Serum Cytokine Responses over the Entire Clinical-Immunological Spectrum of Human Leishmania (L.) infantum chagasi

The clinical-immunological spectrum of human Leishmania (L.) infantum chagasi infection in Amazonian Brazil was recently reviewed based on clinical, DTH, and IFAT (IgG) evaluations that identified five profiles: three asymptomatic (asymptomatic infection, AI; subclinical resistant infection, SRI; and indeterminate initial infection, III) and two symptomatic (symptomatic infection, SI; American visceral leishmaniasis, AVL; and subclinical oligosymptomatic infection, SOI). TNF-α, IL-4, IL-6, and IL-10 serum cytokines were analyzed using multiplexed Cytometric Bead Array in 161 samples from endemic areas in the Brazilian Amazon: SI [AVL] (21 cases), III (49), SRI (19), SOI (12), AI (36), and a control group [CG] (24). The highest IL-6 serum levels were observed in the SI profile (AVL); higher IL-10 serum levels were observed in SI than in SOI or CG and in AI and III than in SOI; higher TNF-α serum levels were seen in SI than in CG. Positive correlations were found between IL-6 and IL-10 serum levels in the SI and III profiles and between IL-6 and TNF-α and between IL-4 and TNF-α in the III profile. These results provide strong evidence for associating IL-6 and IL-10 with the immunopathogenesis of AVL and help clarify the role of these cytokines in the infection spectrum

New Players in the Same Old Game: Disturbance of Group 2 Innate Lymphoid Cells in HIV-1 and <i>Mycobacterium leprae</i> Co-infected Patients

<div><p>Abstract</p><p>Leprosy control is achieved through a fine-tuning of T_H1 and T_H2 immune response pattern balance. Given the increasing epidemiological overlay of HIV and <i>M</i>. <i>leprae</i> infections, immune response in co-infected patients consists in an important contemporary issue. Here we describe for the first time the innate lymphoid cells compartment in peripheral blood of leprosy and HIV/<i>M</i>. <i>leprae</i> co-infected patients, and show that co-infection increases group 2 innate lymphoid whilst decreasing group 1 innate lymphoid cells frequencies and function.</p></div

<i>M</i>. <i>Leprae</i>/HIV-1 co-infected patients present a disturbance of circulating ILC2 cells.

<p><i>A</i>, gate strategy used to define group 2 innate lymphoid cells based on the expression of surface proteins. <i>B</i>, frequencies of circulating ILC2 cells defined as Lin^-CD45⁺CD161⁺CD25⁺CRTH2⁺ from healthy (n = 16), M. leprae-infected (n = 7), HIV-1-infected (n = 11) and co-infected (n = 10) cohorts. <i>C</i>, intracellular staining of IL-4 on Lin^-CD45⁺CD25⁺ cells. Frequencies of IL-4 producing cells within healthy (n = 10), <i>M</i>. <i>leprae</i>-infected (n = 5), HIV-1-infected (n = 10) and co-infected (n = 7) groups. <i>D</i>, intracellular staining of IL-13 on Lin^-CD45⁺CD25⁺ cells. Frequencies of IL-13 producing cells within healthy (n = 10), M. leprae-infected (n = 5), HIV-1-infected (n = 10) and co-infected (n = 7) groups. Each dot represents an individual, and bars indicate medians in the graphs. Statistical analysis was performed using the Kruskal-Wallis test. * p<0.05; ** p<0.001; ***p<0.0001.</p

<i>M</i>. <i>Leprae</i>/HIV-1 co-infection led to a decrease of circulating ILC1 cells.

<p><i>A</i>, gate strategy used to define group 1 and group 3 innate lymphoid cells based on the production of TNF-α and IL-17 by Lin^-CD45⁺CD56⁺ cells. <i>B</i>, Frequencies of TNF-α-producing cells, defined as ILC1 cells, from healthy (n = 10), <i>M</i>. <i>leprae</i>-infected (n = 6), HIV-1-infected (n = 10) and co-infected (n = 10) patients. <i>C</i>, Frequencies of IL-17-producing cells, defined as ILC3 cells, from healthy (n = 10), <i>M</i>. <i>leprae</i>-infected (n = 6), HIV-1-infected (n = 10) and co-infected (n = 10) patients. Each dot represents an individual, and bars indicate medians in the graphs. Statistical analysis was performed using the Kruskal-Wallis test. * p<0.05; ** p<0.001; ***p<0.0001.</p

Novel CD28 antagonist mPEG PV1-Fab’ mitigates experimental autoimmune uveitis by suppressing CD4+ T lymphocyte activation and IFN-γ production

