American Journal of Transplantation 2014; 14: 1109–1119 Wiley Periodicals Inc.

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Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.12656

IgG Response to Intracerebral Xenotransplantation: Specificity and Role in the Rejection of Porcine Neurons E. Mathieux1,2,3, V. Nerrie`re-Daguin1,2,3, X. Le´ve`que1,2,3, D. Michel-Monigadon1,2,3, T. Durand4, V. Bonnamain1,2,3, S. Me´noret1,2,3,5, y I. Anegon1,2,3,5, P. Naveilhan1,2,3,4,*, y and I. Neveu1,2,3,4, 1

INSERM, UMR 1064, Center for Research in Transplantation and Immunology, Nantes, France 2 CHU de Nantes, Institut de Transplantation et de Recherche en Transplantation, ITERT, Nantes, France 3 LUNAM Universite´, Universite´ de Nantes, Faculte´ de Me´decine, Nantes, France 4 INSERM, UMR 913, Nantes cedex, France 5 Platform Rat Transgenesis IBiSA-CNRS, Nantes, France  Corresponding author: Philippe Naveilhan, [email protected] y Both authors contributed equally.

Xenogenic fetal neuroblasts are considered as a potential source of transplantable cells for the treatment of neurodegenerative diseases, but immunological barriers limit their use in the clinic. While considerable work has been performed to decipher the role of the cellular immune response in the rejection of intracerebral xenotransplants, there is much still to learn about the humoral reaction. To this end, the IgG response to the transplantation of fetal porcine neural cells (PNC) into the rat brain was analyzed. Rat sera did not contain preformed antibodies against PNC, but elicited anti-porcine IgG was clearly detected in the host blood once the graft was rejected. Only the IgG1 and IgG2a subclasses were up-regulated, suggesting a T-helper 2 immune response. The main target of these elicited IgG antibodies was porcine neurons, as determined by double labeling in vitro and in vivo. Complement and antiporcine IgG were present in the rejecting grafts, suggesting an active role of the host humoral response in graft rejection. This hypothesis was confirmed by the prolonged survival of fetal porcine neurons in the striatum of immunoglobulin-deficient rats. These data suggest that the prolonged survival of intracerebral xenotransplants relies on the control of both cell-mediated and humoral immune responses. Keywords: Antibodies, humoral rejection, knockout, neural grafts, xenotransplantation

Abbreviations: ADCC, antibody-dependent, cell-mediated cytotoxicity; ANOVA, analysis of variance; D, day; E15, embryonic day 15; FACS, fluorescent-activated cell sorting; Fc, crystallizable fragment; FcgR, Fc gamma receptors; FITC, fluorescein isothiocyanate; G28, gestation day 28; GFAP, glial fibrillary acidic protein; HAR, hyperacute rejection; IFNg, interferon gamma; MCP-1, monocyte chemotactic protein-1; MLR, mixed lymphocyte reaction; NeuN, neuronal nuclei; NF, neurofilament; p, probability; PAEC, pig aortic endothelial cells; PBMC, peripheral blood mononuclear cells; PNC, porcine neural cells; SEM, standard error of the mean; Th, T-helper; TH, tyrosine hydroxylase; Tuj-1, b-III tubulin Received 23 July 2013, revised 25 November 2013 and accepted for publication 14 December 2013

Introduction Launched in the 1980s as a potential treatment for neurodegenerative diseases, the transplantation of neural cells into the brain is still the only curative option for disorders such as Parkinson’s and Huntington’s diseases. Clinical trials performed between 1990 and 2005 provided proof-of-principle that human fetal neurons can be transplanted and function in the striata of patients, thus confirming the results of two decades of animal experimentation (1–3). A direct clinical application was, however, severely hampered by ethical concerns and a shortage of human fetal tissue. Alternative strategies have, therefore, to be considered, including the possibility of transplanting stem cells derived from human tissue, or neuroblasts isolated from animals such as pigs (4). Whereas stem cell grafting incurs the risk of tumors and defective cell differentiation, the transplantation of xenogenic cells into the brain is preempted by a slow but fatal host immune response, which leads to the rejection of porcine cells (5,6). Despite the presence of the blood–brain barrier, and the absence of classic lymphatic drainage in the central nervous system, intracerebral xenografts are targeted by a host-cellmediated immune response, which has been well characterized using the transplantation of porcine neuroblasts into the rat striatum as a model. In the absence of immunosuppressors, fetal porcine neurons are rejected within 5–7 weeks. The rejection process is characterized by a strong 1109

