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. 2015 Aug 15;75(16):3216-26.
doi: 10.1158/0008-5472.CAN-15-0584. Epub 2015 Jun 22.

Tumor-Specific Effector CD8+ T Cells That Can Establish Immunological Memory in Humans after Adoptive Transfer Are Marked by Expression of IL7 Receptor and c-myc

Smita S Chandran  1 , Biman C Paria  1 , Abhishek K Srivastava  1 , Luke D Rothermel  1 , Daniel J Stephens  1 , Udai S Kammula  2
Affiliations

Tumor-Specific Effector CD8+ T Cells That Can Establish Immunological Memory in Humans after Adoptive Transfer Are Marked by Expression of IL7 Receptor and c-myc

Smita S Chandran et al. Cancer Res. .

Abstract

The optimal T-cell attributes for adoptive cancer immunotherapy are unclear. Recent clinical trials of ex vivo-expanded tumor-infiltrating lymphocytes indicated that differentiated T effector cells can elicit durable antitumor responses in some patients with cancer, with their antitumor activity tightly correlated with their persistence in the host. Thus, there is great interest in the definition of intrinsic biomarkers that can predict the conversion of short-lived tumor antigen-specific T effector cells into long-lived T memory cells. Long-term persistence of ex vivo-expanded tumor-specific CD8+ T effector clones has been reported in refractory metastatic melanoma patients after adoptive T-cell transfer. By using highly homogeneous clone populations from these preparations, we performed a comparative transcriptional profiling to define preinfusion molecular attributes that can be ascribed to an effector-to-memory transition. Through this route, we discovered that preinfusion T-cell clones that expressed the IL7 receptor (IL7R) and c-myc were more likely to persist longer after adoptive transfer to patients. The predictive value of these two biomarkers was strengthened by using IL7R protein, IL7-induced pSTAT5, and c-myc mRNA expression to prospectively identify human tumor-specific T effector clones capable of engraftment into immunodeficient mice. Overall, our findings reveal IL7R and c-myc expression as intrinsic biomarkers that can predict the fate of CD8+ T effector cells after adoptive transfer.

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Conflict of interest statement

