus and predominant Th2 inflammation. Innate immune cells may play a role in AD development and are composed of granulocytes, macrophages, innate-like T cells, and innate lymphoid cells. This study investigates the phenotypic and functional profile of circulating CLA+ natural killer (NK) cells and its role in the skin-homing to NK cells infiltrated in adults' skin with AD. We selected 44 AD patients and 27 non-AD volunteers for the study. The results showed increased frequencies of both CLA+CD56bright and CLA+CD56dim NK cell populations in the peripheral blood, mainly in severe AD patients. Upon SEB stimulation, we observed an augmented percentage of CLA+CD56dim NK cells expressing CD107a, IFN-γ, IL-10, and TNF, reinforcing the role of staphylococcal enterotoxins in AD pathogenesis. Additionally, we demonstrated increased dermal expression of both NK cell markers NCAM-1/CD56 and pan-granzyme, corroborating the skin-homing, mostly in severe AD. Further studies are necessary to elucidate the potential role of NK cells in the chronification of the inflammatory process in AD skin, as well as their possible relationship with staphylococcal enterotoxins, and as practicable therapeutic targets.

γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum-infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria-γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs.

An anti-CD6 antibody for the treatment of COVID-19 patients with cytokine-release syndrome: report of three cases.

In COVID-19, the inflammatory cytokine-release syndrome is associated with the progression of the disease. Itolizumab is a monoclonal antibody that recognizes human CD6 expressed in activated T cells. The antibody has shown to be safe and efficacious in the treatment of moderate to severe psoriasis. Its effect is associated with the reduction of pro-inflammatory cytokines release, including IFN-γ, IL-6 and TNF-α. Here, we report the outcome of three severe and critically ill COVID-19 patients treated with itolizumab as part of an expanded access protocol. Itolizumab was able to reduce IL-6 concentrations in all the patients. Two of the three patients showed respiratory and radiological improvement and were fully recovered. We hypothesize this anti-inflammatory therapy in addition to antiviral and anticoagulant therapy could reduce COVID-19 associated morbidity and mortality.

Contributions of IFN-γ and granulysin to the clearance of Plasmodium yoelii blood stage.

P. vivax-infected Retics (iRetics) express human leukocyte antigen class I (HLA-I), are recognized by CD8+ T cells and killed by granulysin (GNLY) and granzymes. However, how Plasmodium infection induces MHC-I expression on Retics is unknown. In addition, whether GNLY helps control Plasmodium infection in vivo has not been studied. Here, we examine these questions using rodent infection with the P. yoelii 17XNL strain, which has tropism for Retics. Infection with P. yoelii caused extramedullary erythropoiesis, reticulocytosis and expansion of CD8+CD44+CD62L- IFN-γ-producing T cells that form immune synapses with iRetics. We now provide evidence that MHC-I expression by iRetic is dependent on IFN-γ-induced transcription of IRF-1, MHC-I and ß2-microglobulin (ß2-m) in erythroblasts. Consistently, CTLs from infected wild type (WT) mice formed immune synapses with iRetics in an IFN-γ- and MHC-I-dependent manner. When challenged with P. yoelii 17XNL, WT mice cleared parasitemia and survived, while IFN-γ KO mice remained parasitemic and all died. ß2-m KO mice that do not express MHC-I and have virtually no CD8+ T cells had prolonged parasitemia, and 80% survived. Because mice do not express GNLY, GNLY-transgenic mice can be used to assess the in vivo importance of GNLY. Parasite clearance was accelerated in GNLY-transgenic mice and depletion of CD8+ T cells ablated the GNLY-mediated resistance to P. yoelii. Altogether, our results indicate that in addition to previously described mechanisms, IFN-γ promotes host resistance to the Retic-tropic P. yoelii 17XNL strain by promoting MHC-I expression on iRetics that become targets for CD8+ cytotoxic T lymphocytes and GNLY.

Interactome analysis of CD5 and CD6 ectodomains with tegumental antigens from the helminth parasite Echinococcus granulosus sensu lato.

