Background Adequate concentration of peptide-MHC-II complexes on the surface of antigen-presenting cells (APCs) is required to drive strong priming and activating signals for CD4⁺ T cells, which are extensively sensitive to antigen expression levels and its subsequent intracellular processing. Antigen that is taken up by activated APCs through endocytic vesicles are subjected to a class of proteases, cathepsins, that stochastically fragment protein material into peptides suitable for loading onto MHC-II.¹ Altering the nature of the up-taken protein has been shown to greatly influence antigen processing and the resultant CD4⁺ T cell activation. Previous work demonstrated that fusion of a model CD4⁺ T cell antigen peptide to a portion of the transferrin receptor (TFR) as opposed to a cytoplasmic-localizing sequence, facilitated re-localization of the construct to endoplasmic vesicles within APCs for efficient MHC-II presentation and CD4⁺ T cell expansion.^2–4 Additional work is required to better understand the mechanisms that drive MHC-II antigen presentation on APCs, as this would allow for 1) unprecedented resolution of how antigen processing and CD4⁺ T cell stimulation alters the native helper T cell response in cancer and 2) the design of thoughtful immunotherapies that leverage these natural biological phenomena.

Methods Using a GEMM of lung adenocarcinoma driven by mutant Kras^G12D and loss of p53 (KP),⁵ we initiated tumors in mice that express CD8⁺ and CD4⁺ T cell neoantigens. Multiple constructs containing unique B, L, and S cathepsin cleavage sequences flanking the CD4⁺ neoantigen were installed into these tumors and the antigen-specific T cell response was assessed at 5- and 12-weeks post tumor initiation by flow cytometry and histology analysis.

Results Enhancing the antigen processing of the CD4⁺ T cell neoantigen with optimized cathepsin cleavage sites resulted in increased Th1 phenotypes of the antigen-specific CD4⁺ T cells which in turn increased the effector functions of antigen-specific CD8⁺ T cells (figure 1). Concordantly, there was an increased infiltration of tumor-infiltrating lymphocytes and an overall decrease in tumor burden.

Conclusions Modulating where a neoantigen is localized and how it is processed in APCs alters the activation of CD4⁺ T cells and the overall anti-tumor immune response. These elements may be universally installed into vaccine-based immunotherapies to improve T cell functionality.

Acknowledgements This research is funded by the Koch Institute for Integrative Cancer Research at MIT.

References

1. Roche P, Furuta K. The ins and outs of MHC class II-mediated antigen processing and presentation. Nat Rev Immunol 2015;15:203–216. https://doi.org/10.1038/nri3818

2. Fernandes DM, Vidard L, Rock KL. Characterization of MHC class II-presented peptides generated from an antigen targeted to different endocytic compartments. Eur J Immunol. 2000 Aug;30(8):2333–43. doi: 10.1002/1521-4141(2000)30:8<2333::AID-IMMU2333>3.0.CO;2-F. PMID: 10940924.

3. Rowe HM, Lopes L, Ikeda Y, Bailey R, Barde I, Zenke M, Chain BM, Collins MK. Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene. Mol Ther. 2006 Feb;13(2):310–9. doi: 10.1016/j.ymthe.2005.08.025. Epub 2005 Nov 4. PMID: 16275163.

4. Diebold SS, Cotten M, Koch N, Zenke M. MHC class II presentation of endogenously expressed antigens by transfected dendritic cells. Gene Ther. 2001 Mar;8(6):487–93. doi: 10.1038/sj.gt.3301433. PMID: 11313828.

5. Jackson EL, et al. The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer research 2005;65:10280–10288, doi:10.1158/0008-5472.CAN-05-2193.

• Download figure
• Open in new tab
• Download powerpoint

Abstract 931 Figure 1

(A) Schematic of the lentiviral construct intratracheally injected into KP mice. The construct contains Cre to tum on the expression of oncogenic Kras along with removal of expression of P53. Sustained expression of a CD8* T cell peptide neoantigen derived from 60S ribosomal subunit L18 (Rpl18) and the CD4+ T cell peptide neoantigen derived from Integrin beta-1 (ltgb1) allows for tracking of an endogenous tumor-immune response. (B) Schematic of the experiment depicted in C-E. The arrowhead in the construct represents the placement of optimized cathepsin cleavage sites (B, L, or S).16 (C) Representative now plots Of the tumor-specific CD4+ T cell response in the draining lymph node (left) at 5 weeks and the tumor-specific CD8+ T cell response in the lung (right) at 12 weeks. Cells were gated on singlets →Live/Dead-→ and CD4+ or CD8+, respectively. (D-E) Number of cells recovered at the indicated timepoints for the tumor-specific CD4+ T cell population in the draining lymph node (top) or CD8+ T cell population in the lung tissue (bottom).