HLA-haploidentical hematopoietic stem cell transplantation (HSCT) using CD34+ selected cells is a widely used procedure, which, however, is complicated by delayed immune reconstitution. We recently developed a new method of graft manipulation based on the physical removal of αβ+ T cells and CD19+ B cells, which permits to leave mature natural killer (NK) cells and γδ+ T cells in the graft. These cells can exert a graft-versus-leukemia (GvL) effect and reduce the risk of infection. In particular, unconventional γδ T cells play a critical role in both innate and adaptive immunity and exert HLA-unrestricted cytotoxicity against both solid and hematological tumors, thus potentially acting as beneficial effector cells in transplanted patients. Moreover, such grafts may limit the risk of graft-versus-host disease and prevent EBV-related lymphoproliferative disease.

We performed phenotypic and functional studies on γδ T cells collected from 20 pediatric patients (pts, 13 males, 7 females, median age 10 years, range 6 months to 16 years) that received this type of allograft. Eighteen pts had acute leukemia and 2 non-malignant disorders. Ex vivo assays of peripheral blood γδ T cell phenotype and function were performed weekly until Hospital discharge and monthly until 6 months after HSCT. Phenotype of γδ T cells was analysed by flow cytometry. Analyses were performed on mononuclear cells labelled with mAb panels (CD3, CD45, pan-γδ, anti-Vδ1, -Vδ2, -Vγ9, CD45RO, CD45RA, CD27, CD16, CD56) allowing the identification of the main γδ+ T cell subsets, including Vδ1+ and Vδ2+ cells, naïve, central memory (CM), effector memory (EM) and terminally differentiated (TD) γδ T cells.

Functional studies were performed using γδ T cells shortly after collection from pts, as well as after in vitro expansion with zoledronic acid and IL-2 for 10 days. Cytotoxic activity of γδ T cells was tested against primary leukemia cells, through CD107a degranulation assay and/or standard 51Cr-release assay.

In the first 4 weeks after HSCT, T cells were consistently of the γδ subset (>90% of CD45+CD3+ cells); by contrast, αβ+ T cells gradually increased over time. In approximately half of the pts, the percentage of αβ T cells exceeded that of γδ T cells already starting from 30 days after HSCT. γδ T cells consisted of Vδ2+Vγ9+ and Vδ1+Vγ9+/- cells, and marginally of the Vδ1-Vδ2-Vγ9- population. Detailed phenotypic characterization of Vδ1+ and Vδ2+ γδ T cells revealed that, at day +20 after HSCT, 44% of Vδ1+ cells were CM (identified as CD45RO+CD27+ cells), 26% naïve (CD45RO-CD27+), 21.4% TD (CD45RO-CD27-) and 6.1% EM (CD45RO+CD27-). Similarly, 55.4% of Vδ2+ γδ T lymphocytes were CM, 9.8% naïve, 11.4% TD and 23.1% EM. The proportion of the different Vδ2+ γδ T cell subset did not change significantly over time, especially when comparing that present at day +20 after HSCT (time point, TP1) with that measured 30 days after the attainment of a 1:1 ratio of αβ-to- γδ T cells (TP2) (Figure 1, left panel). By contrast, by comparing TP1 and TP2, we found that Vδ1+ CM γδ T cells decreased and EM cells increased over time, while naïve or TD Vδ1+ γδ T cells did not change (Figure 1, right panel). In transplanted pts experiencing cytomegalovirus (CMV) reactivation, γδ T cells mostly consisted of Vδ1+ cells (mean 59.8% of γδ T cells), among which 49% were TD, 22.7% EM, 18.9% CM and 10.1% naïve. Noteworthy, in transplanted pts who did not have CMV reactivation, the main γδ T cells showed a Vδ2+ phenotype.

Functional studies revealed that pt-derived γδ T cells consistently expanded in vitro after exposure to zoledronic acid and IL-2, the resulting Vγ9Vδ2 population expressing mainly an EM phenotype. These Vγ9Vδ2 cells exerted cytotoxic activities against primary allogeneic leukemia cells, especially when leukemia cells were pre-treated with zoledronic acid (Figure 2). More importantly, both Vδ1+ and Vδ2+ γδ T cells obtained from transplanted pts showed cytotoxic activity against primary leukemia cells, as assessed by CD107a degranulation assay.

In conclusion, we provide the first phenotypic and functional characterization of γδ T cells, analyzed over time in children transplanted with grafts depleted of αβ+ T cells and of B lymphocytes. Our results support the concept that γδ T cells are important effector cells, which can be expanded and activated after exposure to bisphosphonates and IL-2 with the aim of improving their killing capacity against leukemia cells.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.