Thymosin α1 (Tα1) is a 28-aminoacid thymic peptide classified as a biological response modifier (BRM), being a natural immunomodulant agent with pleiotropic activities. Due to these features, its drug formulation has a clinical history as immunologic adjuvant of more than 30 years, with worldwide therapeutic applications in contexts of immunity dysregulation (eg, cancer, viral infections, chronic inflammations, sepsis, immunodeficiencies, and vaccine non-responsiveness). Among its described activities, Tα1 can promote dendritic cell (DC) maturation and antigen presentation via the toll-like receptor (TLR)-9 and TLR2 stimulation, key linker molecules of innate and adaptive immunity. Tα1 showed to have effects also on restoring CD4+ T, CD8+ T, and/or natural killer (NK) cell counts when concomitantly administered with immunosuppressive cancer therapy or when administered to patients with immunocompromised conditions.
Thus, its pleiotropic effects and its optimal safety profile prompted many investigators to combine Tα1 with different therapeutic strategies. Within the anti-tumor setting, Tα1 has given proof to ameliorate chemotherapy response when administered to metastatic melanoma patients, by improving the duration of therapeutic response and the clinical benefit, with no additional toxicity. A retrospective study investigated whether stage I–III non-small cell lung cancer (NSCLC) patients had benefitted from Tα1 administration alone or combined with chemo(radio)therapy and/or targeted therapy. Results showed that, except for squamous carcinoma patients and targeted therapy-treated patients, Tα1-receiving groups in the adjuvant setting exhibited an increased survival benefit, compared to non-Tα1 groups. Furthermore, in a Phase 2 study, Tα1 displayed beneficial effects for local advanced NSCLC patients by significantly reducing the immune-related adverse events derived from chemoradiotherapy treatments (eg, pneumonitis, lymphopenia), compared to the non-Tα1 control group.
So far, despite the promising results of preclinical studies in murine models, no clinical trial has demonstrated the efficacy of combining Tα1 with the more recent immune-checkpoint blockade (ICB)-based immunotherapy that entirely remodeled the cancer treatment panorama. However, Danielli et al in a retrospective study pointed out that metastatic melanoma patients treated with the anti-CTLA4 monoclonal antibody (mAb) Ipilimumab (IPI) that were previously treated with Tα1 had a prolonged median overall survival (OS) compared to those patients that were not pre-Tα1-treated, with an OS rate at 5 years being 41.2% and 13.0% (p=0.006) for Tα1-IPI and IPI alone patients, respectively.
Although clinical benefits from Tα1 administration in patients are evident among several tumor histotypes, the molecular mechanisms behind Tα1 effects have not been fully elucidated and are sometimes controversial. Besides, whether the clinical efficacy depends on direct effects exerted by Tα1 on immune and also on tumor cells is still unclear. Because of this gap and to better investigate the possible role of Tα1 in improving the effectiveness of immunotherapy-based strategies, the current work aimed to deepen the potential immunomodulatory effect of Tα1 on tumor cell lines of 3 different histotypes (n=13 cell lines) and on distinct immune cell subsets from healthy donors (HDs; n=3). In particular, for the first time to our knowledge, an in-depth study evaluating Tα1 ability to explicate a common modulation of the transcriptional immune landscape of tumor cells was conducted. Additionally, to better understand the role of Tα1 within the immune system, its effects on proliferation and gene expression profile of distinct immune cell subsets (ie, CD4+ T, CD8+ T, B, and NK cells) from HDs were studied by culturing and Tα1-treating cells separately. Thus, Tα1 direct stimulation of different immune populations allowed us to unravel the individual responses of each investigated cell subset to Tα1. Ultimately, whether Tα1 was able to modulate the cytolytic activity of immune effector cells (ie, Lymphokine-activated killer cells (LAK) and Cytotoxic T Lymphocytes (CTL)) was explored.
