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. 2011 Jan 20;117(3):808-14.
doi: 10.1182/blood-2010-05-286286. Epub 2010 Oct 22.

Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy

Christian S Hinrichs  1 , Zachary A Borman, Luca Gattinoni, Zhiya Yu, William R Burns, Jianping Huang, Christopher A Klebanoff, Laura A Johnson, Sid P Kerkar, Shicheng Yang, Pawel Muranski, Douglas C Palmer, Christopher D Scott, Richard A Morgan, Paul F Robbins, Steven A Rosenberg, Nicholas P Restifo
Affiliations

Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy

Christian S Hinrichs et al. Blood. .

Abstract

Cluster of differentiation (CD)8(+) T cells exist as naive, central memory, and effector memory subsets, and any of these populations can be genetically engineered into tumor-reactive effector cells for adoptive immunotherapy. However, the optimal subset from which to derive effector CD8(+) T cells for patient treatments is controversial and understudied. We investigated human CD8(+) T cells and found that naive cells were not only the most abundant subset but also the population most capable of in vitro expansion and T-cell receptor transgene expression. Despite increased expansion, naive-derived cells displayed minimal effector differentiation, a quality associated with greater efficacy after cell infusion. Similarly, the markers of terminal differentiation, killer cell lectin-like receptor G1 and CD57, were expressed at lower levels in cells of naive origin. Finally, naive-derived effector cells expressed higher CD27 and retained longer telomeres, characteristics that suggest greater proliferative potential and that have been linked to greater efficacy in clinical trials. Thus, these data suggest that naive cells resist terminal differentiation, or "exhaustion," maintain high replicative potential, and therefore may be the superior subset for use in adoptive immunotherapy.

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Figures

Figure 1
Figure 1
Naive CD8+ T cells are the most abundant subset of human CD8+ T cells. Cryopreserved leukapheresis samples were thawed and rested overnight. (A) Frequency of TN (CD62L+, CD45RO−), TCM (CD62L+, CD45RO+), and TEM (CD62L−, CD45RO+) CD8+ T-cell subsets in leukapheresis samples from 11 healthy adult donors (*P < .01, **P < .001). (B) Representative flow cytometric dot plot with overlay of FACS-sorted cells from the TN, TCM, and TEM subsets. The purity of each subset is indicated in parentheses. The dot plot is on log axes.
Figure 2
Figure 2
Effector cells derived from naive rather than memory precursors express the genetically engineered TCR with greater frequency. Cryopreserved leukapheresis samples were thawed and rested overnight. CD8+ T-cell subsets were isolated then stimulated with allogeneic feeders, anti-CD3, and IL-2. Cells were transduced to express the 1G4-α-95:LY mutant TCR 48 hours after stimulation, and flow cytometric analysis was performed 10-16 days after stimulation. (A) Expression of Vβ13.1 (the 1G4 β-chain clonotype) and binding to HLA-A2-NY-ESO-1157-165 tetramer by transduced cells from 2 representative patients. Gate frequencies are displayed. (B) Frequency of Vβ13.1+ cells following transduction of samples from 9 patients (**P < .001).
Figure 3
Figure 3
Naive cells display greater in vitro expansion than memory cells. Cryopreserved leukapheresis samples were thawed and rested overnight. CD8+ T-cell subsets were isolated then stimulated with allogeneic feeders, anti-CD3, and IL-2 (A) or stimulated with CD3/CD28 beads and IL-2 (B-C), then transduced to express the 1G4-α-95:LY mutant TCR 48 hours after stimulation. (A) Fold expansion of each culture was determined 10-16 days following stimulation. The fold expansion of CD8+ T-cell subsets from 12 patients is displayed (*P < .01, **P < .001). (B) Cells were pulsed with H-TdR 48 hours after stimulation, and incorporation as determined 16 hours later is shown. N = 6. (*P < .01, **P < .001). (C) Cell division was determined by CFSE dilution 3 days after stimulation (shaded histograms). The frequency of cells within each gate is indicated. Open histograms are undivided controls. The x-axis is on a log scale and the y-axis is on a linear scale. Data are representative of 4 patients. (D) One day after transduction, cells were labeled with 7AAD and annexin V and flow cytometry was performed. Gate frequencies are displayed. Results are representative of 6 patients.
Figure 4
Figure 4
Effector cells derived from naive precursors manifest less acquisition of effector function. Cryopreserved leukapheresis samples were thawed and rested overnight. CD8+ T-cell subsets were isolated then stimulated with allogeneic feeders, anti-CD3, and IL-2. Cells were transduced to express the 1G4-α-95:LY mutant TCR 48 hours after stimulation. The following data were obtained 10-16 days after stimulation. (A-B) Expression of CD45RO, CD62L, and CCR7. Gate frequencies are indicated. Data are representative of 6 patients. (C) EOMES expression by real-time RT-PCR. Expression is relative to ACTB then normalized to the average relative expression for each patient. Data from 5 patients are shown. (D-E) IFN-γ production from 16-hour coculture with NY-ESO-1+ HLA-A2+ tumor targets as determined by intracellular staining and supernatant concentrations, respectively. Coculture with 2361R (NY-ESO-1− HLA-A2+) results in < 0.2 ng/mL of IFN-γ production in all groups. (F) Specific cell killing as determined by 51Cr release assay against the 624.38 tumor line.
Figure 5
Figure 5
Naive-derived effector cells demonstrate resistance to terminal differentiation. Cryopreserved leukapheresis samples were thawed and rested overnight. CD8+ T-cell subsets were isolated then stimulated with allogeneic feeders, anti-CD3, and IL-2. Cells were transduced to express the 1G4-α-95:LY mutant TCR 48 hours after stimulation. The following data were obtained 10-16 days after stimulation. (A) KLRG1 expression by real time RT-PCR. Expression is relative to ACTB then normalized to the average relative expression for each patient. Data from 5 patients are shown. (B) Flow cytometric analysis of CD57 expression. Gate frequencies are indicated. Data shown are representative of 3 patients.
Figure 6
Figure 6
Naive-derived effector cells display indicators of enhanced proliferative potential and efficacy. Cryopreserved leukapheresis samples were thawed and rested overnight. CD8+ T-cell subsets were isolated then stimulated with allogeneic feeders, anti-CD3, and IL-2. Cells were transduced to express the 1G4-α-95:LY mutant TCR 48 hours after stimulation. The following data were obtained 10-16 days after stimulation. (A) Expression of CD27 as determined by flow cytometry. Gate frequencies are shown. Data are representative of 6 patients. (B) Telomere length relative to average length for each patient. Four individual patients are represented by different shapes (*P < .05).
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