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. 2022 Jul 5;119(27):e2118529119.
doi: 10.1073/pnas.2118529119. Epub 2022 Jun 29.

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

Kimberly G Laffey  1 , Robert J Stiles  2 , Melissa J Ludescher  2 , Tessa R Davis  2 , Shariq S Khwaja  2 , Richard J Bram  2   3 , Peter J Wettstein  2   4 , Venkataraman Ramachandran  5 , Christopher A Parks  2 , Edwin E Reyes  2 , Alejandro Ferrer  2 , Jenna M Canfield  1 , Cory E Johnson  6 , Richard D Hammer  6   7 , Diana Gil  1   5   8 , Adam G Schrum  1   5   8
Affiliations

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

Kimberly G Laffey et al. Proc Natl Acad Sci U S A. .

Abstract

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.

Keywords: MHC; NOTCH; T-ALL; TCR; leukemia.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
EADN thymocytes exist naturally in mouse and human. Flow cytometry analysis of thymocytes used to identify EADN cells in (Α) B6 murine thymus and (B) human thymus, where the right-most offset panel represents TCRβ x Side Scatter for CD8 single-positive thymocytes used to identify mature TCRβ surface level. Numbers represent % cells in the gate. (C) Quantification of EADN cells in mice with different genetic lesions affecting T cell generation. One-way ANOVA with Tukey’s posttest, **P < 0.01 ***P < 0.001.
Fig. 2.
Fig. 2.
OT-1 TCR transgenic system provides a model for EADN cell development and response to MHC. (A) Representative flow cytometry analysis of surface CD25 and c-Kit expression from wild-type B6 and OT-1 thymocytes gated on CD4−CD8a− DN, EADN cells. Average frequencies of each subset from 4 mice are presented with SEM. (B) Flow cytometry analysis for surface expression of CD34, CD5, CD1a, and CD28 when gating on human EADN cells. After gating out non-T lineage cells, EADN cells are identified as CD4− CD8a− CD45dim CD7+ αβTCR+. Numbers represent % cells in the gate. One-way ANOVA with Tukey’s posttest. (C) Nur77 expression in EADN cells of wild-type or MHC knockout (I-Ab-/-.b2m-/-), non-TCR transgenic mice, shown as median fluorescence intensity (mFI) from intracellular staining and flow cytometry. Unpaired, two-tailed Student’s t test. (D) OT-1.RAG2−/−.β2M−/− fetal thymic lobes were cultured in the presence or absence of antigenic peptides with exogenously added β2M for 24 h. CD69 up-regulation is shown for stringently gated CD4−CD8− DN cells. Each point represents an individual thymic lobe. (E) SPADE of B6 Thy1+ DN thymocytes. Thymocytes are pregated as Thy1.2+CD4−CD8α− before performing SPADE using 12 other surface markers (Materials and Methods). **P < 0.01 ***P < 0.001.
Fig. 3.
Fig. 3.
EADN represents phenotypically earliest stage in a human T-ALL case. (A) Surface CD4/CD8 profiles of peripheral blood T cells from 2 T-ALL patients. (B) CD8SP, variable CD4/CD8 expressing, and DN cells are further analyzed for surface CD3 expression. CD7 is used as a T-lineage marker. Quadrants are set using isotype controls. (C) Expression of CD45 on neoplastic and healthy T cells in the CD7+CD3+ gated cells from (B). Numbers in flow cytometry plots represent % cells in respective gates. (D) Immunohistochemistry staining for αβTCR (Left) and γδTCR (Right) of patient cells (bone marrow, original magnification ×600).
Fig. 4.
Fig. 4.
Spontaneous EADN T-ALL transformation in OT-1 mice. (A) Lymphoid organs in leukemic mice are enlarged compared to healthy littermates. Top to Bottom: spleen, thymus, gut lymph nodes, various peripheral lymph nodes. (B) Frequency of T-lineage cells (Thy1.2+) in healthy OT-1 littermate (Left) and primary leukemic (Right) spleens. (C) Flow cytometry analysis comparing the size (FSC) and granularity (SSC), (D) OT-1 TCR surface expression, and (E) CD4/CD8 expression of healthy OT-1 and (F–H) primary OT-1 T-ALLs assessed in splenocytes. Numbers in flow cytometry plots represent frequency of cells in respective gates. (I) Western blot of lysates prepared from wild-type B6 thymocytes, CBP-null thymocytes, OT-1 T-ALL tumors as named, and an OT-1 T-ALL tumor cell line grown in vitro (Tot-1si). Lysates were immunoblotted for NOTCH1 expression. Actin was assessed as a loading control, where B6 thymocytes were intentionally overloaded to amplify NOTCH1 detection from that sample. (J) Tot-1si cells (2.5 × 105) were seeded and cultured in the presence of DMSO vehicle control or the gamma-secretase inhibitor, Compound E. Unpaired, two-tailed Student’s t test, ***P < 0.001.
Fig. 5.
Fig. 5.
OT-1 T-ALL transformation occurs at the immature, EADN stage. Hierarchical clustering of gene expression data of Tot-1si leukemic cells (label highlighted in blue) and wild-type progenitor and mature T cell subsets (31). Branches of subsets that bear the closest similarity to the tumor represent immediate-post-β-selection stages, and their branches are colored blue. DN, CD4−CD8− double negative. ETP, early thymic progenitor. ISP, immature single positive. DP, CD4+CD8+ double positive. NKT, natural killer T cell. GDT, γδ T cell. SP, single positive.
Fig. 6.
Fig. 6.
EADN-stage OT-1 T-ALL cells are capable of coreceptor independent MHC engagement. (A) Tot-1si was derived from the most immature-appearing tumor cells adapted to culture after serial transplantation in B6 recipients. Tot-1si was analyzed for surface CD4/CD8 and OT-1 TCR expression. Tot-1si DN subset categorization was determined by CD44/CD25 expression, and post-β-selection status was determined by CD28 expression level. (B) Surface level of CD69 induced in Tot-1si cells incubated with null (FARL), weak (Q4H7), or strong (OVA) antigenic peptides presented by APCs. Cells were cultured overnight with 2 μM peptide and APCs, with anti-CD8 blocking antibody or irrelevant IgG control. Numbers in flow cytometry plots represent frequency of cells in respective gates. Error bars represent SEM. Unpaired, two-tailed Student’s t test, *P < 0.05 ***P < 0.001.
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