Echinococcus granulosus sensu lato (s.l.) is a cestode parasite affecting both human and livestock health. Recombinant ectodomains of human scavenger receptors CD5 (rshCD5) and CD6 (rshCD6) were previously reported to bind its tegumental antigens and to exert prophylactic effects in a murine model of infection. Although the properties of mammalian scavenger receptors include the binding to diverse pathogen-derived structures, their interaction with helminth parasites has been scarcely explored. Therefore, we report here a search for CD5 and CD6 interactors within E. granulosus s.l. antigens. Mass spectrometry analysis of pull-downs from soluble tegumental components with biotinylated rshCD5 and rshCD6 resulted in 17 and 11 overrepresented interactors, respectively, 8 of which were shared. The interactors included previously reported protective molecules against E. granulosus s.l. and/or other helminths. Similar studies performed with 11-mer peptides mapping to each of the three extracellular scavenger domains of CD5 and CD6 allowed an estimated molecular topology of the interactions. In conclusion, the fact that most helminth interactors identified for rshCD5 and rshCD6 were already reported as vaccine candidates or pharmacological targets against different helminthiases, supports the view that their beneficial effects in experimental infection results from binding to multiple relevant tegumental antigens.

BAY 41-2272 inhibits human T lymphocyte functions.

BAY 41-2272 increases guanosine 3', 5'-cyclic monophosphate (cGMP) levels by stimulating soluble guanylate cyclase (sGC). In this study, we evaluated the effect of BAY 41-2272 on human T lymphocyte functions. Pretreating T cells for 24 h with BAY 41-2272 at 3 µM and 30 µM, followed by activation with 90 nM phorbol myristate acetate (PMA), inhibited interferon-gamma (IFN-γ) production, with 3 µM and 30 µM BAY causing 16.5-fold and 12.1-fold inhibition, respectively, compared to PMA alone (p < 0.05, one-way ANOVA followed by Tukey's test). We also observed suppressive effects on the expression of CD69, with 30 µM BAY causing 3.55-fold lower expression than PMA/ionomycin (p < 0.001 one-way ANOVA followed by Tukey's test), and T-bet, with 30 µM BAY causing 1.47-fold lower expression than PMA/ionomycin (p < 0.05, one-way ANOVA test followed by Tukey's test). Additionally, T lymphocyte proliferation was reduced 2.13-fold and 4.3-fold, respectively, by 3 µM BAY and 30 µM BAY compared to PMA/ionomycin (p < 0.01, p < 0.001, one-way ANOVA followed by Tukey's test). BAY 41-2272 inhibits human T lymphocyte function and may be explored as an immunomodulatory drug in patients with autoimmune/inflammatory diseases and lymphoproliferative syndromes.

Mutant GNLY is linked to Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare severe cutaneous adverse reactions to drugs. Granulysin (GNLY) plays a key role in keratinocyte apoptosis during SJS/TEN pathophysiology. To determine if GNLY-encoding mutations might be related to the protein's functional disturbances, contributing to SJS/TEN pathogenesis, we performed direct sequencing of GNLY's coding region in a group of 19 Colombian SJS/TEN patients. A GNLY genetic screening was implemented in a group of 249 healthy individuals. We identified the c.11G > A heterozygous sequence variant in a TEN case, which creates a premature termination codon (PTC) (p.Trp4Ter). We show that a mutant protein is synthesised, possibly due to a PTC-readthrough mechanism. Functional assays demonstrated that the mutant protein was abnormally located in the nuclear compartment, potentially leading to a toxic effect. Our results argue in favour of GNLY non-synonymous sequence variants contributing to SJS/TEN pathophysiology, thereby constituting a promising, clinically useful molecular biomarker.

CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice.

BACKGROUND: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. METHODS: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. RESULTS: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFß1 (p = 0.038). CONCLUSION: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

The role of GNLY gene polymorphisms in psoriasis pathogenesis.

