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αβTCR signaling acts as a tumor suppressor
regulation of the Vβ5.1 transcript (Figure 1D). This sug- gests an active counter-selection of leukemic (or pre- leukemic) thymocytes bearing the transgenic TCR. The latency of tumor onset was significantly increased in [OT- II x Rag1–/– x Ptendel] mice compared to Ptendel mice, consis- tent with the time that is likely required for selection of TCRneg cells, while in the enforced absence of TCR ([ Rag1–/– x Ptendel] mice), latency was significantly reduced (Figure 1E), evoking a potential tumor suppressor role of TCR signaling in leukemogenesis.
To rule out transgenic-specific effect, we also tested the impact of selection in the H-Y mouse model, which expresses a transgenic TCR recognizing the male H-Y antigen in the context of MHC-I molecules. In female H- Y mice, negative selection is not operating, and positively selected mature H-Y+ TCR T cells differentiate as CD8 SP.20 H-Y mice were crossed with Ptendel mice, on a Rag- proficient background to allow non-transgenic TCR com- petitive formation and development. [H-Y x Ptendel] females developed TCRαβ+ T-ALL (Figure 2A and B). Remarkably, tumors were typically CD4 SP (never CD8 SP), and none of them expressed the transgenic H-Y TCR (Figure 2B). Thus, regardless of the model used, the expression by (pre-)tumoral thymocytes of a fit TCR is counter-selected during T-ALL development. In disease- free thymi and spleens of young female [H-Y x Ptendel] mice (and thus before clinical tumor manifestation), we detected a severe reduction of H-Y+ TCR cells at the CD8 SP stage compared to control H-Y mice, and this was already apparent at the DP stage of thymocyte develop- ment (Figure 2C). In addition, this counter selection of H- Y+ thymocytes occurs post β-selection, after immature sin- gle positive (ISP) stage (Online Supplementary Figure S4).
These data suggest that Pten-deficient H-Y+ TCR thy- mocytes are eliminated instead of positively selected, and that this counter-selection occurs before full malignant transformation, preventing leukemia development.
Disruption of final maturation of Pten-deficient SP cells
Since most mature thymocytes with fit H-Y TCR are eliminated in young female disease-free [H-Y x Ptendel] mice, we analyzed the remaining H-Y TCR negative SP cells, using the CD69, CD62L and CCR9 markers of T-cell differentiation. Developmental sequence of CD4 SP thy- mocyte maturation is usually described as SP1 (CD69+,CD62LLow/med, CCR9+), SP2 (CD69+, CD62LLow/med, CCR9Neg) and SP3 (CD69Neg, CD62LHigh, CCR9Neg).21,22 For CD8 SP cells, the most immature cells are CD69+ and CD62LLow/med while the more mature are CD69Neg and CD62LHigh. We observed for both [H-Y x Ptendel] and Ptendel models a partial block of positively selected cells at the immature CD69+CD62Llow stage (Figure 2D). Such differ- entiation arrest could provide an additional opportunity for malignant transformation.
Fit TCR signaling acts as a tumor suppressor
With the premise that Pten-deficient cells with fit TCR are counter-selected, we next assessed whether cells carry- ing low affinity (unfit) TCR were prone to leukemia devel- opment. OT-II TCR originates from CD4+ I-Ab-restricted T-cell hybridoma,18 thus positive selection is optimal in I- Ab/b background and sub-optimal in I-Ab/d background. C57BL/6 (I-Ab) [OT-II x Rag1–/– x Ptendel] mice were crossed with BALB/C (I-Ad) mice to generate Rag-proficient [OT-II x Ptendel] mice on I-Ab/d background. In the spleens from OT-II control mice, percentages of CD4+ T cells dropped from approximately 78% in syngeneic I- Ab/b background to approximately 7% in allogenic I-Ab/d background (Figure 3A). [OT-II x Ptendel] Rag-proficient mice developed T-ALL with a similar latency as Ptendel mice (approx. 11 weeks), irrespective of the backgrounds (I-Ab/b or I-Ab/d). Leukemic blasts in the spleen were mostly CD4+ (Table 1). On the I-Ab/b background, while all T-ALL analyzed (n=9) expressed a TCRαβ, none of them
AB
Figure 3. Thymocytes harboring unfit TCRαβ sig- naling develop T-cell acute lymphoblastic leukemias (T-ALL). (A) Flow cytometry analysis of typical spleens from [OT-II x Ptenflox] (Control) and tumoral [OT-II x Ptendel] mice bred either on I-Ab/b (left) or I-Ab/d (right) backgrounds (1 representative of n=9). Leukemic cells from [OT-II x Ptendel] I-Ab/b mouse was further screened by cytometry using a Vβ panel and (B) the result indicates that this T- ALL expresses a TCRVα2Vβ14 receptor (see also Table 1).
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