Communicated by Ernest Beutler, The Scripps Research Institute, La Jolla, CA, July 8, 1997 (received for review May 5, 1997 ABSTRACTHuman hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid͞scid (NOD͞SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34 ؊ CD19 ؉ (B-lymphoid) and CD34 ؉ (myeloid) colonyforming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains Ϸ5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38 ؊ and CD38 ؉ subsets of CD34 ؉ CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34؉ CD38؊ human CB cells in serum-free medium containing f lt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD͞SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.

Tisagenlecleucel is an autologous anti-CD19 chimeric antigen receptor-T cell therapy with clinically meaningful outcomes demonstrated in patients with relapsed/refractory (r/r) B-cell lymphoma. In a previous pilot study of tisagenlecleucel in r/r follicular lymphoma (FL), 71% of patients achieved a complete response (CR). Here we report the primary, prespecified interim analysis of the ELARA phase 2 multinational trial of tisagenlecleucel in adults with r/r FL after two or more treatment lines or who relapsed after autologous stem cell transplant (no. NCT03568461). The primary endpoint was CR rate (CRR). Secondary endpoints included overall response rate (ORR), duration of response, progression-free survival, overall survival, pharmacokinetics and safety. As of 29 March 2021, 97/98 enrolled patients received tisagenlecleucel (median follow-up, 16.59 months; interquartile range, 13.8-20.21). The primary endpoint was met. In the efficacy set (n = 94), CRR was 69.1% (95% confidence interval, 58.8-78.3) and ORR 86.2% (95% confidence interval, 77.5-92.4). Within 8 weeks of infusion, rates of cytokine release syndrome were 48.5% (grade ≥3, 0%), neurological events 37.1% (grade ≥3, 3%) and immune effector cell-associated neurotoxicity syndrome (ICANS) 4.1% (grade ≥3, 1%) in the safety set (n = 97), with no treatment-related deaths. Tisagenlecleucel is safe and effective in extensively pretreated r/r FL, including in high-risk patients.

SummaryA high proportion of the CD34+CD38 -cells in normal human marrow are defined as longterm culture-initiating cells (LTC-IC) because they can proliferate and differentiate when cocultured with cytokine-producing stromal feeder layers. In contrast, very few CD34+CD38 -cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC), although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38 -cells in the latter type of culture showed that Flt3-1igand (FL), Steel factor (SF), and interleukin (IL)-3 were both necessary and sufficient to obtain an N30-fold amplification of the input LTC-IC population within 10 d. As single factors, only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly, a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of >500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38 -cells. However, this required the presence oflL-6 and/or granulocyte/colonystimulating factor and/or nerve growth factor 13 in addition to FL, SF, and IL-3. Also, for this response, the most potent single-acting factor tested was IL-3, not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facihtate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols.