Treatment With Cladribine Selects IFNγ+IL17+ T Cells in RRMS Patients – An In Vitro Study

Abstract

BackgroundMultiple sclerosis (MS) is an incurable autoimmune disease mediated by a heterogeneous T cell population (CD3+CD161+CXCR3−CCR6+IFNγ−IL17+, CD3+CXCR3+CCR6+IFNγ+IL17+, and CD3+CXCR3+IFNγ+IL17− phenotypes) that infiltrates the central nervous system, eliciting local inflammation, demyelination and neurodegeneration. Cladribine is a lymphocyte-depleting deoxyadenosine analogue recently introduced for MS therapy as a Disease Modifying Drug (DMD). Our aim was to establish a method for the early identification and prediction of cladribine responsiveness among MS patients.MethodsAn experimental model was designed to study the cytotoxic and immunomodulatory effect of cladribine. T cell subsets of naïve relapsing-remitting MS (RRMS) patients were analyzed ex vivo and in vitro comparatively to healthy controls (HC). Surviving cells were stimulated with rh-interleukin-2 for up to 14days. Cell proliferation and immunophenotype changes were analyzed after maximal (phorbol myristate acetate/ionomycin/monensin) and physiological T-cell receptor (CD3/CD28) activation, using multiparametric flow cytometry and xMAP technology.ResultsEx vivo CD161+Th17 cells were increased in RRMS patients. Ex vivo to in vitro phenotype shifts included: decreased CD3+CCR6+ and CD3+CD161+ in all subjects and increased CD3+CXCR3+ in RRMS patients only; Th17.1 showed increased proliferation vs Th17 in all subjects; CD3+IL17+ and CD3+IFNγ+IL17+ continued to proliferate till day 14, CD3+IFNγ+ only till day 7. Regarding cladribine exposure: RRMS CD3+ cells were more resistant compared to HC; treated CD3+ cells proliferated continuously for up to 14 days, while untreated cells only up to 7 days; both HC/RRMS CD3+CXCR3+ populations increased from baseline till day 14; in RRMS patients vs HC, IL17 secretion from cladribine-treated cells increased significantly, in line with the observed proliferation of CD3+IL17+ and CD3+IFNγ+IL17+ cells; in both HC/RRMS, cladribine led to a significant increase in CD3+IFNγ+ cells at day 7 only, having no further effect at day14. IFNγ and IL17 secreted in culture media decreased significantly from ex vivo to in vitro.ConclusionsCD3+ subtypes showed different responsiveness due to selectivity of cladribine action, in most patients leading to in vitro survival/proliferation of lymphocyte subsets known as pathogenic in MS. This in vitro experimental model is a promising tool for the prediction of individual responsiveness of MS patients to cladribine and other DMDs.

Cytokine Secretion Dynamics of Isolated PBMC after Cladribine Exposure in RRMS Patients

“…The isolation method was very effective, but adding CLD to the culture did not decrease IL-17 secretion in any group, irrespective of CLD exposure duration. This finding is consistent with Dobreanu et al, (2021), who concluded that CLD targets the Th17 population [ 14 , 39 , 40 , 41 , 42 ], which was indirectly confirmed in this study by the nonsignificant change in IL-17 secretion in both groups after CLD exposure. Therefore, an initial increase in IL-17 secretion by PBMCs in RRMS patients could indicate CLD treatment avoidance.…”

“…Therefore, these cytokines can be used as biomarkers of differential MS activity during different disease stages and to monitor the immune system response to DMTs. Heterogeneity in the immunologic pathway influences responses to DMTs, as seen in the cytokine profiles of RRMS patients in previously published studies [ 14 , 15 ]. The pathogenic initiators of MS are periphery-activated CD4 T cells secreting cytokines such as IFN-γ, TNF-α, and IL-17 [ 13 ].…”

“…The changes in absolute lymphocyte number reflected cell proliferation or depletion in response to the cytotoxic action of CLD. The results concerning the cytotoxic effect of CLD on T cells were previously published by our team (Dobreanu et al [ 14 ]. Here are our findings: the initial T cell proliferation after 48 h of CLD exposure was lower for treated cells from both HC and RRMS patients, compared to untreated cells.…”