Early expression of mature αβ TCR in CD4−CD8− T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations

During normal T cell development in mouse and human, a low-frequency population of immature CD4⁻CD8⁻ double-negative (DN) thymocytes expresses early, mature αβ T cell antigen receptor (TCR). We report that these early αβ TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αβ T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy arising from transformed immature T cell precursors (1). It represents ∼15% of pediatric and ∼25% of adult ALLs (2). Identification of subgroups with varied biological features, including overall relapse risk and responses to standard therapies, has allowed stratification of patients to the most appropriate therapeutic regimens that maximize efficacy, and has led to generally improved survival. However, prognosis remains poor for patients with treatment-refractory primary disease or relapse (3, 4). Clinical T-ALL can show inter- and intrapatient heterogeneity in the differentiation stage of tumor cells, implying that multiple pathways of cancer development exist (1, 5). Despite the heterogeneity, a unifying oncogenic network hub required for most or all T-ALL in humans and mice is hyperactivated (often mutated) NOTCH (6). Among the best understood causal drivers is developmentally early CD3 signaling at the pre-T cell antigen receptor (TCR)/γδTCR lineage bifurcation checkpoint, without a role for major histocompatibility complex (MHC)-based ligand (7–9). In contrast, a requirement for MHC and mature αβTCR to drive thymic leukemogenesis, resulting in mutant NOTCH-bearing tumors, has not been previously demonstrated.This makes sense based on the known relationship between T-ALL and T cell development. While there is overlap in NOTCH and developmentally early CD3 signals, cessation of NOTCH prior to MHC-restricted positive/negative selection signals mediated by αβTCR largely prevents simultaneous activity of the two receptors. Most conventional thymocytes rearrange first TCRβ and later TCRα loci in separate, ordered developmental stages. NOTCH signaling is required for early CD4⁻CD8⁻ double negative (DN) thymocyte development (10) while rearrangement of TCRβ and pre-TCR expression mediate β-selection, clonal expansion, and advancement to CD4⁺CD8⁺ double-positive (DP) stage (11). NOTCH signaling is then turned off (12), while DP thymocytes rearrange TCRα, express mature αβTCR, and test self-peptide-MHC reactivity in positive/negative selection (13).However, outside of this well-described sequence of events, a low-frequency, natural subset of DN thymocytes was once shown to rearrange and prematurely express the full αβTCR in wild-type mice (14). The cells were first detected in pre-Tα^−/− mice, where early TCRα replaced pre-Tα to provide β-selection signaling to generate DP cells. Conventional αβ T cell development potential was retained as proven in positive selection assays, but subsequent to the initial report little information on biological roles for these cells has followed. While pursuing developmental stages and signals in T-ALL leukemogenesis, we found that early αβTCR-expressing DN (EADN) cells can be generated in mouse and human thymus at a similar rate, and they are susceptible to T-ALL transformation in both species. We present a mouse model in which EADN oncogenesis requires MHC to drive development of T-ALL bearing NOTCH-mutations, highlighting a novel developmental state with unique signaling rules for a cancer phenotype that appears to be represented in the clinical, human disease spectrum.