TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active p38 MAPK

<p>Abstract</p> <p>Background</p> <p>Cytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.</p> <p>Results</p> <p>TGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.</p> <p>Conclusions</p> <p>We suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.</p

Cell Cycle Analysis and Relevance for Single‐Cell Gating in Mass Cytometry

Cell cycle analysis by mass cytometry (MC) is hampered by the poor resolution of the Iridium‐labeled DNA intercalator compared to DNA‐specific fluorescent dyes. We report here a minimum cell cycle panel for MC consisting of Ir‐intercalator (DNA content), IdU (S phase), anti‐pS28HistoneH3 (mitosis), anti‐CDT1 (G1 phase) and anti‐Geminin (non‐G1 phases). Cell cycle distributions obtained by MC were not significantly different from fluorescence flow cytometry results (r2 = 0.98, P < 0.001). Further subdivision of the G1 and G2 phases could be done with anti‐pS780RB1 (late G1) and anti‐PLK1 (late G2), respectively. A disadvantage of MC is that aggregates of cells cannot easily be removed while retaining all single cells. We have developed an analysis pipeline including unsupervised clustering by FlowSOM and subsequent single‐cell gating. When performed on cells stained with the cell cycle panel, this analysis pipeline successfully identified debris, dead/apoptotic cells, nonsingle‐cell populations and the major cell cycle phases. The presented cell cycle panel and analysis pipeline thus achieves true single‐cell analysis at the same time as any additional channels in the panel are open for phenotyping and cell cycle‐resolved expression or modification analysis. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry

Reduced polyfunctional T cells and increased cellular activation markers in adult allergy patients reporting adverse reactions to food

Background The underlying cellular mechanisms causing adverse reactions to food are complex and still not fully understood. Therefore, in this study we aimed to identify functional and/or phenotypical immune cell signatures characteristic for adult patients reporting adverse reactions to food. By mass cytometry, we performed high-dimensional profiling of peripheral blood mononuclear cells (PBMC) from adult patients reporting adverse reactions to food and healthy controls. The patients were grouped according to sIgE-positive or sIgE-negative serology to common food and inhalant allergens. Two broad antibody panels were used, allowing determination of major immune cell populations in PBMC, as well as activation status, proliferation status, and cytokine expression patterns after PMA/ionomycin-stimulation on a single cell level. Results By use of data-driven algorithms, several cell populations were identified showing significantly different marker expression between the groups. Most striking was an impaired frequency and function of polyfunctional CD4+ and CD8+ T cells in patients reporting adverse reactions to food compared to the controls. Further, subpopulations of monocytes, T cells, and B cells had increased expression of functional markers such as CD371, CD69, CD25, CD28, and/or HLA-DR as well as decreased expression of CD23 in the patients. Most of the differing cell subpopulations were similarly altered in the two subgroups of patients. Conclusion Our results suggest common immune cell features for both patient subgroups reporting adverse reactions to food, and provide a basis for further studies on mechanistic and diagnostic biomarker studies in food allergy

Reduced polyfunctional T cells and increased cellular activation markers in adult allergy patients reporting adverse reactions to food

Background The underlying cellular mechanisms causing adverse reactions to food are complex and still not fully understood. Therefore, in this study we aimed to identify functional and/or phenotypical immune cell signatures characteristic for adult patients reporting adverse reactions to food. By mass cytometry, we performed high-dimensional profiling of peripheral blood mononuclear cells (PBMC) from adult patients reporting adverse reactions to food and healthy controls. The patients were grouped according to sIgE-positive or sIgE-negative serology to common food and inhalant allergens. Two broad antibody panels were used, allowing determination of major immune cell populations in PBMC, as well as activation status, proliferation status, and cytokine expression patterns after PMA/ionomycin-stimulation on a single cell level. Results By use of data-driven algorithms, several cell populations were identified showing significantly different marker expression between the groups. Most striking was an impaired frequency and function of polyfunctional CD4+ and CD8+ T cells in patients reporting adverse reactions to food compared to the controls. Further, subpopulations of monocytes, T cells, and B cells had increased expression of functional markers such as CD371, CD69, CD25, CD28, and/or HLA-DR as well as decreased expression of CD23 in the patients. Most of the differing cell subpopulations were similarly altered in the two subgroups of patients. Conclusion Our results suggest common immune cell features for both patient subgroups reporting adverse reactions to food, and provide a basis for further studies on mechanistic and diagnostic biomarker studies in food allergy

TIGIT and PD-1 mark intratumoral T cells with reduced effector function in B-cell non-Hodgkin lymphoma

Checkpoint blockade can reverse T-cell exhaustion and promote antitumor responses. Although blocking the PD-1 pathway has been successful in Hodgkin lymphoma, response rates have been modest in B-cell non-Hodgkin lymphoma (NHL). Coblockade of checkpoint receptors may therefore be necessary to optimize antitumor T-cell responses. Here, characterization of coinhibitory receptor expression in intratumoral T cells from different NHL types identified TIGIT and PD-1 as frequently expressed coinhibitory receptors. Tumors from NHL patients were enriched in CD8+ and CD4+ T effector memory cells that displayed high coexpression of TIGIT and PD-1, and coexpression of these checkpoint receptors identified T cells with reduced production of IFNγ, TNFα, and IL2. The suppressed cytokine production could be improved upon in vitro culture in the absence of ligands. Whereas PD-L1 was expressed by macrophages, the TIGIT ligands CD155 and CD112 were expressed by lymphoma cells in 39% and 50% of DLBCL cases and in some mantle cell lymphoma cases, as well as by endothelium and follicular dendritic cells in all NHLs investigated. Collectively, our results show that TIGIT and PD-1 mark dysfunctional T cells and suggest that TIGIT and PD-1 coblockade should be further explored to elicit potent antitumor responses in patients with NHL