<div><p>Autoimmune Uveitis is an important chronic inflammatory disease and a leading cause of impaired vision and blindness. This ocular autoimmune disorder is mainly mediated by T CD4⁺ lymphocytes poising a T_H1 phenotype. Costimulatory molecules are known to play an important role on T cell activation and therefore represent interesting therapeutical targets for autoimmune disorders. CD28 is the prototypical costimulatory molecule for T lymphocytes, and plays a crucial role in the initiation, and maintenance of immune responses. However, previous attempts to use this molecule in clinical practice achieved no success. Thus, we evaluated the efficacy of mPEG PV1-Fab’ (PV1), a novel selective CD28 antagonist monovalent Fab fragment in the treatment of Experimental Autoimmune Uveitis (EAU). Here, we showed that PV1 treatment decreases both average disease score and incidence of EAU. A decrease in the activation profile of both T CD4⁺ and T CD8⁺ eye-infiltrating lymphocytes was evidenced. In the periphery, T CD4⁺ cells from PV1-treated mice also showed a decrease in their activation status, with reduced expression of CD69, CD25, and PD-1 molecules. This suppression was not dependent on Treg cells, as both their frequency and absolute number were lower in PV1-treated mice. In addition, frequency of CD4⁺IFN-γ⁺ T cells was significantly lower in PV1-treated group, but not of IL-17-producing T cells. Moreover, after specific restimulation, PV1 blockade selectively blocked IFN-γ production by CD4⁺ lymphocytes Taken together, our data suggest that mPEG PV1-Fab’ acts mainly on IFN-γ-producing CD4⁺ T cells and emphasize that this specific CD28 blockade strategy is a potential specific and alternative tool for the treatment of autoimmune disorders in the eye.</p></div

mPEG PV1-Fab’ treatment decreases T_reg population.

<p>Female B10.RIII mice were immunized with 50 μg/animal of 161–180 IRBP in CFA, plus 500 ng/animal of PTx boost. Starting on day 9, mice were treated every 4 days with CD28 antagonist, PV1 (10mg/Kg; ip), or left untreated. On day 14 mice were sacrificed and dLN were collected for immunophenotyping of regulatory T cells. (A) Representative plot showing gate strategy for defining T_reg population. Foxp3 expression was defined by using a Fluorescence Minus One (FMO) control. (B) Frequency and (C) total number of CD3⁺CD4⁺CD25⁺Foxp3⁺ cells in dLN of B10.RIII mice. (D) Frequency and (E) total numbers of CD3⁺CD4⁺CD25⁺Foxp3⁺ cells in spleen of B10.RIII mice. Data are representative of three independent experiments; 5-mice per group. Median and range are depicted. **, p<0.01, two-tailed Mann-Whitney test.</p

PV1-treated mice exhibited less activated eye-infiltrating lymphocytes.

<p>B10.RIII mice were immunized with 50 μg/animal of 161–180 IRBP in CFA, plus 500 ng/animal of PTx boost. Starting on day 9, mice were treated every 4 days with CD28 antagonist, PV1 (10mg/Kg; ip), or left untreated. On day 14 mice were sacrificed and eyes were collected for immunophenotyping of eye-infiltrating leukocytes. (A) Total count of eye-infiltrating leukocytes. (B) Frequency of CD4⁺ and CD8⁺ T lymphocytes infiltrating the eyes of B10.RIII mice. (C) Total number of CD4⁺ T lymphocytes and (D) CD8⁺ T lymphocytes. (E) Representative plots display CD44 and CD62L expression by CD4⁺ and CD8⁺ cells. (F) T_effector/T_naïve ratio (as defined by CD44 and CD62L expression) for CD4⁺ and (G) CD8⁺ T lymphocytes. (H) Frequency of CD4⁺CD25⁺ T cells in uveitic eyes. (I) Frequency of CD4⁺PD-1⁺ T cells uveitic eyes. Data combined from three independent experiments; 5–10 mice per group. In (A), (C), (D), (F) and (G), median and range are depicted. In (B), (H) and (I) Mean ± SD are depicted.*, p<0.05, two-tailed Mann-Whitney test.</p

mPEG PV1-Fab’ dampens IFN-γ production by CD4⁺ T lymphocytes.

<p>B10.RIII mice were immunized with 50 μg/animal of 161–180 IRBP in CFA, plus 500 ng/animal of PTx boost. Starting on day 9, mice were treated every 4 days with CD28 antagonist, PV1 (10mg/Kg; ip), or left untreated. On day 14 mice were sacrificed and dLN were collected for immunophenotyping and evaluation of cytokine production. For the intracellular staining of IFN-γ and IL-17 cells were collected from dLN (3 mice/group), plated at 1x10⁶ cells/well concentration and stimulated overnight with 100 ng/mL of PMA and 500 ng/mL of ionomycin, plus GolgiPlug at manufacturer’s recommended concentrations. (A) Representative plots show IFN-γ, IL-17 and IL-2 production by CD3⁺CD4⁺ cells. (B) Pie charts (C) and absolute frequency of CD4⁺IFN-γ⁺ cells. In brief, each subpopulation depicted in the bar graph is also depicted in the pie chart as pie slices, following the same color code. Overall IFN-γ, IL-17 and IL-2 production is displayed as the outer arcs in the pie charts. (D) Total numbers of CD4⁺IFN-γ⁺ cells. Mean ± SD are depicted in (C) and (D). **, p<0.01; ***, p<0.0001, two-tailed Mann-Whitney test.</p