Immunological effects of CD19.CAR-T cell therapy in systemic sclerosis: an extended case study

Abstract

Objective: The high potential of CD19.CAR-T cells to treat autoimmune diseases such as Systemic Sclerosis (SSc) supposedly relies on the disappearance of autoantibodies. Here we investigated effects of CAR-T cells on the innate immune system which is an important contributor to pathology in SSc.

Methods: Longitudinal analysis of peripheral blood mononuclear cells from an Scl70 + SSc patient treated with CAR-T cells sampled over 18 months by 29-color spectral flow cytometry, in vitro experiments using sera from patient cohorts.

Results: In the patient treated with CAR-T cells, the substantial clinical improvement was paralleled by dynamic changes in innate lymphoid cells, namely Fcγ-receptor IIIA-expressing natural killer (NK) cells. NK cells adopted a more juvenile, less activated, and less differentiated phenotype. In parallel, the potency of serum to form Scl70-containing immune complexes that activate Fcγ-receptor IIIA decreased over time. These observations suggested a mechanistic link between reversal of adaptive autoimmunity and recovering Fcγ-receptor IIIA-expressing innate immune cells after CAR-T cell therapy via regressing immune complex activity. Experiments with sera from the non-CAR-T-treated SSc cohort confirmed that Scl70-containing immune complexes activate Fcγ-receptor IIIA-expressing NK cells in a dose-dependent manner, substantiating the relevance of this link between adaptive and innate immunity in SSc.

Conclusion: This report describes for the first time the phenotypic recovery of innate Fcγ-receptor-expressing cells in an SSc patient treated with CAR-T cells. Decreasing autoantibody levels associated with a reduced ability to form functional immune complexes, the latter appearing to contribute to pathology in SSc via activation of Fcγ receptor IIIA + cells such as NK cells.

Keywords: CAR-T cell therapy; Fcγ receptor; NK cells; NKG2A; Pulmonary fibrosis; Systemic sclerosis.

Fig. 1

Shifts in Lymphocyte populations during the treatment course in the case study. Thawed PBMCs from the SSc patient treated with CD19.CAR-T cells were analyzed by spectral flow cytometry. A UMAPs of cell types within PBMCs based on high-dimensional spectral flow cytometry test results. The right UMAP shows concatenated PBMC samples from timepoints before vs. after CAR-T cell infusion. B Lymphocyte subset proportions were determined using manual standard gating strategies. C The expression profiles of all surface markers included in the 29-parameter panel were analyzed on total NK cells. Except for CD16 and NKG2A that are shown in the graph, no clear tendencies could be observed during the treatment course. On the right, manually gated major NK subsets (CD56^dimCD16 + vs. CD56^brightCD16-) are shown

Fig. 2

High-dimensional analysis of NK cell dynamics during the treatment course. Thawed PBMCs were analyzed by spectral flow cytometry as shown in Fig. 1. Analyses of pre-gated NK cells are shown. A tSNE was used for unbiased dimensionality reduction of NK cells; the graph shows individual time points. B tSNE overlay of concatenated samples from before and after CAR-T cell therapy shown in panel A. C AI-sustained unbiased Phenograph cluster exploration yielded in 18 NK cell subclusters. The bar diagram compares samples before and after CAR-T therapy within each cluster. Filling of bars denotes after CAR-T therapy and colored bars represent clusters overrepresented before and after CAR-T cell therapy, respectively. The same color-code is used in E. D Frequencies of NK cell subcluster #14 characterized by a bright expression of the lymphocyte activation marker CD69. E Analysis the top three overrepresented subclusters before (top) and after (bottom) CAR-T cell therapy. Top three subclusters were visually identified within the ten most frequent NK cell subclusters. All top three overrepresented NK cell subclusters after CAR-T therapy were characterized by strong expression of NKG2A, an inhibitory receptor and marker of early developmental stages in NK cells. Subcluster #3 corresponds to CD56^brightCD16- NK cells, which are believed to be precursors of more mature CD56^dimCD16+ NK cells. Subcluster #7 is an intermediate stage being NKG2A^bright and CD16 positive, but not CD16^bright. Given the overall expression profile of this subset, these “CD56^dimCD16^dimNKG2A^brightCD57^neg” NK cells most likely represent less mature, juvenile NK cells. CD16+ CD57^bright NK cell subcluster #2 represents terminally differentiated NK cells

Fig. 3

Scl70 antigen and anti-Scl70 autoantibodies form soluble immune complexes (sICs) that bind and activate CD16 on NK cells. A Bioactivity of sICs in serum from the described CAR-T cell patient, SLE patients (n = 3) and healthy controls (n = 4) in the presence of 0.05 µg/ml Scl70 antigen. Data are shown as mean ± SEM and are presented as fold over CD16 bioactivity in the absence of Scl70 antigen. For the CAR-T cell patient, serum samples before (blue) and after (orange) CAR-T cell treatment are shown. B Longitudinal measurement of CD16 activation by sICs (squares) in the presence of 0.05 µg/ml Scl70 antigen before (blue) and after (orange) CAR-T cell treatment and serum anti-Scl70 autoantibody concentration (triangles). Anti-Scl70 values below 5 U/ml are considered negative. C PBMC were stimulated with serum from 5 different Scl70 + SSc patients in the presence of the indicated concentrations of Scl70 antigen. Upregulation of the activation marker CD69 of NK cells, B cells and T cells was determined by flow cytometry. D left and middle: In the same experiment shown in (C), CD56^dim and CD56^bright NK cells were gated separately. Shown is the frequency of CD69-positive cells as a technical confirmation of activation of Fc-receptor-bearing CD56^dim NK cells by Scl70-containing immune complexes. Right; from the same PBMCs, NK cells were in parallel isolated and their degranulation without (0) and with (5 µg/ml) Scl70 antigen in the same five sera from Scl70 + SSc patients as shown in C and D left/middle was determined. CD107a + NK cells represent degranulated NK cells, expressed as percentage of all NK cells. These data confirm functionally relevant activation of NK cells by Scl70-containing immune complexes