BACKGROUND: Psoriasis is a systemic inflammatory disorder that involves complex pathogenic interactions between the innate and adaptive immune systems. The most accepted mechanism in the etiopathogenesis of psoriasis is the induction of inflammation with keratinocyte hyperproliferation. Granulysin (GNLY) is a cytolytic antimicrobial peptide (AMP) that is secreted together with granzyme and perforin from the granules of human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It has been immunohistochemically proven that the expression of granulysin is increased in lesions of psoriasis. OBJECTIVE: This study aimed to investigate the relationship between psoriasis disease and granulysin gene polymorphisms. METHODS: GNLY rs7908 and rs10180391 polymorphisms were studied by PCR-RFLP in 100 psoriasis patients under treatment in the Dermatology Polyclinic of Bulent Ecevit University. In addition, 100 healthy individuals with similar age and sex distribution were used as a control group. RESULTS: In the control group, GNLY rs7908 CC genotype was significantly higher than in psoriasis patients (P= 0.031; OR= 0.305; Cl= 0.305 (0.121 - 0.773). In our study, the genotype distributions in patients and control groups were GNLY rs7908 (SNP) GG (51%, 37%), GC (41%, 44%), CC (8%, 19%); GNLY rs10180391 (SNP) from the CC (41%, 44%), CT (42%, % 41), TT (17%, 15%). STUDY LIMITATIONS: The study only included Turkish patients. CONCLUSION: Our findings showed that GNLY rs7908 CC genotype and C allele had a protective effect against psoriasis and decreased the disease severity (according to PASI score), whereas rs10180391 SNP did not show any effective role in psoriasis pathogenesis.

Group 2 innate lymphoid cells exhibit progressively higher levels of activation during worsening of liver fibrosis.

INTRODUCTION: The interleukin-33/interleukin-13 pathway is involved in the immunopathology of liver fibrosis and recently characterized group 2 innate lymphoid cells (ILC2) were identified as profibrotic immune cells in the liver of mouse models. Our aim was to elucidate whether ILC2 might be present in human liver tissue and whether ILC2 contribute to liver fibrosis. MATERIALS AND METHODS: To identify ILC2 in liver tissue and blood, we purified mononuclear immune cells from needle biopsies, cirrhotic explant specimen, and paired peripheral blood samples. Cell suspensions were incubated with specific markers for ILC2 and analyzed by flow cytometry. The CD69 marker was included to assess the activation level of ILC2. In addition, we determined the IL-33 plasma level. RESULTS: Results were correlated with the METAVIR fibrotic score of patients enrolled in this study. We detected ILC2 in a higher percentage of CD45+ cells in liver tissue than in paired peripheral blood. The number of ILC2 was significantly increased in fibrotic tissue, but only slightly increased in paired peripheral blood. A higher percentage of CD69+ ILC2 was observed in fibrotic tissue, and this increase correlates positively with aggravation of liver fibrosis measured by fibrotic METAVIR score. A higher level of plasma IL-33 was only detected in samples obtained from cirrhotic patients. CONCLUSION: Our study indicates that ILC2 are present in the human liver and are activated in tissue contributing to the immunopathology of human liver fibrosis, independently of the etiology; which might be a potential new therapeutic target.

The role of GNLY gene polymorphisms in psoriasis pathogenesis

Abstract BACKGROUND: Psoriasis is a systemic inflammatory disorder that involves complex pathogenic interactions between the innate and adaptive immune systems. The most accepted mechanism in the etiopathogenesis of psoriasis is the induction of inflammation with keratinocyte hyperproliferation. Granulysin (GNLY) is a cytolytic antimicrobial peptide (AMP) that is secreted together with granzyme and perforin from the granules of human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It has been immunohistochemically proven that the expression of granulysin is increased in lesions of psoriasis. OBJECTIVE: This study aimed to investigate the relationship between psoriasis disease and granulysin gene polymorphisms. METHODS: GNLY rs7908 and rs10180391 polymorphisms were studied by PCR-RFLP in 100 psoriasis patients under treatment in the Dermatology Polyclinic of Bulent Ecevit University. In addition, 100 healthy individuals with similar age and sex distribution were used as a control group. RESULTS: In the control group, GNLY rs7908 CC genotype was significantly higher than in psoriasis patients (P= 0.031; OR= 0.305; Cl= 0.305 (0.121 - 0.773). In our study, the genotype distributions in patients and control groups were GNLY rs7908 (SNP) GG (51%, 37%), GC (41%, 44%), CC (8%, 19%); GNLY rs10180391 (SNP) from the CC (41%, 44%), CT (42%, % 41), TT (17%, 15%). STUDY LIMITATIONS: The study only included Turkish patients. CONCLUSION: Our findings showed that GNLY rs7908 CC genotype and C allele had a protective effect against psoriasis and decreased the disease severity (according to PASI score), whereas rs10180391 SNP did not show any effective role in psoriasis pathogenesis.

