Balancing Anti-Tumor Immunity and Autoimmunity:

Elucidating the Interplay between Immune Checkpoint Inhibitor Therapy and T follicular helper cells in the Pathogenesis of Immune-Related Adverse Events (IRAEs)

Author: Kristy Kim

Area of Concentration (Petal): Basic, Clinical, and Translational Research

Specialty (if any): Immunology, Oncology

Keywords: IRAEs (immune-related adverse events), T follicular helper (Tfh) cells, immune checkpoint inhibitor (ICI)

Background: Immune checkpoint inhibitor (ICI) therapy has revolutionized cancer treatment by leveraging the body’s immune system to target cancer, however it can lead to off-target immune responses against healthy tissues, known as immune-related adverse events (IRAEs).1 IRAEs can affect any organ system, manifesting as colitis, thyroiditis, hepatitis, and may lead to life-threatening complications such as pneumonitis and myocarditis. Up to 60% of patients treated with ICI therapy will develop autoimmune toxicity, some so severe warranting the cessation of ICI altogether and compromising anti-tumor efficacy.2 Previous work by our lab and others have shown enrichment of T follicular helper (Tfh) cells, a specialized subset of CD4+ T cells, in tissues from patients who developed IRAEs, suggesting their involvement.3-5 However, the role of Tfh cells in the pathogenesis of IRAEs is an area of ongoing investigation.

Objective: The purpose of this study is to elucidate the role of Tfh cells in the pathogenesis of IRAEs, thereby uncovering potential targets to offset autoimmune toxicities. By understanding the balance between anti-tumor immunity and autoimmunity in the context of Tfh cell regulation, we can enhance the safety of ICI therapy for patients while leveraging the efficacy of these potent cancer therapies.

Methods: Human PBMCs from the University of California, Los Angeles Virology Core were isolated by density gradient centrifugation using Ficoll Paque Plus. CD4+ T cells were positively selected using Human Naïve CD4+ T cell Isolation Kit (Miltenyi Biotec) according to manufacturer’s protocol. PBMCs or CD4+ T cells were induced into T follicular helper phenotype by plate-bound aCD3 (5ng/mL), soluble aCD28 (5ng/mL), Activin A (100ng/mL), and IL-12 (50ng/mL) and treated with Pembrolizumab (10ug/mL), Ipilumimab (10ug/mL), or Ruxolitinib (10mM) for 72 hrs. Samples were stained for Tfh surface markers– CD4, PD1, ICOS, CXCR5. Intracellular staining was done for transcription factors such as BCL6. Data analysis was performed by GraphPad Prism 10. One-way ANOVA was used to compare multiple (>2) groups with one independent variable. p values <0.05 were considered statistically significant.

Results: Our previous studies collected fine-needle aspirate (FNA) from patients with ICI-induced thyroiditis showed enriched populations of CD4+ T follicular helper (Tfh) cells via single-cell RNAseq analysis. Tfh populations from ICI-thyroiditis patients produce cytokines such as CXCL13 and IL-12, which are critical for tissue honing and T cell recruitment. ICI therapy significantly increases the Tfh induction in human PBMCs (p < 0.0001) suggesting a role of ICI therapy in the initial Tfh programming, which can predispose patients to IRAEs. Literature has shown Tfh differentiation occurs through activation of the JAK/STAT pathway.6 Ruxolitinib (FDA-approved JAK/STAT inhibitor) completely inhibits Tfh induction in both whole PBMCs and isolated CD4+ T cells (p < 0.001).

Conclusions: This study demonstrates ICI therapy significantly increases human Tfh induction. Enriching the Tfh population can lead to an increased inflammatory response via IL-21 and CXCL13 cytokine release and increased recruitment of cytotoxic CD8+ T cells to healthy tissue, resulting in IRAEs. This finding prompted us to investigate potential therapeutic strategies to mitigate an ICI-induced increase in Tfh differentiation. This study shows Ruxolitinib, an FDA-approved JAK/STAT inhibitor, completely inhibits Tfh induction via direct effect on CD4+ T cells. Our results provide a promising therapeutic strategy for decreasing IRAE development in patients receiving ICI therapy, thereby improving the safety profile of potentially life-saving cancer treatments.

References

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4. Zhu, C. et al. Increased frequency of follicular helper T cells in patients with autoimmune thyroid disease. J. Clin. Endocrinol. Metab. 97, 943–950 (2012).

5. Herati, R. S. et al. PD-1 directed immunotherapy alters Tfh and humoral immune responses to seasonal  influenza vaccine. Nat. Immunol. 23, 1183–1192 (2022).

6. Locci, M., Wu, J. E., Arumemi, F., Mikulski, Z., Dahlberg, C., Miller, A. T., & Crotty, S. (2016). Activin A programs the differentiation of human TFH cells. Nature immunology, 17(8), 976–984. https://doi.org/10.1038/ni.3494