Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapy

Abstract

In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen-specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA- CD62 ligand- (CD62L-) CC chemokine receptor 7- (CCR7-) interleukin-7 receptor alphaLo (IL-7RalphaLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Ralpha in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen-specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+)CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L- CCR7- profile, which may explain in part their ability to mediate tumor destruction.

Figure 1. Ex vivo–expanded T cells used for treatment contain tumor antigen–specific clones with late-stage effector phenotype

(A) Tumor antigen–specific CD8⁺ T cells are detectable in TIL samples using MART-1:26–35(27L) peptide containing HLA-A*0201 tetramer complexes and anti-CD8α antibody. Alternatively, melanoma antigen–specific T-cell clones were identified in patients 9 and 21 TIL using Vβ7- or Vβ22-specific antibodies, respectively. (B) Cell surface expression of costimulatory, lymphoid-homing, and homeostasis-associated molecules on pretreatment PBL and TIL-derived tumor antigen–specific CD8⁺ T cells prior to ACT. The percentage of marker-positive events within the gated tetramer or Vβ-stained, CD8α⁺ population are provided. Pt indicates patient; ND, not done.

Figure 2. High levels of tumor antigen–reactive clones persist in the blood of ablated patients after adoptive transfer

(A) Elevated numbers of lymphocytes in the peripheral blood of select patients following cell transfer contract to normal levels. Approximately 1 week after transfer, absolute lymphocyte counts (ALCs) were measured from peripheral blood from patients 9 (▲), 10 (▧), 20 (◆), 21 (●), 23 (x), and 28 (■). The x-axis represents days relative to cell transfer, with day 0 representing the day of cell infusion. Latest time points analyzed for patients 9, 10, and 23 were days 124, 524, and 161 after infusion, respectively. (B) CD8⁺ T-cell frequencies are generally elevated after cell transfer and diminish temporally. The frequency of CD8⁺ cells was measured on gated viable lymphocytes. (C) High frequencies of tumor antigen–specific CD8⁺ T cells persist in the peripheral blood of select patients. Tumor antigen–specific CD8⁺ T cells were identified using MART-1: 27–35(27L) peptide containing HLA-A*0201 tetramer complexes or Vβ-specific antibodies. (D) High numbers of melanoma-specific CD8⁺ T cells persist in the blood of metastatic melanoma patients receiving ACT. Absolute numbers of tumor antigen–specific CD8⁺ T cells were calculated as number of CD8⁺ lymphocytes per mm³ times percent tetramer or Vβ-specific antibody positive. (E–F) PBLs from treated patients maintain tumor antigen–specific reactivity. Posttreatment PBL from patients 9 (d 56), 10 (d 524) 20 (d 56), 21 (d 63), 23 (d 28), and 28 (d 97) was tested for IFN-γ secretion in response to T2 targets cells pulsed with 1 μM gp100:209–217 or MART-1:27–35 (M1) peptide, or 10 μM MART-1:27–35 (M10) peptide (E) or melanoma cell lines 888 (HLA-A2⁻ MART-1⁺), 2098 (HLA-A2⁺ MART-1⁻), 526 (HLA-A2⁺ MART-1⁺), or 624 (HLA-A2⁺ MART-1⁺) by standard ELISA assay (F).

Figure 3. Adoptively transferred tumor antigen–specific CD8⁺ T cells persist in the peripheral blood of patients as effector memory T cells

(A) Compared with TIL, the frequency of tetramer or Vβ-positive staining CD8⁺ T cells expressing CD45RA increased in the peripheral blood of all patients more than nearly 2 months after cell infusion, while positive CD45RO expression was maintained. Expression was evaluated on tumor antigen–specific CD8⁺ T cells from patients 9 (△), 10 (×), 20 (◇), 21 (○), 23 (x), and 28 (□), and the average of all values is shown (●). (B) CD45RA expression is temporally reacquired with concomitant CD45RO reduction on tumor antigen–reactive T cells in vivo. Representative dot plots show the expression of CD45 isoforms on gated MART-1⁺ CD8⁺ T cells from patient 28 TIL or peripheral blood 63 days after cell administration. (C) TIL and posttransfer PBL-derived tumor antigen–specific CD8⁺ T cells lack expression of the lymphoid homing markers, CD62L and CCR7. Expression of CD62L and CCR7 was not detected. (D) CD62L and CCR7 expression is absent on gated MART-1⁺ CD8⁺ T cells from patient 28 TIL or day-63 posttransfer PBLs and limited to endogenously reconstituted CD8⁺ T cells. TuAg indicates tumor antigen.

