Background/Purpose: Static biomarkers have largely failed to predict patient response to biologic therapies in autoimmune disease. We hypothesized that dynamic immune responses under controlled ex vivo perturbation would reveal functional differences between responders and non-responders. We focused on T cell-mediated autoimmune diseases, including psoriatic arthritis (PsA) and inflammatory bowel disease (IBD), where initial response rates to TNF inhibitors (TNFi) are approximately 50%, with durable responses in a smaller subset of patients. A predictive assay to identify non-responders prior to treatment initiation would have significant clinical and economic impact.

Methods: To address this, we developed an ex vivo peripheral blood mononuclear cell (PBMC) assay in which patient T cells are activated using superantigen in the presence of autologous antigen-presenting cells, with or without TNF blockade. Biologic-naïve, banked PsA and IBD samples with known TNFi outcomes were analyzed alongside healthy controls. Immune responses were quantified using multiplex cytokine profiling (48-plex) and high-dimensional spectral flow cytometry of cellular phenotypes. All measurements were normalized to vehicle controls on a per-patient basis to capture TNF-dependent immune modulation.

Results: Preliminary analysis identified a four-cytokine composite signature that robustly separates TNFi responders from non-responders (p=0.0001). Notably, responders exhibit a response pattern closely resembling healthy controls, consistent with preserved, canonical T cell activation under TNF perturbation. In contrast, non-responders display an aberrant activation profile, suggesting underlying TNF-independent inflammatory circuitry. Complementary cellular analyses demonstrate that TNFi selectively reduces PD-1⁺CD25⁺CD39⁺ exhausted CD8⁺ T cell populations in responders but not in non-responders, implicating differential regulation of T cell exhaustion pathways.

Conclusion: These findings support a model in which TNFi responsiveness reflects preserved immune network architecture. Furthermore, the data suggest that PD-1 agonism may synergize with TNFi by restoring a responder-like immune state in non-responders. Ongoing studies will expand validation cohorts in PsA and IBD and extend this framework to rheumatoid arthritis. Collectively, this work establishes a functional immune assay platform with potential to both predict therapeutic response and guide mechanism-based combination strategies in autoimmune disease.

Figure 1. Ex-vivo immune functional assay work-flow. Pre-biologic, banked, and outcome annotated patient derived PBMCs are cultured with superantigen in the presence or absence of therapeutic biologics. Following a three-day incubation, supernatant and cell pellets are harvested. Cytokines are measured in the supernatant with a 48-plex Luminex based assay. Cell pellets are analyzed via multidimensional spectral flow cytometry with a 35-color immune phenotype exploratory panel in NYU’s immune monitoring lab core facility.

Figure 2. Normalized Ex-Vivo Immune Functional Assay Predicts TNFi Response. A) Cytokine measurement from the ex-vivo immune functional assay supernatant from both PsA and IBD patient samples. Data is represented as a Log2 fold change compared to vehicle on a per-patient basis. IL-1β, Il-2, IL-4, and IL-5 display trends towards responder vs non-responder separation. A composite z-score that computes the directionality of these four cytokine measurements significantly separates responders from non-responders across both PsA and IBD samples. B) Multidimensional spectral flow cytometry measurements from the ex-vivo immune functional assay cell pellet from PsA patient samples. Data is represented as Log2 change from the vehicle condition. TNFi induced a PD-1⁺CD25⁺CD39⁺ exhausted CD8⁺ T cell depletion in the Humira responder, but not in the non-responders. Further validation of this response signature with additional patient samples is required. **** p<0.0001.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/an-ex-vivo-functional-immune-assay-predicts-tnf-inhibitor-response-across-t-cell-mediated-autoimmune-diseases/