Background:The autoimmunity and inflammation phenomena observed in systemic sclerosis (SSc) lead to the dysregulation of type V collagen (Col V) and extracellular matrix (ECM) deposition of Col V, collagen I (Col I) and collagen III (Col III) fibers, characteristic of pulmonary fibroproliferative disease. T cells are the predominant inflammatory infiltrate in the lungs and are believed to produce cytokines that drive the synthesis of matrix proteins by fibroblasts, resulting in excessive fibrosis. Although most studies to date have focused on CD4+ T and CD20+ B immune cells in some SSc subsets, cytotoxic CD8+ T cells are also involved in the pathogenesis of SSc. We previously reported that reactivity to the ColV-self antigen predicts late SSc lung fibrosis in mice immunized with Col V after 120 days; however, the profile of underlying immune matrices in early SSc lung fibrosis remains unclear.Objectives:To quantify T and B-cells and collagen fibers in the days following immunization with Col V in a mouse model mimicking human lung involvement in SSc.Methods:Female C57BL/6 mice (n=18) were immunized subcutaneously with human Col V (150 μg) in complete Freund´s adjuvant, followed by two intramuscular boosters (IM-COLV). The control group (n=18) did not receive Col V. Three groups of animals (n=6 each) were euthanized on days 15, 30 and 45 after immunization. The lung sections were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 4 µm. Immunohistochemistry, immunofluorescence and histomorphometry were performed to phenotype and quantify CD4 and CD8 T-cells, CD20 B-cells, and Col I, III and V, respectively.Results:We found that T-cells (CD4⁺), cytotoxic T cells (CD8⁺), and B-cells (CD20⁺) were present in high densities in the lungs of IM-COLV mice, with variation in the absolute abundance of each of these immune cell lineages at 15, 30 and 45 days after immunization with Col V. In fact, a significantly higher density of CD4⁺, CD8⁺, and CD20⁺ was detected early along the connective matrix of alveolar septa at 15 days after Col V immunization. In addition, higher density of Col I, III and V fibers were also observed early (Table 1). Although early SSc-lung fibrosis is generally considered to be linked to inflammatory cytokines, we also infer that cytotoxic T-cells may induce alveolar cells apoptosis by triggering kinases on macrophages and then accelerate fibrotic scar.Conclusion:Our study quantitatively establishes that the densities of CD4+ CD8+ and CD20+, as well as Col I, Col III and Col V can be characterized as markers of early lung fibrosis in SSc. These findings suggest that cytotoxic T cells may induce apoptosis and secrete profibrotic cytokines to induce the deposition of collagen fibers in the lungs, inferring that early B cell depletion may improve early lung fibrosis in SSc.Post-Col V Immunization (Days)153045ControlIM-COLVControlIM-COLVControlIM-COLVImmune CellsCD4+ cells/mm² (%)13.85 ± 0.4634.14 ± 1.55*12.63 ± 0.5623.38 ± 0.8314.32 ± 0.5223.62 ± 0.40CD8+ cells/mm² (%)13.84 ± 0.6039.40 ± 0.99**12.42 ± 0.5222.87 ± 0.8512.93 ± 0.5723.07±1.00CD20+ cells/mm² (%)12.12 ± 1.0133.84±1.43^#11.70 ± 1.1415.23 ± 6.7012.46 ± 0.5028.24 ± 0.67Collagen FibersCol V (area fraction)0.05 ± 0.0030.12±0.010.08 ± 0.0030.14±0.01^††0.07 ± 0.0030.11±0,003Col I (area fraction)0.16 ± 0,010.38±0.05^##0.14 ± 0.010.25±0.030.15 ± 0.020.24±0.01Col III (area fraction)0,16 ± 0,0030.28±0.01^†0.14 ± 0.0040.25±0.050.13± 0,0070.16±0.02Total Col µg/mg2.7 ± 0.293.99 ± 0.583.15 ± 0.193.77 ± 0.272.87 ± 0.304.99 ± 0.60Data are shown as mean ± SD. Comparisons were done using ANOVA/Dunn´s Multiple comparison testP<0.05). *15 vs 30 and 45 days; **15 vs 30 and 45 days; ^#15 vs 30 and 45 days; ^## 15 vs 30 and 45 days; ^† 15 vs 45 days; ^†† 30 vs 45 days. Col I, collagen type I; Col III, collagen type III; Col V, collagen type V.Disclosure of Interests:Vitoria Elias: None declared, Zelita Queiroz: None declared, Sergio Catanozi: None declared, Antonio Santos Filho: None declared, Sandra Fernezlian: None declared, Ana Paula Velosa: None declared, Percival D. Sampaio-Barros Speakers bureau: Actelion, Boehringer Ingelheim, Abbvie, Lilly, Novartis, Consultant of: Abbvie, Bayer, Boehringer Ingelheim, Lilly, Novartis, Pfizer, Vera Capelozzi: None declared, Walcy Teodoro: None declared