Inhibiting Adenosine Receptor Signaling Promotes Accumulation of Effector CD4+ T Cells in the Lung Parenchyma During Severe Tuberculosis.

BACKGROUND: Tuberculous pneumonia, necrotic granulomatous lesions, and bacterial dissemination characterize severe forms of mycobacterial infection. METHODS: To evaluate the pulmonary CD4+ T-cell response during severe tuberculosis, C57BL/6 mice were infected with approximately 100 bacilli of 3 hypervirulent mycobacterial isolates (Mycobacterium tuberculosis strain Beijing 1471 and Mycobacterium bovis strains B2 and MP287/03) or the H37Rv M tuberculosis strain as reference for mycobacterial virulence. Because high expression of both CD39 and CD73 ectonucleotidases was detected on parenchymal CD4+ T cells, we investigated whether CD4+ T-cell suppression in the context of severe disease was due to the extracellular adenosine accumulation that resulted from tissue damage. RESULTS: Lowest expression of CD69, which is an activation marker implicated in maintaining cells in tissues, was observed in lungs from mice displaying the most severe pulmonary pathology. Reduced interferon (IFN)γ-producing CD4+ T cells were also found in the lung of these mice. Intranasal administration of the adenosine receptor antagonist caffeine substantially enhanced the frequency and number of parenchymal CD4+ T cells as well as both CD69 expression and IFNγ production. CONCLUSIONS: These results indicate that adenosine, which may be generated by extracellular adenosine triphosphate degradation, impairs the parenchymal CD4+ T-cell response and contributes to the development of severe tuberculosis.

CD4+CD69+ T cells and CD4+CD25+FoxP3+ Treg cells imbalance in peripheral blood, spleen and peritoneal lavage from pristane-induced systemic lupus erythematosus (SLE) mice

Abstract Background: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. Methods: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. Results: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4+ CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038). Conclusion: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.

The ectodomains of the lymphocyte scavenger receptors CD5 and CD6 interact with tegumental antigens from Echinococcus granulosus sensu lato and protect mice against secondary cystic echinococcosis.

BACKGROUND: Scavenger Receptors (SRs) from the host's innate immune system are known to bind multiple ligands to promote the removal of non-self or altered-self targets. CD5 and CD6 are two highly homologous class I SRs mainly expressed on all T cells and the B1a cell subset, and involved in the fine tuning of activation and differentiation signals delivered by the antigen-specific receptors (TCR and BCR, respectively), to which they physically associate. Additionally, CD5 and CD6 have been shown to interact with and sense the presence of conserved pathogen-associated structures from bacteria, fungi and/or viruses. METHODOLOGY/PRINCIPAL FINDINGS: We report herein the interaction of CD5 and CD6 lymphocyte surface receptors with Echinococcus granulosus sensu lato (s.l.). Binding studies show that both soluble and membrane-bound forms of CD5 and CD6 bind to intact viable protoscoleces from E. granulosus s.l. through recognition of metaperiodate-resistant tegumental components. Proteomic analyses allowed identification of thioredoxin peroxidase for CD5, and peptidyl-prolyl cis-trans isomerase (cyclophilin) and endophilin B1 (antigen P-29) for CD6, as their potential interactors. Further in vitro assays demonstrate that membrane-bound or soluble CD5 and CD6 forms differentially modulate the pro- and anti-inflammatory cytokine release induced following peritoneal cells exposure to E. granulosus s.l. tegumental components. Importantly, prophylactic infusion of soluble CD5 or CD6 significantly ameliorated the infection outcome in the mouse model of secondary cystic echinococcosis. CONCLUSIONS/SIGNIFICANCE: Taken together, the results expand the pathogen binding properties of CD5 and CD6 and provide novel evidence for their therapeutic potential in human cystic echinococcosis.

Human CD8 T-cell activation in acute and chronic chikungunya infection.

There is a need for more detailed elucidation of T-cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin-17A, interleukin-10 and CD95 ligand, and co-expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.

Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes.

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8+ T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8+ T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I7. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax-infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax-infected reticulocytes in a human leukocyte antigen-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax-infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the P. vivax-infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy.

Natural killer cells are pivotal for in vivo protection following systemic infection by Sporothrix schenckii.

