Author Of 1 Presentation
YI01.02 - BTK inhibition results in preferential reduction of pro-inflammatory B-cell responses through modulation of B-cell metabolism
Abstract
Background
Results of B-cell depleting therapies have underscored important antibody-independent functions of B cells in multiple sclerosis (MS) pathogenesis. Bruton’s Tyrosine Kinase (BTK) is a key down-stream signaling molecule of the B cell receptor, and BTK inhibitors (BTKi) have been used for treating various B-cell malignancies. BTKi are now being pursued as a ‘next generation’ non-depleting approach to B-cell targeting in MS. However, the contributions of BTK to MS-relevant functional B-cell responses, and the impact of BTKi on such responses, remain largely unknown.
Objectives
We would like to assess the impact of BTK inhibition on MS implicated B-cell functions and explore underlying mechanisms.
Methods
We applied a series of functional assays and RNA sequencing to human B cells, isolated from peripheral blood of healthy individuals or MS patients, as well as from human tonsils, to study influence of BTKi on B-cell survival, activation, proliferation, antibody production, antigen presenting functions, cytokine production and metabolism.
Results
As expected, BTKi strongly decreased B-cell activation with minimal effects on B cell survival. BTKi also significantly limited the induction of co-stimulatory molecules (CD80, CD86) expression on activated B cells, which in turn resulted in a decreased capacity of the B cells to support both polyclonal as well as antigen-specific T-cell activation. Interestingly, BTKi treatment preferentially reduced pro-inflammatory B-cell cytokine (GM-CSF, TNFα and IL-6) secretion with only marginal influence on B-cell IL-10 production, resulting in a decreased capacity of the B cells to promote myeloid cell pro-inflammatory responses. Unbiased transcriptomic analysis of either vehicle- or BTKi-treated B cells suggested that BTKi could limit metabolism-related pathways, and subsequent seahorse analyzer experiments confirmed that BTKi decreased B-cell mitochondrial respiration and glycolysis. Metabolic manipulation further revealed that the level of mitochondrial respiration could control the balance between pro- and anti-inflammatory B cell responses.
Conclusions
BTKi preferentially limits pro-inflammatory B-cell responses that are implicated in MS pathophysiology. Our study also reveals a fundamental role for metabolism in regulating B-cell functions, and points to a novel therapeutic strategy targeting the balance between pro- and anti-inflammatory responses of B cells through modulation of their energy-utilization pathways.
Presenter Of 1 Presentation
YI01.02 - BTK inhibition results in preferential reduction of pro-inflammatory B-cell responses through modulation of B-cell metabolism
Abstract
Background
Results of B-cell depleting therapies have underscored important antibody-independent functions of B cells in multiple sclerosis (MS) pathogenesis. Bruton’s Tyrosine Kinase (BTK) is a key down-stream signaling molecule of the B cell receptor, and BTK inhibitors (BTKi) have been used for treating various B-cell malignancies. BTKi are now being pursued as a ‘next generation’ non-depleting approach to B-cell targeting in MS. However, the contributions of BTK to MS-relevant functional B-cell responses, and the impact of BTKi on such responses, remain largely unknown.
Objectives
We would like to assess the impact of BTK inhibition on MS implicated B-cell functions and explore underlying mechanisms.
Methods
We applied a series of functional assays and RNA sequencing to human B cells, isolated from peripheral blood of healthy individuals or MS patients, as well as from human tonsils, to study influence of BTKi on B-cell survival, activation, proliferation, antibody production, antigen presenting functions, cytokine production and metabolism.
Results
As expected, BTKi strongly decreased B-cell activation with minimal effects on B cell survival. BTKi also significantly limited the induction of co-stimulatory molecules (CD80, CD86) expression on activated B cells, which in turn resulted in a decreased capacity of the B cells to support both polyclonal as well as antigen-specific T-cell activation. Interestingly, BTKi treatment preferentially reduced pro-inflammatory B-cell cytokine (GM-CSF, TNFα and IL-6) secretion with only marginal influence on B-cell IL-10 production, resulting in a decreased capacity of the B cells to promote myeloid cell pro-inflammatory responses. Unbiased transcriptomic analysis of either vehicle- or BTKi-treated B cells suggested that BTKi could limit metabolism-related pathways, and subsequent seahorse analyzer experiments confirmed that BTKi decreased B-cell mitochondrial respiration and glycolysis. Metabolic manipulation further revealed that the level of mitochondrial respiration could control the balance between pro- and anti-inflammatory B cell responses.
Conclusions
BTKi preferentially limits pro-inflammatory B-cell responses that are implicated in MS pathophysiology. Our study also reveals a fundamental role for metabolism in regulating B-cell functions, and points to a novel therapeutic strategy targeting the balance between pro- and anti-inflammatory responses of B cells through modulation of their energy-utilization pathways.
