Classic Anti-Myeloma Treatments Alter Circulating Immune Cell Subsets with Potential Implications for T Cell Immunotherapies in Multiple Myeloma
Authors: Zhou Xiang , Wiethe Sarah , Wolff Angelika , Steinhardt Max J , Afrin Nazia , John Mara , Riedhammer Christine , Xiao Xianghui , Munawar Umair , Mersi Julia , Han Seungbin , Waldschmidt Johannes , Riedel Angela , Topp Max S. , Duell Johannes , Einsele Hermann , Kortüm K. Martin , Rasche Leo 
Published: December 2024

Background
A growing body of evidence suggests the crucial role of patient's baseline immune status prior to chimeric antigen receptor-modified T cell (CAR-T) therapy. Recently, low CD4/CD8 ratio has been reported as a negative prognostic factor and is associated with increased risk of manufacturing failure in CAR-T therapy (Jo et al, BJHaem 2023). Moreover, persistence of CD4+ CAR-T is related to long-term response in CLL (Melenhorst et al, Nature 2022). Thus, in relapsed/refractory (RR) multiple myeloma (MM), there is a clear need to elucidate the impact of classic anti-MM drugs on CD4/CD8 ratio and CD4+ T cell count for planning CAR-T therapy. As T cell immunotherapies are moving to the frontline, it is of interest to analyze the dynamics of immune cells from monoclonal gammopathy of undetermined significance (MGUS) to RRMM to tailor the optimal sequencing strategy for T cell immunotherapies. We therefore performed the current study to address these issues.
Methods
We collected peripheral blood (PB) of patients with MGUS, smoldering MM (SMM), newly diagnosed (ND) and RRMM, retrospectively between September 2009 and October 2022 as well as prospectively since November 2022. Immune status was determined by flow cytometry using the following markers: CD45, CD3, CD4, CD8, CD19 and CD56, and then combined with clinical data.
Results
As of July 2024, 1886 sequential PB samples (MGUS=91, SMM=61, NDMM=47, RRMM=1687) from 459 patients (MGUS=42, SMM=22, NDMM=43, RRMM=352) were included. The median age at sampling was 66 years (range 31-90). The RRMM patients had been treated with a median of 1 line of therapy (range 1-11), and 250 (71.0%) patients had received high-dose melphalan and autologous stem cell transplant (HD-Mel).
Firstly, we compared the immune status in MGUS, SMM, NDMM and RRMM. We noticed significantly lower total T cell (CD3+CD19-), B cell (CD19+CD3-), CD4+ T cell (CD3+CD4+), NK cell (CD56+CD3-) counts and CD4/CD8 ratio in RRMM than MGUS, SMM and NDMM (all P&lt;0.001). Moreover, CD8+ T cells (CD3+CD8+) and NK T cells (CD56+CD3+) increased from MGUS to SMM (P&lt;0.001) and remained constant from SMM to NDMM. RRMM showed lower NK T cell count than NDMM (P&lt;0.001), while CD8+ T cell counts were similar in NDMM and RRMM.
Secondly, we evaluated the prognostic value of immunologic parameters in RRMM. In multivariate cox regression model, CD4+ T cells &lt; 400/µl (HR: 7.8, 95%CI 1.9-33.1, P=0.005) and CD4/CD8 ratio &lt;0.70 (HR: 2.3, 95%CI 1.1-4.7, P=0.023) were negative prognostic factors for progression free survival. Moreover, CD4+ T cells &lt; 400/µl (HR: 2.3, 95%CI 1.2-4.2, P=0.008), NK cells &lt; 200/µl (HR: 1.7, 95%CI 1.0-3.0, P=0.049) and CD4/CD8 ratio &lt;0.70 (HR: 1.8, 95%CI 1.1-3.0, P=0.02) were associated with inferior overall survival.
Thirdly, we analyzed the impact of classic anti-MM treatments on circulating immune cell subsets, particularly the immunologic features with prognostic relevance, using multivariate generalized linear model. Carfilzomib (CFZ) pretreatment negatively correlated with CD4+ T cell count (n/µl) (β=-103.6, P&lt;0.001) and CD4/CD8 ratio (β=-0.4, P&lt;0.001). Interestingly, prior bortezomib therapy showed positive correlation with CD4/CD8 ratio (β=0.5, P=0.001), possibly due to the reversible proteasome inhibition in contrast to CFZ. Prior HD-Mel resulted in lower CD4+ T cell (β=-184.7, P&lt;0.001), higher CD8+ T cell counts (β=109.1, P&lt;0.001) and lower CD4/CD8 ratio (β=-1.7, P&lt;0.001). As expected, treatment-free interval (days) positively correlated with CD4+ T cell count (β=0.09, P&lt;0.001). The number of prior lines of therapy negatively correlated with CD4+ T cell count (β=-21.4, P&lt;0.001) and CD4/CD8 ratio (β=-0.06, P&lt;0.001). Surprisingly, older patients showed higher CD4+ T cell count (β=3.3, P&lt;0.001) and CD4/CD8 ratio (β=-0.03, P&lt;0.001), probably due to the more intensive therapy in younger patients, e.g. HD-Mel.
Conclusion
Our findings suggest the changes of immune system from MGUS to RRMM and the potential impact of classic anti-MM drugs on immune status. The known biomarkers for a fit immune system, i.e. sufficient CD4+ T cell count and CD4/CD8 ratio, are associated with favorable survival outcome in RRMM, and the immune status recovers after a treatment-free interval. To achieve better anti-MM effect and to reduce the risk of manufacturing failure, leukapheresis for CAR-T should be considered prior to HD-Mel and CFZ due to their negative impact on CD4+ T cells and CD4/CD8 ratio.