Comparison of naïve and central memory derived CD8+ effector cell engraftment fitness and function following adoptive transfer

Abstract

Human CD8⁺ effector T cells derived from CD45RO⁺CD62L⁺ precursors enriched for central memory (T_CM) precursors retain the capacity to engraft and reconstitute functional memory upon adoptive transfer, whereas effectors derived from CD45RO⁺CD62L^- precursors enriched for effector memory precursors do not. Here we sought to compare the engraftment fitness and function of CD8⁺ effector T cells derived from CD45RA⁺CD62L⁺ precursors enriched for naïve and stem cell memory precursors (T_N/SCM) with that of T_CM. We found that cytotoxic T cells (CTLs) derived from T_CM transcribed higher levels of CD28, FOS, INFγ, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of γ-chain cytokines compared to CTLs derived from T_N/SCM. Higher frequencies of CTLs derived from T_CM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RγC^null mice, CD8⁺ T_CM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8⁺ T_CM derived CD19CAR⁺ CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8⁺ T_N/SCM derived counterparts. These studies support the use of T_CM enriched cell products for adoptive therapy of cancer.

Keywords: Antitumor activity; adoptive therapy; central memory T cell derived CTLs; engraftment fitness; naïve T cell derived CTLs.

Figure 1.

Frequency and phenotypic attributes of CD8⁺ T_N/SCM and CD8⁺ T_CM from healthy donor peripheral blood. (A) Representative gating/sorting strategy for CD8⁺CD45RA⁺CD62L⁺ T_N/SCM vs. CD8⁺CD45RO⁺CD62L⁺ T_CM. (B) Analysis of PBMC from eight different healthy donors gated on the CD8⁺ population and analyzed for CD45RA⁺CD62L⁺ T_N/SCM and CD45RO⁺CD62L⁺ T_CM by multicolor flow cytometry. (C) Percentages of immunoreactive cells in each population are indicated (mean + SEM, n = 8). * p< 0.05 when comparing T_N/SCM and T_CM using a paired Student’s t-test.

Figure 2.

Effector cells derived from CD8⁺ T_CM precursors expressed higher levels of CD28 and IL-2 production upon stimulation. Purified CD8⁺ T_N/SCM and CD8⁺ T_CM were subject to 14 d in vitro REM stimulation in the presence of rhIL-2 (50U/mL). (A) Phenotypic analysis of differentiated effector cells derived from CD8⁺ T_N/SCM (T_N/SCM CTL) and CD8⁺ T_CM (T_CM CTL) following expansion_. Mean percentages of immunoreactive cells + SEM from 4 different donors are presented (**p < 0.01, *p < 0.05). (B) Representive flow cytometric analysis of CD28 on the CTLs derived from CD8⁺ T_N/SCM and CD8⁺ T_CM. (C) Supernatants were collected after overnight co-incubation of either unstimulated CD8⁺ T_N/SCM and CD8⁺T_CM or stimulated CD8⁺ T_N/SCM and CD8⁺T_CM with OKT3 expressing LCL. Cytokine levels (means +SEM of triplicate wells) were determined using cytometric bead array. **p < 0.01. Representative data of 4 experiments are depicted. (D) After 14 d of stimulation, RNA was extracted and analyzed for genes that confer differentiation, survival, and apoptotic qualities of effector T cells and 4 control genes including housekeeping genes, RT control and positive PCR control. Medians of fold change of CD28, FOS, IFNγ, Eomes and BCL2L11 mRNA from cells derived from CD8⁺ T_CM (T_CM CTL) vs. CD8⁺ T_N/SCM (T_N/SCM CTL) from 4 individual donors are presented. **p < 0.01. (E) Effector T cells derived from CD8⁺ T_N/SCM (T_N/SCM CTL) (blue) and CD8⁺T_CM (T_CM CTL) (red) were stained for intracellular phosphorylated AKT (pAKT). Fluorochrome conjugated isotype matched antibody stained cells are shown in black. (F) Percentages of AKT⁺ cells from 4 donors are presented.

Figure 3.

