Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells

Abstract

Central memory CD8+ T cells (T(CM)) and effector memory CD8+ T cells (T(EM)) are found in humans and mice; however, their relative contributions to host immunity have only recently been examined in vivo. Further, the ability of T(CM) to treat an established tumor or infection has yet to be evaluated. To address the therapeutic potential of different tumor-reactive CD8+ T cell memory subsets, we used an established model for the in vitro generation of T(CM) and T(EM) by using IL-15 and IL-2, respectively. Adoptively transferred T(CM) exhibited a potent in vivo recall response when combined with tumor-antigen vaccination and exogenous IL-2, leading to the eradication of large established tumors. By contrast, T(EM) were far less effective on a per-cell basis. Microarray analysis revealed that the signature of highly in vivo effective antitumor T cells included the overexpression of genes responsible for trafficking to secondary lymphoid tissues. This gene expression profile correctly predicted the in vitro and in vivo lymphoid-homing attributes of tumor-reactive T cells. Furthermore, we found that homing to secondary lymphoid tissue is required for optimal tumor treatment. Our findings indicated that highly in vivo effective antitumor T cells were those that initially targeted secondary lymphoid tissue, rather than tumor sites, as had previously been postulated. Thus, tumor-reactive CD8+ T cell populations with the phenotypic and functional attributes of T(CM) may be superior to T(EM)/effector T cells for adoptive immunotherapies using concomitant tumor-antigen vaccination.

Fig. 1.

Trafficking of tumor-reactive CD8⁺ T cells to 2° lymphoid tissues is required for antitumor treatment. (A)WT or L Tα^-/- mice bearing 9-day s.c. B16 tumors were left untreated as controls (• and ○, respectively) or received 1 × 10⁶ pmel T_EM, rFPhgp 100 vaccination, and exogenous IL-2 (▪ and □, respectively). (B) CD62L expression by adoptively transferred tumor-reactive CD8⁺ T cells is required for optimal antitumor treatment. WT mice bearing 9-day s.c. B16 melanoma tumors were treated with nothing (•), pmel-CD62L^-/- cells (□), or age-matched pmel-CD62L^+/+ cells (▪) in combination with rFPhgp100 and exogenous IL-2. (C) Redundancy of lymphoid-homing molecules. Splenocytes from pmel-CD62L^-/- or age-matched pmel-CD62L^+/+ controls were stimulated with 1 μM hgp 100_25–33 peptide and cultured under T_EM conditions. Cells were cytofluorometrically analyzed on day 6 for the expression of integrin αE, integrin β_7, and CCR7. Numbers represent the percentage of gated cells in each quadrant after gating on propidium iodide-negative and CD8⁺ lymphocytes. (D) Tumor treatment fails in β₂M^-/- hosts, but can be rescued by cotransfer of BM-derived DCs. WT or β₂M^-/- mice bearing 9-day B16 tumors were left untreated as controls (• and ○, respectively) or received 1 × 10⁶ T_EM pmel, rFPhgp100, and exogenous IL-2 (▪ and □, respectively) ± cotransfer of mature BM-derived DCs (▴).

Fig. 2.

Gene expression profiling and FACS analysis of self/tumor-reactive CD8⁺ T_CM vs. T_EM. (A) A selected summary of microarray results. Shown in green are the genes involved in trafficking to 2° lymphoid organs; the hatched green bar indicates a gene (mcl1) that has antiapoptotic activity; shown in red are genes involved in effector functions; and hatched red bars indicate proapoptotic genes. (B) Cytofluorometric validation of microarray results of pmel T_CM and T_EM. Surface expression of CD62L and integrin αE are shown with the percentages of CD8⁺ lymphocytes in each quadrant or the mean fluorescence intensity (MFI) for intracellular granzyme B.

Fig. 3.

LN-homing attributes of tumor Ag-specific CD8⁺ T_CM vs. T_EM. (A and B) Pmel T_CM but not T_EM roll efficiently on substrates coated with glycam-1, a ligand for CD62L. Calcein-acetomethyl-labeled pmel T_CM, T_EM, or CD8-enriched naïve cells were injected at 1.5 dynes per cm² into a parallel plate flow chamber in which the bottom plate was coated with glycam-1, E-selectin, or BSA. After 4 min, rolling cells were photographed (four to six random fields per condition) and counted in a blinded fashion (mean ± SD). (C and D) In vivo LN homing of pmel T_CM vs. T_EM. CFSE-labeled, pmel-thy1.1 T_CM and T_EM were adoptively transferred into separate Thy1.2 WT mice, and LNs were extracted 24 h later. (C) Cryostat sections of inguinal LNs 24 h after ACT of 4 × 10⁶ CFSE-labeled (green) pmel T_CM (Lower) or T_EM (Upper). High endothelial venules were identified by staining for PNAd followed by Cy5-conjugated secondary antibody (blue). (D) Cytofluorometric analysis for CFSE⁺CD8⁺ cells from homogenized peripheral LNs and Peyer's patches of recipient mice 24 h after ACT.

Fig. 4.

Enhanced in vivo recall response of tumor-Ag-specific CD8⁺ T_CM over T_EM. (A–C) Sublethally irradiated WT mice bearing 9-day established B16 tumors were left untreated as controls (•) or received the tripartite combination of rFPhgp100, exogenous IL-2, and 1 × 10⁶ pmel T_CM (▪)orT_EM (□). Absolute numbers of adoptively transferred pmel-1 cells (identified by CD8⁺Vβ13⁺ lymphocytes) were enumerated in the spleens or blood of treated animals as a function of time. Each data point represents the average of at least two mice per group. For comparison, the mean absolute lymphocyte count (± SEM) of five nonirradiated tumor-bearing WT mice is shown (▴). (D) Comparison of the intrinsic proliferative capacity of restimulated pmel T_CM vs. T_EM. Pmel-thy1.1 cells expanded under T_CM (light lines) or T_EM (bold lines) conditions were CFSE-labeled, then restimulated with irradiated WT splenocytes pulsed with hgp100_25–33 peptide in complete media containing IL-2. CFSE dilution was determined by daily FACS analysis after gating on CD8⁺thy1.1⁺ lymphocytes.

Fig. 5.

Tumor-reactive CD8⁺ T_CM are superior to T_EM in the treatment of B16 melanoma. (A) Sublethally irradiated (5 Gy) WT mice bearing 9-day B16 tumors were left untreated as controls (•) or received rFPhgp100 vaccination, exogenous IL-2, and either pmel T_CM (▪) or T_EM (□). (B) Enhanced tumor regression in mice receiving pmel T_CM correlated with statistically prolonged animal survival, compared with mice receiving T_EM.