The thymus is a central organ responsible for T-cell differentiation, maturation, and both positive and negative selection. These processes ensure the generation of functionally competent and self-tolerant T lymphocytes, which play a pivotal role in cellular immunity, including the recognition of tumor cells, foreign antigens, and pathogens. Recent studies have expanded the thymus’s role beyond T-cell development, suggesting its contribution to memory B-cell differentiation through an unconventional pathway independent of external antigen exposure (1). Together with medullary thymic epithelial cells (TECs), dendritic cells, and macrophages, these B cells support negative T-cell selection, immune homeostasis, and the regulation of autoimmunity, particularly during aging (2).

The thymus is highly sensitive to various forms of stress, both via neural regulation (3) and direct physical or chemical injury (4–6). Such insults result in profound structural and functional impairment, disrupting normal T-cell development and leading to increased susceptibility to cancer, autoimmune, allergic, and infectious diseases, as well as accelerated immunosenescence. Nevertheless, the thymus possesses a remarkable capacity for self-renewal due to the presence of thymic stem cells (TSCs), which are key to structural regeneration and functional recovery under both physiological and stress-induced conditions (7, 8).

However, despite the long history, the mechanisms contributing to endogenous thymic regeneration were not well understood. Recent studies on mouse models have reported multiple pathways of thymic regeneration and the molecular mechanisms that trigger these pathways following various damaging treatments (9). These investigations have demonstrated also the important role of regulatory T (Treg)-cell balance in the thymic recovery via expression of various regenerative factors, in particular, the cytokine amphiregulin (10). Furthermore, an analogous population of Treg cells (CD39⁺ICOS⁺) was identified by these authors also in the human thymus, inciting their new function and potential in therapeutic applications associated with aging- and treatment-induced immunosuppression.

T-cell development occurs within specialized thymic microenvironments known as niches, where immature thymocytes interact with TECs and other stromal elements. These interactions guide thymocyte differentiation through distinct developmental stages, ensuring the generation of mature, functional, and self-tolerant T cells (11–13). However, age-related epithelial defects limit thymic function (14, 15) and impair regeneration following injury (16).

While current research has primarily focused on TECs and thymic epithelial stem cells (TESCs) as the principal components responsible for thymic renewal (12, 16–26), intrathymic T-lymphocyte precursors (TLPs) exhibiting stem-like properties have received comparatively little attention in the context of thymic recovery. Some investigators have proposed that the bone marrow contains a population of universal dormant hematopoietic stem cells (HSCs), which can be activated under severe stress and serve as progenitors not only for TLPs but also for epithelial and stromal compartments, depending on local epigenetic cues (27–29). This hypothesis is supported by several experimental evidence showing the generation of epithelial tissues in the lungs, liver, and intestine from transplanted highly purified bone marrow or cord blood HSCs (29–34). Although intriguing, this concept requires further verification, as most researchers support the view that discrete stem cell populations exist for lymphoid and stromal-epithelial lineages.

Thymocyte development begins with early CD25^-CD44⁺ TLPs, also referred to as CD4^-CD8^- double-negative 1 (DN1) cells, which originate from multipotent HSCs migrating from embryonic sources such as the aorta–gonad–mesonephros region, yolk sac, and fetal liver, and later from the adult bone marrow. Intrathymic TLPs are localized mainly in the subcapsular cortical zone, although some studies also indicate their presence near the paracortical region (8, 13).

Entry of these progenitors into the thymus requires expression of CCR7 and CCR9 and responsiveness to Notch signaling (13, 35, 36). Early T-cell maturation occurs primarily in the thymic cortex through interactions with cortical TECs, which produce chemokines CCL25 and CXCL12, cytokines interleukin-7 (IL-7) and stem cell factor (SCF), and the Notch ligand Dll4 - all essential for thymocyte survival and differentiation (7, 20, 37, 38). At the DN1-DN2 stages, survival is regulated by IL-7 and SCF through their receptors IL-7R and c-kit (CD117), respectively, activating anti-apoptotic Bcl-2 signaling (13, 39). DN2 TLPs (CD4^-CD8^-CD25⁺CD44⁺) proliferate, downregulate CD44, and transition into DN3 cells (CD4^-CD8^-CD25⁺CD44^-), where TCRβ rearrangement occurs and B-cell potential is lost (40, 41). Subsequent DN4 cells (CD4^-CD8^-CD25^-CD44^-) proliferate in the subcapsular zone, migrate to the cortex, and differentiate into double-positive (DP) CD4⁺CD8⁺ thymocytes (13, 42), which express the mature TCRαβ/CD3 complex and undergo negative selection. DP thymocytes then move into the medulla, where positive selection yields single-positive (SP) naïve T cells that subsequently migrate to the periphery (8, 40, 41).

Both αβ and γδ T-cell lineages arise in the thymus from common uncommitted early T-cell precursors (ETPs), derived from bone marrow HSCs and represented by DN1 TLPs (CD44⁺CD25^-CD4^-CD8^-B220^-CD11b^-CD11c^-NK1.1^-TCRβ^-TCRγδ^-), including both CD117⁺ and CD117^-/lo subpopulations (43). Among these, only the CD117⁺ DN1 fraction represents true TLPs capable of progressing to DN2 and DN3 stages and generating DP αβ thymocytes (42). These cells retain limited NK potential (44) and express transcriptional regulators associated with stemness and early T-cell identity (45). Divergence of αβ and γδ T-cell lineages occurs at the DN3 stage (43).

Based on CD24 expression, CD117⁺ DN1 ETPs are subdivided into CD24^- (DN1a) and CD24^lo (DN1b) subsets, thought to have a precursor–progeny relationship (44). Within the ETP pool, a CD63⁺Ly6c⁺ subpopulation has been identified as a granulocyte-committed precursor lacking T-cell potential (43). The γδ lineage is believed to diverge from the main developmental pathway at the DN2–DN3 transition, when TCRβ/γ/δ gene rearrangement occurs; this bipotency is lost by DN3 (46). The current model of early T-cell development posits that ETPs (DN1 thymocytes) represent the most immature thymic population, progressing to DN2 where lineage commitment toward αβ or γδ T cells is initiated, and finalized at DN3 during β-selection (expression of functional TCRβ/pre-Tα complex) or γδ TCR dimer formation (43).

Recent studies show that some γδ T cells may also arise from CD117^- DN1 thymocytes. Single-cell transcriptomic analyses revealed multiple DN1 subpopulations with preferential differentiation toward IL-17- or IFNγ-producing γδ T cells (43). These CD117^- DN1 cells can be further subdivided into CD117^loCD24^hi (DN1c), CD117^-CD24^hi (DN1d), and CD117^-CD24^- (DN1e) subsets (43), previously not considered part of the canonical T-cell developmental pathway. However, it was demonstrated that IL-17-producing γδ T cells derive from Sox13⁺ DN1d thymocytes rather than from ETPs, bypassing the classic ETP–DN2–DN3 sequence (47). Furthermore, TCR signal strength influences γδ T-cell fate: weak signaling promotes IL-17A, whereas strong signaling induces an IFNγ phenotype (48).

A particularly intriguing and underexplored aspect of thymic biology is the existence of radioresistant stem cells found among both TLP and TESC populations (49–64). These radioresistant TSCs can survive and maintain their regenerative capacity even after exposure to lethal irradiation, demonstrating self-renewal, plasticity, and multilineage differentiation potential (7, 49, 53–55, 57), highlighting the critical role of radioresistant TSCs in the early autonomous restoration of thymic architecture following irradiation damage.

Modern studies, focusing on the radioresistance mechanisms of thymic stromal cells and TESCs, demonstrated the activation of DNA repair mechanisms, antioxidant defenses, and stress-response signaling pathways (9, 56–63). It was reported that proteins such as p53 and ATM, as well as antioxidant enzymes like superoxide dismutase, play key roles in cellular protection (56, 59). Additionally, the activation of survival pathways PI3K/AKT and MAPK, along with stress-response regulators ATF4, promotes resistance to radiation-induced apoptosis (60).

As early as the 1960s, it was recognized that certain thymic TLPs exhibit substantial radioresistance (65). In 1975, Kadish and Basch reported the existence of a local population of radioresistant intrathymic TLPs capable of driving post-irradiation thymic regeneration independently of precursors migrated from bone marrow (49). These radioresistant TLPs represented a minor subset of CD4^-CD8^- DN intrathymic TLPs (early referred to as the L3T4^-Lyt2^- TLPs), located primarily in the subcapsular zone of the thymic cortex and capable of differentiating into both CD4⁺ and CD8⁺ T cells (4, 49, 52, 54, 55, 66–70).

While radioresistant intrathymic TLPs have now largely remained outside researchers’ attention in contrast to the stromal-epithelial thymic compartment, two fundamental properties of these TLPs, stem cell-like potential and functional resilience under damaging conditions, underscore their biological significance within the thymic microenvironment. These TLPs contribute to thymic regeneration through cytokine secretion and intercellular interactions, impacting the stromal-epithelial compartment, as well as through their differentiation into mature thymocytes (8, 37, 52, 71–73).

In 1983, we established two transformed thymic cell lines, TC-SC-1/1.1 and TC.SC-1/2.0, with a phenotype characteristic of intrathymic TLPs, expressing stem cell 1 antigen (SC-1) (74). These cell lines, under stimulation with gamma-irradiation, produced an unknown growth activity, identified by us in 1984 and later named a thymocyte growth factor (THGF) (50, 53, 72, 75, 76). These cell lines, as well as their secretory product, THGF, and its target cells were intensively explored from 1983 to 1991, and several additional important data were obtained later, in 1999. The results of these research findings were presented in detail in four doctoral theses, and the main data were published in scientific journals, primarily in Russian-language journals, and remain poorly accessible to broad scientific discussion. TC-SC-1/1.1 and TC.SC-1/2.0 cell lines were registered and stored in the Official Cell Line Collection at the Institute of Cytology, Russian Academy of Sciences (St. Petersburg, Russia) since 1987.

This article aims to conduct a retrospective analysis of our findings and reinterpret them through the lens of modern knowledge in immunology and stem cell biology in the context of the presumable role of THGF activity and its comparison with other cytokines. The updated publication of the combined data may motivate further research, also by independent research groups, which were interrupted due to a range of critical circumstances, and lead to a deeper understanding of TLP radioresistance mechanisms and their functional roles in the endogenous thymic regeneration after damage treatments, especially irradiation injury. In turn, this could open new avenues for therapeutic interventions and radioprotection in the context of thymic dysfunction and thymic-associated immunosenescence. The discussed experimental data are related mainly to the TC.SC-1/2.0 cell line, which was used as the basic experimental model of intrathymic TLPs.

Historically, the SC-1 antigen, initially identified using rabbit antisera against mouse brain, was used as a marker of HSCs and intrathymic TLPs (77–79). In some TLP populations, it was co-expressed with the Thy-1 antigen (80–82). Other TLP identifying markers included glycan receptors for the galactose-specific lectin peanut agglutinin (PNA-R) (67, 83, 84) and interleukin-2 (IL-2) (85), along with the absence of mature T-cell markers L3T4 (CD4) and Lyt-2 (CD8) (37, 86) (Table 1).

Since the anti-SC-1 immune serum was used before the development of anti-SC-1 monoclonal antibodies, it is necessary to clarify the relationship between SC-1⁺ TLPs, identified with the immune antisera and Sca-1⁺ and Sca-2⁺ TLPs, which were identified later with monoclonal antibodies. Sca-1 and Sca-2, named due to their expression by mouse bone marrow stem cells, were also evaluated for expression within the thymus. Sca-1 is expressed by cells in the thymic medulla and by some subcapsular blast cells. Sca-2 expression is limited to the thymic cortex and associated with large cycling thymic blast cells. Both Sca-1 and Sca-2 are expressed on a subpopulation of CD4^-CD8^-TLPs (87).

Sca-1 is currently one of the most commonly used markers of normal mouse stem cells, which was reported as a cell surface marker of HSCs (88, 89) and cells with increased tumorigenic potential (90), suggesting that Sca-1 may be an important factor in the maintenance of malignant stem cells. Sca-1 is an 18-kDa surface protein coded by the Ly6a gene (91). Sca-1 can interact with other proteins on the cell surface to form complex signaling pathways. This protein interacts with the TGF-β receptors and ligands, which modulate the downstream signaling in multiple organs (92). In particular, TGF-β signaling regulates Sca-1 expression, tumorigenicity, and plasticity in the mammary epithelial and cancer stem cells (88). Besides HSCs and TLPs, Sca-1 is expressed on the surface of myeloid cells and peripheral B and T lymphocytes (87–89), that is predominantly CD4⁺ T helper (Th) cells (87). Sca-1⁺ cell population may also serve as progenitors for endothelial, epithelial, and mesenchymal cells (88, 93), suggesting their high heterogeneity and plasticity, as well as multipotency, at least some of them. Sca-1 is involved in the regulation of T and B cell responses and c-kit (CD117) expression, and is believed to play roles in the differentiation, proliferation, and survival of hematopoietic and progenitor stem cells, as well as maintaining their stemness (94).

Sca-2⁺ population is the earliest known intrathymic precursor. High expression of Sca-2 is found at day 14 of mouse fetal development (95). Sca-2⁺ population is characterized by expression of an intermediate level of heat-stable antigen, a very low level of Thy-1, and a high level of CD44 antigens. It is negative for B-cell, granulocyte, macrophage, and erythrocyte markers (B220, Gr-1, Mac-1, and TER-119, respectively) (96). Within the T cell lineage, upregulation of Sca-2 expression coincides with the transition from a multipotential bone marrow stem cell to an intrathymic TLPs, suggesting an important function for Sca-2 in early thymopoiesis (97). The thymic Sca-2⁺ population is similar to bone marrow HSCs in surface antigenic phenotype and is preferentially in an activated state rather than a quiescent cell state (98). Sequence analysis of the Sca-2 protein showed that Sca-2, as well as Sca-1, is a glycosylphosphatidylinositol-anchored molecule that shares some characteristics with the members of the Ly-6 multigene family. Sca-2 is likely identical to the mouse thymocyte shared antigen-1 (TSA-1), as the protein sequence of TSA-1 is the same as that of Sca-2 (97, 99).

Remarkably, the SC-1⁺ TLPs share properties with the Sca-1⁺ and Sca-2⁺ cell populations. Therefore, SC-1⁺ TLPs likely include both Sca-1⁺ and Sca-2⁺ TLP populations and may be presented as Sca1⁺/Sca2⁺ TLPs, which may be analogous also to DN1 ETPs or CD117^-/lo DN1 subtypes (100). In addition, the SC-1⁺ population includes the thymus-committed HSCs, described as the SC-1⁺Thy-1⁺ TLP2 subtype, which may overlap with early bone marrow HSCs, described as the SC-1⁺Thy-1^- TLP1 subtype, but yet thymus-uncommitted (73, 81, 82). CD117 receptor is the ligand for SCF, identifying HSCs, as well as the early intrathymic TLPs/ETPs (100). The thymus-committed CD117^lo HSC subset (likely the SC-1⁺Thy-1⁺ TLP2) is enriched with multipotent precursors and crucial for thymic regeneration and thus, also can be included in DN1 ETPs. Therefore, the SC-1⁺Thy-1^+/- TLP subtype is likely analogous to the combined Sca1⁺/Sca-2⁺ subtype, composing the DN1 ETP population including CD117^-/lo DN1 subtypes and in combination may be submitted as SC-1⁺(Sca-1⁺/Sca-2⁺)Thy-1^+/- CD117^+/- DN1 TLPs/ETPs (Table 1).

In the modern view, the DN ETPs of leukemic cells express at least one stem cell marker (CD34, CD117), and/or myeloid marker (CD11b, CD13, CD33, HLA-DR, CD65). This population consists of at least 25% leukemic blast cells and is characterized by the absence or dim expression of CD5. Additionally, ETPs lack the T-cell maturation markers CD1a and cytoplasmic CD3 (cCD3), while expressing CD7, which is one of the earliest antigens appearing on T-lymphocytes and the clinical marker of acute T-lymphocyte leukemia (101, 102).

The TC.SC-1/2.0 transformed thymic cell line was generated by multiple injections of human IL-2 into BALB/c mice. This treatment led to an increase in SC-1⁺ blast cells in the thymus, which subsequently underwent malignant transformation, possibly facilitated by a naturally occurring C-group lymphotropic retrovirus (74, 103). IL-2 administration increased the SC-1⁺ fraction up to ~10%, correlating with the emergence of cells expressing tumor-associated antigens (TAA) and clonogenic potential, absent in untreated mice (104).

These observations, together with the literature data on spontaneous and chemically induced thymic tumors in AKR mice (105–108), as well as data on acute T-cell leukemia in human (101, 102) suggest that the thymic microenvironment plays a critical role in neoplastic transformation of Thy-1⁺SC-1⁺/Sca-1⁺ TLP2 (likely CD117^+/- CD7⁺ ETPs), targeting them in the transition state from non-activated SC-1⁺/Sca-1⁺CD44⁺CD25^- DN1 to SC-1⁺/Sca-1⁺CD44⁺CD25⁺ activated DN1 state, at which they possibly expressing also Sca-2, before become CD44⁺CD25⁺ DN2 subtype. Lymphotropic retroviruses may contribute to the transformation of susceptible cells at a sufficient viral load in the thymic niche, as well as an adequate quantity of these CD44⁺CD25⁺ DN1 cells (Figure 1).

This suggestion is supported by the observation that transformed TLPs in AKR mice appeared only after thymic migration from bone marrow, with no transformation detected in marrow-resident cells (105, 106). This unique intrathymic niche effect is potentially mediated by stromal-epithelial signals, local cytokines, and cell–cell contacts. At this, the IL-2/IL-2R signaling appears also critical for the expansion of immature radioresistant triple-negative (CD3^-CD4^-CD8^-) intrathymic SC-1⁺ TLPs for post-irradiation regeneration, likely via receptor upregulation, which activates downstream STAT5, PI3K/AKT, and MAPK pathways that promote TLP survival and proliferation (52).

From a mechanistic perspective, SC-1⁺ (Sca-1⁺/Sca-2⁺) DN1-DN2 TLPs may activate anti-apoptotic programs through Bcl-2 and Mcl-1, stress-response pathways including p53/ATM and ATF-4, and antioxidant defenses such as SOD and catalase, collectively supporting survival under genotoxic or oxidative stress (52, 56, 59, 60). These pathways likely confer resilience during irradiation or chronic inflammatory stress, explaining the accumulation of TLPs (likely CD34⁺/CD117^+/- ETPs) in the thymic oncogenesis model. Furthermore, split-dose irradiation, used in cancer therapy, can induce virus-independent malignant transformation of DN TLPs (ETPs), which may be accumulated due to radiation-induced maturation arrest at the triple-negative CD3^-CD4^-CD8^- stage (109). Constitutive secretion of IL-7 and SCF by thymic epithelial cells may further support survival and expansion, potentially facilitating radiation-induced leukemogenesis (7, 110). These observations integrate historical and modern knowledge, providing a framework for understanding thymic TLP transformation and highlighting targets for further experimental validation.

