B cells sustein Trypanosoma cruzi specific CD8+ T cell response via IL-17

F FIOCCA VERNENGO; CG BECCARIA; L ALMADA; C ARAUJO FURLAN; CL MONTES; EV ACOSTA RODRIGUEZ; A GRUPPI

Cancún, Quintana Roo

Congreso; XII Congress of the Latin American Association of Immunology and XXIII Congress of the Mexican Society of Immunology; 2019

Introduction and Objetives: Anti-CD20 therapy has been widely used to treat different diseases such as autoimmune disorders or B cell malignancies. Although B lymphocytes are the only cells capable of producing antibodies, antigen presentation and cytokine production has been attributed to these cells. Many reports highlight the role of B cells in promoting cellular immunity; however, if B cells affect CD8 responses remain uncharacterized. To test this cell interaction we used the experimental model of T. cruzi infection, where the CD8+T cell response play a central role in the defense against this parasite.Methods: For this, 8 days before intraperitoneal (ip) infection with 5000 trypomastigotes of T.cruzi Tulahuén strain, C57BL/6 mice were ip injected with anti-CD20 (BcD mice), to deplete B cells, or with control isotype. At 20 days post infection (dpi), tissue parasitic DNA quantification was assessed by real time PCR and T.cruzi-specific CD8+T cell response was measured by FACS using tetramers loaded with the parasite immunodominant peptide Tskb20. For in vivo cytotoxicity assay unpulsed, Tksb20, Tskb18 or PA8 pulsed spleen cells were stained with different dyes, transferred to treated or control infected animals and studied in the spleen, liver and blood cells 4 hours later by FACS. For in vitro cytokine production assay total splenocytes from either control or depleted animals were cultured for 5 hours with Tskb20, PMA-Ionomicin or leftunstimulated Phenotype and function of CD8+ T cells were also analyzed by FACS. In some experiments B cell depletion was assessed 12 days after T cruzi infection. Murine recombinant IL-17 was used to treat BcD mice.Results: Infected BcD mice exhibited higher parasitism in the spleen, liver and heart than controls. Further, infected BcD mice had a significant lower frequency and number of total and Tskb20+CD8+T cells in blood, spleen and liver (p<0.01, p=0,002 and p=0,02 respectively). Interestingly BcD mice presented lower frequencies of short-lived (CD44+KLRG1+CD107-) (p=0,03) and memory (CD62L-CD44+) (p=0,02) effector cells, and a significant higher frequency of naïve (CD62L+CD44-) CD8+T cells, than infected controls. Total and T.cruzi-specific CD8+T cells from infected BcD mice exhibited a lesser extent of activation but higher levels of inhibitory receptors such as LAG-3, TIGIT and PD-1. Additionally, CD8+T cells from BcD mice express lower levels of the proliferation marker Ki67 and where more apoptotic than the control counterparts. Moreover, Bcl6 expression, which can regulate CD8 T cell proliferation, was decreased in CD8+T cells from BcD mice. When functionality was studied, CD8+T cells from infected BcD mice presented reduced cytotoxicity (p=0,03), degranulation, IFNf and TNF production (p<0,001 ). Accordingly to the IFNf production, T-bet expression was also diminished in CD8+T cells from BcD mice (p=0,001). When infected mice with a settled down specific-CD8+T cell response (12dpi) were depleted from B cells, interestingly, they exhibited the same characteristics than those depleted before infection. Considering that B cells produce IL-17 during T. cruzi infection, we measured IL-17 production in the spleen cells of BcD mice and found a significative reduction of these cells.Finally, injection of recombinant IL-17 rescued the frequency, phenotype and function of CD8+T cells generated in B cell absence.Conclution: The results indicate that B cells are key for T. cruzi specific CD8+T cell maintenance and function, but are not necessary for their induction. Considering B cells produce IL-17 in T. cruzi infection probably its function on CD8+ T cells depends on IL-17.