Gradual Decline in Malaria-Specific Memory T Cell Responses Leads to Failure to Maintain Long-Term Protective Immunity to Plasmodium chabaudi AS Despite Persistence of B Cell Memory and Circulating Antibody

The mechanisms responsible for the generation and maintenance of immunological memory to Plasmodium are poorly understood and the reasons why protective immunity in humans is so difficult to achieve and rapidly lost remain a matter for debate. A possible explanation for the difficulty in building up an efficient immune response against this parasite is the massive T cell apoptosis resulting from exposure to high-dose parasite Ag. To determine the immunological mechanisms required for long-term protection against P. chabaudi malaria and the consequences of high and low acute phase parasite loads for acquisition of protective immunity, we performed a detailed analysis of T and B cell compartments over a period of 200 days following untreated and drug-treated infections in female C57BL/6 mice. By comparing several immunological parameters with the capacity to control a secondary parasite challenge, we concluded that loss of full protective immunity is not determined by acute phase parasite load nor by serum levels of specific IgG2a and IgG1. Abs, but appears to be a consequence of the progressive decline in memory T cell response to parasites, which occurs similarly in untreated and drug-treated mice with time after infection. Furthermore, by analyzing adoptive transfer experiments, we confirmed the major role of CD4(+) T cells for guaranteeing long-term full protection against P. chabaudi malaria. The Journal of Immunology, 2008, 181: 8344-8355.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazi

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