Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: Human T cell lines used in this study.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Generated, Cell Culture

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: Continuous TGF-β exposure augmented CD25 expression and enabled superior IL-2–driven growth of long-term FOXP3 ± T cell cultures. (A, B) CD4 + T cells were purified from PBMCs and were activated for 3 d with PHA and 1 nM IL-2 in the presence or absence of 10 nM TGF-β to initiate two long-term lines. TGF-β–cultured T cells and control T cells were then passaged every 3 to 7 d in 1 nM IL-2 with or without 10 nM TGF-β respectively for a total of 38 d. Cells were stained with anti-CD3 PerCP-Cy5.5 (HIT3a), anti-CD4 BV421 (OKT4), anti-CD8 AF700 (SK1), anti-CD25 PE (M-A251), and anti-HLA-DR BUV395 (G46-6). Histograms (A) and bar charts (B) show flow cytometric analyses of cell size (forward scatter) together with CD3, CD4, CD25, and HLA-DR expression (n = 2). (C) Nonfractionated PBMCs (left) and purified CD4 + PBMC T cells (right) were activated and maintained as in panel A for a total of 20 d. To assess IL-2 responsiveness in proliferative assays, TGF-β–cultured T cells and control T cells were incubated with 10 pM–1 nM IL-2 (x-axis) with or without TGF-β in flat-bottomed 96-well plates. Cells were pulsed with [ 3 H]thymidine during the last 24 h of a 72-h culture (counts per minute [CPM]). (D) Independently derived T cell lines were initiated by a 3-d stimulation with PHA (rows 1, 2, 3, and 5) or without mitogen (autologous, row 4). T cells were cultured with 1 nM IL-2 in the presence or absence of 10 nM TGF-β (as designated) from day 0 until the day of analysis (day 12, 26, 38, or 47). Cells were stained with anti-FOXP3 clone PCH101 Alexa Fluor 488. (E) A nonfractionated T cell line was initiated as in panel A followed by continuous propagation in IL-2 and TGF-β for 19 d. Cells were gated on CD3 + and stained with the anti-FOXP3 clone 150D Alexa Fluor 488. Lines used in these experiments were CL2 (A, B), CL1 (C), NL1 (D, rows 1 and 2), CL1 (D, row 3), CL4 (D, row 4), CL2 (D, row 5), and P2 (E). * P < 0.05, ** P < 0.01. FMO, fluorescence minus one; FSC-A, forward scatter area; ns, not significant.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Expressing, Purification, Cell Culture, Control, Staining, Incubation, 96 Well Plates, Derivative Assay, Analysis, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: Continual TGF-β exposure supported stable IL-2–dependent expansion of FOXP3 ± T cells. (A–C) PBMCs from 3 donors (P1, P2, and P3) were activated for 3 d with PHA and IL-2 in the presence or absence of 10 nM TGF-β. The respective cell lines were then propagated in IL-2 plus TGF-β versus IL-2 alone per the original culture conditions. Mitogens were not included after the first culture. Cells were passaged at equivalent densities of approximately 100,000 cells/mL. Day 0 represents purification and initiation of PBMC cultures, and each data point represents a cell count and a passage. Cell counts were recorded at each passage until the end of the experiment (day 80) or until cultures were lost due to cell death (A). The calculated expansion from a theoretical single cell (y-axis) is displayed against the number of days in culture (x-axis). The fold expansion for each passage was multiplied across all passages to calculate the total cell expansion from a theoretical single cell. Histograms show flow cytometric analysis on days 11 or 18 (B) and day 65 (C) after staining cells with CD3 NovaFluor Blue 610-70S (SK7), CD4 BV421 (OKT4), CD8 AF700 (SK1), CD25 APC-Fire750 (BC96), CD69 PE (FN50), CD80 BV711 (2D10), CD86 BV650 (FUN-1), CTLA4 AF647 (BN13), PDL2 PE-Cy7 (MIH18), PD1 PE-Cy5 (EH12.2H7), HLA-DR BUV395 (G46-6), and FOXP3 AF488 (PCH101). (D) Nonfractionated PBMCs from a healthy volunteer were seeded at 50,000 cells/well in LymphoOne medium in a flat-bottomed 96-well plate (n = 4) and cultured with 10 nM IL-2, 1 nM IL-4, and 1 nM GM-CSF with or without 10 nM TGF-β. Cells were passaged with new media and cytokines after approximately 7 d. Cell counts were initiated once growth was visually evident, and lines were passaged into T12.5 or T25 flasks for the remainder of experiment with 1 nM IL-2 with or without 10 nM TGF-β. IL-4 and GM-CSF were discontinued after 2 wk of culture. T cell expansion calculations were performed as in panel A. (E) The surviving T-APC line at day 40 in panel D is the same line portrayed in panel E (red diamonds). This culture exhibited sustained exponential growth through day 40 and was subsequently propagated through day 120 for a total 10 22 -fold expansion. (F) CD3 + CD4 + JL3.C2 T-APCs and human PBMCs (frozen stocks from a healthy human volunteer) were stained with CD3 BV510 (SK7), CD4 BV421 (OKT4), CD8 SB574 (SK1), CD19 StarBright UV795 (HIB19), CD25 APC-Fire 750 (BC96), FOXP3 Alexa Fluor 488 (150D), and BCL6 Alexa Fluor 647 (IG191E/A8). T-APCs were compared with CD3 + CD4 + and CD3 − CD19 + gated T and B cell PBMC subsets, respectively. (G, H) The phenotypes of a control CD4-purified T-APC line (day 33) and the CD8 + T-APC line from panel E (day 83) were assessed with CD3 NovaFluor Blue 610-70S (SK7), CD4 BV421 (OKT4), CD8 AF700 (SK1), CD25 APC-Fire750 (BC96), HLA-A2 NovaFluor Yellow 730 (BB7.2), FOXP3 AF488 (150D), BCL6 AF647 (K112-91), FASL BUV615 (NOK-1), FAS BUV737 (DX2), CD86 BV650 (FUN-1), CXCR5 BV605 (MU5UBEE), CD69 PE (FN50), and PD1 PE-Cy5 (EH12.2H7). Lines used in these experiments were P1, P2, and P3 (A–C), JL3.C2 (F), V1 (E, H), and V3 (G). FMO, fluorescence minus one; FSC-A, forward scatter area.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Purification, Cell Characterization, Single Cell, Analysis, Staining, Cell Culture, Control, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: A neutralizing anti-CD4 mAb inhibited autologous MHCII-restricted autoantigen recognition and IL-2–dependent growth of CD4 ± T-APCs. (A–G) Distinct CD4 + purified (A, D–G) and CD8 + purified (B) T-APC lines were initiated by a 3-d stimulation with PHA in the presence of 1 nM IL-2 and 10 nM TGF-β. Cells were passaged every 3 to 7 d in the presence of TGF-β and IL-2 for a total of 22 d (A), 80 d (B), 10 to 15 d (D-E), 78 d (F), 23 d (G, left two panels), or 36 d (G, right two panels). Human T cells (A, B, D–G) (50,000 cells/well, unless designated otherwise) or mouse SJL-PLP.1 T cells (C) (10,000 cells/well) were cultured with 10 pM, 100 pM, or 1 nM IL-2 (x-axis) in the presence or absence of anti-CD4 mAbs (OKT4 or RPA-T4) or isotype controls (MPC-11 or MOPC-21) in the presence or absence of 10 nM TGF-β. mAb concentrations were 20 μg/mL unless designated as 10 μg/mL. Cultures were pulsed with [ 3 H]thymidine during the last 24 h of a 5-d or 6-d culture unless designated otherwise. T-APC lines used in these experiments were CL5 (A, D), P2 (B), murine SJL-PLP.1 (C), CL6 (E, F), or CL7 (G). Two-way analysis of variance with a Tukey’s multiple comparisons test was used to assess significance. *** P < 0.001, and **** P < 0.0001 for comparisons of the designated mean value(s) at the same IL-2 concentration. CPM, counts per minute; ns, not significant.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Purification, Cell Culture, Analysis, Concentration Assay