Author Of 2 Presentations
P0383 - Resistance of CD11c+ B cells to anti-CD20 depletion with treatment initiation and early preferential repopulation of anti-inflammatory B cells in MS (ID 1210)
Abstract
Background
Anti-CD20 therapy is highly efficacious in limiting new disease activity in multiple sclerosis (MS), which depletes most circulating B cells and a small subset of T cells. However, relatively little is known about how anti-CD20 therapy affects T cells.
Objectives
We aimed to define phenotypic and functional profiles of B cells during depletion and early reconstitution following anti-CD20 antibody initiation.
Methods
Peripheral blood mononuclear cells (PBMC) were serially isolated and cryopreserved using strict standard operating procedures prior to treatment, early (3-4 months) and/or later (approx. 6 months). Following anti-CD20 (ocrelizumab) treatment initiation, in 18 previously treatment-naive MS patients. Functional immune phenotyping was performed in batch using multi-parametric flow cytometry panels developed and validated for use with cryopreserved PBMC.
Results
In addition to plasmablasts which, as expected, were not fully depleted, CD11c+ B cells appeared less efficiently depleted after treatment initiation. By 6 months post-treatment, B cells were partially repopulated, though to differing extents across individuals. In general, CD10+ transitional B cells (implicated as anti-inflammatory), and a subset of memory B cells, were preferentially repopulated. The repopulating B cells exhibited increased proliferation, though they expressed lower levels of activation markers and higher levels of regulatory markers. Ratios of IL-6/IL-10-producing B cells were significantly diminished in the reconstituting population, as compared to the treatment-naïve baseline.
Conclusions
The abnormal pro-inflammatory/anti-inflammatory imbalance of B cells seen in untreated MS patients appears improved in reconstituting B cells even after an initial cycle of ocrelizumab, though with a considerable degree of heterogeneity across patients. Unexpectedly, CD11c+ B cells, that have been implicated as pro-inflammatory in other systemic autoimmune diseases, appeared less susceptible to depletion. Of interest is whether a particular imbalance between CD11c+ B and other B cell subsets may underlie the infrequent episodes of disease activity observed early after treatment initiation.
P0952 - Characterization of age-related changes in circulating T cells in multiple sclerosis and normal controls: a pilot study (ID 975)
Abstract
Background
Immunosenescence (ISC) is characterized by age-associated changes in immune system composition and function. Multiple sclerosis (MS) is a lifelong illness, hence the disease process is superimposed on, and may interact bi-directionally with, ISC such that ISC may alter disease activity, while cumulative inflammatory events in MS may influence ISC. Since the T cell compartment is markedly affected by ISC, we hypothesized that T-cell aging may differ between MS patients and normal controls (NCs).
Objectives
To characterize age-related changes in circulating T cells in treatment-naïve multiple sclerosis patients compared to NCs.
Methods
Frequencies of circulating T-cell subsets were determined using multiparametric flow cytometry of peripheral blood mononuclear cells from 50 NC (Mean Age 48.6, Range 20 – 84) and 40 treatment-naïve MS (Mean Age 43.3, Range 18 – 72). Age-related changes in T cell subsets, and differences in T cell ISC between NC and MS, were determined using linear mixed effects models.
Results
Age-related changes in circulating T-cell subsets in the NCs recapitulated known features of ISC, including reductions in recent thymic emigrants and reciprocal changes in CD4 and CD8 T cells as well as in naïve and memory T cells. While most aspects of T cell ISC in MS patients were similar to those observed in NC, MS patients experienced early and persistent redistribution of the naïve and memory CD4 T cell compartment, such that at any given age, frequencies of circulating naïve (CCR7+CD45RA+) CD4 T cells were 16.7% lower and frequencies of effector memory (CCR7-CD45RA-) CD4 T cells were 14.5% greater on average than NC. Further, aged MS patients exhibited a relative increase in activated (HLA-DR+CD38+) and cytotoxic (CCL5+EOMES+) CD4 T cells compared to NC, while they did not exhibit increased CLA+ CD4 T cells. Lastly, aged MS patients exhibited altered immune checkpoint-molecule expression, wherein frequencies of CTLA-4+ CD8 T cells did not increase with age as was seen in NC.
Conclusions
Most T-cell subsets followed similar aging trajectories in MS patients and NCs, indicating normal ISC is largely conserved in MS. Nonetheless, key differences suggest that aged MS patient T cells exhibit increased propensity for immune activation and effector function compared to NC, which may reflect ongoing inflammation and injury throughout the lifespan. Further elucidation of ISC in MS may inform management of immune therapies in aging MS patients.