BV421-conjugated mouse anti-human CD45, FITC-conjugated mouse anti-human CD3, APC-H7-conjugated mouse anti-human CD8, PE-conjugated mouse anti-human CD4, BV650-conjugated mouse anti-human CD20, and BV421-conjugated mouse anti-human PD-L1 were purchased from BD Biosciences (NJ, USA); PE-conjugated mouse anti-human CD56 and the anti-human CD3ε antibody (clone OKT3) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Alexa fluor488-conjugated mouse anti-human HLA-ABC, PE-conjugated mouse anti-human CD54, and PE-conjugated mouse anti-human Melan-A (MART-1) were purchased from Biolegend (CA, USA), Beckman Coulter (CA, USA), and Santa Cruz Biotechnology (TX, USA), respectively. Interleukin (IL)-2 was purchased from Clinigen (PA, USA). Tα1 was kindly provided by SciClone Pharmaceuticals Inc (CA, USA).
Mel146, Mel195, Mel261, Mel275, Mel313, Mel514, Mel599, and Mel601 cutaneous melanoma (cMM), SiGBM56 and SiGBM71 glioblastoma (GBM), Meso4, Meso6, and Meso7 pleural mesothelioma (PM) cell lines originated, respectively, from patients’ metastatic tissue of melanoma, primary GBM tumor lesions, and pleural effusions of mesothelioma. Patient-derived cell lines were established in our laboratory, as follows. In detail, cMM and GBM tissues were processed within 60 min following surgical removal to be dissected into fragments by mechanical digestion (1–2 mm 3), and subsequently cultured in the appropriate medium (RPMI Medium 1640; Biochrom, Berlin, Germany); whereas mesothelioma pleural effusions were collected by thoracentesis or paracentesis, centrifuged for 10 min at 150 g, and cell pellets were resuspended in the ad hoc medium (HAM’s F-12; Euroclone, Milan, Italy). Culture media were supplemented with 20% heat-inactivated fetal bovine serum (FBS; Biochrom, Berlin, Germany), up to the 5 th passage of in vitro culture, and with 10% FBS for subsequent ones. For the experiments reported below, continuous immortalized tumor cell lines, established after at least 20 passages of in vitro culture, were used.
In addition, the LN-18 GBM cell line (American Type Culture Collection (ATCC) company) was cultured in 5% FBS-DMEM medium (Sigma-Aldrich, MO, USA); the FO-1 melanoma and the DBTRG-05MG GBM (ATCC) cell lines were cultured in 10% FBS-RPMI 1640 medium; the LoVo colorectal adenocarcinoma cell line (ATCC) was maintained in 10% FBS-HAM’s F-12 medium; and the K562 erythroleukemia cells were grown in 10% FBS-Iscove’s Modified Dulbecco’s Medium (Euroclone, Milan, Italy).
All cell media were supplemented with 2mM L-glutamine and 100 µg/µL penicillin/streptomycin (Biochrom, Berlin, Germany). Cells were incubated at 37°C and 5% CO 2 and passaged at 80–90% confluency.
Human peripheral blood mononuclear cells (PBMCs) from HDs, purchased from ZenBio (NC, USA; catalog number SER-PBMC-200-F), were processed to isolate specific immune cell subsets. In detail, PBMCs went through the MACS® Column-based cell separations following the CD19, CD56, CD8, and CD4 MicroBeads human protocols (Miltenyi Biotec, Bergisch Gladbach, Germany), to isolate B, NK, CD8+ T, and CD4+ T cell subsets, respectively. An aliquot of 0.5×10 6 isolated immune cells was used to assess the purity of each cell subset by multiparametric flow cytometry, with a percentage (%) threshold of positive cells to specific subset markers (ie, CD3−CD20+, CD56+, CD3+CD8+, and CD3+CD4+) >80% (Supplementary Files 1 and 2). The remaining immune cell subsets were cultured in vitro under the following specific conditions: B cells were activated for 14 days with 50 IU/mL IL-4 and human CD40-Ligand (CD40L) Multimer Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) in the StemMACS™ HSC Expansion Media XF supplemented with 5% human serum (Euroclone, Milan, Italy), according to the B Cell Expansion Kit guidelines (Miltenyi Biotec, Bergisch Gladbach, Germany); resting CD4+ and CD8+ T cells were cultured for 24 hours in 10% human serum-Iscove’s Modified Dulbecco’s Medium with 100 U/mL IL-2; activated CD4+ and CD8+ T cells were maintained for 3 days in 10% human serum-Iscove’s Modified Dulbecco’s Medium with 100 U/mL IL-2 and 50 ng/mL anti-CD3 antibody; activated NK cells were cultured for 24 hours with 600 U/mL IL-2 in 10% FBS-RMPI 1640 medium.
