Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice - PubMed (original) (raw)

Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice

Xiuli Wang et al. Blood. 2011.

Abstract

In clinical trials of adoptive T-cell therapy, the persistence of transferred cells correlates with therapeutic efficacy. However, properties of human T cells that enable their persistence in vivo are poorly understood, and model systems that enable investigation of the fate of human effector T cells (T(E)) have not been described. Here, we analyzed the engraftment of adoptively transferred human cytomegalovirus pp65-specific CD8(+) T(E) cells derived from purified CD45RO(+)CD62L(+) central memory (T(CM)) or CD45RO(+)CD62L(-) effector memory (T(EM)) precursors in an immunodeficient mouse model. The engraftment of T(CM)-derived effector cells (T(CM/E)) was dependent on human interleukin-15, and superior in magnitude and duration to T(EM)-derived effector cells (T(EM/E)). T-cell receptor Vβ analysis of persisting cells demonstrated that CD8(+) T(CM/E) engraftment was polyclonal, suggesting that the ability to engraft is a general feature of T(CM/E.) CD8(+) T(EM/E) proliferated extensively after transfer but underwent rapid apoptosis. In contrast, T(CM/E) were less prone to apoptosis and established a persistent reservoir of functional T cells in vivo characterized by higher CD28 expression. These studies predict that human CD8(+) effector T cells derived from T(CM) precursors may be preferred for adoptive therapy based on superior engraftment fitness.

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Figures

Figure 1

Figure 1

Frequency of CD8+ T-cell memory subsets in human peripheral blood. (A) Flow cytometric analysis of PBMCs from 4 different human donors gated on the lymphoid population by forward and side scatter (left panel) and analyzed for CD45RO, CD62L, and CD8 expression. CD45RO+ lymphocytes were then gated for CD8+CD62L+ TCM and CD8+CD62L− TEM (middle and right panels) and analyzed by multicolor flow cytometry with anti-CCR7, anti-perforin, or anti-granzyme A mAbs (B). Percentage of cells in each gate (red) is indicated, and mean percentage of the CD8+ TCM or TEM cells that were CCR7, perforin, or granzyme A positive (± SE; n = 3 donors) is indicated. *P < .05, CD8+ TCM versus TEM cells (unpaired Student t test). (C) Cytokine production profiles of the freshly isolated CD8+ TCM and TEM. Supernatants were collected after overnight coincubation with LCL-OKT3, and cytokine levels (mean ± SE of triplicate wells) were determined as described in “Cytokine production assays.” *P < .0001, cytokine levels of CD8+ TCM versus TEM cells (unpaired Student t test).

Figure 2

Figure 2

TE cells derived from TCM and TEM in vitro are similar in phenotype and function. (A) Schematic of methods for deriving CMV-specific TCM/E and TEM/E. Purified TCM, TEM, and pp65-expressing vAPCs were generated from the same CMV-seropositive donor's PBMCs. (B) CD45RO and CD62L staining of TCM (top) and TEM (bottom) after sorting from PBMCs. (C) CD8 and pp65-tetramer staining of gated PBMCs (left panels) and of CMV-specific TCM/E and TEM/E at day 7 (middle panels) and 21 (right panels) after stimulation with vAPCs. Histogram quadrants are based on staining with isotype and negative tetramer controls, and percentage of double-positive cells is indicated. (D) pp65 tetramer and intracellular IFN-γ staining of TCM/E and TEM/E before infusion, after overnight coincubation with LCL-pp65. (E) Fold expansion of pp65-tetramer+ cells was determined by multiplying the total number of cells by the percentage pp65tet+ (determined as shown in panel C) found at days 0, 7, 14, and 21 of vAPC stimulation and 14 days after the first and second anti-CD3 (REM) stimulations; these values were then normalized to the input cell number (day 0). (F) Expression of CD62L, CD127, CD28, CCR7, and CD8 on the TCM/E and TEM/E cell products. (G) Cytotoxic activity of TCM/E and TEM/E cell products against auto-LCLs loaded with either an HLA-A2-restricted control peptide (cLCL) or CMV pp65 peptide (pp65LCL). Mean percentage of 51Cr release (± SD) of triplicate wells is depicted. (H) Cytokine production by TCM/E and TEM/E. Supernatants were collected after coincubating T cells overnight with CMV pp65 peptide-loaded auto-LCLs, and mean (± SD of triplicate wells) cytokine levels were determined using cytometric bead array.

Figure 3

Figure 3

IL-15-dependent engraftment of CMV-specific TCM/E cells in NOG mice is greater than that of TEM/E. (A) Schematic of the experiment. (B) Mean percentage (± SE) of human T cells (CD45+ CD8+) in peripheral blood lymphocytes (PBLs) of mice engrafted with TCM/E (squares) or TEM/E (circles) was determined by flow cytometry (n = 5). *P < .05, TCM/E versus TEM/E cell engraftment in the presence of NS0-IL-15 cells (unpaired Student t test). (Inset) Mean levels of human IL-15 (± SE) in day 7 serum of NOG mice that had received 3 intraperitoneal injections of 1.5 × 107 irradiated NS0-IL-15 cells (n = 6) or in control mice (n = 10). (C) Mean percentage of human T cells (CD45+ CD8+) plus or minus SE in mouse PBL, bone marrow, and spleen at day 21. *P < .05, TCM/E cell engraftment in each organ versus that of TEM/E in the presence of NS0-IL-15 cells. (D) TCR Vβ repertoire of the CMV-specific TCM/E and TEM/E before (Input) and after (d21) engraftment. Percentage of CD3+ cells (Input) or CD45+ CD3+ cells (d21) that were positive for the indicated TCR Vβ genes was determined by flow cytometry.

