Altered donor P2X7 activity in human leukocytes correlates with P2RX7 genotype but does not affect the development of graft-versus-host disease in humanised mice

Abstract

Graft-versus-host disease (GVHD) is a life-threatening consequence of allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. The ATP-gated P2X7 receptor channel is implicated in the development of GVHD. P2X7 activity on human leukocytes can be influenced by gain-of-function (GOF) and loss-of-function (LOF) single nucleotide polymorphisms (SNPs) in the P2RX7 gene. In this study, the P2RX7 gene was sequenced in 25 human donors and the P2X7 activity on subsets of peripheral blood T cells, natural killer (NK) cells and monocytes was measured using an ATP-induced dye uptake assay. GOF and LOF SNPs representing 10 of the 17 known P2RX7 haplotypes were identified, and correlated with P2X7 activity on all leukocyte subsets investigated. Notably, invariant (i) NK T cells displayed the highest P2X7 activity amongst all cell types studied. To determine if donor P2X7 activity influenced the development of GVHD, immunodeficient NOD-SCID-IL2Rγ^null (NSG) mice were injected with human peripheral blood mononuclear cells isolated from donors of either GOF (hP2X7^GOF mice) or LOF (hP2X7^LOF mice) P2RX7 genotype. Both hP2X7^GOF and hP2X7^LOF mice demonstrated similar human leukocyte engraftment, and showed comparable weight loss, GVHD clinical score and overall survival. Donor P2X7 activity did not affect human leukocyte infiltration or GVHD-mediated tissue damage, or the relative expression of human P2X7 or human interferon-γ (hIFNγ) in tissues. Finally, hP2X7^GOF and hP2X7^LOF mice demonstrated similar concentrations of serum hIFNγ. This study demonstrates that P2X7 activity correlates with donor P2RX7 genotype on human leukocyte subsets important in GVHD development, but does not affect GVHD development in a humanised mouse model of this disease.

Keywords: Bone marrow transplantation; Humanised mice; Leukocyte; P2RX7 gene single nucleotide polymorphism; P2X7 receptor; Purinergic receptor; Xenogeneic graft-versus-host disease.

Fig. 1

Donor P2X7 activity correlates with P2RX7 genotype on human T cell subsets. a-k hPBMCs, from donors of either GOF or LOF P2RX7 genotype, resuspended in NaCl medium were incubated with 1 μM YO-PRO-1²⁺ in the absence or presence of 1 mM ATP for 5 min at 37 °C. YO-PRO-1²⁺ uptake into human T cell subsets was analysed by flow cytometry using the gating strategy shown. a Single cells were gated by forward scatter area (FSC-A) and height (FSC-H). b Viable single lymphocytes were gated using FSC-A and side scatter (SSC-A). Single viable cells were then used to identify c CD3⁺ T cells, d CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells and e CD3⁺CD4⁺CD25⁺CD127^lo Tregs. Single, viable cells were also used to gate f CD3⁺CD19⁻ cells to subsequently identify g CD3⁺Vα24⁻Jα18⁺ iNKT cells. h P2X7 activity was determined by comparing YO-PRO-1²⁺ uptake in the absence (blue histograms) and presence (orange histogram) of ATP; histograms for CD3⁺ T cells are shown as examples. P2X7 activity in i CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells and Tregs was determined in multiple donors and P2X7 activity in j iNKT cells and corresponding CD3⁺ T cells was determined in some donors. k hPBMCs from three donors were pre-incubated with 60 μM JNJ-47965567 (JNJ) at 37 °C for 15 min to block P2X7 before incubation in the absence or presence of 1 mM ATP. Symbols correlate to symbols in Table 2 and represent i–j mean values of duplicate ATP-induced YO-PRO-1²⁺ uptake measurements into CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ (GOF, n = 19; LOF, n = 6), Tregs (GOF, n = 11; LOF, n = 3) and iNKT cells (GOF, n = 5; LOF, n = 3); *P < 0.05, ***P < 0.001 compared to corresponding LOF donors; ††P < 0.01, †††P < 0.001, compared to corresponding CD3⁺ T cells. k Data represents group means ± SEM (n = 3), ****P < 0.0001 compared to hPBMCs incubated with ATP and JNJ. GOF, gain-of-function; iNKT cell, invariant natural killer T cell; LOF, loss-of-function; Tregs, regulatory T cells

