Naïve CD8(+) T cell derived tumor-specific cytotoxic effectors as a potential remedy for overcoming TGF-β immunosuppression in the tumor microenvironment

Abstract

Despite of the potential implications for cancer immunotherapy, conventional approaches using in vitro expanded CD8(+) T cells have suboptimal outcomes, mostly due to loss of functionality from cellular exhaustion. We therefore investigated the phenotypic and functional differences among in vitro activated CD8(+) T cells of three different sources, namely naïve (NTeff), memory (MTeff) and tumor-infiltrating lymphocytes (TILeff) from human and mice, to better understand mechanisms behind potent effector functions and potential for overcoming current limitations. In line with the greater proliferation activity and longer telomere lengths of NTeff populations, cells of naïve origin exhibited significantly less amounts of T cell exhaustion markers than those of MTeff and TILeff, and moreover, acquired distinct expression patterns of memory-promoting transcription factors, T-bet and Eomes, induced in a rapid and sustainable manner. NTeff cells appeared to have lower expression of Foxp1 and were refractory to apoptosis upon TGF-β conditioning, implying better survival potential and resistance to tumor-induced immune suppression. Of CD8(+) T cell pools activated to tumor-specific CTLs, naïve cell generated effectors possessed the most potent cytotoxic activity, validating implications for use in rational design of adoptive immunotherapy.

Figure 1. Characteristics of human naïve, memory, and TIL effector CD8⁺ T cells.

(A) Isolation of CD8⁺ T cell subsets and proliferation assays. MACS isolation and FACS Aria analyses displayed in vitro expansion of naïve (CCR7⁺CD45RA⁺), memory (CD45RA⁻), and TIL (CD44⁺) subpopulations, 5 days post TCR stimulation. CFSE assay showed positive proliferation results after 3 to 5 days of culture. (B) Activation markers of effector cell subsets. Effector naïve and memory CD8⁺ cells (NT_eff and MT_eff, respectively) are characterized by CD62L^-CD25⁺CD44⁺OX40⁺ expression. Re-stimulated CD8⁺ TIL (TIL_eff) populations also showed similar phenotypes. (C) OX40⁺ cell percentage of effector cell subsets. NT_eff showed significantly higher values compared to MT_eff and TIL_eff (*P < 0.05 vs TIL_eff, **P < 0.005 vs MT_eff). (D) Telomere length comparison showed the longest telomeres in NT_eff and shortest in TIL_eff cells (*P < 0.05). RTLs of effector cells were measured against CCRF-CEM control cells (1:1 ratio, total 5.0 × 10⁵ cells) with a DAKO Telomere PNA Kit. Data are representative of three to four independent experiments and are presented as mean ± SD.

Figure 2. Exhaustion phenotypes of effector CD8⁺ T cell subpopulations in human and mice.

Effector cells (OX40⁺) derived from naïve cells (NT_eff) expressed lower levels of exhaustion markers PD1, CTLA4, KLRG1, and LAG3 compared to effectors from memory cells or TILs (MT_eff or TIL_eff) (A) in human (*P < 0.05 for PD-1, **P < 0.005 for CTLA4) and (B) in mice at 5 days post-stimulation with anti-CD3/CD28 and anti-CD2. MFI levels are the average result of three experiments and are presented as mean ± SD. (C) In vitro functional analysis of human effector cells by cytokine production. An equal number of progenitors (2 × 10⁵ cells per subtype) activated to OX40⁺ effectors were characterized by flow cytometry for direct comparison among subtypes. NT_eff cells demonstrated a greater increase in perforin⁺ granzyme B⁺ populations compared to MT_eff and TIL_eff subsets at two time points (day 3 and 5 post-stimulation). Data are representative of three independent experiments.

Figure 3. Expression of T-box transcriptional factors and characteristics of human effector CD8⁺ T cells under TGF-β conditioning.

(A) Intracellular staining of T-bet and Eomes expressed on TCR stimulated effectors NT_eff, MT_eff, and TIL_eff. MFI levels are shown for day 0 (initial peak) and day 5 (right side peak) post-stimulation. For TILs, day 0 peaks are lost due to insufficient cell amount pre-expansion. (B) Western blot analysis of naïve and memory T cells, and TILs on days 0, 3, 5 and 8 post TCR activation. Kinetics of T-bet and Eomes expression in differentiating CD8⁺ T cells suggest rapid and sustained stimulation for NT_eff populations. Effector groups from day 5 post-stimulation are treated with TGF-β (10 ng/mL) for 48 hours and checked for Foxp1 expression (day 7 post-stimulation) by (C) intracellular FACS staining and (D) Western blot. Increase in Foxp1 MFI levels pre and post TGF-β conditioning was minimal for naïve effector cells and NT_eff showed the least Foxp1 protein expression, although not statistically significant. MFI levels and protein density ratios are the average result of three experiments and shown as mean ± SD. (E) Annexin V assay by flow cytometry. Quadrant plots show the percent distribution of early and late apoptotic cells (first quadrant) with or without 48-hour exposure to TGF-β. Naïve cell derived effectors (NT_eff) showed minimal increase in apoptotic cell populations relative to MT_eff and TIL_eff subgroups. All experiments were repeated three times and are presented by one representative figure.

Figure 4. Tumor-specific CTL function of in vitro human and in vivo murine CD8⁺ T cells.

(A) In vitro comparison of tumor-specific CTL function of IL-2 primed human CD8⁺ T cells against U266 MM cells by IFN-ɤ ELISPOT assay. CTL alone represents the control group and U266 refers to cytotoxic activity of effectors against U266 target cells (E:T ratio of 10:1) in the absence of MHC class I restriction, expressed as the number of IFN-ɤ spots observed. Naïve T cells activated under high-dose IL-2 for 14 days (Naïve D14) displayed highest activity against U266 target cells. (B) LDH cytotoxicity assay of IL-2 primed human CD8⁺ T cells. Specific cytotoxicity lysis percentages against U266 MM target cells were highest for Naïve D14 effectors compared to other groups when E:T ratios were at least 10:1 (*p < 0.05). (C) In vivo CTL activity in the murine model. Following injection of NT_eff, MT_eff, and TIL_eff CD8⁺ T cells, OVAp-expressing EL4-EG7 cells (8 × 10⁵cells/200 L) were inoculated subcutaneously in B6 recipient mice in a 1:2 ratio of target tumor to effector cells. All B6 mice injected with TIL_eff had positive mass (1 cm³) formation after 28 days, whereas tumor growth was suppressed in NT_eff harboring B6 mice for longer than 35 days. All data are representative of three independent experiments and shown as mean ± SD.