Conflicts of Interest: None

Figures

Figure 1
Figure 1. Heterogeneous engraftment of CD8+ T effector clones after adoptive transfer in humans
(A.) Dichotomous long term engraftment of M1 and M4 MART specific CD8+ T effector clones as determined by the absolute number of CD8+Tetramer+Vβ+ T cells per microliter of blood at time points relative to infusion in respective melanoma patients. Arrow denotes the day of clone infusion. (B.) Heterogeneous persistence of infused effector clones (n=10 clones infused into 9 patients) as assessed by % CD8+Tetramer+Vβ+ cells identified in PBMC at day 30 post infusion. Non-persisting (NP) clones were defined to have engraftment levels of ≤0.1% of CD8+ T cells; Persisting (P) clones had engraftment levels >0.1% of CD8+ T cells. (C.) Pre-infusion phenotype of NP (n=4) and P (n=6) effector clones as determined by FACS expression of the indicated differentiation markers (% of cells). (D.) Cognate peptide avidity and (E.) reactivity against 526 Mel (A2+) tumor cell line for NP (n=4) and P (n=5) effector clones. Shown are supernatant IFN-γ levels after subtracting background reactivity against either control peptide or 888 Mel (A2−) tumor cell line. Statistical comparison performed by unpaired T test; ns, non-significant. Bar on graphs represents mean.
Figure 2
Figure 2. Comparative gene expression profiling reveals pre-infusion transcriptional differences between persisting and non-persisting CD8+ T effector clones
(A) Affymetrix cDNA microarray comparison of NP (n=4) and P (n=6) clones. Volcano plot highlights selected overexpressed transcripts (p <0.05 and fold difference >2) in NP and P effector clones. (B) Ingenuity Pathway Analysis of the 112 differentially expressed transcripts between NP and P effector clones; shown are functional categories ranked by statistical significance. (C) Relative mRNA quantitation by RMA normalized intensity of selected genes expressed in NP (n=4) and P (n=6) effector clones. Each dot represents an individual infused CD8+ T cell clone. *P< 0.05, ** P< 0.01, ***P< 0.001; unpaired T test. Bar on graphs represents mean.
Figure 3
Figure 3. Pre-infusion expression levels of IL-7R and c-myc by CD8+ T effector clones correlate with their level of persistence after adoptive transfer in humans
(A) Correlation between pre-infusion IL-7R cell surface expression (MFI) on infused CD8+ T effector clones (n=9) and peripheral blood persistence (% CD8+Tet+) at day 30 after adoptive transfer. (B) Representative STAT5 phosphorylation (pSTAT5) assay performed upon effector clones with low and high IL-7R expression. FACS plots show expression of CD8 and IL-7Rα in effector clones. Numbers in FACS plots represent % CD8+IL-7Ra+ cells compared to isotype control staining. pSTAT5 was measured by FACS before (no cytokine) and after clones were exposed to either 10ng/ml of rIL-2, rIL-15, or rIL-7 cytokine for 15 min. Numbers in histograms represent % of cells induced to express pSTAT5 by respective cytokine compared to no cytokine baseline. (C) Correlation between cytokine induced pSTAT5 (% of cells) in infused CD8+ T effector clones (n=7) and peripheral blood persistence (% CD8+Tet+) at day 30 after adoptive transfer. (D) Correlation between pre-infusion c-myc mRNA (per 105 b-actin copies) expressed in infused CD8+ T effector clones (n=9) and peripheral blood persistence (% CD8+Tet+) at day 30 after adoptive transfer.
Figure 4
Figure 4. Prospective characterization of effector CD8+ T cell clones based upon by IL-7R and c-myc expression
(A) Phenotype of individual MART specific CD8+ T effector clones (n=7) isolated by limiting dilution from the peripheral blood of a metastatic melanoma patient. Numbers in FACS plot represent % CD8+Tetramer+ cells (upper panel) and % CD45RO+CD62L− cells (lower panel) compared to isotype. (B) Telomere length of isolated effector clones. (C) Cell surface IL-7R expression (MFI) and c-myc mRNA (per 105 b-actin copies) expression in isolated effector clones. (D) Cytokine induced pSTAT5 expression (% of cells) in isolated CD8+ effector clones (n=7). Numbers in histograms represent % of cells induced to express pSTAT5 by respective cytokine compared to no cytokine baseline.
Figure 5
Figure 5. Pre-infusion expression levels of IL-7R and c-myc by CD8+ T effector clones predict their level of persistence after adoptive transfer in NSG mice
(A) Experimental xenograft model to evaluate the persistence of human CD8+ effector T cell clones after adoptive transfer into immunodeficient NSG mice. (B) Persistence in NSG mice of human MART specific CD8+ T effector clones with varying pre-infusion expression levels of IL-7R, induced pSTAT5, and c-myc. FACS dot plots demonstrate the % of huCD3+huCD8+ cells within the harvested spleens of mice (n=3 mice per clone group; shown in columns) at day 21 post transfer. Each FACS plot represents splenocytes harvested from an individual replicate animal. (C) Respective correlation analyses between pre-infusion IL-7R MFI expression, IL-7 induced pSTAT5 expression, and c-myc mRNA (per 105 b-actin copies) expression in infused CD8+ T effector clones and mean clonal persistence (% huCD3+huCD8+) at day 21 after adoptive transfer into NSG mice (n=3). Linear regression plots demonstrate mean clonal persistence ± SEM (error bars). Experiment is representative of 2 independently performed adoptive transfer experiments.
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References

    1. Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science. 2002;298:850–854. - PMC - PubMed
    1. Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U, et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol. 2008;26:5233–5239. - PMC - PubMed
    1. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res. 2011;17:4550–4557. - PMC - PubMed
    1. Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science. 2014;344:641–645. - PMC - PubMed
    1. Robbins PF, Dudley ME, Wunderlich J, El-Gamil M, Li YF, Zhou J, et al. Cutting edge: persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy. J Immunol. 2004;173:7125–7130. - PMC - PubMed

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