Supplementary Data. Treg phenotype

Transcription

1 Supplementary Data Additional Experiment An additional experiment was performed using cryopreserved peripheral blood mononuclear cells (PBMC) derived from five renal cell carcinoma (RCC) patients [see Lamers et al. (2013a); and following approval by the local medical ethical review board] in order to (1) provide extra information on effect of Treg status (in patients) on outgrowthoftcellswithtregphenotypeinrelationtotcell activation method, and (2) to directly compare activation by scd3 + CD28 mab, with MACSibead CD3/28 and Dynabead CD3/28. PBMC were activated using soluble CD3 + CD28 mabs, MACSi CD3/28 beads, and Dynal CD3/28 beads (see Table 1) according to supplier s instructions. T cell culturesweretransducedwiththemc2tcrandexpandedin IL-2-supplemented culture medium till culture day 15 and evaluated as described in Materials and Methods. Treg phenotype For the healthy donor PBMC used in the main study, the proportion of Treg (CD25 ++CD127 low within CD4 T cells) was 4.5% (median value, range %; n = 12); for an RCC patient cohort previously described in Lamers et al. (2013a), the proportions of Treg were significantly higher, that is, 8.3% (median value, range ; n = 11); t-test p-value In PBMC from five of these patients (median proportion of Treg 10.0%; range %), Treg phenotype was evaluated following activation, transduction, and expansion. At culture day 15, the proportions of CD4 T cells with a Treg phenotype had significantly increased, but the proportions of Treg prior to activation showed an equal relative outgrowth following activation within each activation condition; see Supplementary Fig. S1. Yet the proportion of Treg was significantly lower following activation using soluble CD3 + CD28 mabs (median 21.0%; range Supplementary Table S1. Flow Cytometry Stainings and Antibodies Antibody Fluorochrome Clone Company Tube 1: MC2/A2 TCR expression antitcr-vb2 FITC MPB 2D5 Immunotech MC2/A2 pmhc Biotin palkvdveerv Sanquin Streptavidin PE BD Biosciences CD3 PERCP SK7 BD Biosciences CD8 APC SK1 BD Biosciences Tube 2: CAIX CAR expression NuH82 (IgG1) Unlabeled Purified from culture sup In-house validated GaM IgG1 PE Southern Biotech Tube 3: major lymphocyte subsets CD3 FITC SK7 BD Biosciences TCRgd FITC 11F2 BD Biosciences CD56 PE C5.9 Dako Cytomation CD45 PerCP 2D1 BD Biosciences Tube 4: major T lymphocyte subsets/t-regs CD3 APC SK7 BD Biosciences CD127 PE hil-7r-m21 BD Biosciences CD4 PerCP SK3 BD Biosciences CD25 APC 2A3 BD Biosciences CD8 APC-Cy7 SK1 BD Biosciences Tube 5: T lymphocyte differentiation (1) CD3 FITC SK7 BD Biosciences CCR7 PE R&D Systems CD8 PerCP SK1 BD Biosciences CD45RA PE-Cy7 L48 BD Biosciences CD28 APC CD28.2 BD Biosciences CD27 APC-H7 M-T271 BD Biosciences Tube 6: T lymphocyte differentiation (2) CD3 APC SK7 BD Biosciences CD8 PerCP SK1 BD Biosciences CD45RA FITC L48 BD Biosciences CD27 APC-Cy7 M-T271 BD Biosciences CD45RO PE-Cy7 UCHL-1 BD Biosciences CD62L PE SK11 BD Biosciences

2 %) versus both bead activations, that is, 38.3% (median; range %) for MACSi CD3/28 beads (ttest p-value ) and 33.8% (mean; range %) for Dynal CD3/28 beads (t-test p-value 0.009). Extension activation methods, including Dynal CD3/CD28 beads Results from this experiment using patient-derived PBMC confirmed previous results in PBMC from healthy donors with regard to levels of measurements and activation by soluble CD3 + CD28 mabs outperforming MACSi CD3/ 28 bead activation on tested parameters. Activation by Dynabead CD3/28 performed differently from MACSIbead CD3/28. PBMC activation by Dynabead CD3/28 versus scd3 + CD28 mabs induced a more vigorous T cell proliferation; however, this feature went along with a lower transduction efficiency and hence lower transgene-mediated effector functions; see Supplementary Fig. S2. Regarding T cell phenotype (at culture day 15) Dynabead CD3/28 versus scd3 + CD28 mab activation resulted in similar proportions of general T cell subsets (CD4, CD8, TCRgd, CD25, CD127), slightly lower proportions of NK and CD56 + T cells and higher proportions of CD4 T cells with Treg phenotype and higher levels of expression of CD45RA, CD27, and CD28 (not of CCR7) on CD8 T cells; hence, the proportions of the T CM T cells were higher and the T LE T cells lower; that is, resulting is less terminally differentiated T cells; see Supplementary Fig. S3. SUPPLEMENTARY FIG. S1. Activation condition but not proportion of Treg prior to activation determines outgrowth of T cells with Treg phenotype. PBMC derived from five RCC patients were activated using soluble CD3 + CD28 mabs (scd3-28), MACSi CD3/28 beads (MbCD3-28), and Dynal CD3/28 beads (DbCD3-28). T cell cultures were transduced with the MC2 TCR and expanded in IL-2-supplemented culture medium till culture day 15 and evaluated for proportions of Treg phenotype as described in Materials and Methods. The proportion of Treg was significantly lower following activation using soluble CD3 + CD28 mabs (median 21.0%; range %) versus both bead activations, that is, 38.3% (median; range %) for MACSi CD3/28 beads (t-test p-value ) and 33.8% (mean; range %) for Dynal CD3/ 28 beads (t-test p-value 0.009). PBMC, peripheral blood mononuclear cells; RCC, renal cell carcinoma.

