Longitudinal Cytokine Expression in Aggressive Lymphoma Patients Treated with CAR-T: Applications of Immune Profiling to CAR-T
Authors: Reynolds Gemma K , Williams Lewis , Anderson Mary Ann , Dowling Mark R , Minson Adrian , Dickinson Michael J. , Thursky Karin A , Harrison Simon J , Slavin Monica A , Doerflinger Marcel , Teh Benjamin W 
Published: December 2024

Introduction
Comprehensive immune profiling post chimeric-antigen receptor T-cell (CAR-T) treatment may help better elucidate the interplay between the CAR-T product, adjuvant immune suppression for treatment-related adverse events, and the host-immune system. This exploratory study longitudinally evaluated in vitro cytokine expression in CAR-T treated patients with aggressive lymphoma.
Methods
Patients (pts) with diffuse large B-cell lymphoma (DLBCL) treated with standard-of-care CAR-T between 2019 - 2023 were consented and included (HREC/71984/PMCC). Demographic and treatment-related variables were collected. Cytokine release syndrome (CRS) and immune-effector-cell-associated-neurotoxicity (ICANS) were graded according to CTCAE criteria, with grade ≥ 1 considered clinically actionable. Cumulative exposure to dexamethasone (mg) and tocilizumab (mg) was recorded.
Blood samples were collected at apheresis (baseline) and day 30 (D30) post CAR-T infusion. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved. After thawing, PBMCs were either re-stimulated with phorbol myristate acetate (PMA) and ionomycin or left untreated for 48 hours. Cytokines were recorded on a Luminex xMAP System and quantitated via comparison to a standard curve.
Individual cytokines were analysed, and then combined according to function. For example, the sum of cytokines contributing to the Th1 pathway (TNF-alpha, IFN-gamma, IL2) was analysed. Following log transformation for normality, ANOVA assessed differences in mean cytokine expression following PMA/ionomycin stimulation between baseline and D30, as a function of a treatment-related complications. Cumulative tocilizumab and dexamethasone were entered as covariates. P-values were adjusted for multiple comparisons. Untransformed medians are reported in text to illustrate the direction of effect.
Results
20 pts (70% male) received axicabtagene (n=10) or tisagenlecleucel (n=10) for treatment of DLBCL, with a median of 3 prior therapies (IQR:3 - 4.25). 70% had clinically actionable CRS, and 15% developed ICANS. Median tocilizumab exposure was 510mg (IQR:0-892mg) and dexamethasone was 0mg (IQR:0-17mg).
Overall, cytokine expression in response to stimulation was significantly lower at D30 compared to baseline, for cytokines associated with T-cell response (494 pg/mL vs. 532pg/mL, p = 0.008) and Th1 response (737pg/mL vs. 1122 pg/mL, p = 0.006). This effect controlled for tocilizumab exposure, dexamethasone exposure, and did not differ by CAR-T product (p &gt; 0.05).
In patients with CRS, Th1-associated cytokines were significantly lower at D30 in response to stimulation (21 pg/mL vs. 882 pg/mL, p = 0.007), controlling for cumulative exposure to tocilizumab (median tocilizumab in model 772mg), than in patients without CRS (885 pg/mL vs. 1314 pg/mL). This interaction was driven by lower levels of IL-2 (p = 0.003), IL-12 (p &lt; 0.001), and IL-15 (p = 0.003) in response to stimulation at D30.
Patients who experienced ICANS had significantly higher Th1 responses to stimulation at D30 compared to baseline (2899 pg/mL vs. 1637 pg/mL, p = 0.007), controlling for cumulative dexamethasone exposure (p = .796), compared to patients without ICANS (541pg/mL vs. 822pg/mL). This significant interaction was not observed for individual cytokines within the Th1 pathway (all p &gt; 0.003).
Discussion
Our study is the first to demonstrate that in vitro cytokine expression following stimulation at D30 was significantly lower across multiple cytokine groups when compared to baseline. Other studies have documented increases in the absolute numbers of immune cells following CAR-T,1 but none have evaluated function. The finding that IL-15 levels at D30 was significantly lower in patients experiencing CRS is salient, as IL-15 may be essential to enduring CAR-T expansion.2,3 Additionally, our results suggest that the pro-inflammatory milieu observed during neurotoxicity may extend beyond resolution of the clinical syndrome.4,5 Our study is limited by sample size, particularly the small numbers of patients evaluable for ICANS. Our findings generate interesting hypotheses around the interaction between CAR-T product and host immunity, which should be validated in further translational studies.