Abstract Background: Pregnancy-associated breast cancer (PABC) is BC diagnosed during pregnancy or within 2 years after pregnancy. Although PABC is a rare subgroup of BC, the outcome of PABC is worse than BC developed in the same age group unrelated to pregnancy. The proliferation and involution of mammary gland during and after pregnancy create a unique immune microenvironment in the breast. Comprehensive profiling of the tumor immune microenvironment in PABC is important but lacking. Methods: We retrospectively identified PABC patients from electronic medical records and their tumor blocks from pathology department in our institution. Age- and stage-matched BC patients were selected as control group. NanoString Breast Cancer 360 Gene Expression panel and Opal Polaris 7 color immunohistochemical (IHC) were utilized to profile PABC from transcriptome and protein levels. Result: PABC (N=26), BC developed 2 years (BC>2Y, N=54) after pregnancy, and matched non-pregnant BC patients (NPBC, N=27) were included in the final analysis respectively. Samples from BC>2Y (remote influence from pregnancy) and NPBC (pregnancy did not contribute to BC) both served as control groups for PABC patients in our study. PAM50 subtyping categorized patients into luminal A(N=42), luminal B(N=26), basal(N=12), and HER2 (N=25) enriched subgroups. In multiplex IHC analysis, CD4+ cells, CD8+ cells, CD20+ cells, and CD68+/CD163+ (M2) cells were all statistically higher in the tumor samples from PABC and BC>2Y than from NPBC. This suggests a potential contribution of pregnancy in the immune microenvironment. In the luminal A subgroup, CD8+ cells, CD20+ cells, and CD68+/CD163+ cells remain statistically higher in the PABC than in the NPBC samples. In NanoString BC360 transcriptomic profiling, immune cell abundance was evaluated from representative gene signatures. Mast cells were significantly lower in the PABC group than in the NPBC group. Cytotoxic T cells are significantly higher both in the PABC and BC>2Y groups than in the NPBC group. Conclusion: We are the first study to profile immune microenvironment using both transcriptomic and IHC methods. Our study is also unique in using NPBC and BC>2Y patients as control groups for PABC patients. CD8+ T cells are statistically higher in PABC patients than in NPBC patients using either transcriptomic profiling or multiplex IHC analysis. M2 macrophages, which promote tumor growth, are abundant in all the PABC and the luminal A PABC samples. Citation Format: I-Chun Chen, Ching-Hsuan Chen, Chia-Lang Hsu, Tzu-Pin Lu, Yen-Shen Lu, Ching-Hung Lin. Immune microenvironment profiling of pregnancy associated breast cancer: A transcriptomic and multiplex immunohistochemical analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6852.