Figure 4

Figure 4

Human CD8+ TCM/E persist long-term (100 days) in huIL-15 NOG mice and remain functional. TCM/E and TEM/E (107) were injected intravenously at day 0, and irradiated NS0-IL-15 cells (1.5 × 107) were administered 3 times a week starting at day 0, until mice were killed at day 100. (A) Mean percentage of human CD45+CD8+ cells (± SE) in mouse PBL, bone marrow, and spleen at day 100 was determined by flow cytometry (n = 5). (B) TCR Vβ repertoire of the input and long-term engrafted TCM/E and TEM/E. Bone marrow was pooled from mice, and human CD45+ cells were sorted and expanded by stimulation with anti-CD3. The percentage of CD3+ cells positive for the indicated TCR Vβ genes was determined by flow cytometry. (C) Bone marrow harvested at day 100 from mice engrafted with TCM/E and TEM/E was analyzed by flow cytometry for expression of human CD45, CD62L, CCR7, and CD28. Gating was based on staining with isotype control mAb, and the percentage of double-positive cells is indicated. (D) IL-2 production from CD45+ T cells derived from day 100 bone marrow of mice engrafted with TCM/E and TEM/E. Supernatants were collected after T cells were coincubated overnight with LCL-pp65, and IL-2 levels were determined using cytometric bead array. (E) Cytotoxic activity of human T cells derived from day 100 bone marrow of mice engrafted with TCM/E and TEM/E, and stimulated with anti-CD3 mAb. Target cells included OKT3-expressing LCLs, auto-LCLs or LCL-pp65. Mean percentage of 51Cr release (± SD) of triplicate wells. (F) Intracellular IFN-γ staining of human T cells derived from day 100 bone marrow of mice engrafted with TCM/E and TEM/E and coincubated overnight with LCL-pp65, LCL-OKT3, or auto-LCLs.

Figure 5

Figure 5

Differential cytokine receptor expression, IL-15–mediated proliferation, and caspase activity of TCM/E and TEM/E. (A) IL-15Rα, IL-2Rβ, or IL-2Rγ expression by TCM/E and TEM/E. Mean fluorescence intensity was normalized to that of isotype control staining in each case to determine ΔMFI. (B) Proliferation of TCM/E and TEM/E was determined after 48-hour incubation with different concentrations of rhIL-15 using a standard [3H]-thymidine incorporation assay. (C-D) CFSE-labeled TCM/E and TEM/E (107) were injected intravenously into mice at day 0, and irradiated NS0-IL-15 cells (1.5 × 107) were administered 3 times a week starting at day 0, until mice were killed at either day 9 or day 12. (C) CFSE profiles of the input and engrafted TCM/E and TEM/E in day 9 PBL was assessed by flow cytometry. Percentage of CFSE-diluted cells that fall within the first log are indicated. (D) Engraftment of the CD45+ human T cells in the PBL, bone marrow, and spleen was assessed on days 9 and 12 by flow cytometry. (E) FL-1 profiles of CD45+ human T cells in the PBL were assessed on day 9 as a readout for cleavage of the caspase substrate D2R. The percentage of cells with cleaved D2R is depicted.

Figure 6

Figure 6

Adoptively transferred CMV-specific CD8+ TCM/E exhibit a better response to antigen challenge in vivo than TEM/E. (A) Schematic of in vivo antigenic stimulation of engrafted CMV-specific TCM/E and TEM/E. (B) Engraftment of CMV-specific TCM/E (squares) or TEM/E (circles) was carried out with (black) or without (white) administration of irradiated CMV pp65-expressing LCLs at days 3, 10, and17; and mean percentage (± SE) of human T cells (CD45+ CD8+) in mouse PBL was determined by flow cytometry (n = 6). *P < .05, engraftment of TCM/E alone versus pp65-driven TCM/E engraftment (unpaired Student t test). (C) On euthanasia at day 28, PBLs were harvested and analyzed by flow cytometry for percentage of Ki-67+ cells in the human T-cell population (left) and for the ability of CD45+ human T cells to cleave the caspase substrate D2R (right).

Figure 7

Figure 7

Adoptively transferred CMV-specific CD8+ TCM/E exhibit superior protection from tumor challenge. (A) Schematic of the in vivo tumor challenge experiment. (B) Engraftment of pp65+ ffLuc+ LCL in animals treated with or without CMV-specific TCM/E or TEM/E was determined by Xenogen imaging; and mean (± SE) of total flux levels of luciferase activity are shown for each group (n = 5). *P < .05, animals treated with TCM/E versus either untreated or TEM/E-treated animals (analysis of variance). (C) Mean percentage (± SE) of human CD45+CD8+ cells in day 10 mouse PBL was determined by flow cytometry. *P < .05, TCM/E versus TEM/E engraftment (unpaired Student t test).

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