Thymosin Alpha-1 in Immune Model Research: What the Published Studies Report

Thymosin alpha-1 (Tα1) is a 28-amino-acid acetylated thymic peptide that has been characterised in the literature as pleiotropic, with reported effects on T cells, dendritic cells (DCs), antibody responses, cytokine and chemokine output, and steroid-induced thymocyte apoptosis, alongside activation of indoleamine 2,3-dioxygenase (IDO) [11]. A review of this literature also noted that the peptide has been evaluated in a phase I/II clinical trial in recipients of HLA-matched sibling T cell-depleted stem cell transplants, where immune reconstitution and survival were among the reported endpoints [11]. Because no single specific receptor has been established, one review of biophysical work described experimental models in which Tα1 interacted preferentially with negatively charged membrane regions and phosphatidylserine-exposing vesicles, adopting a two-helix conformation and diffusing laterally before engaging membrane receptor complexes; the authors proposed this as a structural basis for the peptide's pleiotropy [9]. Much of the immune-model literature is preclinical and tumour-focused. In three nonclinical murine models — B16 lung metastasis, B16 tumour growth, and cecal ligation and puncture (CLP) sepsis — Tα1 alone was associated with a 32% decrease in lung metastases, decreases in tumour growth of 34% to 46% depending on the amount administered, and a positive trend toward increased survival and decreased bacterial load in the sepsis model; combinations with an anti-PD-1 antibody produced fewer metastases than vehicle [1]. In an earlier murine B16 melanoma study, combined chemo-immunotherapy incorporating Tα1 was reported to delay tumour relapse, extend median survival time, increase splenocyte cytotoxicity against YAC-1 and autologous B16 cells, and shift CD3, CD4, CD8, B220 and IL-2Rβ-positive splenocyte percentages relative to tumour-bearing controls [5]. In DBA/2 mice challenged with Friend erythroleukemia cells, protocols pairing Tα1 with interleukin-2 or with interferon alpha,beta after cyclophosphamide were reported to produce subcutaneous tumour regression, increased spleen cell cytotoxicity, and lymphoid infiltration at tumour sites, with antitumour activity abrogated by in vivo depletion of asialo-GM1, CD4, or CD8-positive cells [18]. Related mouse work reported that in vitro or in vivo pretreatment with Tα1 increased IL-2-induced cytotoxic activity of spleen lymphocytes from normal, cyclophosphamide-suppressed, and tumour-bearing animals against both NK-sensitive and NK-resistant cell lines [8]. Myeloid and antigen-presenting compartments have also been examined in mice. In a murine T-cell lymphoma (Dalton's lymphoma) model, intraperitoneal Tα1 was associated with activation of tumour-associated macrophages (TAM), which showed increased IL-1, TNF, reactive oxygen intermediates and nitric oxide production together with greater pinocytosis, phagocytosis, antigen presentation and tumour cytotoxicity; in vitro treatment of TAM with Tα1 produced a comparable activated state [4]. A companion study in the same model reported that DCs generated from TAM of Tα1-administered tumour-bearing mice showed increased antitumour activity in vitro, produced more IL-1 and TNF-α, and, when used for adoptive transfer, were associated with delayed tumour growth and longer survival in tumour-bearing mice [7]. Not all preclinical results point the same direction. In a Lewis lung cancer model, Tα1 given as a single agent elevated CD8+ T cells but did not inhibit tumour growth, and myeloid-derived suppressor cells responded with heightened Arginase 1 production dependent on TLR/MyD88 signalling; blocking MyD88 abrogated that upregulation [14]. In two immunocompetent murine multiple myeloma models, Tα1 slightly reduced proliferation of murine and human myeloma cell lines in vitro but had no measurable impact on disease development in vivo, and did not alter lymphocyte reconstitution in a xenogeneic transplantation model [10]. In a Lewis-type contrast, combination work in prostate cancer reported that zoledronic acid plus Tα1 suppressed growth of androgen-independent allograft tumours in mice with increased cytotoxic CD8+ T-cell infiltration, while a retrospective clinical analysis in patients with advanced or metastatic prostate cancer described altered therapeutic outcomes and increased frequency of T cells in the combination group; mechanistic assays implicated MyD88/NF-κB signalling in tumour cells, macrophages and T cells [16]. Tα1 has also been used as one of two clinical-grade adjuvants in a neoantigen hydrogel vaccine tested with dual PD-1 and CTLA-4 blockade in preclinical mouse liver metastasis models, where the combination was reported to increase neoantigen-specific CD8+CD69+ T-cell infiltration [19]. Several studies address non-tumour immune models and human cells. In a rat model of severe acute pancreatitis induced with sodium taurocholate, Tα1-treated animals showed lower serum enzyme, procalcitonin and cytokine levels than untreated model animals within 24 hours, higher CD3+, CD4+ and CD8+ T-cell levels and CD4+/CD8+ ratio, reduced pancreatic and lung histological damage, and lower mortality within the observation window [13]. In CLP septic mice, Tα1 intervention was associated with a decreased percentage of CD4+CD25+Foxp3+ T lymphocytes, an increased apoptosis rate of CD4+CD25+ cells, modulation of IL-2, TNF-α, IL-10 and TGF-β, and an improved 72-hour survival rate [15]. In a murine model of immune checkpoint inhibitor-induced colitis, Tα1 was reported to prevent intestinal immunopathology via an IDO1-dependent tolerogenic pathway, without inducing IDO1 in the tumour microenvironment, and to invert the CD8+/Treg ratio in tumour-infiltrating T cells, an effect linked to DC differentiation and chemokine expression [6]. Human cell work includes a study of colonic lamina propria lymphocytes from eighteen surgical specimens, in which Tα1 and thymosin beta 4 suppressed thymidine incorporation but did not alter incorporation in phorbol ester- and ionomycin-stimulated cells or ornithine decarboxylase activity in Con A-stimulated cells [2]. A more recent in vitro study of cultured human CD8+ T cells reported that Tα1 alone moderately increased proliferation and activation, that combined Tα1 and CD3/CD28 stimulation raised proliferation index and CD69, CD25 and HLA-DR expression along with IL-2, IFN-γ, TNF-α and IL-10 secretion, and that in a repeated-stimulation exhaustion model PD-1, TIM-3 and LAG-3 expression was reduced after Tα1 treatment [17]. Colon cancer work combining cell and mouse experiments reported that Tα1 upregulated CD1d, CD80 and B7H2 on colorectal cancer cells, that iNKT cell activation depended on CD1d rather than the costimulatory molecules, that colon cancer stem cells expressed higher CD1d, and that Erk/MAPK inhibition attenuated the CD1d upregulation [20]. A separate literature places Tα1 within thymic-neuroendocrine research. Two reviews summarised animal and cell-model reports of thymosin fraction 5, thymosin alpha-1 and thymosin beta-4 activity on beta-endorphin, ACTH, glucocorticoid, LHRH and LH secretion, and discussed age-related changes in pituitary responsiveness to thymic hormones [3][12]. Those reviews framed the thymus as a component of a bidirectional immune-neuroendocrine circuit and discussed thymic involution in relation to homeostatic decline during ageing in animal models [3][12].