Efficacy of murine selective CD28 antagonist for the treatment of experimental autoimmune uveitis.

A uveíte autoimune é uma doença inflamatória crônica, caracterizada pela resposta imune a antígenos oculares. É mediada por linfócitos T CD4+ com perfil TH1, e responsável por uma parcela significativa de casos de deficiências visuais e cegueira. Embora efetivos, os tratamentos disponíveis estão associados a efeitos adversos importantes. Logo, a busca de novos alvos terapêuticos mais específicos tem sido o objetivo principal no campo da imunoterapia. Nesse trabalho foi avaliada a eficiência do antagonista seletivo de CD28, mPEG PV1-Fab´(PV1), no tratamento da uveíte autoimune experimental (EAU). Camundongos tratados com PV1 exibiram menores graus de doença quando comparados a controles não tratados. Tal achado foi acompanhado de uma diminuição da ativação de linfócitos T, tanto nos olhos quanto nos órgãos linfoides periféricos desses animais. Mais ainda, o tratamento com PV1 levou a uma diminuição da população de linfócitos T reguladores e de células do tipo TH1. Portanto, concluiu-se que PV1 é eficaz no tratamento da EAU por agir em linfócitos T efetores.Autoimmune uveitis is a T-cell mediated disease that targets mainly the posterior eye pole. Similar to human uveitis, experimental autoimmune uveitis (EAU) is mostly dependent on T cells with a TH1 phenotype. Although many treatment strategies are available, most of them focus on general immunossuppression, resulting in undesirable side effects. Thus, the development of more specific therapies is the major aim in the field of immunotherapy. Here we evaluated the efficacy of mPEG PV-1-Fab´ (PV1), a specific CD28 antagonist, in the treatment of EAU. Our results indicate that PV1 blocks T cell activation by decreasing expression of different costimulatory molecules. Furthermore, PV1 treatment led to a decrease of Treg cell population in peripheral lymphoid organs. Also, IFN-g production by CD4+ cells and TH1 lymphocytes population were decreased. Altogether, our results raise this CD28 blockade strategy as a potential tool for the treatment of autoimmune disorders in the eye, and indicate that mPEG PV1-Fab acts mainly on IFN-g production and TH1 polarization

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

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

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