Impact of Toll - like receptor 2 deficiency on immune responses to mycobacterial 1 antigens . 2 3

25 In the present study, we addressed the question whether the Toll-like receptor 2 (TLR2)26 mediated innate immunity can contribute to the development of acquired immune responses. We 27 immunized TLR2 and wild-type (WT) mice three times subcutaneously with the mycobacterial 28 antigens 19kDa (TLR2 ligand) or Ag85A (not TLR2 ligand). One week after the last immunization, 29 serum and spleens were collected. To evaluate cellular responses, we measured IFNafter in vitro 30 re-stimulation of spleen cells with antigen alone, antigen-pulsed bone marrow derived macrophages 31 (BMMAg) or pulmonary macrophages (PuMAg). Antibody responses were comparable in the two 32 mouse strains but we observed differences in the cellular responses. Recall responses to Ag85A 33 were similar in the two strains but responses to 19kDa given alone or presented by BMM or PuM 34 were lower in TLR2 compared to WT mice. The largest differences in cellular responses were 35 observed when 19kDa was presented by PuM. To understand this, we analyzed phenotypic and 36 functional differences between BMM and PuM upon stimulation with various ligands. Generally 37 PuM had a lower response to the TLR2 ligand Pam3Cys-Ser-(Lys)4 trihydrochloride and to anti38 CD40 than BMM, as measured by cytokine secretion and up-regulation of co-stimulatory molecules. 39 This might provide a partial explanation for the lower capacity of PuM when pulsed with 19kDa, 40 also a TLR2 ligand. Altogether, our results revealed weaknesses in theT cell and APC compartments 41 of the 19kDa immunized TLR2 mice but indicated that specific immune responses could be 42 generated in the absence of TLR2 regardless of the characteristics of the antigen used. 43 44 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom INTRODUCTION 45 Toll like receptors (TLRs) are pattern recognition receptors that recognize 46 microbe/pathogen-associated molecular patterns, contribute to the activation of the innate responses 47 (30) and are involved in the collaboration between the innate and the adaptive branches of the 48 immune system (13, 24). Engagement of TLRs increases co-stimulatory molecule expression, 49 enhances antigen presentation, and induces pro-inflammatory cytokine production (24). TLRs are 50 differentially expressed on hematopoietic and non-hematopoietic cells. Mononuclear phagocytes 51 and dendritic cells express the widest TLR repertoire (21, 37). Moreover, in recent years, it has been 52 shown that TLRs on human T cells act as co-stimulatory receptors and participate in the 53 maintenance of the T-cell memory by enhancing proliferation and/or cytokine production by the 54 activated T cells (15, 17). 55 Mycobacterium tuberculosis (Mtb) contains many TLR ligands where TLR2 and TLR4 have 56 been recognized as the most commonly involved in Mtb-mediated intracellular signaling (20, 32). 57 The interaction of mycobacterial components with TLRs on macrophages may be a critical early 58 step of macrophage activation and production of pro-inflammatory cytokines in a TLR-dependent 59 manner. Mycobacterial cell-wall components such as the 19kDa lipoprotein (Rv3763), the 26kDa 60 lipoprotein (Rv1411), and the lipoglycan lipoarabinomannan, bind to TLR2 and activate 61 intracellular signaling pathways leading to the production of IL-12 and thus polarizing the immune 62 system towards a Th1 type of response (4). 63 It is suggested that TLR2 may be of importance for the control of mycobacterial infection. 64 We have shown that deficiencies in TLR2 signaling increase susceptibility at an early stage of 65 infection in the respiratory tract. This susceptibility was correlated to impaired pro-inflammatory 66 responses measured as TNF and IFNproduction by lung mononuclear cells upon stimulation with 67 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom various mycobacterial components (31). TNF is required for the control of mycobacterial growth 68 and the formation of protective granulomas (10, 16) and IFN-γ is necessary for the activation of 69 macrophages and generation of protective immunity (6, 35). 70 Vaccine antigens coupled with TLR agonists have been proven to be effective in increasing 71 immune responses and protection (34, 38). In mouse models of tuberculosis (TB), vaccination with a 72 mycobacterial fusion protein composed of a TLR2 agonist, Rv1411, and early secretory antigenic 73 target-6 (ESAT-6) exhibited enhanced T cell responses and protection (33). 74 A better understanding of the role of TLR2 in mycobacterial antigen recognition and 75 presentation to T cells for the development of adaptive immune responses is needed for novel 76 therapies and vaccines. In order to investigate these factors, we used two mycobacterial antigens 77 namely, 19kDa a known TLR2 agonist and Ag85A, not considered to be a TLR2 agonist. Ag85A is 78 one of the leading vaccine candidates already tested in preclinical and clinical studies and found to 79 be promising for future TB vaccine development (11, 25). A number of studies (1, 2, 4, 19, 23) 80 regarding the TLR2-mediated effects of 19kDa on phagocytes have been performed but the role of 81 19kDa as antigen has not been equally evaluated. 82 In this study, we sought to determine the contribution of TLR2-mediated innate immunity 83 for the induction and maintenance of adaptive immune responses. A comparison between wild-type 84 (WT) and TLR2 mice revealed that immunization with Ag85A or 19kDa induced antigen-specific 85 humoral and cellular immune responses in both strains of mice. However, we found weaknesses in 86 both the antigen-presenting cell (APC) and the T cell compartments of the TLR2 mice immunized 87 with 19kDa, the TLR2 ligand. Recall responses to Ag85A were similar in the two strains but 88 responses to 19kDa given alone or presented by bone marrow derived macrophages (BMM) or 89 pulmonary macrophages (PuM) were lower in TLR2 compared to WT mice. The largest 90 differences were observed when 19kDa was presented by PuM. Due to the central role of PuM in 91 on S etem er 3, 2017 by gest http/iai.asm .rg/ D ow nladed fom the defense of the respiratory tract, we analyzed phenotypic and functional differences between 92 BMM and PuM upon stimulation with various ligands. PuM were generally lower responders than 93 BMM to some of the stimuli both in the amount of cytokines produced and in up-regulation of co94 stimulatory molecules. The importance of these differences in APC/T cell collaboration is discussed. 95