Yu Qin Li

A clathrin/dynamin- and mannose-6-phosphate receptor–independent pathway for granzyme B–induced cell death

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B–mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin–overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

Fucosyl transferase (H) transgenic heart transplants to Gal-/- mice.

Background. Wehave previously described the rejection of Gal+ mouse hearts by mice lacking Gal&agr;(1,3)Gal (Gal-/-) and demonstrated this to be a model of xenogeneic hyperacute rejection (HAR) which would occur in pig-to-human/primate xenotransplantation, where Gal+ antibody (Ab) and complement (C′) mediate HAR. To reduce the amount of Gal present we used fucosyl transferase (H) as a transgene, H transferase competes for the same substrate as Gal transferase and reduces Gal expression by >90%. Methods. Gal-/- mice received a heart graft from C57BL/6 Gal+ or H transgenic mice and additional Gal Ab and C′ provided; HAR was monitored by direct observation for up to 90 min, or by palpation thereafter. When grafts were rejected they were examined macro- and microscopically. Results. H transgenic mice were used as donors to Gal-/- mice; it was found that: 1) C57BL/6 or H transgenic hearts were not rejected by Gal-/- recipients within 90 min in the absence of additional Gal Ab. 2) If additional Gal Ab and C′ were provided as fresh normal human serum (NHS), Gal+ (C57BL/6) grafts were rejected by Gal-/- mice in ∼34 min, whereas H transgenic hearts mostly lasted up to 17 hr, but were then rejected. The histological appearances showed features of both Arthus and Shwartzmann phenomena. 3) Mice hyperimmunized with Gal with anti-Gal titers of >1:20,000, rejected Gal+ grafts in 31 min; the survival was prolonged to 75 min with the H transgenic hearts. Conclusion. The presence of the H transgene in donor hearts transplanted to naïve Gal-/- mice delays the onset of HAR, but rejection ultimately occurs; if the mice are hyperimmune earlier rejection occurs. The expression of the H transgene alone is insufficient to avoid HAR in the Gal-/- mouse model; the presence of other transgenes and techniques will be required to give an appropriate increase in survival of pig-to-human/primate grafts.

CD46 protects pig islets from antibody but not cell-mediated destruction in the mouse.

Abstract:  We have previously reported that islets present in cultured fetal pig pancreas (FPP) are resistant to destruction by Galα(1,3)Gal antibodies and complement, but are susceptible to the ‘secondary’ antibody response which occurs on transplanting pig islet tissue to Galo/o murine recipients. In a model of antibody‐mediated graft rejection, we tested the resistance of porcine islets to antibody. Using FPP from human CD46 transgenic pigs, we now report that the complement regulator, CD46, affords protection from antibody‐mediated rejection when mouse anti‐pig serum (MAPS) was administered to scid mice bearing FPP grafts from either CD46 transgenic or normal pigs. Indeed, whereas normal pig islets were destroyed by an intraperitoneal (i.p.) injection of 0.1 to 0.2 ml of MAPS antibody, destruction of CD46‐expressing transgenic islets required 0.5 ml, i.e. up to five times the amount. In contrast, there was no prolongation of the survival of CD46 transgenic mouse skin or heart major histocompatibility complex‐compatible or ‐incompatible allografts – rejected by predominantly cellular immune mechanisms, as opposed to xenograft rejection. Although complement regulators have been examined for their protective role in hyperacute rejection of vascularized xenografts, it is clear that they also have protective effects in the later, antibody‐mediated responses, but are unlikely to effect the inflammatory response in cell‐mediated rejection.

Inhibition of hyperacute transplant rejection by soluble proteins with the functional domains of CD46 and FcgammaRII.

BACKGROUND Recombinant soluble forms of complement regulatory molecules, including the human complement regulatory protein CD46 (rsCD46), have been shown to inhibit hyperacute transplant rejection (HAR) and protect against complement-mediated inflammatory tissue damage. Similarly, recombinant soluble forms of the immunoglobulin receptor FcgammaRII (rsFcgammaRII) can attenuate antibody-mediated inflammatory responses. We have produced and tested the function of novel recombinant chimeric proteins that incorporate the functional domains of both CD46 (membrane cofactor protein, MCP) and the low affinity human IgG receptor FcgammaRII (CD32). METHODS Two recombinant soluble chimeric proteins (CD46:FcR and FcR:CD46) were designed and produced using a human cell expression system. Their ability to protect cells against complement-mediated lysis (through the CD46 domain) and bind human IgG (through the Fc receptor domain) was assessed in vitro. They were also tested in vivo in the rat reverse passive Arthus reaction and a murine model of hyperacute cardiac transplant rejection. RESULTS In vitro, the functional domains of the chimeric proteins each retained their activity. In vivo, the serum half-life of the recombinant chimeric proteins in mice was more than either rsCD46 or rsFcgammaRII. In the rat reverse passive Arthus reaction, intradermal injection of each recombinant protein substantially reduced inflammatory skin edema (>50%) and polymorphonuclear neutrophil infiltration (>90%). In the hyperacute rejection model, i.v. treatment with FcR:CD46 prevented complement-mediated rejection, macroscopic bruising, edema, and thrombosis more effectively than rsCD46. CONCLUSIONS CD46/FcgammaRII bifunctional proteins have an improved ability to control complement-mediated hyperacute graft rejection and have therapeutic potential in other conditions involving antibody-mediated inflammation.

Local expression of IDO, either alone or in combination with CD40Ig, IL10 or CTLA4Ig, inhibits indirect xenorejection responses.

Abstract:  Background:  To overcome cell‐mediated xenorejection by transgenic expression of immunomodulatory molecules by a graft, it is likely that expression of multiple molecules will be required. Previous studies support the use of the immunomodulatory agents indoleamine 2,3‐dioxygenase (IDO), CD40Ig, interleukin 10 (IL10), and CTLA4Ig for suppression of rejection responses. We examined the effects of local expression of these molecules by a porcine cell line (PIEC) on indirect murine xenorejection responses in vitro and in vivo.

Dendritic cells expressing soluble CTLA4Ig prolong antigen-specific skin graft survival

Dendritic cells (DCs) and CTLA4Ig are important in regulating T‐cell responses and therefore represent potential therapeutic tools in transplantation. In this study, CTLA4Ig was expressed in a C57BL/6 murine DC line (JAWS II) by lentiviral transduction and these cells were used to examine T‐cell immunomodulatory effects in vitro and in vivo. A lower stimulation index to C57BL/6 was observed with splenocytes from BALB/c mice primed with JAWS II‐CTLA4Ig compared with control JAWS II‐green fluorescent protein (JAWS II‐GFP). Mice primed with JAWS II‐CTLA4Ig cells had significantly prolonged antigen‐specific C57BL/6 skin graft survival compared with either JAWS II‐GFP‐primed or naïve mice (median 13, 11 and 11 days, respectively, P=0.0001). Furthermore, JAWS II‐CTLA4Ig‐primed mice that had been previously transplanted with skin grafts were re‐transplanted with skin grafts 6 months later without immune manipulation. These mice demonstrated specific prolongation of second‐set rejection responses, indicating systemic immune modulation induced by genetically modified DC. The mechanism was not due to expression of indoleamine 2,3‐dioxygenase or induction of circulating regulatory T cells as assessed by flow cytometry of the peripheral blood lymphocytes. This potent effect demonstrated with skin grafts and second‐set responses highlights the potential use of this strategy for transplantation more generally.

LFA-1 and ICAM-1 antibody-idarubicin conjugates separately prolong murine cardiac allograft survival.

Drug antibody conjugates can enhance the activity of monoclonal antibodies (MoAb) and idarubicin-MoAb conjugates have led to tolerance induction with antibodies which are inactive when used alone. It has been reported that, in mice, antibodies to ICAM-1 and LFA-1 have to be used together to induce tolerance to cardiac allografts; here we show that these monoclonal antibodies, conjugated to idarubicin, can lead to tolerance induction to cardiac allografts when used alone.

Effect of IL-4 deletion on cardiac allograft survival in the BALB/c to 129Sv × C57BL/6 strain combination

To investigate the effect of IL‐4 deletion on cardiac allograft survival, vascularized BALB/c cardiac allografts were placed in C57BL/6, 129Sv × C57BL/6 (IL‐4 +/+) or 129Sv × C57BL/6 IL‐4 knockout mice (IL‐4−/−). Untreated recipients rejected allografts in < 15 days while isografts survived indefinitely (> 100 days). Treatment with anti‐CD4 (GK1.5) for 4 days at the time of allografting increased mean survival to > 100 days in C57BL/6, 90±16 days in 129Sv × C57BL/6 (IL‐4 +/+) and 68 ± 36 days in 129Sv × C57BL/6 (IL‐4−/−) recipients. Although there was a trend towards shorter survival times in the IL‐4−/− mAb‐treated mice, survival in the three recipient groups was not significantly different (P = 0.07). A 30‐day course of anti‐CD4 did not further prolong BALB/c heart survival. All long‐surviving hearts had histological evidence of parenchymal damage and transplant vascular disease. None of these recipients developed antigen‐specific tolerance, since both donor and third party skin graft challenges were rejected when challenged at> 60 days post‐graft and all primary grafts failed by 120 days. Thus the effects of IL‐4 deletion were subtle and were seen only with low doses of immunosuppression in this high responder strain combination.