Effector cells derived from CD8⁺T_CM precursors displayed greater ability to persist and expand in vitro. Purified CD8⁺ T_N/SCM and CD8⁺ T_CM were expanded 14 d in vitro using a REM in the presence of rhIL-2 (50U/mL). (A) After 14 d of in vitro stimulation, the effector cells from each population (T_N/SCM CTL and T_CM CTL) were stained with antibodies to the indicated cytokine receptor and analyzed by flow cytometry. Histograms show the mean fluorescence intensities (MFIs) of γ_-chain cytokine receptor positive cells (black) after subtraction from the isotype controls (open). Representative data of 4 experiments are depicted. (B) Positivity of IL-2 receptors from four different donors is presented. *p < 0.05, ***p < 0.001. (C) Percentages of IL-15Rα from a cohort of four donors are presented. (D) After the initial expansion, the T_N/SCM CTL and T_CM CTL cells were maintained in rhIL-2 (50U/mL) (left) or rhIL-15 (10ng/mL) (right). Cytokines were supplemented every other day. Viable cell numbers were determined by Guava ViaCount at the indicated time points.

Figure 4.

Effector cells derived from CD8⁺T_CM precursors exhibited superior engraftment fitness in NSG mice. After 14 d of in vitro stimulation, effector cells derived from CD8⁺ T_N/SCM (T_N/SCM CTL) and CD8⁺T_CM (T_CM CTL) (10⁷) were infused i.v. into huIL-15-replete NSG mice. (A) Three weeks after adoptive transfer, human T cells in the peripheral blood, BM and spleen of recipient mice were determined by flow cytometric analysis using antibodies specific for human CD45 and CD8. Means+SEM of a total of 6 mice per T cell subset in a representative experiment are depicted. **p < 0.01, using a Mann–Whitney test. (B) Percentages of CD62L, CD27, CD28, and IL7Rα positive cells on gated human CD45⁺ positive cells of pooled peripheral blood, BM and spleens are indicated. *p < 0.05 **p < 0.01 using a Mann–Whitney test. (C) TCR vβ repertoire of the effectors derived from CD8⁺ T_N/SCM and CD8⁺T_CM before and after infusion. Percentage (%) of human CD3⁺ cells that were positive for the indicated TCR vβ genes was determined by flow cytometry. (D) For in vivo stimulation, 10⁷ irradiated OKT3-expressing LCL (+OKT3) were injected i.v. into mice that had been engrafted (3 days) with CD8⁺ T_N/SCM or CD8⁺T_CM derived CTLs. Human T cells in peripheral blood were determined 7 d post in vivo challenge. ***p < 0.001. **p < 0.01.

Figure 5.

Engineered CD19 specific T cells derived from CD8⁺ T_CM (T_CM-CD19R) displayed superior engraftment fitness and antitumor activity as compared to that from CD8⁺ T_N/SCM (T_N/SCM-CD19R). CD8⁺ T_N/SCM and CD8⁺ T_CM that had been transduced to express a CD19-specitic CAR (CD19R:CD28:ζ) and huEGFRt selection marker, were enriched for EGFRt⁺ cells and further expanded by REM stimulation in the presence of rhIL-2 and rhIL-15. (A) Purified EGFRt⁺ populations after 3 cycles of REM were flow cytometrically analyzed for expression of CD8⁺ as well as the CAR and EGFRt transgenes (using anti-Fc and Erbitux reagents, respectively) (B) Fold expansion of the purified EGFRt⁺ T_N/SCM-CD19R and T_CM-CD19R cells within the third cycle of REM stimulation. (C) Relative telomere length of the expanded cells. (D) The expanded T_N/SCM-CD19R and T_CM-CD19R cells (10⁷) were adoptively infused into huIL-15 reconstituted NSG mice. Human T cells in the BM were analyzed by flow cytometry at indicated time points (3 mice per time point). ND, not detectable. (E) CD19⁺ffluc⁺LCLs (10⁶) were inoculated into NSG mice i.v. on day -4. Expanded T_N/SCM-CD19R and T_CM-CD19R cells (10⁷) were adoptively transferred (i.v.) into the huIL-15-reconstituted, tumor-bearing mice on day 0. Tumors were monitored with biophotonic imaging. Representative mouse images and mean photon flux ± SEM of each group (N = 6) are depicted.*, p < 0.05 using Mann–Whitney t-test. Representative data from two separate experiments are presented.