Islet xenograft destruction in the hu-PBL-severe combined immunodeficient (SCID) mouse necessitates anti-CD3 preactivation of human immune cells

Abstract

Introduction of the hu-PBL-SCID mouse model has yielded a potentially useful tool for research in transplantation. The aim of this study was to define the conditions necessary for a reconstituted human immune system to destroy in a consistent manner rat islet xenografts in the alloxan-diabetic hu-PBL-SCID mouse. We examined different time points of hu-PBL reconstitution, different transplantation sites of the islets and several hu-PBL reconstitution protocols. Major differences in graft destruction were observed between the different hu-PBL reconstitution protocols, irrespective of timing of hu-PBL reconstitution or site of transplantation. Although preactivation of hu-PBL did not improve the level of hu-PBL chimerism, histological and immunohistochemical analysis of the grafts revealed a severe human lymphocytic infiltration and beta cell destruction only in the grafts of mice receiving preactivated hu-PBL. This beta cell injury resulted in impaired glucose tolerance, with in some animals recurrence of hyperglycaemia, and decreased insulin and C-peptide levels after glucose stimulation. Therefore, we conclude that activation of hu-PBL prior to transfer is essential in achieving xenograft infiltration and destruction in hu-PBL-SCID mice. The need for immune manipulation suggests that interactions between hu-PBL and xenografts in this model may be hampered by incompatibilities in cross-species adhesion and/or activation signals.

Fig. 1

Human chimerism in peripheral blood of untreated (n = 5) or antiasialo GM1 (ASGM1)-treated (n = 14) hu-PBL-SCID mice. Chimerism was tested weekly after hu-PBL reconstitution. Natural killer (NK) cell depletion resulted in a clear increase in hu-PBL engraftment levels in the peripheral blood of SCID recipients 2 and 3 weeks after reconstitution compared with untreated hu-PBL-SCID mice (P < 0·05). No rise in chimerism was seen in the absence of NK cell depletion. Data are presented as an absolute count of human CD3⁺ cells ( × 10⁶)/ml peripheral blood. Means are indicated by a horizontal line.

Fig. 2

In vitro reactivity of splenocytes from hu-PBL-SCID mice compared with naive SCID mice. (a) Reactivity of splenocytes of hu-PBL-SCID mice harvested 4 weeks after hu-PBL reconstitution. Responder cells were incubated in vitro with irradiated splenocytes from naive SCID mice or from BB rats or with irradiated allogeneic hu-PBL in a 5-day proliferation assay, as described in Materials and methods. (b) Reactivity of splenocytes of the hu-PBL-SCID mice harvested 5 weeks after hu-PBL reconstitution. Responder cells were incubated in vitro with irradiated splenocytes from naive SCID mice or from BB rats or with irradiated allogeneic hu-PBL in a 5-day proliferation assay, as described in Materials and methods. These data represent one of three independent experiments.

Fig. 3

Glycaemia of alloxan-diabetic SCID mice transplanted with rat islets. (a) Glycaemia levels of unreconstituted (n = 5, •), hu-PBL reconstituted 5 days before (n = 5, Δ) and 5 days after islet transplantation (n = 5, ▾) SCID mice. Timing of hu-PBL reconstitution had no effect on islet graft survival. (b) Glycaemia levels of kidney-transplanted (n = 5, ○) and spleen-transplanted (n = 5, □) hu-PBL-SCID mice. The site of islet transplantation did not change the incapacity of unstimulated hu-PBL to induce rejection. (c) Glycaemia levels of unboosted (n = 5, ▪), 1 × boosted (n = 6, ▴) and 2 × boosted (n = 5, ♦) hu-PBL-SCID mice. Pre-activation of hu-PBL prior to transfer did not induce recurrence to hyperglycaemia.

Fig. 4

Histology and immunohistochemistry of islet grafts from unreconstituted SCID mice (a,b,c), unboosted (d,e,f) and 1 × boosted (g,h,i) hu-PBL-SCID mice 4 weeks after hu-PBL reconstitution. Haematoxylin and eosin (a,d,g), insulin staining (b,e,h) and staining with anti-human CD45 antibody (c,f,i). Mag. × 200 (a–f), × 100 (g–i). Only the grafts from the boosted hu-PBL-SCID mice (g,h,i) show severe human CD45⁺ T cell infiltrates and clearly reduced insulin-positive cells. No infiltration and high insulin-positive cells are observed in grafts of the unreconstituted SCID mice (a,b,c). No infiltrates were seen in the grafts of unboosted hu-PBL-SCID mice (d,e,f).

Fig. 5

Immunohistochemistry of liver and kidney from 1 × boosted hu-PBL-SCID mice 4 weeks post-reconstitution. Staining with anti-human CD45 antibody (liver (a) and kidney (b)). Mag. × 100 (a,b). No human CD45⁺ T cell infiltrates were detected in the liver or kidney tissue.

Fig. 6

Metabolic evaluation of unreconstituted SCID mice (n = 5, •), unboosted (n = 4, ▪) and 1 × boosted (n = 6, ▴) hu-PBL-SCID mice after glucose challenge. Mice received an i.p. glucose (2 g/kg body wt) injection weekly after hu-PBL reconstitution and were bled 30 min after glucose challenge. Post-glucose glycaemia (a), plasma insulin (b) and C-peptide (c) levels were measured as described in Materials and methods. Note that severe metabolic impairment was present in the boosted hu-PBL-SCID mice, as reflected by return to hyperglycaemia, reduced insulin and C-peptide levels. *P < 0·05 versus initial levels; †P < 0·05 versus unreconstituted SCID mice.