Generation of adult human T-cell progenitors for immunotherapeutic applications.

To the Editor:

Prolonged T-cell deficiency with subsequent high risk of infection and relapse is a major complication of non-HLA identical hematopoietic stem cell transplantation (HSCT). Complete restoration of a polyclonal T-cell repertoire takes up to 2 years and might never reach pretransplantation levels. Even in T cell–replete HSCTs, homeostatic cytokines trigger a first peripheral wave of CD8+ T cells, leading to a skewing of the T-cell receptor (TCR) repertoire, and reconstitution of a fully functional T-cell compartment relies on production of new naive T cells. Adoptive transfer of in vitro–generated T lymphoid precursors provides a promising approach to overcome this hurdle and ameliorate T-cell reconstitution after HSCT.

Considering the known role of Notch signaling in T-cell lineage differentiation in mice and human subjects, we have implemented a feeder cell–free culture system based on the immobilized Notch ligand delta-like (DL) 4 in combination with a specific set of cytokines. Compared with previously described feeder-free systems, our DL-4 culture is more efficient and highly reproducible when applied to cord blood (CB) hematopoietic stem and progenitor cells (HSPCs). Moreover, our system is currently in the process of being approved for clinical application.

Mobilized peripheral blood (mPB) is currently the main source of HSPCs in allogeneic HSCTs because adult HSPCs are available in large quantities and exhibit several advantages over CB grafts in the clinical setting. However, their intrinsic properties of differentiation, survival, and proliferation have been investigated poorly thus far. Here we demonstrate the production of T-cell precursors from adult mPB HSPCs in a feeder cell–free DL-4–based in vitro system. All experimental procedures were performed as described in the Methods section in this article's Online Repository at www.jacionline.org.

Peripheral blood–derived CD34+ cells from granulocyte colony-stimulating factor–mobilized (G-CSF) healthy donors were cultured with DL-4 protein, and the phenotype of emerging cell populations was analyzed. As early as day 3 in adult HSPC cultures (Fig 1, A), expression of CD34 and CD7 marked the presence of an early thymic progenitor (ETP)-like population not detected in control Fc cultures (see Fig E1 in this article's Online Repository at www.jacionline.org). In concordance with further T-lineage commitment, precursors derived from adult HSPCs downregulated CD34 at day 7, giving rise to a CD34−CD7++ cell pro-T1–like cell population (Fig 1, A and B). At day 7, the total number of CD7+ cells was 2.5-fold greater than at day 3 (Fig 1, C).

Fig 1

Generation of T-cell progenitors from mPB CD34+ HSPCs. A-C, Phenotype, frequency, and cell numbers of day 3 and day 7 DL-4 cultures. D, Quantification of T-cell potential by T-cell differentiation on OP9/DL-1 cells in limiting dilution conditions. E-I, Gene expression profile of day 0 CD34+ cells and day 3 and day 7 CD7+ cells by using quantitative RT-PCR (Fig 1, E-G) or day 3 and day 7 CD7− and CD7+ cells (Fig 1, H and I). *P < .05 and **P < .01.

Fig E1

Phenotype of adult CD34+ HSPCs exposed to 3 or 7 days of DL-4–Fc fusion protein compared with Fc alone.

We also quantified T-lineage potential by culture of day 0, 3, and 7 DL-4 progenitors on OP9/DL-1 in limiting dilution assays (LDA) (Fig 1, D). Based on the presence of CD4+CD8+ double-positive cells or CD3+ cells, the frequency of T-cell precursors assessed by LDA reached mean ± SEM values of 1/13.5 ± 1/3.5 at day 3 and 1/18.9 ± 1/6.6 at day 7. These values were similar to values observed in day 7 CB DL-4 cultures and much greater than the value in uncultured CD34+ cells (1/9000).

