Abstract Breast cancer is one of the leading causes of death in women worldwide. Hence, it is important to develop and improve (immuno)therapies for breast cancer patients. Adoptive T-cell therapy with autologous CD8+T cells is a promising method that has already succeeded in melanoma patients. A possible combination with antibody-dependent cell-mediated cytotoxicity dependent (ADCC) antibodies such as Trastuzumab might be beneficial for relapsed late-stage patients. We developed an in vitro protocol to generate antigen-specific CD8+T cells by priming on the HER2+ breast cancer cell line JIMT-1. Subsequently, these primed CD8+T cells were tested in a 2D and a 3D immune cell killing assay in a life cell imaging device in combination with an endpoint viability assay. The activity was compared with unspecific CD8+T cells activated by phytohemagglutinin (PHA) or αCD2/αCD3/αCD28 beads. All T cells were tested as monotherapy and in combination with trastuzumab in JIMT-1 and SKBR-3. The antigen specific CD8+T cells displayed a significantly improved killing potential of JIMT-1 cells compared to unspecific activated or non-activated CD8+T cells. Along these lines, those cells showed the highest TNF-alpha secretion and expression of CD69 (determined by flow cytometry) among all T cell lines. Antigen-specific CD8+T cells from different settings displayed a significantly improved killing potential towards JIMT-1 than non-antigen-specific activated or non-activated CD8+T cells. The use of peripheral blood monocytes (PBMC) instead of isolated CD8+T cells did not influence the activity against JIMT-1 significantly. Interestingly, antigen-specific CD8+T cells activated as PBMC increased the activity of Trastuzumab significantly as compared to unspecific activated CD8+T cells. Testing of different donors revealed a donor dependent degree of killing, however in all donors the activity pattern was the same with antigen specific CD8+T cells being the most active. A direct comparison of fresh vs frozen immune cells from the same donors indicated no significant differences in the killing activity of unspecific activated CD8+T cells. Yet, there were substantial differences in antigen-specific immune cells: the killing potential of the freshly isolated immune cells was significantly higher than of the frozen. In summary, we have developed a protocol to produce antigen-specific tumor-infiltrating CD8+T cells primed to the heterologous JIMT-1 breast cancer cell line. The antigen-specific CD8+T cells showed an increased killing potential over non-specific CD8+T cells. In addition, they were able to increase ADCC-mediated antitumoral activity of trastuzumab. The 2D as well as the 3D immune cell killing assay proved to be robust and amendable for mid throughput screening. The impact of donor variability and fresh vs frozen immune cells must be further evaluated. Citation Format: Ina Rohleff, Matthias Bleisch, Kanstantsin Lashuk, Julia Schueler. Modulation of Trastuzumab antitumoral activity by co-culture with different T cell subtypes in 2D and 3D T cell killing assays targeting HER2+ breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4065.