3 SUPPLEMENTARY FIG. S2. Effects of type of T cell activation beads on T cell proliferation, transduction, and function. PBMC derived from five RCC patients were activated as described in legend to Supplementary Fig. S1, cultured till day 15, and evaluated as described as follows: T cells were monitored for proliferation, expressed as fold increase in cell number from start of transduction, that is, between days 2 and 15 (A and B); transduction efficiency expressed as % TCRVb3 + T cells (C) or % TCRVb3 +, MC2-pMHC + Tcells(D), proportions of CD3 -,CD56 + NK cells (E); CD8+ (F); CD3 +, CD56 + (G); TCRcd T cells (H). T cells were monitored for cytolytic activities, using the CD107-degranulation assay, and results are expressed as % CD107 + of CD8 + T cells following incubation with medium (I), and target cells (corrected for medium) Daudi (J), Daju (K), BSM loaded with irrelevant gp100 or relevant MC2 peptide (L). In addition, T cells were monitored for IFNc production expressed as pg/ml following incubation with medium (M), and target cells (corrected for medium) Daudi (N), Daju (O), and BSM loaded with irrelevant gp100 or relevant MC2 peptide (P). Cell recovery following activation (day 0 2) was for soluble CD3 + CD28 mabs 41% (median, range 25 57%, n = 5), for MACSibead CD3/CD28 beads 27% (median, range 22 32%, n = 5), and for Dynabead CD3/28 beads 36% (median, range 30 50%, n = 5). Graphs represent individual and median values (bars). *p < 0.05; **p < 0.01; ***p < using two-sided paired t-tests.

4 SUPPLEMENTARY FIG. S3. Effects of type of T cell activation beads on T cell differentiation. T cell cultures (n = 5), activated and transduced with the MC2/A2 TCR, as described in legend to Supplementary Fig. S6, were monitored at culture day 15 for the following lymphocyte differentiation markers CD45RA, CD27, CD28, and CCR7 (A); and CD8 + T cell differentiation stages, defined as described for Fig. 2 (B); in contrast to experiments presented in Fig. 2, in this experiment presented differentiation stages did not cover over 70% of the total CD8 + T cell population. Now significant proportions of CD45RA CCR7 - and CD45RA CCR7 - were measured, and when included, differentiation stages present as in Supplementary Fig. S7C; see also legend to Supplementary Fig. S6.

5 SUPPLEMENTARY FIG. S4. Effect of cytokine concentrations on T cell proliferation, transduction, function, and differentiation. Healthy donor T cells (n = 6) were activated with scd and transduced at days 2 and 3 with the MC2 TCR as described in legend to Fig. 1. From days 4 to 15, T cells were expanded in medium supplemented with (1) IL2, (2) IL7 + 15, or (3) IL In addition, T cells were expanded in medium supplemented with IL (50 ng + 50 ng/ml, n = 4); IL (50 ng + 50 ng/ml, n = 4); and IL (10 ng + 50 ng/ml, n = 2). T cell cultures were monitored as described in the legends to Figs. 1 and 2: T cell proliferation (A); % CD8 + T cells (B); %Vb3 +, MC2 pmhc +, T cells (C); cytolysis of Daju cells (D); and IFNc production following incubation with Daju cells (E). Data are expressed relative to the IL2, day 4 condition (our current standard) and presented in box-and-whisker plots. The mean value of the condition IL2 added from day 4 onward is shown. Significant differences between addition of cytokines at day 0 versus day 4 are shown: *p < 0.05; **p < 0.01; ***p < using two-sided paired t-tests. SUPPLEMENTARY FIG. S5. Effect of timing of cytokine addition on T cell differentiation according to a three-marker definition. T cell cultures (n = 6) generated as described in legend to Fig. 5 were monitored for T cell differentiation stages using a three-marker definition (Kaneko et al., 2009; Wang et al., 2011), T-naïve (T N = CD45RA L + ) (A); T- central memory (T CM = CD45RA L + ) (B), T-effector memory (T EM = CD45RA L + ) (C), and T-late effector (T LE = CD45RA - / L - / + ) (D). Data are expressed relative to the IL2, day 4 condition (our current standard) and presented in box-and-whisker plots. Significant differences between addition of cytokines at day 0 versus day 4 are shown; see legend to Fig. 1.

6 SUPPLEMENTARY FIG. S6. Effect of timing of cytokine addition on T cell proliferation, transduction, and function. T cell cultures (n = 6) generated as described in legend to Fig. 5. T cell cultures were monitored for proliferation, transduction efficiency, and function as described in the legends to Fig. 1. Data are presented as box-and-whisker plots. Significant differences between addition of cytokines at day 0 versus day 4 are shown; see legend to Fig. 1.

7 SUPPLEMENTARY FIG. S7. Effect of timing of cytokine addition on lymphocyte subsets and T cell differentiation markers. T cell cultures (n = 6) generated as described in legend to Fig. 5. T cell cultures were monitored for T cell subsets and T cell differentiation markers CD45RA, CD45RO, CD27, and CD62L as described in legend to Fig. 2. Data are presented as box-and-whisker plots. Significant differences between addition of cytokines at day 0 versus day 4 are shown; see legend to Fig. 1.