CD1a⁻ T-ALL cells from poor outcome T-ALL are DEX-resistant. (A) T-ALL (5178) was a diagnostic sample from a patient who did not experience clinical relapse. NSG mice were injected intrafemorally with 5 × 10⁵ 5178 T-ALL cells in the RF. Engraftment was confirmed 12 weeks later by left femur BM aspirate showing more than 25% human CD45⁺ cells. NSG recipients were treated with DEX or DSMO vehicle daily for 14 days. Flow cytometric analysis of BM cells recovered 1 week after completion of treatment was performed. Panels show human CD45⁺ cells from xenografts (top row contour plots) coexpressing CD1a (bottom row histograms). Light gray outline in histograms represents isotype control, dark gray outline represents T-ALL sample stained with PE-conjugated anti-CD1a antibody. T-ALL 5178 engrafted well in NSG xenograft (top row, left panel) and DEX treatment resulted in elimination of most leukemia cells from treated NSG1 and NSG2 xenografts (top row, middle and right panels). The high percentage of CD1a⁻ cells in the patient sample (bottom row, left panel) was maintained in nontreated (bottom row, middle left panel) and DEX-treated xenografts (bottom row, middle right and right panels). (B) T-ALL 90205 was a diagnostic sample from a patient who later relapsed. NSG mice were injected intrafemorally with 5 × 10⁵ T-ALL cells. Engraftment was confirmed 12 weeks later by left femur BM aspirate showing more than 25% human CD45⁺ cells. NSG xenografts were treated with DEX or DSMO vehicle daily for 14 days. The mice were killed 1 week after completion of treatment. Flow cytometric analysis of patient and xenograft BM cells recovered 1 week after treatment revealed CD45⁺ cells (top row contour plots) coexpressing CD1a (bottom row histograms). Light gray outline in histograms represents isotype control, dark gray outline represents T-ALL sample stained with PE-conjugated anti-CD1a antibody. The patient T-ALL had a high percentage of CD1a⁺ cells (bottom row, left panel) that was maintained in the NSG (bottom row, middle left panel) and DMSO-treated (bottom row, middle right panel) xenografts. The DEX-treated NSG xenograft showed a high percentage of residual leukemia cells (top row, right panel) that did not coexpress CD1a (bottom row, right panel). (C) Flow cytometric analysis was used to determine the percentage of CD1a⁻ cells in patient T-ALL compared with nontreated, DMSO-treated, and DEX-treated NSG xenografts. This was multiplied by the total cell dose to determine the number of CD1a⁻ cells injected into NSG recipients (starting dose) or the total BM cell counts to determine the absolute number of CD1a⁻ cells in nontreated, DMSO-treated, and DEX-treated NSG xenografts. Each dot represents the total number of CD1a⁻ cells from each xenograft, with the bar indicating the mean percentage for each treatment group. The number of CD1a⁻ cells in the BM of DMSO- and DEX-treated xenografts increased compared with nontreated xenografts. (D) PCR-based analysis of TCRG gene rearrangements of patient 90205 T-ALL compared with DEX-treated T-ALL NSG xenografts. The arrow indicates Vγ9,11+Jγ1/2 PCR product found in the 90205 patient sample that was not found in the treated recipients. (E) PCR-based analysis of TCRG gene rearrangement of 90205 patient T-ALL compared with sorted 90 205 CD34⁻CD1a⁻ and CD34⁺CD1a⁻ subsets, revealing PCR products of identical size in the CD1a⁻ subsets with the patient sample.