LAK cells were obtained by PBMCs from the same HDs cultured in 10% FBS-RMPI 1640 supplemented with 1000 U/mL IL-2 for 4 days. HLA-A2-restricted gp100-peptide-specific CTL, cultured in 10% human serum-Iscove’s Modified Dulbecco’s Medium with 1000 U/mL IL-2, were generated and characterized as previously described.
All immune cell media were supplemented with 2mM L-glutamine and 100 µg/µL penicillin/streptomycin. B and NK cells were plated at a density of 1×10 6 cells/mL; LAK, CD4+ T, CD8+ T, and CTL cells at a density of 2×10 6 cells/mL. Cells were incubated at 37°C and 5% CO 2.
Cell proliferation was assessed after treatments with scalar doses of Tα1 in tumor cMM (ie, Mel146, Mel195, Mel261, Mel514, Mel599, and Mel601), GBM (ie, LN-18, DBTRG-05MG, SiGBM56, and SiGBM71), and PM (ie, Meso4, Meso6, and Meso7) cell lines (1 µM, 10 µM, 100 µM) and in HD’s immune cell subsets (30 nM, 300 nM, 3 µM), by the WST-1 assay (Roche, Molecular Biochemicals, Mannheim, Germany). Briefly, 5×10 3 tumor cells and 2.5×10 5 immune cells were seeded in triplicate or quintuplicate, according to cell availability, in a 96-well culture plate in a final volume of 200 µL/well culture medium and treated with Tα1 following the appropriate treatment schedule. After 48 hours, 1:10 WST-1 reagent was added to cell plates, and spectrophotometer lectures were performed after 3 and 4 hours for tumor cell lines and immune cell subsets, respectively. Untreated cells that underwent the same experimental conditions represented the assay controls. The blank consisted of the culture medium and an equal amount of the WST-1 reagent. Absorbance was measured using the Benchmark™ Plus microplate reader (Bio-Rad, CA, USA) at 450 nm, subtracting wavelength at 655 nm. The % of cell proliferation was calculated by optical density (OD) results, as follows: (OD treatment – OD blank)/(OD control – OD blank) × 100.
Tα1 in vitro cell treatments were performed on both tumor and immune cells according to the following schedules. Tumor cell lines cMM (ie, Mel146, Mel195, Mel261, Mel514, Mel599, and Mel601), GBM (ie, SiGBM56, SiGBM71, DBTRG-05MG, and LN-18), and PM (ie, Meso4, Meso6, and Meso7) were seeded on day 0 and treated with 10 µM Tα1 on day 1.
Immune cells (ie, immune cell subsets, LAK, and CTL), cultured/activated according to their in vitro cell culture conditions, were seeded and treated with 3 µM Tα1 on day 0.
Tumor and immune cells were harvested 48 hours from drug exposure for the following in vitro experiments. Untreated cells underwent the same experimental conditions and were used as controls.
Cell total RNA was isolated following the Trizol reagent (Invitrogen, CA, USA) protocol and quantified by the NanoDrop™ One spectrophotometer (Thermo Fisher Scientific
