Differential expression of CD39 on CD4+ T cell subsets. PMBC from healthy donors were surface stained for CD4, CD3, CD25, CD127 and CD39 followed by intracellular staining for FoxP3. (A) Representative dot plots showing expression of CD39 as assessed by flow cytometry on live CD3 gated lymphocytes. The frequency (%) of CD4 T cells gated is shown according to their dual expression of CD39 and FOXP3. (B) Two distinct CD39-expressing CD4 T cell subsets are identified and further delineated by CD25 and CD39 expression: CD39+FOXP3+CD25hi/+(green) and CD39+FoxP3-CD25- T cells (blue). (C) FoxP3+CD25+ gated CD4 T cells (green) were further characterized by CD25 and CD39 expression. (D) Plot shows the frequency of CD4+ T cell subsets defined by CD39 and CD25 in four representative healthy donors. (E) Stability of CD39+CD25- and CD39+CD25+ T cells as a percentage of total CD4 T cells among 4 representative healthy donors at 2–3 time points approximately 4–6 months apart. At least 200,000 lymphocyte events were counted and collected for each sample.

Differential function of CD39 expressing CD4+ T cells. Purified CD4+ T cell subsets were isolated from PBMC from healthy donors by cell sorting to a purity >95% as demonstrated in Fig 1A. (A, B) Representative plots shows 50,000 CFSE-labeled CD39-CD25- responder (T_Res) T cells co-cultured in duplicate wells together with an equal number of CD39+CD25- (T_Ind), CD39+CD25+ (T_Sup) or unlabeled control CD39-CD25- (T_Res) T cells for 4–5 days in the presence of soluble anti-CD3/anti-CD28. Plots show proliferation of responder T_Res cells as determined by CFSE dilution. (C) Similar results were obtained in six separate experiments from six different donors. The statistical difference was deemed significant using a Mann Whitney U test analysis if p<0.05. (D, E) In some experiments T_Res cells were co-cultured at different ratios with T_Ind cells or T_Sup cells or a combination of both in the absence or presence of IL-2. Data in D, E are of a representative donor from two separate experiments with two different donors. (F) Graph shows the co-culture of T_res cells with supernatants derived from 4 day stimulated (anti-CD3/anti-CD28 mAb mixture) individual CD4+ T cells subsets from a representation donor. Similar results were observed in three separate experiments from three different donors.

Detection of human cytokines in cell supernatants in anti-CD3/anti-CD28 stimulated (A) co-cultures of responder T_Res cells with either 1= CD39+CD25+ (T_Sup1); 2= CD39-CD25+ (T_Sup2), 3= CD39+CD25- (T_Ind); 4= CD39-CD25- (T_Res); or 5= unstimulated CD39-CD25- (T_Res) T cells. Data is presented from a single donor with background subtracted and is a representation of three separate experiments (from three separate donors). Standard curves were performed for each biomarker using the mixed standards provided with the kit. (B) Cell sorted, purified individual CD4+ T cell subsets alone from three donors is presented. Data is the average of duplicate wells. (C) Specificity of a purified anti-human CD39 (clone BU61) mAb. PBMC from a healthy donor were incubated with either isotype control IgG antibody (blue line) or purified anti-human CD39 (clone BU61) mAb (red line) prior to staining with a conjugated anti-CD39 mAb (clone eBioA1) antibody. (D) CFSE labeled CD39-CD25- responder (T_Res) T cells co-cultured in duplicate wells together with an equal number of indicated CD4 T cell subsets in the presence of either purified anti-CD39 (clone BU61) mAb or isotype IgG control. Cells were stimulated with anti-CD3/CD28 antibody mixture and cultured for 4 days before assessment of CFSE dilution. Data is a representation of two separate experiments from two different donors. (E) Plot shows expression of CD39 and CD25 by purified cell sorted CD39-CD25- responder (T_Res) obtained from a representative healthy donor following anti-CD3/anti-CD28 stimulation for 12 hours. Similar results were obtained in 4 day cultures.