To further characterize adult-derived T-cell precursors, we performed gene expression analysis of day 0 mPB HSPCs and sorted CD7+ fractions on days 3 and 7 of DL-4 cultures, focusing on genes implicated in the Notch pathway, early lymphocyte development, thymus homing, and T-lineage differentiation. Major Notch target genes, such as DTX1, NRARP, and HES1, were induced after 3 days of DL-4 culture and remained stable thereafter (Fig 1, E). Gene expression analysis revealed that exposure of mPB HSPCs to DL-4 led to (1) gradual downregulation of multipotency and early lymphoid development genes, such as CD34, IKAROS, MYB, and KIT; (2) silencing of PAX5; and (3) induction of crucial T-lineage differentiation genes, such as BCL11B, IL7RA, and TCF7, all of which confirmed the proper induction of a T-lineage program in CD7+ cells not detected in CD7− cells (Fig 1, F-H).

Regarding expression of homing molecules, CXCR4 was already expressed in adult CD34+ HSPCs before culture (data not shown). After 3 and 7 days of DL-4 culture, major chemokine receptors potentially implicated in human thymus homing, such as CCR7, CCR9, and CXCR4, were expressed at significantly higher levels in the CD7+ population when compared with the CD7− fraction (Fig 1, I).

Finally, the in vivo T-cell potential of adult DL-4 precursors was tested by means of transplantation into nonirradiated newborn NOD/SCID/γc−/− (NSG) mice. Human thymic engraftment was accelerated greatly, occurring at only 4 weeks in 3 of 3 mice injected with day 7 DL-4 cells and persisting thereafter (Fig 2, A, and see Table E1 in this article's Online Repository at www.jacionline.org). In contrast, we did not observe engraftment before 11 weeks after transplantation in recipients of untreated CD34+ cells or day 3 DL-4 cells (Fig 2, A, and see Table E1). Active human thymopoiesis was further demonstrated by the presence of human CD4+CD8+ double-positive cells and enlarged thymic lobes compared with recipients of uncultured adult HSPCs (Fig 2, B and C). Mature CD3+TCRαβ+ T cells displaying a polyclonal TCR repertoire were detected in the thymi and spleens of recipient mice 8 weeks after transplantation (Fig 2, C and D). Moreover, we detected a small population of CD25+CD4+Foxp3+CD127− regulatory T cells in the spleens of injected mice (see Fig E2 in this article's Online Repository at www.jacionline.org).

Fig 2

In vivo reconstitution potential of T-cell precursors derived from mPB. A, Human thymic engraftment evaluated by the frequency of human CD45+(hCD45) and mouse CD45+(mCD45) lymphocytes in NSG recipients 6 weeks after transplantation with CD34+ cells before (day 0) or after 3 or 7 days of culture in DL-4 conditions. B, Presence of mature CD4+CD8+ double-positive cells in thymi of recipients transplanted with day 7 DL-4–cultured progenitors. The picture shows a representative photograph of recipient thymi injected with day 7 DL-4 cultured cells and day 0 HSPCs 8 weeks after transplantation. C, Presence of CD3+TCRαβ+ T cells in thymi and spleens of recipients injected with day 7 DL-4 cultured progenitors (gated on hCD45+ cells 8 weeks after transplantation). D, TCRβ VJ diversity in splenocytes from NSG mice transplanted with day 7 DL-4–cultured cells (8 weeks after transplantation).

Table E1

Kinetics of thymic engraftment of DL-4–cultured progenitors after transplantation in NSG mice

	Weeks after transplantation
Transplantation (no. of recipients)	4-5	6-7	8-10	11-16
Day 0 CD34+ cells (n = 9)	0/1	0/2	1/2	4/4
Day 3 DL-4 precursors (n = 5)	ND	1/2	0/1	4/4
Day 7 DL-4 precursors (n = 7)	3/3	1/2	1/1	1/1

ND, Not determined.

Fig E2

Presence of human CD25+CD4+Foxp3+CD127− regulatory T cells in the spleens of NSG recipients 8 weeks after transplantation of adult day 7 DL-4 precursors. Cells were first gated on 7AAD− live human CD45+ cells (left panel) and then on CD4+CD25+ cells (right panel).

Overall, our results show that adult HSPCs provide an effective source of in vitro–cultured T-cell precursors harboring all the necessary requirements for in vivo reconstitution of a functional T-cell compartment. Based on these results, we have established a clinical grade protocol to guide immunotherapeutic strategies using T-cell precursors to fasten T-cell recovery in the adult transplantation setting, where HSPCs from mPB are more available and more frequently used than HSPCs from CB.