Peripheral blood cellular profile at pre-lymphodepletion is associated with CD19-targeted CAR-T cell-associated neurotoxicity

Abstract

Background: Infusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS.

Methods: This is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation.

Results: Multivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3⁺CD8⁺ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8⁺CD45RA⁺CD57⁺ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14⁺ and CD45⁺ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8⁺ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients.

Discussion: Our data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8⁺ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history.

Trial registration: ClinicalTrials.gov NCT04892433.

Keywords: chimeric antigen receptor; inflammation; myeloid activation; neurotoxicity; senescence.

Figure 1

Cytofluorimetric analysis. (A) Gating strategy used to identify CD8⁺ T cell subsets. A lymphocyte gate was set based on the CD45⁺ and SSC parameters. Among CD3⁺CD4⁺ or CD3⁺CD8⁺ compartments, different subsets were analyzed: Naive (CD45RA⁺62L^-), central memory (CM) (CD45RA^-62L⁺), effector memory (EM) (CD45RA^-62L^-), terminally differentiated effector (TEMRA) (CD45RA⁺62L^-) T cells; Among CD3⁺CD8⁺ compartment, SIP⁺ cells were measured as percentage of CD45RA⁺28^-57⁺ whereas CD45RA⁺28⁺, CD45RA^-57⁺ and CD45RA^-28⁺ among CD8⁺ T cells were defined as non-SIP. The box plots show the changes in the percentage of (B) TEMRA, (C) non-SIP, (D) SIP, (E) SIP:non-SIP ratio among CD3⁺CD8⁺ T cell compartment in ICANS and no-ICANS patients. Comparisons between 2 groups were made using the non-parametric, unpaired Mann-Whitney test. *p <0.05; **p <0.01; ns = not significant.

Figure 2

Plasma markers analysis. (A) Box plots report the expression levels of miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p at pre-LD in ICANS and no-ICANS patients. Plasma levels of (B) GDF-15, (C) IL-15, (D) CXCL-9 and (E) NfL at pre-LD in ICANS and no-ICANS patients. (F) Range from min to max of the Mean Fluorescence Intensity (MFI) for each plasma EVs marker. Plasma from patients experiencing ICANS in red and no-ICANS patients in green; values have been normalized to blank control. Comparisons between 2 groups were made using t test. *p = <0.05; ns = not significant.

Figure 3

Cytofluorimetric analysis. (A) Gating strategy used to identify the M- and PMN-MDSC in a patient before receiving CAR-T cell infusion. The CD14⁺HLA-DR^−/low cell subset for M-MDSC or CD15⁺HLA-DR^-/low for PMN-MDSC was gated, and the proportion of CD11b⁺CD33⁺ was evaluated. The box plots report the percentage of (B) Monocyres, (C) M-MDSC, (D) PMN-MDSC, and plasma levels of (E) IL-6, (F) IL-10 at pre-LD in ICANS and no-ICANS patients. Comparisons between 2 groups were made using the non-parametric, unpaired Mann-Whitney test. *p <0.05; ns = not significant.