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: The anti-CD4 mAb RPA-T4 promoted a distinct CD25 low subset that had a diminished T-APC signature. A CD4 + purified T cell line was initiated by a 3-d stimulation with PHA in the presence of 1 nM IL-2 and 10 nM TGF-β. These T cells were passaged in the presence of TGF-β and IL-2 for a total of 53 d (A–F) or 100 d (G). During the last 2 wk, T cells were cultured in the presence of 20 μg/mL RPA-T4 mAb or the isotype control mAb, in the presence of 1 nM IL-2 and 10 nM TGF-β. Cells were stained with CD3 PerCP-Cy5.5 (HIT3a), CD4 BV421 (OKT4), CD8 AF700 (SK1), CD25 PE (M-A251), and FOXP3 AF488 (150D) (A–F), and/or antibodies against HLA-DR, DP, DQ SparkBlue574 (Tu39), CD86 BV650 (FUN-1), FAS BUV737 (DX2), CTLA4 AF647 (BN13), GITR BV711 (108-17), CXCR5 BV605 (MU5UBEE), PD1 PE-Cy5 (EH12.2H7), CD69 PE (FN50), CD25 APC-Fire750 (BC96), and LAG3 BUV563 (3DS223H). (A, B) Representative FOXP3 × CD25 dotplots are shown with the respective CD25 histograms. (C) Bar charts show mean forward scatter MFI values (left) and CD25 high percentages (right). (D–F) FOXP3 histograms of CD4-gated and CD25 high /CD25 low gated subsets are shown for RPA-T4–treated versus control T cell cultures together with the respective FOXP3 MFI values. (G) Cultures treated with 20 μg/mL RPA-T4 or isotype control mAbs were also compared for other markers in CD25 high versus CD25 low subsets. CD4 + purified T cells from the P3 line (A–F) or the CL1 line (G) were used in these experiments. Analyses were 2-tailed unpaired t tests (C) or 2-way analysis of variance with Tukey’s multiple comparisons tests (F, G). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. FSC, forward scatter; ns, not significant.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Purification, Cell Culture, Control, Staining, Analysis