Fig. 2

Donor P2X7 activity correlates with P2RX7 genotype on human monocyte subsets and NK cell subsets. a–i hPBMCs, from donors of either GOF or LOF P2RX7 genotype, in NaCl medium were incubated with 1 μM YO-PRO-1²⁺ in the absence or presence of 1 mM ATP for 5 min at 37 °C. YO-PRO-1²⁺ uptake into human monocyte and NK cell subsets was analysed by flow cytometry using the gating strategy shown. a Single cells were gated by forward scatter area (FSC-A) and height (FSC-H) and b viable cells gated using FSC-A and side scatter (SSC-A). Single viable cells were then used to identify c CD14⁺CD16⁻, CD14⁺CD16⁺ or CD14⁻CD16⁺ monocyte subsets. Single, viable cells were used to gate e CD3⁻ cells to subsequently identify f CD16⁺CD56⁻, CD16⁺CD56⁺ or CD16⁻CD56⁺ NK cell subsets. d, g P2X7 activity on these cells was determined by comparing YO-PRO-1²⁺ uptake in the absence (blue histograms) and presence (orange histogram) of ATP; histograms for d CD14⁺CD16⁻ monocytes and g CD16⁻CD56⁺ NK cells are shown as examples. h, i P2X7 activity in h monocyte subsets and i NK cell subsets was determined in multiple donors. Symbols correlate to Table 2 and represent mean values of duplicate ATP-induced YO-PRO-1²⁺ uptake tests from different donors (GOF, n = 7; LOF, n = 3); ***P < 0.001 compared to corresponding LOF donors. GVHD, graft-versus-host disease; GOF, gain-of-function; NK cell, natural killer cell; LOF, loss-of-function

Fig. 3

Engraftment of human leukocytes is similar in hP2X7^GOF and hP2X7^LOF mice. NSG mice were injected (i.p.) with 10 × 10⁶ hPBMCs isolated from donor of either GOF (hP2X7^GOF mice) or LOF (hP2X7^LOF) P2RX7 genotype (four donors per group). hP2X7^GOF and hP2X7^LOF mice were checked for the engraftment of human leukocytes in a–c the blood at 3 weeks post-injection and d–g spleen at endpoint. a, d hCD45⁺ leukocytes are expressed as a percentage of total mCD45⁺ and hCD45⁺ leukocytes. b, e hCD3⁺ cells are expressed as a percentage of total hCD45⁺ leukocytes. c, f hCD4⁺ and hCD8⁺ cells are expressed as a percentage of hCD3⁺ cells. g hCD4⁺hCD25⁺ hCD127^lo Tregs are expressed as a percentage of CD4⁺ T cells. Data is presented as a group mean ± SEM; symbols represent individual mice; ****P < 0.0001 compared to hCD8⁺ T cells. GOF, gain-of-function; LOF, loss-of-function

Fig. 4

Clinical and histological GVHD is similar in hP2X7^GOF and hP2X7^LOF mice. NSG mice were injected (i.p.) with 10 × 10⁶ hPBMCs isolated from either a GOF (hP2X7^GOF mice) or LOF (hP2X7^LOF) P2RX7 genotype donor (four donors per group). a–c Mice were monitored for up to 10 weeks post-hPBMC injection for clinical GVHD development including a weight loss, b GVHD clinical score and c survival. Data represents a, b group means ± SEM or j percentage survival (GOF, n = 30; LOF, n = 37). d At endpoint, tissue sections (liver, small intestine and skin) from hP2X7^GOF mice (top panel) and hP2X7^LOF mice (bottom panel) were stained with haematoxylin and eosin, and viewed by microscopy. Representative images from four mice per group are shown; bars represent 100 μm. GOF, gain-of-function; LOF, loss-of-function

Fig. 5

Relative hIFNγ expression is significantly increased in the livers of hP2X7^GOF mice compared to hP2X7^LOF mice. a–g NSG mice were injected (i.p.) with 10 × 10⁶ hPBMCs isolated from either a GOF or LOF P2RX7 genotype donor (four donors per group). a–f Relative expression of a–c hP2X7 and d–f hIFNγ in a, d spleen, b, e liver and c, f small intestine at endpoint was examined by qPCR. g Concentrations of hIFNγ in serum of humanised mice were analysed by ELISA. a–g Data represents group mean ± SEM (a–f, n = 12–18; g GOF, n = 30; LOF, n = 36); symbols represent individual mice; *P < 0.05 compared to hP2X7^LOF mice. hIFNγ, human interferon-γ; GOF, gain-of-function; LOF, loss-of-function