Abstract A089: Methotrexate preserves anti-PD1 anti-tumor benefits while reducing arthritic inflammation in a tumor and ICI-arthritis combination mouse model
Authors: Papadopoulos Theodore , Santinon Francois , Naranjo Carolina Lopez , Hudson Marie , Del Rincon Sonia Victoria 
Published: February 2025

Abstract
Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer and it is now estimated that up to 50% of cancer patients are eligible for ICIs. ICIs are associated with high rates of immune toxicities known as immune related adverse events (irAEs) which in up to 7% of patients occur as ICI-arthritis. ICI-arthritis is empirically treated with steroids and drugs such as methotrexate (MTX), but evidence is mounting that steroids may diminish the survival benefit conferred by ICIs. With the increasing use of ICIs in cancer, it is critical to determine how immune suppressive treatments used in the treatment of irAEs and ICI-arthritis affect the anti-tumor benefits conferred by ICIs. We developed the first arthritis and tumor combination model of ICI-arthritis. We focused on MTX, a common treatment of rheumatoid arthritis and second line therapy for ICI-arthritis. B16-PDL1 or MC38 tumor bearing mice were treated with vehicle or MTX +/- anti-PD1 and tumor outgrowth was followed. Arthritis was induced in mice following tumor formation and paw inflammation was measured. Tumors were collected at endpoint and immune phenotyped and paws were collected for histological scoring. In vitro exhaustion of CD8 T cells was performed by repeated stimulation. Exhausted CD8 T cells were collected to assess exhaustion marker expression levels and RNA was extracted for bulk RNAseq. We first found that MTX did not enhance tumor growth or diminish the anti-tumor benefits of anti-PD1 in both B16-PDL1 and MC38 tumor models. We observed that MTX +/- anti-PD1 significantly reduced paw swelling and hastened the resolution of arthritis. Furthermore, anti-PD1 increased peak paw inflammation significantly in comparison with vehicle and MTX treated mice. To assess the effects of MTX on anti-tumor immune responses, we used flow cytometry to phenotype tumor infiltrating lymphocytes (TILs). Unexpectedly, we found that MTX+/- anti-PD1 significantly enhanced the formation of memory CD8 TILs. We hypothesized that MTX diminished CD8 TIL exhaustion and thus promoted memory cell formation. We found that MTX reduced the expression of immune checkpoint receptors and exhaustion markers TIM3 and LAG3 on PD1+ CD8 TILs. Modeling exhaustion in vitro, we again found that MTX reduced CD8-T-cell exhaustion. RNA sequencing of MTX treated exhausted CD8 T cells revealed an upregulation in pathways associated with CD8-T-cell activation when compared to vehicle treated CD8 T cells. In conclusion, we have developed a tumor and arthritis combination model to examine the effects of immune suppressive treatments on ICI-mediated anti-tumor immunity. We found that MTX does not diminish anti-PD1 efficacy in B16-PDL1 and MC38 tumor-bearing mice and reduces arthritic inflammation. We further identified that MTX reduces CD8 T cell exhaustion and promotes memory CD8-T-cell formation in tumors.
Citation Format: Theodore Papadopoulos, Francois Santinon, Carolina Lopez Naranjo, Marie Hudson, Sonia Victoria Del Rincon. Methotrexate preserves anti-PD1 anti-tumor benefits while reducing arthritic inflammation in a tumor and ICI-arthritis combination mouse model [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A089.