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: T-APC lines presented agonistic self-antigens and elicited polyclonal Tcon responses while seeding new T-APC lines. (A) JL3 Tcons were derived from freshly isolated PBMCs that were activated with Con-A and 1 nM IL-2 for 4 to 5 d. Tcons were passaged in 1 nM IL-2 in cRPMI media every 4 to 7 d for an additional 29 d. JL2.C5 T-APCs were derived in the presence of GM-CSF, IL-4, IL-2, and TGF-β (1 nM each). JL2.C5 cells were propagated in IL-2 and TGF-β for a total of 92 d (total expansion 10 7.4 -fold). Cells were stained with CD3 BV510 (SK7), CD4 BUV496 (SK3), CD8 Spark Blue 574 (SK1), CD25 PE-Fire 700 (MA251), HLA-DR Spark PLUS UV395 (L243), and CD40L PE (24-31). (B) JL2.C5 T-APCs (day 102) and JL3 Tcons (day 41 postactivation) were cultured for 24 h in the presence or absence of the anti-MHCII mAb L243 or the isotype control (10 μg/mL). (C) Similarly, JL2.C5 T-APCs (day 136) were cultured for 24 h in the presence or absence of anti-MHCII L243, anti-CD40L 5C8, or the isotype control (10 μg/mL). (D, E) Shown are the growth curves for the JL2.C5 T-APCs and the JL3 Tcon lines. (F) Designated densities (x-axis) of senescent nonexpanding JL2.C5 T-APCs (day 129) were added to 96-well plates with JL3 Tcons (day 16, 10 5 /well) in LymphoOne and 1 nM IL-2. Cells were passaged into 24-well plates on day 11. JL3 Tcon expansion was enumerated on day 18 with CountBright Absolute Counting Beads. (G) Cultures derived from panel F were expanded to T25 flasks, and after 2 to 4 wk 3 of these 18 cultures exhibited blastogenic T cell outgrowth. The initiation of the JL3 Tcon/JL2.C5 T-APC coculture (F) was designated as day 0 for initiation of the JL3.B1, JL3.C2, and JL3.T1 lines. (H) To assess whether the JL3.C2 T cells exhibited T-APC activity, JL3 Tcons (10 5 /well, day 39 out of activation) were cultured with designated densities (x-axis) of irradiated JL3.C2 T-APCs (5 Gy) for 5 d in the absence of exogenous mitogen or antigen. Cultures were pulsed with [ 3 H]thymidine during the last 48 h of culture. (I) T-APC lines CL2.2G4 and JL2.C6 and JL3 Tcon lines were compared with the nascent JL3.C2 T-APC line (day 32) for CD40L expression. (J) JL3.C2 T-APCs were cultured without exogenous TGF-β through day 58 and then were cultured with or without 1 nM TGF-β for 4 d. Cells were then subjected to a 10-day passage in duplicate cultures with 1 nM IL-2 in the presence or absence of 1 nM TGF-β without and without 10% heat-inactivated FBS, with manual counts on days 1, 3, 5, and 7 to 11. Cultures were supplemented with 1 nM IL-2 on day 5. (K) CD4 + JL3.C2 (day 70), CD8 + P3 T-APCs (day 14), murine BW5147α/β − cells, or SeAx cells were cultured in triplicate in a 2-d proliferative assay ([ 3 H]thymidine pulse during the last 24 h) with 1 nM IL-2 in the presence of designated titrations of heat-inactivated FBS. (B, C, I) Data were assessed with 1-way analysis of variance with Sidak’s or Dunnett’s multiple comparison test, respectively. (F, H, J, K) Data were assessed with a 2-way analysis of variance with Sidak’s multiple comparison test. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Derivative Assay, Isolation, Staining, Cell Culture, Control, 96 Well Plates, Counting, Activity Assay, Activation Assay, Irradiation, Expressing, Analysis, Comparison

