Marrow-Infiltrating Regulatory T Cells Correlate with the Presence of Dysfunctional CD4⁺PD-1⁺ Cells and Inferior Survival in Patients with Newly Diagnosed Multiple Myeloma

PURPOSE: Immune dysregulation is described in multiple myeloma(MM). While preclinical models suggest a role for altered T cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterise marrow infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first line therapy. EXPERIMENTAL DESIGN: We undertook detailed characterisation of T cells from bone marrow(BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression free survival. RESULTS: We found that patients with MM had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared to healthy donors. Patients with a higher frequencies of Tregs (Treghi) had shorter PFS, and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared to Treglopatients. Analysis of CD4 and CD8 effectors revealed that low CD4effector:Treg ratio, and increased frequency of PD-1 expressing CD4effcells were independent predictors of early relapse over and above conventional risk factors such as genetic risk and depth of response. Ex-vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4effPD-1+ cells displayed transcriptional and secretory features of dysfunction. CONCLUSIONS: BM infiltrating T cell subsets, specifically Treg and PD-1 expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies

Increased Immune-Regulatory Receptor Expression on Effector T Cells as Early Indicators of Relapse Following Autologous Stem Cell Transplantation for Multiple Myeloma

The benefit of autologous stem cell transplantation (ASCT) in newly diagnosed myeloma patients, apart from supporting high dose chemotherapy, may include effects on T cell function in the bone marrow (BM). We report our exploratory findings on marrow infiltrating T cells early post-ASCT (day+100), examining phenotype and T cell receptor (TCR) repertoire, seeking correlations with timing of relapse. Compared to healthy donors (HD), we observed an increase in regulatory T cells (CD4+FoxP3+, Tregs) with reduction in CD4 T cells, leading to lower CD4:8 ratios. Compared to paired pre-treatment marrow, both CD4 and CD8 compartments showed a reduction in naïve, and increase in effector memory subsets, suggestive of a more differentiated phenotype. This was supported by increased levels of several immune-regulatory and activation proteins (ICOS, PD-1, LAG-3, CTLA-4 and GzmB) when compared with HD. Unsupervised analysis identified a patient subgroup with shorter PFS (p=0.031) whose BM contained increased Tregs, and higher immune-regulatory markers (ICOS, PD-1, LAG-3) on effector T cells. Using single feature analysis, higher frequencies of marrow PD-1+ on CD4+FoxP3- cells and Ki67+ on CD8 cells were independently associated with early relapse. Finally, studying paired pre-treatment and post-ASCT BM (n=5), we note reduced abundance of TCR sequences at day+100, with a greater proportion of expanded sequences indicating a more focused persistent TCR repertoire. Our findings indicate that, following induction chemotherapy and ASCT, marrow T cells demonstrate increased activation and differentiation, with TCR repertoire focusing. Pending confirmation in larger series, higher levels of immune-regulatory proteins on T cell effectors at day+100 may indicate early relapse

An APRIL based chimeric antigen receptor for dual targeting of BCMA and TACI in Multiple Myeloma

B-cell maturation antigen (BCMA) is a promising therapeutic target for multiple myeloma (MM), but expression is variable, and early reports of BCMA targeting chimeric antigen receptors (CARs) suggest antigen down-regulation at relapse. Dual antigen targeting increases targetable tumour antigens and reduces the risk of antigen negative disease escape. 'A proliferation-inducing ligand' (APRIL) is a natural high affinity ligand for BCMA and transmembrane activator and CAML interactor (TACI). We quantified surface tumour expression of BCMA and TACI on primary MM cells (n=50). All cases tested expressed BCMA and 39(78%) of them also expressed TACI. We engineered a third generation APRIL-based CAR (ACAR), which killed targets expressing either BCMA or TACI (p<0.01 and p<0.05 respectively, cf control, E:T ratio 16:1). We confirmed cytolysis at antigen levels similar to those on primary MM, at low effector to target ratios (56.2±3.9% killing of MM.1s at 48 hours, E:T ratio 1:32, p<0.01) and of primary MM cells (72.9±12.2% killing at 3 days, E:T ratio 1:1, p<0.05, n=5). Demonstrating tumour control in the absence of BCMA, cytolysis of primary tumour expressing both BCMA and TACI was maintained in the presence of a BCMA targeting antibody. Further, using an intramedullary myeloma model, ACAR T-cells caused regression of established tumour within 2 days. Finally, in an in vivo model of tumour escape, there was complete ACAR-mediated tumour clearance of BCMA+TACI- and BCMA-TACI+ cells while a scFv CAR targeting BCMA alone resulted in outgrowth of BCMA negative tumour. These results support the clinical potential of this approach

Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease

Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease

Marrow-Infiltrating Regulatory T Cells Correlate with the Presence of Dysfunctional CD4⁺PD-1⁺ Cells and Inferior Survival in Patients with Newly Diagnosed Multiple Myeloma.

Purpose Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy.Experimental design We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival.Results We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4eff):Treg ratio and increased frequency of PD-1-expressing CD4eff cells were independent predictors of early relapse over and above conventional risk factors, such as genetic risk and depth of response. Ex vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4effPD-1+ cells displayed transcriptional and secretory features of dysfunction.Conclusions BM-infiltrating T-cell subsets, specifically Tregs and PD-1-expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies