Objectives: Tears of the anterior cruciate ligament (ACL) are common in the pediatric athletic population. In spite of trends towards early surgical reconstruction to minimize the risk of secondary meniscus and cartilage injuries, long term disruption of knee joint homeostasis and progression to post- traumatic osteoarthritis (PTOA) occurs in 50-90% of patients - a particularly challenging problem in this young cohort. The exact pathogenesis of PTOA after ACL injury and reconstruction remains unknown. Evidence from animal models of PTOA suggests a critical role of synovial inflammatory infiltration in both the onset and progression of PTOA. However, there is a scarcity of in vivo human data documenting the synovial inflammatory response to ACL injury. One recent study has looked at immune cell composition in synovial fluid, but there are no comparable publications using synovial tissue. Our primary objective was to use flow cytometry with an advanced immune cell panel to provide a snapshot of the cellular composition of immune infiltrates in the synovium of adolescent patients at the time of ACL reconstruction. Our secondary objective was to look for correlations between demographic and injury parameters and inflammatory cell composition. Methods: Patients between the ages of 12 and 18 years who were undergoing primary ACL reconstruction were enrolled in an IRB-approved prospective study. Exclusion criteria included multiligament knee injuries, revision ACL reconstruction, history of prior knee surgery, and history of inflammatory joint disease. Demographic variables and intraoperative data were recorded. At the time of surgery, an arthroscopic synovial biopsy from the prefemoral synovium was obtained. Synovium tissue was enzymatically digested into a single cell suspension for immune profiling by multicolor flow cytometry. Data was acquired on a BD lsrFortessa flow cytometer and analyzed with FlowJo v10. Statistical analyses were performed with GraphPad Prism including parametric ANOVA (one-way or two- way), simple linear regression, Kruskal Wallis analysis of variance, Mann Whitney T-test, and parallel Principal component analyses (Eigenvalue with Monte Carlo simulation). Heat map variable scaling was calculated relative to the mean and standard deviation of all measurements. Results: Twelve patients were prospectively enrolled in the study (33% male, 67% female, n = 12). Mean age was 15.5 +/- 2.02 years. Median time between injury and surgery was 35.5 days (23.3-67.5). Meniscal tears were present in 58.3% of patients (8.3% medial only, 33.3% lateral only, 16.7% both). Cells isolated from synovium were 36.8% fibroblasts, 9.89% fibrocytes, and 36.0% hematopoietic lineage cells (Fig. 1A). Within the hematopoietic lineage, the dominant cell types were monocytes, macrophages, and T-cells (Fig. 1B). Principle component analysis (PCA) revealed three immunophenotypes. Immunotype 1 had higher mast cell content than other groups and fewer inflammatory cells such as T cells, B cells, and macrophages. Immunotype 2 had strong T/B cell infiltration and lower levels of innate immune cells. These patients also had the highest overall levels of inflammatory cells (Figure 2). Immunotype 3 had a strong innate immune response - in particular higher levels of M2 polarized macrophages - and significantly fewer T/B cells. There were no obvious effects of age, skeletal maturity, or the presence of meniscal tears on immunotype (Table 1). There was a trend towards a correlation between delay in surgery and classification within immunotype 1 (p = 0.09, Table 1). Conclusions: Our study provides the first in vivo analysis of inflammatory cell behavior in the synovium of ACL injured knees. In young patients undergoing primary ACL reconstruction, three immunotypes were discovered. Our pilot data suggest a possible relationship between delay in surgery and a high prevalence of synovial mast cells. This would be of particular interest since mast cells are known to play a role in the development of OA and possibly PTOA. In the more acute setting, two separate immunotypes were encountered, one with heavy inflammation and a predominance of T/B cells, and the other with more moderate inflammation and a higher percentage of innate immune cells / M2 polarized macrophages. These were not clearly tied to patient demographic factors, and could represent intrinsic differences in the response to injury. Future work investigating the correlation between initial immunotype and clinical outcomes – in particular the development of PTOA – may provide insight into the role of synovial inflammation in post-ACL joint health and degeneration, as well as provide strategies for personalized treatments in the prevention of PTOA. [Table: see text]