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inflammatory response with extensive activation of microglial cells, and the recruitment of immune effector cells such as T lymphocytes and dendritic cells (7). This late recruitment, which coincides with a marked accumulation of transcripts encoding the monocyte chemoattractant, monocyte chemotactic protein-1 (MCP-1) (8), suggests an indirect T cell activation initiated by dendritic cells and favoring CD4 T-helper (Th) cells over CD8 T-suppressor cells (9). A central role of CD4 T cells is further supported by the prolonged survival of xenografts after CD4 but not CD8 depletion (10), or in the brains of MHC class II but not class Ideficient mice (11). Immunosuppressive treatments with cyclosporine A (12–14), methylprednisolone (15) or minocycline (7) also improve the survival of intracerebral xenografts, but a major role of the cell-mediated immune response does not exclude a critical contribution of the host humoral response, which might have been underestimated due to the absence of hyperacute rejection (HAR). Vascularized pig organs transplanted in primates are rejected within hours due to preexisting anti-porcine antibodies in the sera of human and nonhuman primates. Interactions of these xenoreactive natural immunoglobulins with their primary target, the carbohydrate Galactose-alpha1,3-galactose, on donor endothelial cells leads to complement activation, hemorrhage, edema and intravascular coagulation (16–18). This rapid process, called HAR, is not observed after the transplantation of xenogenic neural cells into the brain, probably due to the fact that the graft is not vascularized. Vascularization of the graft occurs within a few days, but an acute attack is unlikely as the vessels inside the porcine neural grafts are mainly composed of host endothelial cells (19). Contribution of the humoral immune response to the rejection of porcine neural xenografts was suggested by Barker et al (20), who detected IgM and complement binding in the brain of transplanted rats. Although systemic antibody response was not observed, a possible contribution of the host humoral reaction must be taken into account, as antibodies could affect graft survival when they form immune complexes and recruit cytotoxic effector cells by activating complement or crystallizable fragment gamma receptors (FcgR) (21). In the present study, we transplanted porcine neural cells (PNC) into the rat striatum to characterize the humoral immune response and to evaluate its implication in the rejection of intracerebral xenografts. The data presented indicate that rats do not have preexisting antibodies directed against PNC, but their intracerebral transplantation triggers a humoral immune response characterized by the production of specific anti-porcine IgG. These elicited anti-porcine IgG were found both within the rejecting grafts and in the sera of rats that had rejected their transplant. The specific induction of IgG1 and IgG2a isotypes suggests a Th2 immune response, while double labeling of cultured cells and xenografted brains reveals that porcine neurons are major target cells for these elicited antibodies. Finally, an active role 1110

of the humoral response in the rejection of intracerebral transplantation was demonstrated by the long survival of PNC in the brains of immunoglobulin-deficient rats.

Materials and Methods Animals Porcine fetuses (gestation day 28 [G28]) were obtained from Large White pigs (INRA, Nouzilly, France) and Lewis 1A rats from Janvier CERJ (Le Genest saint Isle, France). The homozygous immunoglobulin-deficient rats (IgM KO) were bred and grown in our animal facility (Platform Rat Transgenesis IBiSA-CNRS). These IgM KO rats have virtually no mature B cells and lack detectable IgA, IgE, IgG and IgM (22,23). Animal manipulations were performed in accordance with the ethical guidelines of the French veterinary service. All procedures were conducted under protocols approved by the Departmental Ethics Committee. Every effort was made to minimize animal suffering and the number of animals.

Intracerebral transplantation PNC from the ventral mesencephalon of G28 fetuses were injected (2  105 cells) by stereotaxy into the rat striatum (anterior þ0.7 mm, lateral 2.8, ventral 6 and 5.6, incisor bar 3.3) (24,25). Sera were collected at euthanasia.

Immunohistochemistry Brain sections and immunohistochemistry were prepared as previously described (9). Porcine neurons were stained with an mAb against the porcine neurofilament (NF)70 (clone DP5, 1/500; Dr. Paulin, Universite´ Paris 7, France). Dopaminergic neurons were detected with anti-tyrosine hydroxylase (TH) antibody (1/1000; Covance, Princeton, NJ). R73 and OX42 were used to identify T cells and microglial cells, respectively. Complement and B cells were stained with anti-C4d (1/500; Dr. Baldwin, Johns Hopkins Medical School, Baltimore, MD) and B cell-specific anti-HIS24 antibodies (1/500; BD Biosciences, Le Pont de Claix, France), respectively. After incubation with biotinylated antibodies, staining reactions were performed with the Vector ABC kit (Vector Laboratories, Burlingame, CA) using very intense purple or diaminobenzidine. The binding of IgG to PNC was determined by incubating the brain sections with anti-glial fibrillary acidic protein (GFAP; 1/500; SigmaAldrich, St. Louis, MO) or anti-neuronal nuclei (NeuN; 1/1000; Millipore, Billerica, MA) mAbs. IgG deposits were then detected with a fluorescein isothiocyanate (FITC)-anti-rat IgG antibody (Fc specific; Jackson Immunoresearch, West Grove, PA) whereas Alexa 568-anti-mouse IgG antibody (1/500; Invitrogen, Saint Aubin, France) was used to stain astrocytes and neurons.

Cell cultures Neural cells were prepared from the cortices of G28 porcine fetuses or embryonic day 15 (E15) rat fetuses, respectively (26). The cells were plated overnight in serum-supplemented medium and cultured for 5 days in N2supplemented medium (Invitrogen) to induce their differentiation/maturation. Freshly isolated PNC were not cultured, to preserve their immaturity. Porcine astrocytes were established from the cortices of newborn piglets (26). Astrocytes were grown for 15 days in serum-supplemented medium trypsinized, and stored frozen. Only P1 astrocytes were used. Pig aortic endothelial cells (PAEC) prepared by collagenase digestion of porcine aortas, were cultured in serum-supplemented medium (27,28).

Mixed lymphocyte reaction T cells were isolated from the spleens of IgM KO rats (29). Peripheral blood mononuclear cells (PBMC) were purified from pig blood using a Ficoll

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IgG Response to Intracerebral Xenograft gradient (LymphosepTM; Biowest, Nuaille, F). The mixed lymphocyte reaction (MLR) was performed by adding irradiated porcine PBMC (105 cells/well) to purified T cells (105 cells/well) in 96-well plates. Cell proliferation was determined at day 5 by [3H]thymidine uptake.

Rat serum analyses Flow cytometry: Cells were resuspended in PFN (phosphate-buffered saline 1, 2% fetal calf serum, 0.1% sodium azide) and distributed into 96well, u-bottomed microtiter plates (Nunc Inc., Napervile, IL). Anti-porcine IgG were detected by incubating the porcine cells with the heat-inactivated rat sera (1/100 in PFN), followed by incubation with an FITC-anti-rat Ig. Immunoglobulin isotypes were determined by incubating the PNC with antibodies directed against rat IgG1, IgG2a, IgG2b or IgG2c (Jackson Immunoresearch), and FITC-anti-mouse IgG was used as the secondary antibody. Fluorescent labeling was measured using a Canto cytometer (BD Biosciences) and analyzed with FlowJo1 software (TreeStar, Inc., Ashland, OR). Sera were analyzed independently on three independent cell batches, each time in triplicate.

Immunocytochemistry: Cells were fixed with 4% paraformaldehyde, exposed to heat-inactivated rat serum (1/100), and incubated with an FITCanti-rat IgG (Fc; 1/100, Jackson Immunoresearch). For the double labeling, cells were fixed after the first labeling, exposed to anti-GFAP (1/500; Sigma) or anti-Tuj-1 (1/1000; Sigma-Aldrich) antibodies and incubated with Alexa 568-anti mouse IgG.

Statistics Data are expressed as mean values  SEM. Statistical analyses were performed using the Kruskal–Wallis test followed by the Dunn posttest, twoway analysis of variance (ANOVA) and the Mann–Whitney test.

Results Transplantation of PNC into the rat striatum induces a late humoral immune response The humoral response to intracerebral xenograft was evaluated by analyzing the sera of rats transplanted with PNC. For this purpose, PNC were transplanted into the striata of 46 immunocompetent rats, and sera were collected on day 3 (D3), D28, D42 and D63 (Table 1). Sera immunoreactivity was first tested on PNC that were cultured for 5 days in defined medium to promote their differentiation. Cultured PNC were incubated with rat sera and IgG binding was determined by exposing the cells to FITC-anti-rat IgG. Table 1: Classification of the rat sera

Total number of rats Healthy NF70þ/R73 Rejecting NF70þ/R73þ Rejected NF70/R73þ Scar NF70/R73

D3

D28

D35

D42

D63

Total

6 6 0 0 0

9 6 3 0 0

4 2 0 2 0

14 2 5 5 2

13 0 0 0 13

46 16 8 7 15

Sera were classified according to the delay after the intracerebral transplantation of porcine mesencephalic neural cells into the rat striatum and the immunological status of the graft as determined using anti-pNF70 and R73 antibodies: healthy (NF70þR73), rejecting (NF70þR73þ), rejected (NF70R73þ) and scar (NF70R73). D, day.

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Flow cytometry analyses show a progressive increase in the median fluorescence intensity over time with statistical significance at D42 and D63 (Figure 1A). This increase was specific as it was not observed using rat neural cells (data not shown). We then correlated the presence of anti-PNC IgG with the graft status. The graft status was determined by immunohistochemistry using an anti-NF70 antibody that specifically recognizes porcine neurons (NF70) and an antiabTCR antibody that stains T cells (R73). All grafts were perfectly healthy (no sign of degeneration) with a quite similar appearance in terms of volume and NF70 immunostaining (healthy, NF70þR73) until the sudden and strong invasion of the graft with numerous T cells (rejecting, NF70þR73þ). The next step was the rapid disappearance of the NF70 staining (rejected, NF70R73þ), corresponding to neuronal degeneration. Finally, the T cells completely disappeared and the graft turned into a scar (scar, NF70R73) (Figure S1; Table 1). As illustrated in Figure 1B, the levels of anti-PCN antibodies (black bars) were significantly higher in the sera of rats that had already rejected their transplant (rejected and scar groups) than in the healthy and rejecting groups. Similar increases were observed with freshly isolated PNC (Figure 1B, open bars); however, the median fluorescence value obtained with these immature neural cells was much lower than that obtained with the differentiated cultured PNC (47.45  12.16 vs. 316  47.89 for the scar group). We then characterized the IgG subclasses induced by the transplantation of PNC. As expected, low amounts of IgG1, IgG2a, IgG2b and IgG2c were found in the rat sera collected at D3 (healthy group) (Figure 1C). Higher levels of the IgG subclasses were found at D63 (scar group) but the difference was only significant for the IgG1 and IgG2a isotypes. As a first attempt to characterize the target antigens, Western blots were performed with protein extracts prepared from porcine cortex and striatum. As shown in Figure S2, IgG present in three of the sera collected at D63 recognized several proteins expressed in the porcine brain. Some proteins of similar size were recognized by two or three sera, whereas others were recognized by only one serum. These data highlight the interindividual differences in the humoral response. Neurons are the major cell target of the anti-porcine elicited IgG antibodies Cell specificity of the humoral immune response was investigated by characterizing the cells that bind the antiporcine antibodies. Incubation of porcine astrocytes (Figure 2A) or PAEC (Figure 2B) with the rat sera generated positive signals on flow cytometry, but the median fluorescence values were much lower than the value for the PNC, which were mostly composed of neurons (over 90%) (Figure 1B): PNC versus astrocytes: 316  47.89 versus 34.92  8.06 for the scar group; PNC versus PAEC: 316  47.89 versus 30.79  6.96 for the scar group. Maximum fluorescence was found at the latest stage (D63, scar group) (Figure 2A and B), as observed for the PNC (Figure 1B). These observations were confirmed by 1111

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Figure 1: Analysis of rat sera for the presence of anti-porcine IgG. Rat sera were collected at D3, D28, D35, D42 or D63 following intrastriatal transplantation of PNC. IgG reactivity was tested by incubating heat-inactivated rat sera (1/100) with PNC. Bound antibodies were detected by flow cytometry using fluorescein isothiocyanate anti-rat IgG subtypes. (A) Levels of IgG directed against cultured PNC (n ¼ see Table 1). (B) Levels of IgG directed against freshly isolated PNC, mostly composed of immature cells (open bar), or cultured PNC, mostly composed of neurons (black bar) (n ¼ see Table 1). The results are expressed according to the immunological status of the graft. (C) Levels of anti-porcine IgG1, IgG2A, IgG2B and IgG2C in the rat sera collected at D3 and D63, as determined using cultured PNC (n ¼ see Table 1). Results are expressed as mean value  SEM (Kruskal–Wallis test and Dunn posttest [A and B] and two-way analysis of variance test and Bonferroni posttest [B and C];  p < 0.05,  p < 0.01,  p < 0.001 vs. D3 and healthy, respectively). D, day; PNC, porcine neural cells.

immunocytochemistry. Strong staining was observed in primary cultures of PNC when using D63 rat sera whereas control sera (ungrafted rat) gave negative signals (Figure 3A). A large number of positive cells exhibited a neuronal shape. The neuronal identity of these cells was confirmed by double immunocytochemistry using Tuj-1, which labels the b-III tubulin (Tuj-1) in neurons. Tuj-1þ cells were clearly immunostained with D63 rat sera (Figure 3B), and the co-localization was confirmed by confocal microscopy (Figure 3C). To determine if astrocytes expressed antigens that could be recognized by the anti-porcine elicited IgG antibodies, double immunocytochemistry was performed using CD44. As shown in Figure 3D, CD44þ cells were present in the cultures of PNC, but no co-localization with anti-porcine IgG was observed. Similar results were obtained using anti-GFAP antibodies that also label astrocytes (data not shown).

The humoral immune response was characterized in vivo by analyzing the deposition of complement and anti-donor antibodies in healthy and rejecting grafts (n ¼ 3 per group) at D28 and D42, respectively. A weak and diffuse deposition of IgG was observed around all the healthy grafts (Figure 4A) whereas rejecting transplants were strongly labeled with the FITC-anti-rat IgG antibodies (Figure 4B). Double immunohistochemistry revealed that IgG deposition was mainly in NeuN-positive neurons (Figure 4C), but a co-localization was found in some GFAP-positive astrocytes (Figure 4D). High levels of rat IgG in the rejecting grafts were associated with positive staining for C4d. The activated complement protein was systematically detected on the grafted side (Figure 5A) but never on the contralateral side (Figure 5B). The presence of B cells in the grafted brains was assessed by immunohistochemistry using an anti-His24 antibody. Rarely, positive cells were found in the

Figure 2: Analysis of rat sera for the presence of IgG directed against porcine astrocytes or PAEC. Immunoreactivity of the rat sera was tested on porcine astrocytes (A) or PAEC (B), as described in Figure 1 (n ¼ see Table 1). Results are expressed according to the immunological status of the transplant. Mean values  SEM are presented (Kruskal–Wallis test and Dunn posttest;  p < 0.05,  p < 0.001). PAEC, pig aortic endothelial cells.

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Figure 3: Cell specificity of anti-porcine IgG: immunocytochemical analyses on cultured PNC. (A) Staining of PNC with rat sera collected at D0 (SD0, ungrafted) or D63 (SD63) posttransplantation. Binding of rat IgG to porcine cells was revealed with a fluorescein isothiocyanate anti-rat IgG antibody (B–D), Double staining of PNC with D63 rat sera and with anti-Tuj-1 (B) or anti-CD44 (D) antibodies that label neurons and astrocytes, respectively. Confocal analyses of Tuj-1/SD63 co-staining (C). Scale bars: A, 40 mm; B and D, 20 mm; C, 10 mm. D, day; PNC, porcine neural cells; Tuj-1, b-III tubulin.

healthy grafts (Figure 5C) whereas a large number of His24þ cells were present throughout the rejecting graft with a higher density around the blood vessels (Figure 5D). Rejection is delayed in immunoglobulin-deficient rats Implication of the humoral response in the rejection of intracerebral xenotransplants was investigated by transAmerican Journal of Transplantation 2014; 14: 1109–1119

planting PNC into the striata of IgM KO rats. These rats are B cell and Ig deficient (22,23) but their T lymphocytes are functional, as addition of porcine PBMC induces their proliferation (Figure 6A). Since IgM/ rats have an SPD background, cell survival was controlled in IgMþ/þ rats. The analyses revealed that the PNC survived longer in SPD than in Lewis 1A rats. Indeed, 50% of the IgMþ/þ rats exhibited 1113

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Figure 4: IgG deposits in the brains of transplanted rats. (A and B) IgG deposits in healthy (A) or rejecting grafts (B), as determined using fluorescein isothiocyanate anti-rat IgG antibody. (C and D) IgG deposits are present in NeuN-positive cells (C) and GFAP-positive cells (D), as assessed by double immunohistochemistry. Scale bars: A and B, 200 mm; C, 50 mm; D, 25 mm. GFAP, glial fibrillary acidic protein; Gr, grafts; NeuN, neuronal nuclei.

healthy transplants at D63 posttransplantation (Figure 6B) whereas all the Lewis 1A rats had rejected their grafts at this time point (Table 1). The PNC survived even longer in the IgM/ rats, as 100% of the rats exhibited healthy grafts at D63. These healthy grafts contained numerous porcine neurons, as detected with the anti-pNF70 antibody (Figure 6C). Neurons displaying a dopaminergic phenotype were also observed, as revealed by anti-TH immunostaining (Figure 6D). Infiltration of the grafts with immune cells was evaluated using OX42 and R73 antibodies. As seen in Figure 6E and F, very few activated microglial cells and rare R73þ cells were present within and around the grafts at 1114

D63. No C4d deposits were observed in the brains of grafted IgM KO rats (Figure 6G).

Discussion In the present study, we have shown that the transplantation of PNC into the striata of immunocompetent rats induces a specific humoral immune response, as demonstrated by the deposition of activated complement protein and xenoreactive IgG in the brain parenchyma, and the appearance of elicited, anti-porcine IgG in the rat sera. American Journal of Transplantation 2014; 14: 1109–1119

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Figure 5: Complement deposition and B cell infiltration in the brains of transplanted rats. (A and B) The presence of activated complement proteins in the ipsilateral (A) and contralateral sides (B) was assessed by anti-C4d immunohistochemistry. (C and D) Infiltration of B cells in healthy (C) and rejecting grafts (Gr) (D) was determined by anti-HIS24 immunohistochemistry. Scale bars 200 mm.

At early time points, we did not detect anti-donor antibodies in the rat sera, indicating that the rats did not produce natural antibodies against porcine neurons, porcine astrocytes or PAEC. This observation is in agreement with the results of Barker et al (20), who did not find anti-porcine IgM in the sera of transplanted rats up to 10 days posttransplantation. At late time points, such as D42 or D63, we observed anti-donor antibodies, but individual analyses revealed that their levels in the rat sera remained low until complete rejection of the graft. These results are consistent with previous findings showing that the anti-donor antiAmerican Journal of Transplantation 2014; 14: 1109–1119

bodies present within kidney transplants are detected in the peripheral traffic only after graft rejection (30,31). The timing of these events suggests that the antibodies are trapped inside the graft until its rejection. The fetal ventral midbrain contains neural precursors, neuroblasts and glioblasts, which rapidly differentiate into neurons or glial cells after their transplantation. The immunogenicities of these cell types remain to be clearly determined, but we show here that differentiated neurons are the major target of the anti-porcine IgG induced by the 1115

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Figure 6: Survival of PNC in the striatum of the IgM KO rat. (A) The ability of Ig-deficient rat T cells to respond to porcine cells was assessed by performing an MLR with porcine PBMC. T cell proliferation in the absence (T) or presence (MLR) of porcine PBMC was determined by measuring [3H]thymidine incorporation. (B) Percentage of rats exhibiting pNF70þ/R73 transplants (healthy grafts), 63 days after the transplantation of PNC into the striatum of WT (n ¼ 8) or IgM KO (n ¼ 6) rats. Mean values  SEM are presented (Mann–Whitney test;  p < 0.05). (C–G) At day 63, the grafts (Gr) were analyzed by immunohistochemistry using anti-pNF70 (porcine neurons, C), anti-TH (dopaminergic neurons, D), OX42 (microglial cells, E), R73 (T lymphocytes, F) and anti-C4d (complement deposits, G) antibodies. MLR, mixed lymphocyte reaction; PBMC, peripheral blood mononuclear cells; PNC, porcine neural cells.

transplantation of PNC into the rat brain. This statement is supported by the strong binding of elicited IgG to differentiated porcine neurons in culture, and to most of the pNF70þ neurons present in the transplant. In addition, very low binding was observed with freshly isolated PNC, composed mostly of immature neural cells. Staining of the porcine astrocytes is more difficult to interpret as the low but positive staining observed in vitro using fluorescentactivated cell sorting (FACS) was not detected by immunocytochemistry. This discrepancy is probably due to the low affinity or low concentration of the serum IgG directed against porcine astrocytes, as FACS is more sensitive than immunocytochemistry. Interestingly, IgG deposition was observed in some astrocytes present within the graft, but these were few in number. As rat reactive astrocytes could infiltrate the graft (25), and as IgG could be trapped by reactive astrocytes (32), it is difficult to draw a conclusion regarding this point, but if rat serum IgG specifically bind to particular porcine astrocytes, then it would be of great interest to characterize their phenotypes. The humoral immune response is specific, as only the IgG1 and IgG2a subclasses were significantly increased after the intracerebral transplantation of PNC. In the rat, these two 1116

isotypes are both controlled by Th2 cytokines, known for their role in mediating the activation and maintenance of the humoral response (33–35). Whether Th2 cytokines are involved in the IgG1a and IgG2a up-regulation remains to be determined, but a local increase of IL-10 and transforming growth factor beta expression was detected at 35 days posttransplantation. Interestingly, this increase was preceded by an up-regulation of interferon gamma (IFNg) (8) (Le´veˆque et al, submitted). This balance between Th1 and Th2 cytokines is probably necessary for proper control of the cellular and humoral reactions, as must be the IgG1 and IgG2a up-regulation. The functional meaning of IgG1 and IgG2a up-regulation remains to be determined, but each IgG subclass has a distinct range of potential effector functions. First, they have their own properties in terms of ability to fix or activate the complement system (36). For instance, rat IgG2a has a lower avidity index than rat IgG1, Ig2b and Ig2c, but IgG2a-containing complexes display a remarkable complement-activating capacity (37). Both rat IgG2b and IgG2c bind C1q, whereas only rat IgG2b mediates complement-dependent cytotoxicity (38). Using a model of hamster-to-rat cardiac xenografts, Miyatake et al (39) showed that only some subclasses of elicited IgG induced xenograft rejection in a complement-dependent manner. American Journal of Transplantation 2014; 14: 1109–1119

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Second, IgG subclasses bind with varying affinities and specificities to the various FcgR (40). For instance, the lowaffinity FcgRIII binds IgG1, IgG2a and IgG2b whereas the high-affinity FcgRI binds exclusively IgG2a (41). These specificities are important, as activation of the FcR in immune cells initiates a range of biological responses that may contribute to the rejection of PCN, such as phagocytosis, cytolysis and transcriptional activation of inflammatory cytokines. The role of IgG1 and IgG2a in the rejection of intracerebral xenotransplants is still unclear but several observations suggest an impact on the macrophages/ microglial cells present inside and around the xenograft. First, microglial cells express stimulatory and inhibitory FcgR (42). Second, inflammatory conditions stimulate the expression of FcgR in microglial cells (43,44) and, upon IFNg stimulation, Fc binding on rat microglial cells is even greater than on their peripheral counterparts (43). Third, immune complexes containing IgG1, IgG2a and IgG3, but not IgG2b, induce the expression of macrophage inflammatory protein1a and phagocytosis in human microglial cells through FcgR activation (45). Fourth, incubation of microglial cells with immune complexes induces antibody-dependent, cell-mediated cytotoxicity (ADCC) and oxidative burst (46). Alternatively, IgG1 and IgG2a may act on astrocytes, as these cells are massively activated in and around the graft at the time of rejection (25), express FcgR, and mediate ADCC in vitro (47). Whether the IgG1 and IgG2a elicited in response to the intracerebral transplantation of PNC act via the ADCC or the complement-dependent cell-mediated cytotoxicity pathways remains to be determined, but their presence at the graft site together with activated complement proteins raises the possibility of an active role in the rejection of intracerebral xenografts. The cellular immune response is certainly the major component in the rejection of intracerebral neural xenografts (10,11,24), but here we show that the host humoral response should not be neglected. Indeed, 9 weeks after cell transplantation, 50% of the WT animals had rejected their transplant whereas in Ig-deficient rats, all the grafts were healthy, with pNF70þ and THþ neurons. These data are important, as the functional restorative effect in the rat model of Parkinson’s disease is correlated with the number of THþ neurons, and reversion of the behavioral effect is observed upon graft rejection (48,49). Prolonged survival of PNC was previously observed in the brains of IgM KO mice, but cell rejection finally occurred at 2–4 weeks with an intrastriatal inversion of the CD4/CD8 T cell ratios (50). The authors of this study suggest that, in the absence of Ig, an immune response based on the cytotoxic activity of CD8 cells takes place to ensure cell rejection. The present work does not exclude a similar mechanism in the IgM KO rats at late time points, but the kinetics of rejection are too different to speculate on this possibility. Concerning the role of B cells, their presence in the grafted striatum raises the possibility of a local production of anti-donor antibodies, as observed after the intracerebral delivery of soluble antigens in animals with an intact blood–brain barrier American Journal of Transplantation 2014; 14: 1109–1119

(51,52). Our results indicate that the anti-donor antibodies could interact directly with transplanted cells such as porcine neurons, but this does not exclude a direct role of B cells, as activated B cells can act as antigen-presenting cells and stimulate CD4 T cells. The characteristics of the host humoral response in humans have to be clarified, but anti-HLA antibodies have been detected in the blood of Huntington’s patients transplanted with allogeneic fetal tissue (53). This suggests that both the cell-mediated and humoral immune responses should be controlled for the long-term survival of intracerebral transplants. The administration of steroid (prednisolone), in association with minocycline that targets microglial cells (7,54) and immunosuppressive drugs that target T cells (cyclosporine A) and B cells (azathioprine, mycophenolate mofetil, rituximab), is one potential therapeutic approach. Complementary strategies might be necessary with xenogenic cells. Indeed, even if HAR has never been observed following the transplantation of neural cells into the brains of humans and Old World primates, natural antibodies such as anti-alpha-galactosyl IgG may compromise their survival, in particular during the first 2 weeks when the blood–brain barrier is still damaged (12). This risk might be reduced by performing plasmapheresis (55), or by transplanting fetal neural cells from alpha1,3-galactosyltransferase KO pigs (56–58). Complement activity could be inhibited using mAbs that block C5 cleavage or by transplanting neurons from CD59 transgenic pigs (59,60). These strategies should be tested in Old World primates, which produce large amounts of natural antibodies, as do humans. In the present article, we provide the first evidence that neurons are the main target of anti-donor IgG following the intracerebral implantation of PNC. We also show that deposition of elicited IgG and complement in the brain parenchyma occurs before the detection of anti-donor antibodies in the serum. The host humoral response is specific, as only IgG1 and IgG2a subclasses show significant induction. These data, together with the fact that xenogenic neurons survive longer in the brains of Igdeficient rats, indicate that the humoral immune response is a critical parameter for the long-term survival of xenogenic neurons in the brain.

Acknowledgments This work was supported by the ‘‘Fe´de´ration des Groupements de Parkinsoniens’’: CECAP and Progreffe Foundation (to EM).

Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. 1117

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References 1. Barker RA, Barrett J, Mason SL, Bjo¨rklund A. Fetal dopaminergic transplantation trials and the future of neural grafting in Parkinson’s disease. Lancet Neurol 2013; 12: 84–91. 2. Brundin P, Barker RA, Parmar M. Neural grafting in Parkinson’s disease problems and possibilities. Prog Brain Res 2010; 184: 265–294. 3. Winkler C, Kirik D, Bjo¨rklund A. Cell transplantation in Parkinson’s disease: How can we make it work? Trends Neurosci 2005; 28: 86–92. 4. Le´veˆque X, Cozzi E, Naveilhan P, Neveu I. Intracerebral xenotransplantation: Recent findings and perspectives for local immunosuppression. Curr Opin Organ Transplant 2011; 16: 190–194. 5. Brevig T, Holgersson J, Widner H. Xenotransplantation for CNS repair: Immunological barriers and strategies to overcome them. Trends Neurosci 2000; 23: 337–344. 6. Larsson LC, Widner H. Neural tissue xenografting. Scand J Immunol 2000; 52: 249–256. 7. Michel-Monigadon D, Nerrie`re-Daguin V, Le´ve`que X, et al. Minocycline promotes long-term survival of neuronal transplant in the brain by inhibiting late microglial activation and T-cell recruitment. Transplantation 2010; 89: 816–823. 8. Melchior B, Re´my S, Nerrie`re-Daguin V, Heslan J-M, Soulillou J-P, Brachet P. Temporal analysis of cytokine gene expression during infiltration of porcine neuronal grafts implanted into the rat brain. J Neurosci Res 2002; 68: 284–292. 9. Michel DC, Nerrie`re-Daguin V, Josien R, Brachet P, Naveilhan P, Neveu I. Dendritic cell recruitment following xenografting of pig fetal mesencephalic cells into the rat brain. Exp Neurol 2006; 202: 76–84. 10. Wood MJ, Sloan DJ, Wood KJ, Charlton HM. Indefinite survival of neural xenografts induced with anti-CD4 monoclonal antibodies. Neuroscience 1996; 70: 775–789. 11. Duan W-M, Westerman MA, Wong G, Low WC. Rat nigral xenografts survive in the brain of MHC class II-, but not class Ideficient mice. Neuroscience 2002; 115: 495–504. 12. Brundin P, Widner H, Nilsson OG, Strecker RE, Bjo¨rklund A. Intracerebral xenografts of dopamine neurons: The role of immunosuppression and the blood-brain barrier. Exp Brain Res Exp 1989; 75: 195–207. 13. Larsson LC, Frielingsdorf H, Mirza B, et al. Porcine neural xenografts in rats and mice: Donor tissue development and characteristics of rejection. Exp Neurol 2001; 172: 100–114. 14. Pakzaban P, Isacson O. Neural xenotransplantation: Reconstruction of neuronal circuitry across species barriers. Neuroscience 1994; 62: 989–1001. 15. Duan WM, Brundin P, Grasbon-Frodl EM, Widner H. Methylprednisolone prevents rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in adult rats. Brain Res 1996; 712: 199– 212. 16. Collins BH, Cotterell AH, McCurry KR, et al. Cardiac xenografts between primate species provide evidence for the importance of the alpha-galactosyl determinant in hyperacute rejection. J Immunol 1995; 154: 5500–5510. 17. Galili U. Evolution and pathophysiology of the human natural antialpha-galactosyl IgG (anti-Gal) antibody. Springer Semin Immunopathol 1993; 15: 155–171. 18. Galili U, Anaraki F, Thall A, Hill-Black C, Radic M. One percent of human circulating B lymphocytes are capable of producing the natural anti-Gal antibody. Blood 1993; 82: 2485–2493. 19. Harrower TP, Tyers P, Hooks Y, Barker RA. Long-term survival and integration of porcine expanded neural precursor cell grafts in a rat model of Parkinson’s disease. Exp Neurol 2006; 197: 56–69.

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20. Barker RA, Ratcliffe E, McLaughlin M, Richards A, Dunnett SB. A role for complement in the rejection of porcine ventral mesencephalic xenografts in a rat model of Parkinson’s disease. J Neurosci Off J Soc Neurosci 2000; 20: 3415–3424. 21. Ravetch JV. A full complement of receptors in immune complex diseases. J Clin Invest 2002; 110: 1759–1761. 22. Geurts AM, Cost GJ, Freyvert Y, et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 2009; 325: 433. 23. Me´noret S, Iscache A-L, Tesson L, et al. Characterization of immunoglobulin heavy chain knockout rats. Eur J Immunol 2010; 40: 2932–2941. 24. Re´my S, Canova C, Daguin-Nerrie`re V, et al. Different mechanisms mediate the rejection of porcine neurons and endothelial cells transplanted into the rat brain. Xenotransplantation 2001; 8: 136–148. 25. Michel-Monigadon D, Bonnamain V, Nerrie`re-Daguin V, et al. Trophic and immunoregulatory properties of neural precursor cells: Benefit for intracerebral transplantation. Exp Neurol 2011; 230: 35–47. 26. Sergent-Tanguy S, Chagneau C, Neveu I, Naveilhan P. Fluorescent activated cell sorting (FACS): A rapid and reliable method to estimate the number of neurons in a mixed population. J Neurosci Methods 2003; 129: 73–79. 27. Azimzadeh A, Wolf P, Thibaudeau K, Cinqualbre J, Soulillou JP, Anegon I. Comparative study of target antigens for primate xenoreactive natural antibodies in pig and rat endothelial cells. Transplantation 1997; 64: 1166–1174. 28. Charreau B, Coupel S, Boulday G, Soulillou JP. Cyclosporine inhibits class II major histocompatibility antigen presentation by xenogeneic endothelial cells to human T lymphocytes by altering expression of the class II transcriptional activator gene. Transplantation 2000; 70: 354–361. 29. Bonnamain V, Mathieux E, Thinard R, et al. Expression of heme oxygenase-1 in neural stem/progenitor cells as a potential mechanism to evade host immune response. Stem Cells 2012; 30: 2342–2353. 30. Lee P-C, Terasaki PI, Takemoto SK, et al. All chronic rejection failures of kidney transplants were preceded by the development of HLA antibodies. Transplantation 2002; 74: 1192–1194. 31. Worthington JE, Martin S, Dyer PA, Johnson RW. An association between posttransplant antibody production and renal transplant rejection. Transplant Proc 2001; 33: 475–476. 32. Bernstein JJ, Willingham LA, Goldberg WJ. Sequestering of immunoglobulins by astrocytes after cortical lesion and homografting of fetal cortex. Int J Dev Neurosci 1993; 11: 117–124. 33. Binder J, Graser E, Hancock WW, et al. Downregulation of intragraft IFN-gamma expression correlates with increased IgG1 alloantibody response following intrathymic immunomodulation of sensitized rat recipients. Transplantation 1995; 60: 1516–1524. 34. Gracie JA, Bradley JA. Interleukin-12 induces interferon-gammadependent switching of IgG alloantibody subclass. Eur J Immunol 1996; 26: 1217–1221. 35. Saoudi A, Kuhn J, Huygen K, et al. TH2 activated cells prevent experimental autoimmune uveoretinitis, a TH1-dependent autoimmune disease. Eur J Immunol 1993; 23: 3096–3103. 36. Roos A, Essers M, van Gijlswijk-Janssen D, Bovin NV, Daha MR. Both IgG and IgM anti-pig antibodies induce complement activation and cytotoxicity. Xenotransplantation 2001; 8: 3–14. 37. Medgyesi GA, Miklo´s K, Kulics J, Fu¨st G, Gergely J, Bazin H. Classes and subclasses of rat antibodies: Reaction with the antigen and interaction of the complex with the complement system. Immunology 1981; 43: 171–176.

American Journal of Transplantation 2014; 14: 1109–1119

IgG Response to Intracerebral Xenograft 38. Bru¨ggemann M, Teale C, Clark M, Bindon C, Waldmann H. A matched set of rat/mouse chimeric antibodies. Identification and biological properties of rat H chain constant regions mu, gamma 1, gamma 2a, gamma 2b, gamma 2c, epsilon, and alpha. J Immunol 1989; 142: 3145–3150. 39. Miyatake T, Sato K, Takigami K, et al. Complement-fixing elicited antibodies are a major component in the pathogenesis of xenograft rejection. J Immunol 1998; 160: 4114–4123. 40. Okun E, Mattson MP, Arumugam TV. Involvement of Fc receptors in disorders of the central nervous system. Neuromolecular Med 2010; 12: 164–178. 41. Nimmerjahn F, Ravetch JV. Fcgamma receptors: Old friends and new family members. Immunity 2006; 24: 19–28. 42. Vedeler C, Ulvestad E, Grundt I, et al. Fc receptor for IgG (FcR) on rat microglia. J Neuroimmunol 1994; 49: 19–24. 43. Woodroofe MN, Hayes GM, Cuzner ML. Fc receptor density, MHC antigen expression and superoxide production are increased in interferon-gamma-treated microglia isolated from adult rat brain. Immunology 1989; 68: 421–426. 44. Loughlin AJ, Woodroofe MN, Cuzner ML. Modulation of interferon-gamma-induced major histocompatibility complex class II and Fc receptor expression on isolated microglia by transforming growth factor-beta 1, interleukin-4, noradrenaline and glucocorticoids. Immunology 1993; 79: 125–130. 45. Song X, Shapiro S, Goldman DL, Casadevall A, Scharff M, Lee SC. Fcgamma receptor I- and III-mediated macrophage inflammatory protein 1alpha induction in primary human and murine microglia. Infect Immun 2002; 70: 5177–5184. 46. Ulvestad E, Williams K, Matre R, Nyland H, Olivier A, Antel J. Fc receptors for IgG on cultured human microglia mediate cytotoxicity and phagocytosis of antibody-coated targets. J Neuropathol Exp Neurol 1994; 53: 27–36. 47. Nitta T, Yagita H, Sato K, Okumura K. Expression of Fc gamma receptors on astroglial cell lines and their role in the central nervous system. Neurosurgery 1992; 31: 83–87; discussion 87–88. 48. Huffaker TK, Boss BD, Morgan AS, et al. Xenografting of fetal pig ventral mesencephalon corrects motor asymmetry in the rat model of Parkinson’s disease. Exp Brain Res Exp 1989; 77: 329–336. 49. Galpern WR, Burns LH, Deacon TW, Dinsmore J, Isacson O. Xenotransplantation of porcine fetal ventral mesencephalon in a rat model of Parkinson’s disease: Functional recovery and graft morphology. Exp Neurol 1996; 140: 1–13. 50. Larsson LC, Czech KA, Widner H, Korsgren O. Discordant neural tissue xenografts survive longer in immunoglobulin deficient mice. Transplantation 1999; 68: 1153–1160.

American Journal of Transplantation 2014; 14: 1109–1119

51. Knopf PM, Harling-Berg CJ, Cserr HF, et al. Antigen-dependent intrathecal antibody synthesis in the normal rat brain: Tissue entry and local retention of antigen-specific B cells. J Immunol 1998; 161: 692–701. 52. Cserr HF, DePasquale M, Harling-Berg CJ, Park JT, Knopf PM. Afferent and efferent arms of the humoral immune response to CSF-administered albumins in a rat model with normal blood-brain barrier permeability. J Neuroimmunol 1992; 41: 195–202. 53. Krystkowiak P, Gaura V, Labalette M, et al. Alloimmunisation to donor antigens and immune rejection following foetal neural grafts to the brain in patients with Huntington’s disease. PLoS ONE 2007; 2: e166. 54. Kobayashi K, Imagama S, Ohgomori T, et al. Minocycline selectively inhibits M1 polarization of microglia. Cell Death Dis 2013; 4: e525. 55. Alwayn IP, Basker M, Buhler L, Cooper DK. The problem of anti-pig antibodies in pig-to-primate xenografting: Current and novel methods of depletion and/or suppression of production of antipig antibodies. Xenotransplantation 1999; 6: 157–168. 56. Dai Y, Vaught TD, Boone J, et al. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002; 20: 251–255. 57. Lai L, Kolber-Simonds D, Park K-W, et al. Production of alpha-1,3galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002; 295: 1089–1092. 58. Phelps CJ, Koike C, Vaught TD, et al. Production of alpha 1,3galactosyltransferase-deficient pigs. Science 2003; 299: 411–414. 59. Fodor WL, Williams BL, Matis LA, et al. Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection. Proc Natl Acad Sci USA 1994; 91: 11153–11157. 60. Cicchetti F, Fodor W, Deacon TW, et al. Immune parameters relevant to neural xenograft survival in the primate brain. Xenotransplantation 2003; 10: 41–49.

Supporting Information Additional Supporting Information may be found in the online version of this article. Figure S1: Classification of the graft. Figure S2: Porcine brain proteins recognized by the elicited IgG present in the blood of transplanted rats.

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