Chimeric antigen receptor T cell (CAR-T) therapy has revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM) [1, 2]. Although BCMA CAR-T cell therapy induces deep and durable responses in most patients with RRMM, disease progression ultimately occurs in most cases. Studies in B-cell acute lymphoblastic leukemia and non-Hodgkin lymphoma have demonstrated that distinct CAR-T cell subsets exhibit differential functions in antitumor efficacy and toxicity profiles [3, 4]. Peripheral blood and bone marrow immune profiling of multiple myeloma (MM) patients treated with CAR-T cell therapy and bispecific T-cell engagers has revealed enrichment of CD4+ T cells and memory T-cell populations, along with elevated CD4+ /CD8+ ratios, in treatment responders compared to non-responders [5]. The association between pre-treatment circulating immune cell populations and therapeutic efficacy of BCMA CAR-T-cell therapy in RRMM remains inadequately characterized.

We report the results of the largest study to date examining the association between baseline peripheral blood immune cell profiles and clinical outcomes following BCMA CAR-T cell therapy in patients with RRMM.

Flow cytometric immunophenotyping was performed to assess T cell subsets, B cells, and natural killer cells. The T cell helper/suppressor panel evaluated the following lymphocyte populations with their respective normal reference ranges: CD3+ T lymphocytes (64–82%, 1171–2005 cells/μL), CD3+CD4+ helper /inducer T lymphocytes (39–57%, 720–1348 cells/μL), CD3+CD8+ suppressor/cytotoxic T lymphocytes (17–31%, 318–710 cells/μL), CD19+ B lymphocytes (8–16%, 151–343 cells/μL), and CD56+ natural killer lymphocytes (7–21%, 145–453 cells/μL). The CD4+/CD8+ ratio was calculated with a normal range of 1.00–3.60. For this test, we use BD Multitest 6-color TBNK reagent with TruCount tubes (CD3Fitc/CD16&56Pe/CD45PerCP-Cy5.5/CD4 Pe-Cy7/CD19APC/CD8APC-Cy-7). Both relative percentages and absolute counts were determined for each cell population. CD3 represents the pan-T cell marker identifying all mature T lymphocytes, while CD4 and CD8 distinguish helper and cytotoxic T cell subsets, respectively. CD19 serves as a pan-B cell marker, and CD56 identifies natural killer cells.

Institutional Review Board approval was obtained from the University of Arkansas for Medical Sciences (UAMS) prior to study initiation. The study was performed in accordance with all applicable guidelines and regulations. Patient informed consent was waived given the retrospective nature of the investigation.

A total of 110 patients with RRMM who received BCMA CAR-T therapy at the UAMS were included. The median age at diagnosis and at apheresis was 56.4 and 65.5 years, respectively. Extramedullary disease (EMD) was present in 17 (16%) of patients prior to CAR-T infusion. One-third of patients had high-risk disease at diagnosis as defined by the second revision of the International Staging System for Multiple Myeloma (R2-ISS) (Table 1).

The median number of prior myeloma therapies was 6.5 (range, 1 to 15). Most patients (94%) received at least one autologous stem cell transplantation (ASCT). The majority of patients (74%) had penta-refractory disease, defined as resistance to at least two agents in the immunomodulatory (IMiD) class, two in the proteosome-inhibitor (PI) class and an anti-CD38 monoclonal antibody. Six patients received prior GPRC5D bispecific antibody and 39% received prior anti-BCMA therapy (Supplementary Table 1).

The majority of patients required a single attempt at apheresis (95%). Bridging therapy was administered to 83% of patients. The most commonly used regimens were daratumumab-based regimens (21%), followed by PI-based regimens (16%).

CAR-T cell manufacturing was successful in 92% of patients. The most frequently utilized BCMA CAR-T product was idecabtagene vicleucel (66%), followed by ciltacabtagene autoleucel (34%). One patient received an investigational BCMA CAR-T product (Supplementary Table 2).

Cytokine release syndrome occurred in 87% of patients (mostly grade 1, 66%), while immune-effector cell-associated neurotoxicity syndrome occurred in 16%. Cytopenias were common: neutropenia and anemia (96% each), and thrombocytopenia (88%) (Supplementary Table 3).

Treatment responses were assessed on days 30 and 90 according to the revised criteria by the International Myeloma Working Group (IMWG) [6]. All but one patient had follow-up data on day 30, and 98 (89%) had follow-up data on day 90. The overall response rate was 56% on day 30, improving to 76% on day 90 (Table 2).

Immune profiles were abnormal in more than 50% of patients based on reference ranges. This pattern was consistent prior to apheresis and lymphodepletion (Supplementary Table 4). The most commonly abnormal component was the absolute count of CD3+CD4+ helper /inducer T lymphocytes, with 90% and 74% of patients having abnormal results prior to apheresis and lymphodepletion, respectively.

Immune profiles were analyzed both as continuous variables and as categorical variables dichotomized by the reference ranges (low vs. normal/high). The primary endpoints were day 90 response and day 90 measurable residual disease (MRD) negativity. For categorical analyses, chi-square tests were employed, while binary logistic regression models were used to assess continuous immune profile measures as independent predictors.

