Poster N12 - Investigating Co-Activation of CD4+ and CD8+ T-cells Using an Artificial Lymph Node

Introduction: T-cell-based cancer immunotherapies, such as adoptive cell transfer (ACT), have shown promising results by utilizing autologous antigen-specific cytotoxic CD8+ T-cells to target tumors. However, despite decades of development, these treatments often yield incomplete responses for patients. In recent years, growing evidence has highlighted the importance of helper CD4+ T-cells in cellular anti-tumor responses, prompting efforts to harness their potential for promoting cancer immunity. Here, we introduce the first steps towards developing a novel biomaterials-based platform known as an artificial lymph node (aLN), designed to directly activate naïve CD4+ antigen-specific T-cells alongside CD8+ T-cells to promote enhanced T-cell functionality. Additionally, this unique platform can be utilized both in vitro and in vivo providing an easy path for translation to in situ treatments.

Materials and

Methods: Thiol-modified signals 1 (anti-CD3, I-abOVA, or I-abOVA and KbOVA), 2, (anti-CD28) and 3 (IL-2 antibody) are combined with thiol-modified hyaluoronic acid. PEGDA is added to cross-link the hyaluronic acid and the resulting hydrogel is passed through a mesh to form aLN microparticles (MPs) (Fig. 1A). Transgenic murine OT-II CD4+ T-cells were cultured in vitro with aLN-MPs at various stiffnesses (Fig. 1B) or with varying thiolated signal 1 amounts (Fig. 1C) and expansion was measured on day 7 by cell count. In vivo capabilities were tested by injecting naïve B6 CD8+ T-cells or splenocytes subcutaneously into mice with different aLN-MPs. On day 9, target cells were injected intravenously and cells from the spleen and lymph node were harvested the following day (Fig 1D) to measure in vivo killing.

Results, Conclusions, and Discussions: OT-II CD4+ T-cells were first cultured in vitro for 7 days with non-specific anti-CD3 aLN-MPs (Fig 1A) to confirm the platform’s ability to both activate and expand CD4+ T-cells. Several stiffnesses were tested to determine optimal conditions for T-cell proliferation, which was found to occur at 1 kPa (Fig 1B). The results followed a pattern similar to that observed previously during CD8+ T-cell expansion: both too low and too high stiffnesses resulted in little to no proliferation. We hypothesize this is due to enhanced mechanotransduction properties of TCR signaling at lower aLN stiffnesses. Next, antigen-specific biotinylated I-abOVA was tested at different signal densities by altering the amount of its aLN linking moiety, thiolated streptavidin (SA-SH). As expected, increased amounts of SA-SH showed corresponding increases in CD4+ T-cell expansion and at a concentration of 10 ug/ml, worked equally as well as our standard anti-CD3 system (Fig.1C).

To test in vivo efficacy, we generated aLN-MPs with either KbOVA alone for CD8+ T-cell activation or KbOVA and I-abOVA for co-activation of CD4+ and CD8+ T-cells. Different combinations of aLN-MPs were injected on day 0 with either splenocytes or CD8+ T-cells and target cells were injected on day 9. The splenocytes + KbOVA + I-abOVA group showed the highest percent of in vivo killing in the lymph node (Fig 1D). This shows that our aLN-MPs are not only capable of stimulating both CD4+ and CD8+ T-cells, but that this co-activation has potential therapeutic efficacy.

In conclusion, we have developed a novel biomaterials-based platform for co-activation of CD4+ and CD8+ T-cells. Our findings demonstrate effective CD4+ T-cell expansion with proliferation profiles consistent with our previous T-cell expanding platform, including cellular sensitivity to changes in aLN stiffness and signal 1 density, highlighting the importance of biomechanical cues in T-cell activation and expansion. A preliminary in vivo experiment also showed dual activation of CD4+ and CD8+ T-cells with improved CD8+ T-cell killing capability. These findings lay the groundwork for further exploration and refinement of our aLN-MP platform, with the ultimate goal of improving the efficacy of T-cell-based cancer immunotherapies for the benefit of patients.

Acknowledgements (Optional): ML would like to thank everyone in the Schneck lab for their help. Special thank you to Natalie Livingston for her training and endless support.