Faculty Bibliography

INTRODUCTION/BACKGROUND:The information content of multiparametric flow cytometry experiments is routinely underexploited given the paucity of adequate tools for unbiased comprehensive data analysis that can be applied successfully and independently by immunologists without computational training. METHODS:We aimed to develop a tool that allows straightforward access to the entire information content of any given flow cytometry panel for immunologists without special computational expertise. We used a data analysis approach which accounts for all mathematically possible combinations of markers in a given panel, coded the algorithm and applied the method to mined and self-generated data sets. RESULTS:We developed Flow Plex, a straightforward computational tool that allows unrestricted access to the information content of a given flow cytometry panel, enables classification of human samples according to distinct immune phenotypes, such as different forms of autoimmune uveitis, acute myeloid leukemia vs "healthy", "old" vs "young", and facilitates the identification of cell populations with potential biologic relevance to states of disease and health. CONCLUSIONS:We provide a tool that allows immunologists and other flow cytometry users with limited bioinformatics skills to extract comprehensive, unbiased information from flow cytometry data sets.

A 58-year-old man with a history of recurrent aphthous ulcers since childhood was admitted to the hospital with acute neurological decline characterised by loss of motor dexterity, dysarthria, dysphagia and unsteady gait. MRI brain was significant for symmetrical hyperintense T2 fluid attenuated inversion recovery (FLAIR) in the corticospinal tracts, including parts of the pons and the mesodiencephalic junction. Though initial concern was for neuro-Behçet's disease, brain biopsy ultimately revealed a diagnosis of astrocytoma. This report demonstrates a mimic of neuro-Behçet's disease and the importance of confirming the correct diagnosis prior to initiating therapy.

Background/Purpose: Functionality and immune-phenotypes of the human CD4+ T-cell compartment in Behcet's disease (BD) are under-investigated, but several lines of evidence point to its relevance in the pathogenesis and progression of the disease. We aimed to apply an unbiased single cell approach to dissect the immune-phenotype of CD4+ T cells in prototypical BD in order to identify cell populations of potential pathobiological relevance.
Method(s): We determined single cell expression levels of CD3, CD4, CD8, CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki76, HLA-DR, CD38, CD39 in PBMC by flow cytometry and computed the representation of all possible cell populations within CD4+ starting populations in PBMC from healthy (HD, n=25), BD (n=13), and diseased subjects with non-BD auto-immune uveitis (n=11, VKH, Sarcoidosis, and HLA-B27 associated uveitis). BD subjects met ISG criteria and were Arab or Chinese. 62% had pan-uveitis, 23% major vascular disease, and 7% parenchymal CNS disease. 46% were HLA-B51 carriers.
Result(s): Computation of all populations defined by 8 markers (CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki67, HLA-DR) within the CD4⁺ T cell compartment yielded a total of 6,560 cell populations per subject out of which 45 reached significance (p<=0.000001) differentiating 3 groups (BD, non-BD uveitis, and HD). All of these populations comprised sub-types of the human regulatory T (Treg) cell compartment with strong predominance of non-proliferating, non-activated, FoxP3⁺Helios⁺ Treg carrying central-memory phenotypes (CD45RA-, CCR7⁺). 2-group testing of BD vs non-BD revealed 43 distinct cell populations at a significance level of p <=0.002 representing CD25⁺ non-Treg; comparison of BD vs HD uncovered 58 populations at significance level of p <=0.0001 representing FoxP3⁺Helios⁺ subpopulations, and non-BD vs HD identified 61 populations at p<=0.001, comprising CD25⁺CD127⁺/- FoxP3⁺/-, but consistently HELIOS-, presumably non-Treg populations. A separate analysis using 6 marker combinations (CD38, CD39, CD226, TIGIT, CD45RA, CCR7) within the CD3⁺CD4⁺CD8-CD127-CD25⁺compartment which contains most human Treg, showed 48 populations (p <=0.0001) in 3-way comparison (BD, non-BD uveitis, and HD) pointing to high significance of TIGIT and CD226, and 18 populations with differential expression of CD39⁺ between BD and non-BD diseases subjects. TIGIT and CD226 co-expressing Treg (CD127-CD25⁺) subpopulations also reached significance (p<=0.02) in a longitudinal analysis of 7 BD subjects in active vs inactive disease states, as did 56 out of 6,560 populations within total CD4⁺CD3⁺ cells, mostly representing non-Treg cells in active disease.
Conclusion(s): Differential expression of CD4⁺ Treg and non-Treg cells shapes the immune-phenotype of BD vs HD and non-BD autoimmune diseases that have phenotypic overlap with BD (uveitis). Populations within the HELIOS⁺FOXP3⁺compartment of non-activated, non-proliferating Treg had the highest significance when differentiating BD from HD, suggesting relevance of a true Treg phenotype. Non-Treg CD25⁺ cell populations seem more indicative of BD vs non-BD uveitic disease as well as of clinically active BD while populations with high CD39 expression may indicate non-BD states