In our studies, the TC.SC-1/2.0 thymic cell line was characterized as L3T4^-Lyt2^- (CD4^-CD8^-) transformed TLPs. This was supported by co-expression of the stem cell marker SC-1 and the T-cell marker Thy-1, along with TAA, in more than 95% of cells during early culture stages (days 80-630) (67). At later stages of culture stabilization, the proportion of Thy-1⁺ cells decreased to 18%, and TAA⁺ cells to 29%, whereas SC-1 expression remained stable (104). Conversely, PNA-R expression was initially detected in 49% of TC.SC-1/2.0 cells and dropped to 12% after treatment with the thymic hormone thymosin (67), confirming the immature and heterogeneous nature of the cell population. In the stabilized culture, PNA-R expression increased to 98%, and SC-1 remained at 94% (104).

Importantly, TC.SC-1/2.0 cells expressed IL-2R and responded to IL-2 stimulation in vitro, features characteristic of activated triple-negative (CD3^-CD4^-CD8^-) TLPs (52) and mature CD4⁺ or CD8⁺ thymocytes after activation (111, 112). However, IL-2R expression and proliferative responses varied with the culture stage after reseeding, correlating with the level of spontaneous proliferation. These oscillations reflect cyclical processes of cell maturation, differentiation, and activation that occur within the TC.SC-1/2.0 population between days 1–4 cultures after reseeding (104).

The single-cell clonal analysis revealed that the major pool of SC-1⁺PNA⁺Thy-1^-CD4^-CD8^- TLPs, corresponding presumably to the activated CD117^+/- Sca-1⁺ DN1 ETPs, remained predominant (about 75-80%) and stable in the composition of the TC.SC-1/2.0 cell line. The remaining cells (20-25%) composed the subpopulations of thymocytes at different stages of maturation, preferably, the CD4⁺ T-cell lineage of development (67, 104). This heterogeneity demonstrates the self-renewing potential of TC.SC-1/2.0 cell line and its ability to undergo cycling differentiation preferably along the CD4⁺ T-helper pathway.

The expression of immunoglobulin receptors characteristic of B cells or Fc receptors (FcR) typical for macrophages, B cells, dendritic cells (DCs), granulocytes, innate lymphoid cells type 3 (ILC3), natural killer T (NKT) cells, or CD8⁺ T cells (113, 114) was not detected in TC.SC-1/2.0 cultures. Moreover, the cells lacked cytolytic activity against mouse thymocytes or ⁵¹Cr-labeled YAC-1 lymphoma targets (67). These data confirm the immature T-lineage identity of the TC.SC-1/2.0 cell line (Table 2).

Continued studies demonstrated that the stable in vitro growth of the TLP-derived TC.SC-1/2.0 cell line required the production of a previously unidentified autocrine growth factor, designated THGF (51, 53, 72, 75, 76). Remarkable, intraperitoneal injection of TC.SC-1/2.0 cells into syngeneic BALB/c mice resulted in the formation of ascitic tumors, while some cells migrated into the thymus and developed thymomas. Furthermore, cell lines established from these tumors displayed contrasting properties (Table 3).

The cell line derived from a thymoma (TC.SC-1/2.0-Th) produced THGF, proliferated actively, and was unresponsive to exogenous THGF but responded to IL-2. These characteristics match those of activated TLPs (DN1→DN2 stage) and the original TC.SC-1/2.0 cell line. In contrast, the cell line established from an ascitic tumor (TC.SC-1/2.0-As) neither produced THGF nor responded to IL-2, exhibited slow spontaneous proliferation, but responded strongly to exogenous THGF, especially in combination with IL-2 (104), likely presenting dormant TLPs similar to those which are target cells for THGF within freshly isolated thymocyte populations.

Since the thymus allows entry only to early intrathymic TLPs and committed SC-1⁺Thy-1⁺CD4^-CD8^- bone marrow HSCs (similar to CD117^+/- DN1 ETPs), stimulated by thymic hormones such as thymosin, but restricts the entry of mature T cells (13, 49, 81), these findings support the primary stem-cell nature of the THGF-producing TC.SC-1/2.0 cell line. Its base phenotype likely corresponds to activated SC-1⁺Thy-1^+/-PNA⁺CD25⁺CD4^-CD8^- DN1→DN2 TLPs. Moreover, these findings indicate both autocrine and paracrine functions of THGF, as well as the symbiotic coexistence of at least two interacting cell populations within the TC.SC-1/2.0 cell line composition.

Interestingly, spontaneous and induced thymic tumors in AKR mice also contain cells with varying degrees of differentiation and thymic tropism (105, 108), similar to the TC.SC-1/2.0 TLP line, suggesting common targets and transformation mechanisms underlying lymphoid-type thymic tumors across different mouse strains.

In contrast to intraperitoneal injection, subcutaneous injection of the TC.SC-1/2.0 cell line in syngeneic mice produced local transplantable solid tumors without migration into thymus, when they were returned to culture in vitro (104), confirming their transformed nature.

Notably, the phenotypic heterogeneity of TC.SC-1/2.0 cell line correlated with karyotypic heterogeneity. Particularly, the major cell population remained stable with a modal class of 48 chromosomes, while minor populations demonstrated a chromosomal range from 42 to 52 (the normal mouse karyotype is 40 chromosomes) (103, 104).

This phenotypic and karyotypic heterogeneity suggests creating an in vitro microenvironment that mimics intrathymic conditions and supports complementary autocrine and paracrine interactions within the TLP sublines. Considering the presence of two cell populations with opposing THGF secretion and responsiveness, it is plausible that during TC.SC-1/2.0 cell line evolution a dynamic cooperation emerged between activated THGF-producing DN1-DN2 SC-1⁺CD25⁺ TLPs with high proliferative potential and autocrine THGF utilization, and DN1 SC-1⁺ CD25^- dormant TLPs, activated by THGF in a paracrine manner and maintained as multipotent stem cells. These DN1 cells may subsequently give rise to other subpopulations, progressing through DN2, DN3, and DN4 TLP stages up to DP CD4⁺CD8⁺, and to limited SP thymocytes, which may reflect the abnormal differentiation patterns of transformed TLPs or the absence of sufficient in vitro microenvironmental signals required for conventional cell maturation and differentiation.

Remarkably, the cells of TC.SC-1/2.0 line were capable of producing THGF spontaneously, and its production was markedly enhanced by γ-irradiation, but not by stimulation with mitogens. Among the tested irradiation doses (3–24 Gy), only doses in the range of 10–15 Gy (optimally 12 Gy) strongly activated THGF production (50, 53, 72, 104) (Table 4, Exp. 1, 2). At this, both spontaneous and irradiation-induced THGF secretion by TC.SC-1/2.0 cell line depended on the culture age and cell cycle phase after passage and transfer from serum-containing to serum-free medium. The spontaneous THGF production was minimal in young actively proliferating 1-day cultures. Maximum THGF secretion occurred in 2-day cultures, corresponding to the mature growth phase at the peak cell density. THGF levels declined sharply in 3-day (aging) cultures, and THGF production was virtually absent in 4-day degenerating cultures (53, 72, 104) (Table 4, Exp. 4).

In all culture stages, the level of irradiation-induced THGF secretion substantially exceeded that of spontaneous production. This enhancement reflected the activating effect of the specific 12 Gy dose on de novo THGF synthesis, rather than the release of pre-formed factor from damaged or dying cells. Several observations support this conclusion: a) irradiation doses below or above 12 Gy were far less effective in inducing THGF despite comparable rates of cell death; b) the death rate of irradiated cells only slightly exceeded that of non-irradiated controls in serum-free conditions (53, 72, 104) (Table 4, Exp. 2).

The 48-hour dynamics of THGF secretion after irradiation also substantiated this suggestion (72, 104). Immediately after exposure, cell viability decreased dramatically, while no THGF activity was detected in the supernatant. Minimal THGF activity appeared 4 hours post-irradiation, coinciding with a further drop in cell viability. Over the next 20 hours, THGF activity in the culture medium increased sharply, reaching a maximum at 28 hours, while cell viability remained relatively constant at approximately 27%. Finally, cell viability declined by 48 hours from 80% to 10%. However, no further growth of THGF activity was observed after 28 hours of culturing (Table 4, Exp. 3).

Remarkably, when these irradiated cells were reseeded again into fresh serum-free medium after 24 hours of culturing, no THGF activity was detected in this new supernatant, confirming that THGF is not stored intracellularly but synthesized and secreted actively in response to irradiation (72). These data also indicate a 4-hour latent phase followed by 16 hours of active THGF synthesis and secretion, with minor continued accumulation up to 28 hours. The residual increase likely reflects incomplete synchronization of the irradiated culture before seeding. The data also suggest that approximately 30% of the TC.SC-1/2.0 cell population is active radioresistant THGF producers. Remarkably, under comparable conditions, γ-irradiation did not stimulate IL-2 secretion by the EL-4 cell line (53, 72, 104), emphasizing fundamental differences between THGF- and IL-2-producing cells, as well as between these cytokines themselves.

During the formation of the TC.SC-1/2.0 cell line, a distinct pattern emerged in the ratio between spontaneous and irradiation-induced (12 Gy) THGF secretion. THGF activity was first detected in conditioned medium on day 77 of the cell line formation. Up to approximately 550 days, spontaneous THGF production remained low but increased markedly following irradiation. With further stabilization, spontaneous THGF secretion rose progressively and eventually equaled the level of irradiation-induced production (50, 53, 72, 104). These irreversible changes reflected evolutionary stabilization and cellular diversification within the TC.SC-1/2.0 cell line, during which continuous THGF production functioned likely as a key autocrine-paracrine factor in selective cell survival and expansion.

Collectively, these findings suggest that THGF is not stored in intracellular depots but secreted constitutively during synthesis, similar to most thymic cytokines, and utilized quickly by proliferating cells, likely in autocrine - paracrine manner.

Retrospective analysis of our early published data, summarized in this review, generally supports our conclusions regarding the nature and novelty of THGF and the unique properties of its target cells, presumably dormant radioresistant intrathymic stem cells. This analysis also clarifies the most probable position of THGF-producing and THGF-responding TLPs within the modern intrathymic hierarchy, placing them at the stage of non-activated DN ETPs with the integrated phenotype CD117^-Thy-1⁺Sca-1⁺CD44⁺CD25^-CD4^-CD8^-, which, under THGF activation, express functional IL-2R and, presumably, c-kit receptor for SCF, transferring to DN1-DN2 stages (Figure 3).

The properties of the THGF-dependent long-term STh-870 cell line suggest that THGF induces not only robust DNA synthesis but also intracellular formation of new daughter cells. These processes occur predominantly within the first 15 days after dormant stem cells receive the THGF signal. During this period, the most intense wave of DNA synthesis is observed, yet without an increase in cell number. And what is critically important, this synthesis is fully insensitive to colchicine. At the end of this phase, only single blast-like cells remain visible in culture; however, over the subsequent 10–15 days, they give rise to rosettes and, later, clusters of 30–50 daughter cells. This second phase is accompanied by a moderate increase in DNA synthesis and a rise in viable cell numbers.

The intracellular generation of new THGF-dependent cells may be advantageous under extreme conditions, such as radiation injury, where classical mitosis is impaired. Morphological comparisons, coupled with the colchicine-resistant proliferative response, suggest that large “mother” cells serve as primary recipients of THGF signals, within which daughter cells are formed. In this context, the mother cell functions as a germinal-like center or a spheroid niche, enabling the protected formation of new cells. Such a mechanism could account for the absence of colchicine effects on THGF-driven proliferation, implying that dormant stem cells employ this strategy for rapid, protected expansion before further differentiation.

The proposed mechanism of intracellular or intra-spheroid generation of new daughter cells may involve a combination of defended mitosis or amitotic division in mother cells and defended mitosis or endomitotic processes in daughter cells. This hypothesis helps explain both the colchicine insensitivity and the pronounced radio resistance of THGF-responding cells, as amitotic and endomitotic division are inherently less vulnerable to radiation-induced damage. Such mechanisms may have evolved as ancient anti-radiation strategies preserved in primitive dormant stem cell populations.

Taken together, these observations suggest that mother cells in long-lived THGF-dependent cultures, as well as analogous radioresistant cells, are likely dormant self-renewing intrathymic CD4^-CD8^- multipotent stem cells at the DN1 ETP stage, which can be activated either by THGF or irradiation. Their progeny, the smaller cells, correspond to the DN2-DN4 transition stages. After activation by THGF, these TLPs may acquire responsiveness to SCF, IL-2, and IL-7 and potentially may generate immature DP CD4⁺CD8⁺ and SP CD4⁺ and CD8⁺ thymocytes. However, their continued differentiation requires additional cytokines (IL-2, IL-4, IL-7) and interaction with stromal–epithelial microenvironment that are absent in vitro, eventually leading to the cluster degradation (Figure 3).

Although the concept of intracellular formation of daughter-cell pools may seem unrealistic, it is supported by several analogous observations. Thymic epithelial nurse cells are known to internalize immature CD4⁺CD8⁺ thymocytes and provide intracellular sites for TCRγδ cell maturation (150, 151). Similar rosette-like structures include thymic rosettes formed by thymocytes associated with intrathymic macrophages or dendritic cells (152, 153). While we did not find these cell types in THGF-responding long-term cultures, their possible involvement in THGF-dependent growth and cluster formation should also be brought to attention. Thus, verification of the single cells in the clusters remains a subject of further studies and debate, as well as the nature of “mother cells”.

The proliferative response of the long-lived THGF-dependent STh-870 line to THGF, interleukins, and mitogens was fundamentally similar to those of freshly isolated thymocytes, irradiated long-lived thymocytes, or thymocytes pre-incubated with THGF. However, this response was time-dependent after the addition of a fresh THGF portion, and the level of spontaneous proliferation, which supports the conclusion that THGF induces the expression of high-affinity receptors for other cytokines, and the activation of the THGF signaling pathway is a priority in the chain of further events.