T cells expressing checkpoint receptor TIGIT are enriched in follicular lymphoma tumors and characterized by reversible suppression of T-cell receptor signaling

Purpose: T cells infiltrating follicular lymphoma (FL) tumors are considered dysfunctional, yet the optimal target for immune checkpoint blockade is unknown. Characterizing coinhibitory receptor expression patterns and signaling responses in FL T-cell subsets might reveal new therapeutic targets. Experimental Design: Surface expression of 9 coinhibitory receptors governing T-cell function was characterized in T-cell subsets from FL lymph node tumors and from healthy donor tonsils and peripheral blood samples, using high-dimensional flow cytometry. The results were integrated with T-cell receptor (TCR)-induced signaling and cytokine production. Expression of T-cell immunoglobulin and ITIM domain (TIGIT) ligands was detected by immunohistochemistry. Results: TIGIT was a frequently expressed coinhibitory receptor in FL, expressed by the majority of CD8 T effector memory cells, which commonly coexpressed exhaustion markers such as PD-1 and CD244. CD8 FL T cells demonstrated highly reduced TCR-induced phosphorylation (p) of ERK and reduced production of IFNγ, while TCR proximal signaling (p-CD3ζ, p-SLP76) was not affected. The TIGIT ligands CD112 and CD155 were expressed by follicular dendritic cells in the tumor microenvironment. Dysfunctional TCR signaling correlated with TIGIT expression in FL CD8 T cells and could be fully restored upon in vitro culture. The costimulatory receptor CD226 was downregulated in TIGIT+ compared with TIGIT− CD8 FL T cells, further skewing the balance toward immunosuppression. Conclusions: TIGIT blockade is a relevant strategy for improved immunotherapy in FL. A deeper understanding of the interplay between coinhibitory receptors and key T-cell signaling events can further assist in engineering immunotherapeutic regimens to improve clinical outcomes of cancer patients

BMP-7 induces apoptosis in human germinal center B cells and is influenced by TGF-β receptor type I ALK5

<div><p>Selection and maturation of B cells into plasma cells producing high-affinity antibodies occur in germinal centers (GC). GCs form transiently in secondary lymphoid organs upon antigen challenge, and the GC reaction is a highly regulated process. TGF-β is a potent negative regulator, but the influence of other family members including bone morphogenetic proteins (BMPs) is less known. Studies of human peripheral blood B lymphocytes showed that BMP-6 suppressed plasmablast differentiation, whereas BMP-7 induced apoptosis. Here, we show that human naïve and GC B cells had a strikingly different receptor expression pattern. GC B cells expressed high levels of BMP type I receptor but low levels of type II receptors, whereas naïve B cells had the opposite pattern. Furthermore, GC B cells had elevated levels of downstream signaling components <i>SMAD1</i> and <i>SMAD5</i>, but reduced levels of the inhibitory <i>SMAD7</i>. Functional assays of GC B cells revealed that BMP-7 suppressed the viability-promoting effect of CD40L and IL-21, but had no effect on CD40L- and IL-21-induced differentiation into plasmablasts. BMP-7-induced apoptosis was counteracted by a selective TGF-β type I receptor (ALK4/5/7) inhibitor, but not by a selective BMP receptor type I inhibitor. Furthermore, overexpression of truncated ALK5 in a B-cell line counteracted BMP-7-induced apoptosis, whereas overexpression of truncated ALK4 had no effect. BMP-7 mRNA and protein was readily detected in tonsillar B cells, indicating a physiological relevance of the study. Altogether, we identified BMP-7 as a negative regulator of GC B-cell survival. The effect was counteracted by truncated ALK5, suggesting greater complexity in regulating BMP-7 signaling than previously believed.</p></div

Introduction of truncated ALK5 in the B-cell lymphoma cell line Mino abrogates BMP-7-induced apoptosis.

<p>Mino cells were transduced with truncated ALK5 or truncated ALK4. (A) The cells were stained with biotinylated anti-ALK5, followed by streptavidin PE or by biotinylated anti-ALK4, followed by streptavidin APC, and analyzed by flow cytometry. Receptor expression is compared in GFP⁺ or mCherry⁺ transduced cells vs. GFP^-/mCherry^- non-transduced cells. (B-C) The transduced Mino cells were cultured in serum free media (X-VIVO 15) over night and then left in medium alone (unstim) or stimulated with BMP-2, BMP-7 or TGF-β for 60 min, before detection of phosphorylated (p-) Smad 1/5 or p-Smad 2/3 by flow cytometry. (B) One representative experiment showing p-SMAD1/5 vs. GFP in truncated ALK5-2A-GFP expressing cells. (C) BMP- or TGF-β-induced phosphorylation is shown relative to unstimulated cells, using arcsinh transformation of median fluorescence intensity data. Mean ± SEM, <i>n</i> = 5. (D-E): Transduced Mino cells were cultured in X-VIVO 15 and left unstimulated or stimulated with TGF-β or BMP-7 for 72 hours and stained for active caspase-3 before analysis by flow cytometry. Shown here is active caspase-3 staining of control cells and transduced cells for (D) one representative experiment and (E) mean ± SEM, <i>n</i> = 3. *<i>p</i> < 0.05; two-tailed, paired Student’s <i>t</i>-test.</p