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: Tcon-derived JL3.C2 T-APCs expressed a professional APC phenotype. (A, B) The Tcon-derived JL3.C2 and JL3.B1 T-APC lines were compared with established T-APC lines (JL2.C5 and JL2.C6) and to the parent Tcon line (JL3 Tcon6). (A) Cells were stained with TCRα/β PE/Cy7 (IP26), CD3 BV785 (SK7), CD4 BV421 (OKT4), CD8 Alexa Fluor 700 (SK1), CD28 BV605 (CD28.2), CD25 FITC (M-A251), CD74 PE (LN2), CD80 BV711 (2D10), CD86 BV650 (FUN-1), CD123 PerCP/Fire806 (S18016C), OX40/CD134 PE-Cy5 (ACT35), and HLA-DR Spark PLUS UV395 (L243). (B) Mean MFI values are shown for CD4 and CD8 gated cell lines. Statistical analyses were restricted to CD4 gated values and were assessed by 1-way analysis of variance with Tukey’s multiple comparison test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. (C, D) JL3.C2 T-APCs and PBMCs (day 0, no culture) were stained with TCRα/β PE/Cy7 (IP26), CD3 BV510 (SK7), CD4 BV421 (OKT4), CD8 Spark Blue 574 (SK1), CD11b StarBright UV575 (M1/70), CD11c BV785 (Bu15), CD14 PE/Fire 700 (S18004B), CD19 StarBright UV795 (HIB19), CD25 APC/Fire 750 (BC96), CD74 PE (LN2), CD80 BV711 (2D10), CD86 BV650 (IT2.2), CD278 BV605 (C398.4A), and HLA-DR Spark Plus UV395 (L243). The same flow gain settings were used for comparison of JL2.C2 T-APCs and PBMCs. ns, not significant.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Derivative Assay, Staining, Analysis, Comparison

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: IL-2 and TGF-β are required for respective short- and long-term viability of continuous T-APC lines. A JL3.C2 T-APC subline was thawed on day 31, and parallel cultures were initiated on day 39 with or without 1 nM TGF-β. On day 78, the effects of IL-2 and/or TGF-β withdrawal were assessed via a time course experiment. (A–E) JL3.C2 TGF-β–cultured T-APCs were cultured with or without IL-2 in the presence or absence of 1 nM TGF-β from days 0 to 14 (n = 3). Passages were performed on days 3, 6, and 10. At each time point, cells were stained with CD3 BV510 (SK7), CD4 BV421 (OKT4), CD25 PE-Fire 700 (M-A251), CD86 BV650 (FUN-1), FAS PerCP-Fire806 (DX2), HLA-DR Spark Plus UV396 (L243), and FOXP3 Alexa Fluor 488 (150D). Dotplots (A, D) show representative positive control cultures (with IL-2 and TGF-β) in the top rows versus cytokine-deprived cultures in the bottom rows (CD3 + CD4 + gate). Quadrants depict reference comparators for positive controls versus cytokine-deprived groups, whereas all populations were positive compared with unstained negative controls. (B) IL-2 withdrawal for all groups was assessed on day 6 via a 1-way analysis of variance with Sidak’s multiple comparison test. (C) Mean values on days 1, 3, and 6 were compared by 2-way analysis of variance with a Tukey’s multiple comparison test. (E) TGF-β withdrawal was assessed for the remaining IL-2–supplemented groups on days 10 and 14 with a 1-way analysis of variance with Sidak’s multiple comparison test. (F) Shown are growth curves for JL3.C2 T-APCs and JW4 T-APCs that were cultured with and without TGF-β. ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Cell Culture, Staining, Positive Control, Analysis, Comparison