Figure 4. IL-7Rα and CD28 are highly expressed by melanoma antigen–specific CD8⁺ T cells in the early aftermath of ACT into lymphoablated metastatic melanoma patients

(A) The frequency of tumor-reactive CD8⁺ T cells with elevated IL-7Rα expression levels is increased nearly 1 week after cell infusion. Representative histograms show IL-7Rα expression by TIL or day-7 posttransfer PBL-derived MART-1⁺ or Vβ-22⁺ CD8⁺ T cells from patients 9 and 21, respectively. (B) Tumor antigen–specific T-cell clones rapidly and continuously express IL-7Rα after adoptive transfer. IL-7Rα expression was detected on tumor antigen–specific CD8⁺ T cells from patients 9 (x), 10 (▧), 20 (◇), 21 (□), 23 (○), and 28 (△), and the average of all values is shown (●). Expression by persistent tumor antigen–specific CD8⁺ T cells was assessed about 1, 4, and 8 or more weeks after ACT. (C–D) CD28 expression was immediately enhanced on melanoma-reactive CD8⁺ T cells from most patients after cell transfer. Although low, CD28 expression was shown to temporally increase on Vβ-7⁺ CD8⁺ T cells from patient 10 in vivo in 4 independent flow cytometric analyses.

Figure 5. CD27 expression by melanoma antigen–specific CD8⁺ T cells increases in an indolent manner and identifies a subset of memory cell precursors within the effector cell population

(A) The increase in CD27 by tumor-specific CD8⁺ T cells occurs in an indolent fashion. Dot plot analysis shows that the MART-1:26–35(27L) peptide–specific lymphocyte population from the peripheral blood of patient 9 is composed of Vβ12⁺ cells 55 days after cell transfer (left). Representative histograms show the steady temporal increase in CD27 expression level and frequency by Vβ12⁺ CD8⁺ T cells from patient 9. (B) The frequency of CD27-expressing tumor-reactive T cells decreases in the early aftermath of cell transfer in select patients. Dot plot analysis of CD8⁺ T cells from patient 10 TIL and PBLs demonstrates the early reduction in CD27 expression by the Vβ7⁺ tumor-reactive population at day 7 after transfer, compared with TIL, and the uniformity of CD27 expression 524 days after ACT. (C) The frequency of CD27-expressing melanoma-reactive CD8⁺ T cells increases with continued persistence in vivo. Tumor antigen–specific CD8⁺ T cells from patients 9 (x), 10 (▧), 20 (◇), 21 (□), 23 (○), and 28 (△) were assayed for CD27 expression in the TIL or peripheral blood 1, 4, and 8 or more weeks after ACT. The average of all values is shown (●). (D) CD27 identifies a stable subset of tumor antigen–specific CD8⁺ effector T cells that give rise to the memory T-cell population in most patients. Longitudinal examination of the absolute number of CD28- (◇), CD27- (□), or IL-7Rα– (△) expressing melanoma antigen–specific CD8⁺ T cells compared with the total number of melanoma antigen–specific CD8⁺ T cells (●) in the blood of all 6 patients is shown. Values represent the number of tumor antigen–specific CD8⁺ T cells with the indicated phenotype in the peripheral blood as cells/mm³.

Figure 6. Persisting melanoma antigen–specific CD8⁺ T cells express both CD27 and CD28 in vivo

(A) Expression of CD27 and CD28 by the reconstituting endogenous CD8⁺ T-cell repertoire. Dot plot analysis of gated CD8⁺ MART-1/A2 tetramer-negative T cells from patient 28’s PBLs obtained prior to and after ACT of TIL reveals that the phenotype of pretreatment CD8 T cells is restored in reconstituting CD8 T-cell population. The frequency of CD28⁺ CD27⁻ is low in PBLs prior to lymphodepletion and at day 63 after cell infusion. (B) Transfer of predominantly CD27⁻ CD28⁻ TIL results in the generation of a persisting population of CD27⁺ CD28⁺ tumor antigen–specific CD8⁺ T cells. Representative dot plots show the expression of CD27 and CD28 on tumor antigen–specific CD8⁺ T cells from TIL and posttreatment PBL samples from patients 28 (MART-1⁺), 23 (MART-1⁺), 10 (Vβ7⁺), and 21 (Vβ22⁺). The tumor antigen–reactive T-cell population transitioned through a CD28⁺ CD27⁻ 1 week after transfer before becoming CD27⁺ CD28⁺ memory T cells. (C) CD28-expressing melanoma-reactive CD8⁺ T cells that concomitantly express CD27 persist after ACT in vivo. The absolute number of transferred tumor antigen–specific CD8⁺ T cells expressing both CD27 and CD28 was assessed in PBLs from patients with objective clinical responses (10, 21, and 28) beginning 1 week after infusion and is represented as cells/mm³, depicted in log scale.