Our experimental results show that the target cells of THGF belong to a radioresistant intrathymic stem-cell population with a D₀ exceeding 50 Gy. Irradiation presumably induces the activation of dormant radioresistant DN1 TLPs and secretion of endogenous THGF by these cells, which then regulates their proliferation via an autocrine loop and induces their transition to the CD25⁺ DN2 stage, further controlled by IL-2 and IL-7. This interpretation is consistent with evidence that radioresistant DN2 TLPs proliferate after irradiation in an IL-7-dependent manner, generating conventional thymocytes and using the IL-2/IL-2R pathway during progression (52, 85, 140).

As is known, some DN1-DN2 TLPs possess multipotent potential and can generate not only T cells but also NK cells (154), dendritic cells (155), macrophages, and B cells (156). These cells may secrete IL-7, SCF, and other cytokines that support thymic regeneration in vivo after irradiation. These findings also assume the possibility that THGF-dependent, or macrophage- or dendritic cell-associated rosette-like structures could arise through self-organization from single multipotent TLPs due to their plasticity and multipotency.

A recently described subpopulation of radioresistant TECs (58, 59, 62) may also contribute to the postirradiation restoration of the thymic function by producing essential cytokines and providing signaling pathways for intercommunication with radioresistant TLPs (57, 60, 61), as well as producing chemokines such as CCL19, CCL21, and CCL25, which are important for the attraction of TLPs (20, 122).

Furthermore, innate lymphoid cells 3 (ILC3) and T helper 17 (Th17) cells produce a cytokine (IL-22) that is crucial for the thymic epithelial compartment recovery after high-dose chemotherapy or irradiation damage (136–138). Defects in IL-22 production delay thymus recovery in irradiated mice and decrease the expression of genes Foxn1, Aire, and Kgf, associated with thymic function. In contrast, the administration of IL-22 facilitates the repair of TECs, increases the number of T cells, increases the level of Aire, and increases the proportion of natural regulatory T cells in the thymus (139).

Notably, following total body irradiation or targeted thymus irradiation, which leads to crucial depletion of DP thymocytes, the level of intrathymic IL-22 has been increased, suggesting a link between IL-22 and mechanisms of endogenous recovery (136, 138, 157) similar to the effect of THGF on TLPs. Furthermore, production of IL-22 following damage is related to radioresistant innate thymic LTi/ILC3 cells, whose number increases following thymic insult (20, 136). This is also similar to the THGF effect, increasing SC-1⁺ TLPs in the thymus upon injection into mice (104).

These data suggest the need for further evaluation of THGF-responding/producing cells in comparison with the LTi/ILC3 population, as well as additional identification of THGF, THGF-specific receptors, and key molecular regulators of the THGF-signaling transduction pathways.

The collective evidence presented in this review supports the concept that THGF is a key regulator of early intrathymic stem cells, playing an initiating role in the regeneration of the lymphoid compartment under stress conditions.

THGF-producing and THGF-responding cells are likely localized at the earliest DN1/DN2 stages of thymocyte development and display properties characteristic of primitive, radioresistant intrathymic stem cells.

Their unique response to THGF distinguishes THGF-driven proliferation from classical cytokine-induced mitosis.

THGF presumably initiates the earliest step in a hierarchical sequence of cytokine responsiveness, enabling subsequent sensitivity to IL-7, SCF, IL-2, and other mediators of thymocyte expansion and differentiation.

The ability of γ-irradiation to trigger THGF secretion further highlights its physiological relevance in thymic repair.

Insights into THGF-dependent mechanisms of radio resistance in parallel with recent discoveries concerning radioresistant TEC subsets and LTi/ILC3 populations that contribute to post-irradiation thymic recovery, suggesting that THGF may operate within a broader reparative network.

The proposed models of daughter cell generation in the conditions of defended mitosis or amitotic/endomitotic cell division provide a reasonable explanation for both the exceptional radio resistance and the morpho-proliferative peculiarities of THGF-sensitive TLPs, as well as unresponsiveness to colchicine effect, potentially representing an evolutionarily conserved mechanism for maintaining thymopoiesis and possible hematopoiesis under extreme conditions.

The properties of THGF to stimulate the development of colonies/clusters in long-lived THGF-dependent thymic cultures, as well as colony-formation in spleen, suggest its similarity to other colony-stimulating factors, such as SCF, GM-CSF, and IL-3. However, a range of parameters suggests an independent role of THGF extended beyond the thymus and requires further verification.

Further molecular identification of THGF and its receptors in long-lived thymic cell cultures, induced by irradiation and THGF-associated signaling pathways, as well as of these radioresistant cells nature and interaction of THGF with other cytokine pathways, may provide essential information for verification of THGF biology and understanding of thymic homeostasis in the context of the thymus recovery after irradiation and other injuring actions, providing new potential for developing therapeutic approaches to immune system reconstitution.