Natural killer (NK) cells are one of the first cell types to enter inflammation sites and have been historically known as key effector cells against tumours and viruses; now, accumulating evidence shows that NK cells are also capable of direct in vitro activity and play a protective role against clinically important fungi in vivo. However, our understanding of NK cell development, maturation and activation in the setting of fungal infections is preliminary at best. Sporotrichosis is an emerging worldwide-distributed subcutaneous mycosis endemic in many countries, affecting humans and other animals and caused by various related thermodimorphic Sporothrix species, whose prototypical member is Sporothrix schenckii. We show that following systemic infection of BALB/c mice with S. schenckii sensu stricto, NK cells displayed a more mature phenotype as early as 5 days post-infection as judged by CD11b/CD27 expression. At 10 days post-infection, NK cells had increased expression of CD62 ligand (CD62L) and killer cell lectin-like receptor subfamily G member 1 (KLRG1), but not of CD25 or CD69. Depletion of NK cells with anti-asialo GM1 drastically impaired fungal clearance, leading to a more than eightfold increase in splenic fungal load accompanied by heightened systemic inflammation, as shown by augmented production of the pro-inflammatory cytokines tumour necrosis factor-α, interferon-γ and interleukin-6, but not interleukin-17A, in the spleen and serum. Our study is, to the best of our knowledge, the first to demonstrate that a fungal infection can drive NK cell maturation in vivo and that such cells are pivotal for in vivo protection against S. schenckii.

The anti-CD6 antibody itolizumab provides clinical benefit without lymphopenia in rheumatoid arthritis patients: results from a 6-month, open-label Phase I clinical trial.

Itolizumab is a humanized anti-CD6 monoclonal antibody (mAb) that has previously shown encouraging results, in terms of safety and positive clinical effects, in a 6-week monotherapy clinical trial conducted in rheumatoid arthritis (RA) patients. The current Phase I study evaluated the safety and clinical response for a longer treatment of 12 itolizumab intravenous doses in subjects with active RA despite previous disease-modifying anti-rheumatic drug (DMARD) therapy. Twenty-one subjects were enrolled into four dosage groups (0·1, 0·2, 0·4 and 0·8 mg/kg). Efficacy end-points including American College of Rheumatology (ACR)20, ACR50 and ACR70 response rates and disease activity score in 28 joints (DAS28) were monitored at baseline and at specific time-points during a 10-week follow-up period. Itolizumab was well tolerated up to the highest tested dose. No related serious adverse events were reported and most adverse events were mild. Remarkably, itolizumab treatment did not produce lymphopenia and, therefore, was not associated with infections. All patients achieved a clinical response (ACR20) at least once during the study. Eleven subjects (55%) achieved at least a 20% improvement in ACR just 1 week after the first itolizumab administration. The clinical response was observed from the beginning of the treatment and was sustained during 24 weeks. The efficacy profile of this 12-week treatment was similar to that of the previous study (6-week treatment). These results reinforce the safety profile of itolizumab and provide further evidence on the clinical benefit from the use of this anti-CD6 mAb in RA patients.

Modulation of γδ T-cell activation by neutrophil elastase.

γδ T cells are non-conventional, innate-like T cells, characterized by a restricted T-cell receptor repertoire. They participate in protective immunity responses against extracellular and intracellular pathogens, tumour surveillance, modulation of innate and adaptive immune responses, tissue healing, epithelial cell maintenance and regulation of physiological organ function. In this study, we investigated the role of neutrophils during the activation of human blood γδ T cells through CD3 molecules. We found that the up-regulation of CD69 expression, and the production of interferon-γ and tumour necrosis factor-α induced by anti-CD3 antibodies was potentiated by neutrophils. We found that inhibition of caspase-1 and neutralization of interleukin-18 did not affect neutrophil-mediated modulation. By contrast, the treatment with serine protease inhibitors prevented the potentiation of γδ T-cell activation induced by neutrophils. Moreover, the addition of elastase to γδ T-cell culture increased their stimulation, and the treatment of neutrophils with elastase inhibitor prevented the effect of neutrophils on γδ T-cell activation. Furthermore, we demonstrated that the effect of elastase on γδ T cells was mediated through the protease-activated receptor, PAR1, because the inhibition of this receptor with a specific antagonist, RWJ56110, abrogated the effect of neutrophils on γδ T-cell activation.