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: T-APCs encompassed distinct subsets marked by differential expression of CD25, CD69, and PD1. CD4 + T cells were purified from PBMCs and were activated for 3 d with PHA and IL-2 in the presence of 10 nM TGF-β. TGF-β–cultured T cells were then passaged every 3 to 7 d in IL-2 with TGF-β for 73 d. Cells were washed and cultured in triplicate at 500,000 cells/well in 24-well plates with 100 pM, 1 nM, 10 nM, or 100 nM IL-2 (x-axis) in the presence or absence of 10 nM TGF-β for 7 d. Cells were stained with CD3 NovaFluor Blue 610-70S (SK7), CD4 BV421 (OKT4), CD8 AF700 (SK1), CD25 APC-Fire750 (BC96), CD69 PE (FN50), and PD1 PE-Cy5 (EH12.2H7). These T cells were >99% CD4 + and lacked detectable CD8 + T cells. (A, B) CD3 + CD4 + gated T cells were analyzed in CD25 × CD69 dotplots (A), and the respective frequencies of CD25 low CD69 high T cells (upper left quadrant) are portrayed in the associated line graph (B). (C, D) CD3 + CD4 + –gated T cells were analyzed in PD1 × CD69 dotplots (C), and the respective frequencies of PD1 high CD69 high T cells (upper right quadrant) are portrayed in the associated line graph (D). (E) Histograms of the combined top two quadrants or the combined bottom two quadrants from panels A and C show bimodality of CD25 and PD1 expression as a function of IL-2/TGF-β concentrations and CD69 expression. (F, G) After the 7-d culture with 100 nM IL-2 and 10 nM TGF-β, percentages of CD25 low CD69 high versus CD25 high CD69 low subsets (F) and percentages of PD1 high subsets (G) are shown in the respective right-hand line graphs. T-APCs from the CL6 line were used in these experiments. (B, D, F, G) A 2-way analysis of variance with a Tukey’s multiple comparisons test was used to assess differences between groups. ** P < 0.01, *** P < 0.001, **** P < 0.0001. FSC-H, forward scatter height.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Quantitative Proteomics, Purification, Cell Culture, Staining, Expressing, Analysis

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: High concentrations of IL-2 and TGF-β caused dominance of CD25 low T-APCs even though CD25 high T-APCs maintained an enhanced IL-2 signaling signature. CD4 + T cells were purified from PBMCs and were activated for 3 d with PHA and IL-2 in the presence of 10 nM TGF-β. Cells were then passaged every 3 to 7 d in IL-2 and TGF-β for a total of 53 d. T cells were washed and cultured in triplicate at 500,000 cells/well in 24-well plates with 10 pM, 100 pM, 1 nM, and 10 nM IL-2 in the presence or absence of 10 nM TGF-β for 7 d. Cells were stained (via multiple panels) with CD3 NovaFluor Blue-610 (SK7), CD4 BV421 (OKT4), CD8 AF700 (SK1), CD25 APC-Fire750 (BC96), CD25 PE (MA251), CD28 BV510 (CD28.2), CD69 PE (FN50), CD80 B711 (2D10), CD86 BV650 (FUN-1), CD95/FAS BUV615 (DX2), CD127 PE-Cy5 (A019D5), CTLA4 AF647 (BN13), CD178/FASL BUV737 (NOK-1), CD273/PDL2 PE-Cy7 (MIH18), CD279/PD1 PE-Cy5 (EH12.2H7), GITR BV711 (108-17), FOXP3 AF488 (PCH101), HLA-DR BUV395 (G46-6), GARP PE-Cy7 (7B11), and LAG-3 BUV563 (3DS223H). T cells were >99% CD4 + and lacked detectable CD8 + T cells. (A) Percentages of CD25 high and CD25 low subsets and the respective MFI values of markers (B–T) are portrayed as a function of IL-2 concentration. Note the use of different y-axis scales. (U) CD3 + CD4 + gated T cells were analyzed in CD25 × forward scatter area (FSC-A) plots to distinguish CD25 low versus CD25 high subsets. T cells from the CL7 line were used in these experiments. A 2-way analysis of variance with a Tukey’s multiple comparisons test was used to assess differences between groups. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. FSC-A, forward scatter area.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Purification, Cell Culture, Staining, Concentration Assay, Analysis