OBJECTIVE:Apoptotic cell receptors contribute to the induction of tolerance by modulating dendritic cell function following the uptake of apoptotic cells or microparticles. Dendritic cells that have bound or ingested apoptotic cells produce only low amounts of pro-inflammatory cytokines and fail to prime effector T cell responses. Specifically, ligation of the apoptotic cell receptor CR3 (CD11 b/CD18) on human monocyte-derived dendritic cells (moDC) down-modates proinflammatory cytokine secretion, but the consequences for human Th17 cell homeostasis and effector responses remain unknown. Here, we aimed to establish whether CD11b-ligated moDC modulate Th17 cell effector reponses to assess their potential for future use in moDC-based suppressive immunotherapy. METHODS:We generated a bead-based surrogate system to target CD11b on monocyte-derived human dendritic cells and examined the effects of CD11b ligation on Th17-skewing cytokine secretion, priming, expansion and functional plasticity in DC/T cell co-culture systems at the poly- and monoclonal level. RESULTS:T cell compartment was efficiently constricted by targeting the CD11b receptor on moDC. This tolerogenic capacity was primarily dependent on cytokine skewing. Furthermore, ligation of CD11b on healthy homozygous carriers of the rs11143679 (ITGAM) variant - a strong genetic susceptibility marker for human systemic lupus erythematosus - also down-modulated the secretion of Th17-skewing cytokines. CONCLUSION/CONCLUSIONS:Overall, our findings underline the potential of targeted CD11b ligation on human dendritic cells for the engineering of suppressive immunotherapy for Th17-related autoimmune disorders.

Background: Functionality and immune-phenotypes of the human CD4⁺ T-cell compartment in Behcet's disease (BD) are under-investigated, but several lines of evidence point to its relevance in the pathogenesis, progression and remission of the disease. Objectives: We aimed to apply an unbiased single cell approach to assess protein expression levels of phenotypic and functional markers within the human CD4+ T cell compartment in subjects with prototypical BD in order to identify cell populations of potential biologic relevance. Methods: We determined single cell surface/intra nuclear expression levels of CD3, CD4, CD8, CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki67, HLADR, CD38, CD39 in PBMC by flow cytometry and computed the representation of all mathematically possible cell populations within pre-defined starting populations. PBMC from BD subjects (n=13), healthy donors (HD) (n=25) and diseased subjects with non-BD auto-immune uveitis (n=11; VKH, Sarcoidosis, and HLAB27 associated uveitis) were used. BD subjects met ISG criteria and were Arab or Chinese. 62% had pan-uveitis, 23% major vascular disease, and 7% parenchymal CNS disease. 46% were HLA-B51 carriers. Results: Computation of all populations defined by CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki67, HLA-DR within the CD4+ T cell compartment yielded a total of 6560 (38-1) cell populations out of which 45 reached a significance level of p<=0.000001 in ANOVA testing to differentiate 3 groups (BD, non-BD uveitis, and HD). All of these populations comprised sub-types of the human regulatory T (Treg) cell compartment with a strong predominance of non-proliferating, non-activated, FoxP3+HELIOS+ Treg carrying central-memory phenotypes (CD45RA-, CCR7+). Unpaired testing of BD vs non-BD revealed 43 distinct cell populations at a significance level of p<=0.002 representing CD25⁺ non-Treg; comparison of BD vs HD uncovered 58 populations at a significance level of p<=0.0001 representing FoxP3+HELIOS+ subpopulations, and non-BD vs HD identified 61 populations at p<=0.001, comprising CD25⁺CD127+/- FoxP3+/-, but consistently HELIOS-cells. A separate analysis using CD38, CD39, CD227, TIGIT, CD45RA, CCR7 within the CD3+CD4+CD8-CD127-CD25⁺ compartment that contains most human Treg, showed 48 populations (p<=0.0001) in 3-way comparison pointing to high significance of TIGIT and CD226 staining Treg subpopulations, and 18 populations with differential expression of CD39+ between BD and non-BD diseased subjects. TIGIT and CD226 co-expressing Treg (CD127-CD25⁺) subpopulations also reached significance (p<=0.02) in a longitudinal paired analysis of 7 BD subjects in active vs inactive disease states, as did 56 out of 6560 populations within total CD4+ T cells, mostly representing non-Treg cells in active BD. Conclusions: Differential expression of CD4+ Treg and non-Treg cells shapes the immune-phenotype of BD vs states of health and non-BD autoimmune diseases that have phenotypic overlap with BD (uveitis). The populations with the highest significance for differentiating BD from healthy states seem to exist within the HELIOS+FOXP3+ compartment of non-activated, non-proliferating Treg, suggesting relevance of a true Treg phenoptype. This is likely not specific to BD. Non-Treg CD25⁺ cell populations seem more indicative of BD vs non-BD uveitic disease as well as of clinically active BD while populations with high CD39 expression may signify non-BD states of immune-mediated disease

A 46-year-old Hispanic man presented with fever, genital ulcers, left eye redness and chest pain. Physical examination was notable for a healed oral ulcer and scrotal ulcers, and bilateral superficial thrombophlebitis. He was found to have new-onset pancytopenia. CT of the chest showed pericardial and pleural effusions and rapidly progressing inflammation of the aortic arch and ascending vessels. Although the patient had Behcet's disease (BD)-like symptoms, pancytopenia could not be explained by the diagnosis, prompting a bone marrow biopsy which showed myelodysplastic syndrome. This report highlights the importance of excluding alternate disorders before making a diagnosis of Behcet's disease if atypical, BD-incompatible or incomplete constellations of symptoms and findings are present.

Introduction. Functionality and immune-phenotypes of the human CD4+ T-cell compartment in Behcet's disease (BD) are under-investigated, but several lines of evidence point to its relevance in the pathogenesis and progression of the disease. Aims. We aimed to apply an unbiased single cell approach to assess protein expression levels of phenotypic and functional markers within the human CD4⁺ T cell compartment in subjects with prototypical BD in order to identify cell populations of potential pathobiological relevance. Methods. We determined single cell expression levels of CD3, CD4, CD8, CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki76, HLA-DR, CD38, CD39 in PBMC by flow cytometry and computed the representation of all mathematically possible cell populations within pre-defined starting populations (i.e., CD3⁺CD4⁺CD8-; CD3⁺CD4⁺CD8-CD127-CD25⁺). PBMC from BD patients (n=13), healthy donors (HD) (n=25) and diseased subjects with non-BD auto-immune uveitis (n=11, VKH, Sarcoidosis, and HLA-B27 associated uveitis) were used. BD subjects met ISG criteria and were Arab or Chinese. 62% had pan-uveitis, 23% major vascular disease, and 7% parenchymal CNS disease. 46% were HLA-B51 carriers. Results. Computation of all populations defined by 8 markers (CD127, CD25, CD45RA, CCR7, FoxP3, HELIOS, Ki67, HLA-DR) within the CD3⁺CD4⁺CD8-(=CD4⁺ T cell) compartment yielded a total of 6560 (3'8-1) cell populations per subject out of which 45 populations reached a significance level of p<=0.000001 in ANOVA testing to differentiate 3 groups (BD, non-BD uveitis, and HD). All of these populations comprised sub-types of the human regulatory T (Treg) cell compartment with strong predominance of non-proliferating, non-activated, FoxP3⁺Helios⁺ Treg carrying central-memory phenotypes (CD45RA-, CCR7⁺). 2-group testing of BD vs non-BD revealed 43 distinct cell populations at a significance level of p<=0.002 representing CD25⁺ non-Treg; comparison of BD vs HD uncovered 58 populations at significance level of p<=0.0001 representing FoxP3⁺Helios⁺ subpopulations, and non-BD vs HD identified 61 populations at p<=0.001, comprising CD25⁺CD127⁺/-FoxP3⁺/-, but consistently HELIOS-, presumably non-Treg populations. A separate analysis using 6 marker combinations (CD38, CD39, CD226, TIGIT, CD45RA, CCR7) within the CD3⁺CD4⁺CD8-CD127-CD25⁺ compartment which contains most human Treg, showed 48 populations (p<=0.0001) in 3-way comparison (BD, non-BD uveitis, and HD) pointing to high significance of TIGIT and CD226 staining Treg subpopulations, and 18 populations with differential expression of CD39⁺ between BD and non-BD diseases subjects. TIGIT and CD226 co-expressing Treg (CD127-CD25⁺) subpopulations also reached significance (p<=0.02) in a longitudinal analysis of 7 BD subjects in active vs inactive disease states, as did 56 out of 6560 populations within total CD4⁺CD3⁺ cells, mostly representing non-Treg cells in active disease. Conclusion. Differential expression of CD4⁺ Treg and non-Treg cells shapes the immune-phenotype of BD in comparison to healthy and non-BD autoimmune diseases that have phenotypic overlap with BD (uveitis). The populations with the highest significance for differentiating BD from healthy states seem to exist within the HELIOS+⁺FOXP3+⁺compartment of non-activated, non-proliferating Treg, suggesting relevance of a true Treg phenotype. Non-Treg CD25⁺ cell populations seem more indicative of BD vs non-BD uveitic disease as well as of clinically active BD while populations with high CD39 expression may indicate non-BD states