Several categorical measures were significantly associated with outcomes on day 90 (Supplementary Table 5). Patients with a CD4+/CD8+ ratio of <1 prior to apheresis were less likely to achieve a response on day 90 (p = 0.015, ORR 81% vs 100%). Patients with normal or elevated absolute CD3+CD8+ suppressor/ cytotoxic T lymphocytes prior to apheresis were less likely to achieve a response on day 90 (p = 0.05, ORR 97% vs 81%). For the continuous variables analysis, higher levels of CD3 + CD8+ suppressor/ cytotoxic T lymphocytes were significantly associated with no response on day 90 (p = 0.036, odds ratio (OR) = 0.959). This was also significant after adjusting for receiving prior BCMA therapy, CAR-T product type, and high-risk disease (p = 0.044, OR = 0.956). No continuous or categorical variables were significantly associated with MRD negativity on day 90.

The median follow-up for all patients was 13.7 months after apheresis. Both progression-free survival (PFS) and overall survival (OS) were calculated from time of CAR-T infusion (Supplementary Figs. 1 and 2). Median OS for all patients was not reached. One-year OS rate was 91% (95% CI, 85 to 98). Median PFS was 13.4 months (95% CI, 11.1 to 16.1). Immune cell measures were analyzed as continuous variables in cox regression models to test their effect on OS and PFS. No immune cell measures prior to apheresis significantly affected either OS or PFS.

Our study demonstrated that in 110 patients with RRMM treated with BCMA CAR-T cell therapy, a CD4+/CD8+ ratio of <1 and normal/high levels of CD3+CD8+ suppressor/ cytotoxic T lymphocytes at apheresis and prior to lymphodepletion were associated with a lower likelihood of achieving a response at day 90. In addition, reduced levels of CD19+ B cells prior to lymphodepletion were associated with a lower likelihood of day 90 response.

Pu et al. analyzed peripheral blood lymphocyte subsets in 55 patients with acute B-cell lymphoblastic leukemia at the time of apheresis and developed a predictive model for response to CD19 CAR-T cell therapy. They found that a lower proportion of regulatory and effector T cells, an increased proportion of CD3+ cells, and a higher CD4+/CD8+ ratio (1.15 vs 0.59) were predictive of complete remission [7]. These data suggest that both subsets are essential to response, and the optimal ratio likely approximates1:1. However, it remains unclear whether peripheral blood lymphocyte subsets are consistently associated with response.

Similar studies in MM remain limited. Marumo et al. evaluated T-cell phenotypes at apheresis in 34 RRMM patients receiving idecabtagene vicleucel and observed no significant differences in CD4+/CD8+ ratios or CD3+, CD4+, and CD8+ cell proportions between durable and transient responders [8]. However, they demonstrated that CCR7+CD8+T cells (comprising stem cell memory and central memory cells) were associated with long-term responders and improved PFS. Additionally, IMiD-based conditioning regimens prior to apheresis yielded decreased PD-1+CD8+ cell frequencies compared to proteasome inhibitor-based therapies, with reduced PD-1+CD8+ populations associating with durable responses.

Cohen et al. studied the composition of the leukapheresis product prior to manufacturing of BCMA CAR-T cells in 25 patients with RRMM and found that a higher ratio of CD4+/CD8+ cells was associated with enhanced in vivo CAR-T cell expansion and response, while absolute CD3+, CD4+ and CD8+ T cells were not associated with outcomes [9]. Fischer et al. analyzed the peripheral blood of 27 RRMM patients and found that responders had significantly higher CD8+ compared to CD4+ T cells [10]. These discordant findings suggest that an optimal CD4+/CD8+ ratio threshold (potentially ≥1) exists in RRMM with deviations conferring inferior therapeutic outcomes.

Our study has several limitations. Clinical-grade flow cytometry lacks the granularity to assess T-cell memory subsets and exhaustion markers impacting CAR-T outcomes. Bridging therapy was heterogeneous across patients, potentially confounding pre-lymphodepletion immune profiles. Our selection of day-90 overall response as the primary endpoint prioritized data completeness over prognostic depth, as MRD negativity and survival endpoints better predict long-term outcomes. As an exploratory study with a small sample, we did not adjust for multiple comparisons; thus, findings require validation.

In conclusion, this study represents the largest analysis to date examining baseline immune cell profiles as predictors of BCMA CAR-T therapy outcomes in RRMM patients. Our findings demonstrate that pre-treatment CD4+/CD8+ ratios<1 at apheresis and elevated CD3+CD8+ suppressor/cytotoxic T cells at both apheresis and prior to lymphodepletion, along with low B cell levels prior to lymphodepletion, are associated with inferior therapeutic responses at day 90. These biomarkers may help identify patients at risk for treatment failure and guide personalized therapeutic strategies. The identification of these pre-treatment immune signatures could facilitate patient selection and potentially inform combination approaches to optimize CAR-T cell therapy outcomes in multiple myeloma. Future prospective studies are needed to validate these findings and explore interventions to modulate immune profiles prior to CAR-T cell therapy.