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: T-APC coculture with syngeneic polyclonal Tcons caused an initial activation followed by fratricidal collapse. (A–D) The JL3.C2 T-APC line (day 43, 10 4 /well) and JL3 Tcons (day 39 of rest, 10 5 /well) were cultured alone or together in 96-well plates with 1 nM IL-2 (n = 3). On day 5, cells were passaged to a 24-well plate with 1 nM IL-2. On day 9, cells were subjected to flow cytometric analysis of CD3 BV785 (SK7), CD4 BV421 (OKT4), CD8 Alexa Fluor 700 (SK1), CD25 FITC (M-A251), CD28 BV605 (CD28.2), CD74 PE (LN2), CD86 BV650 (FUN-1), CD134 PE-Cy5 (ACT35), and HLA-DR Spark Plus UV395 (L243). (A) Shown are representative dotplots of the Tcon-derived CD4 + JL3.T1 and JL3.C2 T-APC lines, the combination culture (JL3.C2 T-APCs + JL3 Tcons), and Tcons alone. (B) Mean MFI values are shown for CD4, DN, and CD8 gated cells. Statistical analyses were restricted to comparison of “Both” (i.e. JL3.C2/Tcon cocultures) versus Tcons alone to assess coculture-induced changes in the Tcon phenotype. (C) Cell yields (day 9) were assessed with CountBright Absolute Counting Beads to calculate cell/bead ratios. (D) Manual counting was also used to compare cell yields (day 9) relative to input values for T-APCs (10 4 /well) and Tcons (10 5 /well), given that DN and CD8 + single-positive Tcons comprised approximately 40% of cells by day 9. (E) Total cell yields based on manual cell counting were assessed on days 16, 20, and 24. (F) JL3.C2 T-APCs were cultured without TGF-β for 58 d and then split into parallel cultures with and without 1 nM TGF-β for 3 d. Cells were then tested in a 2-d proliferative assay ([ 3 H]thymidine pulse during the last 24 h) in the presence of 1 nM IL-2 at 10 5 /well (200 µL in LymphoOne) in the presence or absence of 10 µg/mL functional grade anti-CD95 IgM mAb (EOS9.1). (G) CD4 + JL3.C2 T-APCs, CD8 + P3 T-APCs, SeAx T cells, and BW5147α/β − T cells tested in a proliferative assay as described in panel F at 50,000 cells/well with designated dilutions of the EOS9.1 mAb. (E) Data were assessed with an unpaired 2-tailed t test. (C–F) Data were assessed with 1-way analysis of variance with a Tukey’s multiple comparisons test. (B, G) Data were assessed with a 2-way analysis of variance with Sidak’s or Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. CPM, counts per minute. Flow cytometric analyses showed that T-APCs activated resting conventional responder T cells, which subsequently acquired a T-APC phenotype. The activated responder T cells then eliminated the original T-APCs and later exhibited mutual killing, ultimately causing collapse of the culture. Additional experiments showed that T-APCs were highly susceptible to FAS-mediated cell death following treatment with an agonistic cross-linking anti-FAS IgM antibody.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: Activation Assay, Cell Culture, 96 Well Plates, Analysis, Derivative Assay, Comparison, Counting, Cell Counting, Functional Assay

Journal: The Journal of Immunology Author Choice

Article Title: A human self-stimulatory CD4 + T cell antigen-presenting cell subset presents and recognizes agonistic self-antigens to sustain exponential autologous clonal expansion

doi: 10.1093/jimmun/vkag274

Figure Lengend Snippet: Phenotypic relationships of T-APCs with professional APCs, Tregs, and Tfrs.

Article Snippet: PBMCs were cultured as nonfractionated populations or as purified CD4 + and CD8 + T cell subsets magnetically sorted by REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec #130-117-037 or #130-117-036).

Techniques: