Human CD8+ memory and EBV-specific T cells show low alloreactivity in vitro and in CD34+ stem cell–engrafted NOD/SCID/IL-2Rγcnull mice  Simone Thomas, Sebastian Klobuch, Maria Sommer, Reyn van Ewijk, Matthias Theobald, Ralf G. Meyer, Wolfgang Herr  Experimental Hematology  Volume 42, Issue 1, Pages 28-38.e2 (January 2014) DOI: 10.1016/j.exphem.2013.09.013 Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 Alloreactivity of TCRpp65-RNA transfected CD8+ T cell subsets. (A) In vitro expanded CD8+ Tbulk, TN, TM, and TEBV cells were analyzed on day 1 after TCRpp65-RNA (or mock) transfection for IFN-γ ELISPOT reactivity to allogeneic EBV-LCL at a CD8+-to-target cell ratio of 0.3:1. Numbers of SFC obtained from five HLA-A2.1+ healthy donors against-A2.1− (n = 3; left panel) or A2.1+ (n = 3; right panel) EBV-LCL. Horizontal bars represent medians of grouped spot numbers derived from different T cell donors. Values were compared using Wilcoxon signed-rank tests. (B) Individual T cell populations from three donors were prestimulated with EBV-LCL from allogeneic A2.1− individuals in a CD8+-to–EBV-LCL ratio of 1:1. After 1 week of allo-stimulation, IFN-γ ELISPOT reactivity was measured against allogeneic EBV-LCL, CD40L-activated B cells, and mDCs or against autologous counterparts at a CD8+-to-target cell ratio of 0.3:1. To demonstrate HLA class I–restricted recognition, HLA class I blocking mAb W6/32 or IgG isotype control were added. Shown are means ± SEM of pooled data from three different T cell donors. (C) After 1 week of allo-stimulation with EBV-LCL, CD8+ populations were tested at the indicated CD8+ to target cell ratios for cytolytic activity against HLA-mismatched EBV-LCL and mDCs from the allogeneic donor used in prestimulation or against autologous EBV-LCL and mDCs. The percentages of lysis are calculated as means from duplicates ± SEM and are shown for a representative 51chromium-release assay out of two different experiments. **p < 0.01. ns = not significant. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 Designing the adoptive transfer of allo-stimulated CD8+ T cells in CD34-engrafted NSG mice. (A) Mice were first engrafted with human CD34+ stem cells (1.3 × 106 cells/animal). From week 6 on, in vitro–expanded CD8+ TN, TM, and TEBV subsets of an HLA-mismatched donor were prestimulated twice during an additional 12 culture days against irradiated PBMCs of the CD34+ stem cell donor, and were then injected intravenously with IL-2 and FcIL-7 into reconstituted mice at week 8. At week 9, animals were sacrificed and organs were analyzed for human hematopoietic cell infiltration. Monoclonal Ab to HLA alleles mismatched between T cell donor and stem cell donor were used to determine the origin of cells. (B) Mice (n = 19) reconstituted with CD34+ progenitor cells were investigated after 7 weeks in PB for human CD45+ cell engraftment by flow cytometry. The horizontal bar represents mean ± SEM. (C) IFN-γ spot production of allo-stimulated TN-, TM-, and TEBV-derived cells (15,000 cells/well) directly before adoptive transfer into CD34-reconstituted mice. Targets were allogeneic PBMCs from the stem cell donor, autologous PBMCs loaded with A2.1-binding EBV-LMP2(CLG; 426-434) epitope (10−6 mol/L) or unloaded, and K562. HLA class I blocking mAb W6/32 or IgG isotype control was included in some wells. SFC numbers are means of duplicates ± SEM and are representative of four experiments performed with two different T cell–stem cell donor pairs. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 3 Engraftment and proliferation of adoptively transferred CD8+ T cell subsets in humanized NSG mice. (A) In week 8 after injection of CD34+ cells, successfully reconstituted NSG mice were infused intravenously with 1 × 107 CD8+ TN, TM, and TEBV cells that had been prestimulated in vitro against PBMCs of the stem cell donor (for experimental design see Figure 4). After 1 week, mice were sacrificed and percentages of human CD8+ T cells of T cell donor origin (HLA-B7+) were determined on viable cells by flow cytometry using mAb to HLA-B7 and CD8. Shown is the gating strategy in splenocytes of one animal of five. (B) Percentages of human CD8+ HLA-B7+ T cells of T cell donor origin in BM and spleen (n = 5 per group). Flow cytometry gating was performed as shown in (A). Horizontal bars represent medians. Values were compared using Wilcoxon signed rank tests. (C) To analyze in vivo proliferation of transferred CD8+ TN and TM cells, mice were injected intraperitoneally with BrdU on day 3–5 after T cell transfer. Cell proliferation was measured in splenocytes of BrdU-treated or untreated mice 1 week after adoptive transfer of T cells by BrdU-specific staining in flow cytometry. Shown density plots are gated on CD3+ HLA-B7+ T cells. As controls, CD8+ TN and TM cells were infused into BrdU-treated mice that were not reconstituted with CD34+ progenitor cells (without CD34+). Shown are representative flow cytometry data out of two independent experiments with two different T cell–stem cell donor pairs. *p < 0.05. ns = not significant. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 4 Alloreactivity of CD8+ T cell subsets against human hematopoietic cells in NSG mice. (A) Splenocytes and BM cells of human CD34+ engrafted mice were harvested 1 week after intravenous injection of CD8+ TN, TM, and TEBV cells, which had been prestimulated in vitro against PBMCs of the HLA-disparate CD34+ donor over 12 days. Untreated mice (without T cells) were included as a control group. Frequencies of CD45+HLA-B7− hematopoietic cells of stem cell donor origin were determined in individual mice (n = 5 per group) by flow cytometry. Horizontal bars represent medians. Values were compared using Wilcoxon signed rank tests. (B) Similar to (A), frequencies of CD19+ B cells derived from the stem cell donor were measured. (C) Specificity analysis of CD8+ TN-, TM-, and TEBV-derived cells upon mouse passage. One week after injection into human CD34+ engrafted animals, mice were sacrificed and cells from two homogenized spleens per group were pooled for in vitro restimulation against PBMCs of the original stem cell donor. After two weekly stimulations (day 12 of culture), IFN-γ ELISPOT reactivity of expanding cells (6 × 104 cells/well) was determined against PBMCs of the CD34+ donor and against murine splenocytes. Data are from one representative experiment of two. *p < 0.05. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 Multiple transfers of TCR-RNA transfected CD8+ T cell subsets in humanized NSG mice. (A) CD8+ TN-, TM-, and TEBV-derived cells prestimulated in vitro against PBMCs from the HLA-mismatched stem cell donor twice during 12 days were transfected with either TCRpp65-RNA or without RNA (mock) and were analyzed 1 day later for reactivity in IFN-γ ELISPOT assay (15,000 cells/well). Target cells were allogeneic PHA-activated T cell blasts of the stem cell donor, autologous PBMCs, T2 cells loaded with A2.1-binding pp65(NLV; 495-503), or EBV-BMLF-1 (GLC; 280-288) epitope (10−6 mol/L) or unloaded. SFC numbers are means of duplicates ± SEM. (B) In week 8–10 after transplantation with CD34+ cells, successfully engrafted NSG mice were injected weekly (three times) and intravenously each with 1 × 107 allo-stimulated and TCRpp65-RNA or mock transfected CD8+ TN, TM, and TCRpp65-RNA transfected TEBV cells. One week after the third T cell transfer (week 11), mice were analyzed for the engraftment of human T cells of T cell donor origin (HLA-A2+) by flow cytometry using mAb to CD8 and HLA-A2 (for gating strategy see Fig. 3A). Hematopoietic cells of the HLA-mismatched stem cell donor were HLA-A2−. Shown are percentages (and medians) of HLA-A2+ T cells in spleen and BM of mice (n = 5 per group). (C) In week 11, splenocytes and BM cells of CD34-reconstituted and CD8+ T cell treated mice were also analyzed for engraftment with CD45+ HLA-A2− hematopoietic cells of stem cell donor origin (%). Horizontal bars represent medians. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure 1 Sorting and phenotypic characterization of CD8+ T cell subpopulations. (A) Immunomagnetically selected CD8+ T cells (Tbulk) were stained with mAb to CD3, CD8, CD28, and CD95/Fas to identify and sort CD28+Faslow TN and CD28+/−Fashigh TM fractions by flow cytometry. Sorted CD8+ Tbulk, TN, and TM cells had a purity of 99% before in vitro expansion. (B) EBV-specific CD8+ T cell lines (TEBV) were generated by in vitro stimulation of CD8+ Tbulk with LMP2(426-434) peptide-loaded autologous PBMCs over 14 days, followed by immunomagnetic cell selection using APC-labeled LMP2(426-434)A2.1 pentamers and anti-APC beads (left dot plot: peptide-stimulated CD8+ Tbulk before selection, right dot plot: peptide-stimulated CD8+ Tbulk after enrichment). (C) Immunophenotypes of individual CD8+ T cell populations (Tbulk, TN, TM, TEBV derived) were analyzed by flow cytometry for expression of CD45RO, CD45RA, CD62L, and CCR7 after in vitro expansion over 14 days by anti-CD3/CD28 beads (Tbulk, TN, TM) or LMP2-peptide loaded autologous PBMCs (TEBV), respectively. Shown are representative flow cytometry data from five independent experiments. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure 2 TCRpp65 expression and CMV-specific cytotoxicity by redirected CD8+ T cell populations. (A) After in vitro expansion for 2 weeks, CD8+ Tbulk-, TN-, TM-, and TEBV-derived subsets were electroporated with TCRpp65 RNA (or without; mock) and were then monitored for pp65(495-503)A2.1 tetramer binding 1, 4, and 6 days later in flow cytometry. Percentages of Tetpp65+ T cells are shown for a representative fluorescence-activated cell sorting staining out of three experiments with three different T cell donors. (B) Cytotoxicity of TCRpp65 RNA (or mock) transfected CD8+ T cell subpopulations were analyzed against CMVpp65+- (CMV) or CMVpp65--infected (CMV without pp65) A2.1+ allogeneic fibroblasts, and A2.1+ autologous EBV-LCL in 51chromium-release assays at a CD8+-to-target ratio of 20:1 at the indicated time points after RNA transfection. Data from controls (CMV without pp65, autologous EBV-LCL, and mock) are shown for day 1, but were observed at similar levels on days 4 and 6 (not shown). The percentages of lysis are calculated as means from duplicates ± SEM and are shown for a representative assay out of three experiments with three different T cell donors. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Supplementary Figure 3 Engraftment and alloreactivity of CD8+ T cell subsets in humanized NSG mice in an HLA-A2–disparate donor model. (A) Seven weeks after intravenous injection of CD34+ stem cells (1.4 × 106 cells/animal) of a HLA-A2− donor into NSG mice (n = 20), frequencies of human CD45+ cells in murine peripheral blood (PB) was measured by flow cytometry. Horizontal bar represents mean ± SEM. (B) On week 8, successfully engrafted NSG mice were infused intravenously with 1 × 107 CD8+ TN-, TM-, and TEBV-derived cells from a HLA-A2+ healthy individual, which had been prestimulated in vitro against PBMCs of the stem cell donor as described in Figure 2. One week later, mice were sacrificed, and percentages of human HLA-A2+ T cells in BM and spleen were determined by flow cytometry (n = 4 per group). Control mice were left untreated (without T cells). Horizontal bars represent medians ± ranges. (C, D) Splenocytes and BM cells of humanized mice (n = 4) harvested 1 week after adoptive T cell transfer were also analyzed for frequencies of HLA-A2− CD45+ hematopoietic cells (C) and CD19+ B cells (D) of stem-cell donor origin by flow cytometry. Values were compared using Wilcoxon signed rank tests. *p < 0.05. ns = not significant. Experimental Hematology 2014 42, 28-38.e2DOI: (10.1016/j.exphem.2013.09.013) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions