A Distinct Th1 Immune Response Precedes the Described Th2 Response in Islet Xenograft Rejection

Pregnant sows from a local stock were killed using a slaughtering mask at 70 ± 5 days of gestation (full-term is 115 days). The fetuses (8–16 per litter) were immediately collected from the uterus and placed on ice during transport to the laboratory. After aseptic removal, the pancreatic glands were minced into fragments measuring 1–2 mm³ in cold Hanks’ solution and then treated with collagenase (10 mg/ml; Boehringer Mannheim, Mannheim, Germany) with vigorous shaking, as previously described (12). The digested tissue was washed and explanted into culture dishes that allowed cellular attachment (Nunclon 90 mm Ø; Nunc, Kamstrup, Denmark). The culture medium was RPMI 1640 (11.1 mmol/l glucose; Flow Laboratories, Irvine, U.K.) supplemented with 10% heat-inactivated human AB serum (vol/vol) (Blood Center, Huddinge Hospital, Huddinge, Sweden) and 10 mmol/l nicotinamide (Sigma, St. Louis, MO). The culture dishes were kept at 37°C in humidified air containing 5% CO₂, and the culture medium was changed every other day. On day 4 of culture, most of the ICCs were free-floating or could easily be detached by gently flushing the culture medium. All free-floating fragments (diameter <0.7 mm) were considered to be ICCs and were harvested without any further purification step.

Male inbred normal Lewis (LEW) and athymic (LEW nu/nu) rats (Møllegaard Breeding Center, Ry, Denmark) were used as recipients. The animals had free access to tap water and pelleted food throughout the study. All animals were anesthetized with 0.1 ml/1,000 g body wt Hypnorm (10 mg/ml fluonisonum + 0.2 mg/ml fentanylum; Janssen Pharmaceutica, Belgium) i.m. and transplanted with two 2-μl grafts of ICCs (∼2 × 200 ICCs), via a braking pipette, under the left kidney capsule. The animals were killed at 1 (six LEW), 3 (six LEW and three LEW nu/nu), 5 (six LEW and four LEW nu/nu), or 12 (six LEW and three LEW nu/nu) days after transplantation. When they were killed, the two ICC grafts were clearly visible as whitish spots beneath the kidney capsule. One ICC graft from each rat was removed for histological evaluation, and the other one was used for quantification of cytokine mRNA expression. The ICC grafts subsequently used for histological evaluation were excised from the kidneys with a margin of ∼3 mm, whereas the grafts used for mRNA analyses were peeled off of the kidneys with minimal amounts of adjacent kidney tissue. Regional lymph nodes (draining the graft-bearing kidneys), peripheral lymph nodes (collected from the right iliaca vessel regions), and control kidney tissue (from the non–graft-bearing kidneys) were also collected and, along with the ICC grafts, were immediately snap-frozen in liquid nitrogen and stored at −70°C. Kidney tissue and peripheral and kidney-draining lymph nodes were also collected from three nontransplanted LEW rats to obtain day 0 values for the subsequent RT-PCR analyses.

Serial sections (6-μm thick) were cut in a cryostat (−20°C), air dried, and then stored at −70°C. After storage, the slides were fixed in cold acetone diluted 1:2 in distilled water for 30 s, followed by final fixation in cold acetone (100%) for 5 min. Sections from all of the animals were stained with hematoxylin for the histology studies.

Slides were fixed as described in the research design and methods section on histology, and the following steps were carried out using Dako Tech Mate 500 Plus software, version 1.11b. Endogenous peroxidase was blocked by incubation in 0.3% H₂0₂ for 15 min, and the sections were then washed 3 × 5 min in phosphate-buffered saline (PBS). The slides were subsequently incubated for 30 min with monoclonal antibodies, as specified in Table 1 (with anti-iNOS from Sigma and the others from Serotec, Oxford, U.K.), followed by washes for 3 × 5 min. Next, the slides were incubated for 20 min with rabbit anti–mouse IgG antibody (Dako, Glostrup, Denmark) and 1% normal rat serum, followed by 4 × 5 min washes. After a 20-min incubation with monoclonal mouse peroxidase–anti-peroxidase (PAP) reagent (Dako) and 5 × 5-min washes, the peroxidase reaction was developed by 3 × 5-min incubations in a carbazole-containing buffer. The slides were finally counterstained with hematoxylin and mounted in glycerine gelatin. Control experiments were performed by omitting the primary antibodies.

Slides were fixed as described in the research design and methods section on histology. Fluorescein-labeled rabbit anti–rat IgG and IgM (Serotec) were used for direct immunofluorescence staining using the Dako Tech Mate 500 Plus software, version 1.11b. A goat anti–rat C3 (Organon Teknika, Westchester, NY) followed by fluorescein-labeled rabbit anti–goat Ig (Dako) was used for indirect staining. All incubations were carried out for 30 min. The slides were mounted with Mounting Medium (Immuno Concepts) and analyzed for green fluorescence with a Zeiss UV microscope.

Slides were fixed as described in the research design and methods section on histology. Eosinophils were detected by histochemical visualization of cyanide-resistant endogenous peroxidase activity (13). Briefly, tissue sections were incubated for 8 min at room temperature in PBS buffer supplemented with 3.3-diaminobenzidine tetrahydrochloride (60 mg/100 ml; Sigma Chemicals), H₂0₂ (0.3 ml 30% / 100 ml; Kebo Lab), and NaCN (120 mg/100 ml; Aldrich). After rinsing with water, slides were counterstained with hematoxylin and mounted in glycerine gelatin. Eosinophils were identified by their dark brown reaction product.

Apoptotic cells were detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method (In situ Cell Death Detection Kit; Boehringer Mannheim). The assay uses terminal deoxynucleotidyl transferase to label free 3′OH strand breaks in genomic DNA with fluorescein-dUTP to discriminate apoptosis from necrosis (14). All TUNEL stainings were performed in accordance with the manufacturer’s instructions. In brief, slides were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. After a 10-min wash in PBS, the tissue sections were permeabilized by incubating in 0.1% sodium citrate containing 0.1% Triton X-100 for 2 min on ice. The slides were then rinsed twice with PBS before they were incubated for 60 min at 37°C in the dark with TUNEL reaction mixture (terminal deoxynucleotidyl transferase and fluorescein-dUTP). Sections incubated in TUNEL reaction mixture without terminal deoxynucleotidyl transferase served as negative controls. All of the slides were mounted in PBS-glycerol and analyzed for green fluorescence with a Zeiss UV microscope.

The tissue samples were lysed in 350 μl extraction buffer (100 mmol/l Tris-HCl, pH 8.0, 10 mmol/l EDTA, 5 mmol/l dithiothreitol (DTT), 1% lithium dodecyl sulfate, and 500 mmol/l LiCl) and homogenized by means of a syringe and needle. cDNA synthesis was performed when mRNA had been isolated with oligo(dT)-coated manifold supports (15). Briefly, the mRNA in the tissue extract was immobilized on oligo(dT)-coated manifold supports and transferred to 50 μl cDNA synthesis reactions (50 mmol/l Tris-HCl, pH 8.3, 75 mmol/l KCl, 3 mmol/l MgCl₂, 10 mmol/l DTT, 0.5 mmol/l dNTP, 0.1 μg/μl bovine serum albumin [BSA], 0.5 μmol/l random hexamers, 0.5 units/μl human placental ribonuclease inhibitor [HPRI; Amersham Pharmacia Biotech], and 4.2 units/μl SuperScript II RNase H⁻ reverse transcriptase [Life Technologies]). The reactions were incubated for 10 min at 42°C followed by 50 min at 37°C, and the newly synthesized cDNA was then released from the supports by incubating the manifolds in 70 μl water for 5 min at 95°C. A Prism 7700 Sequence Detection System (ABI, Foster City, CA) was used for running 5′ nuclease assays for quantitative analysis of the generated cDNA. The cDNA sequences for the genes of interest were obtained from GenBank, and the primer and probe sequences, which are specified in Table 2, were designed using PrimerExpress software (PE Applied Biosystems). To avoid amplification of genomic DNA, the primer-probe sets were designed to span exon-exon borders. The probes were labeled with FAM at the 5′ end and TAMRA at the 3′ end. PCR amplifications were performed in a total volume of 25 μl containing 5 μl cDNA sample; 800 nmol/l of each primer; 100 nmol/l of the corresponding probe; 200 μmol/l dNTP; 0.625 units AmpliTaq Gold (PE Applied Biosystems); 50 mmol/l KCl; 10 mmol/l Tris HCl (pH 8.3); 10 mmol/l EDTA; 60 nmol/l Passive Reference 1; and 1, 2, or 5 mmol/l MgCl₂ (1 mmol/l for IL-2, IL-4, IL-10, IFN-γ, and β-actin; 2 mmol/l for TNF-α; and 5 mmol/l for IL-1β and IL-12p40). For each reaction, the polymerase was activated by preincubating at 95°C for 10 min, and amplification was then performed by 50 cycles of switching between 95°C for 15 s and 58°C for 60 s. The results were represented as threshold cycle values (C_t values), which are estimates of the amplification cycle number when the fluorescence exceeds a specified threshold value (16,17). All of the tissue samples were run in triplicates for the cDNA synthesis and in the following PCR amplifications. Known amounts of amplicons, generated by the different primer pairs, were diluted and run in all PCR amplifications. Standard curves were then created by plotting C_t values versus the log of the amount of cDNA template in the respective dilution. These were then used to calculate the initial quantity of cDNA template in the tissue samples. No-template controls (i.e., cDNA substituted with water) and RT⁻ controls (where reverse transcriptase had been left out in the earlier cDNA synthesis) were run together with the unknown samples in all PCR amplifications to screen for possible contamination and genomic amplification. Data are given as the means ± SE.

Rat anti-ICC antibodies were measured before transplantation and at the end of the observation period with an enzyme-linked immunosorbent assay (ELISA) (18) using solubilized membrane fractions from porcine ICC as antigens. Briefly, ELISA plates (Costar 3590; Costar, Cambridge, MA) were coated with a solubilized antigen solution at 4°C overnight. After blocking nonspecific binding and then washing, the various rat sera were applied in a dilution of 1:50 and incubated for 3 h at room temperature. After washing, the secondary antibodies (goat anti–rat IgM and IgG conjugated to alkaline phosphatase; Southern Biotechnology, Birmingham, AL) were applied for 2 h at room temperature. The wells were then washed, and substrate solution was added. The optical density was measured in a microplate autoreader at 405 nm. Optical density values were presented as the means ± SE. The statistical significance of the data were determined by Student’s paired t test. P < 0.05 was considered to be significant.

No IL-2 or IL-4 and only occasional low-gene expression of the other studied cytokines was detected in the contralateral kidney of the xenotransplanted LEW rats. The ICC xenografts in the LEW nu/nu rats were intact throughout the study, despite a moderate infiltration of macrophages and occasional-to-few dendritic and NK-cells (Table 3). Cytokine mRNA expression in the grafts and in the regional and peripheral lymph nodes of the LEW nu/nu rats were markedly lower than in the LEW rats at all time points (Fig. 1).

On day 0 (nontransplanted rats), transcripts of the cytokines studied were either not detected or detected only at very low levels in both the regional and peripheral lymph nodes as well as in kidney tissue. On day 1, the ICC xenografts were roughly intact, and only occasional immunocompetent cells were found infiltrating the grafts. No expression of β-actin and the studied cytokines, or in some cases only very low levels of expression, were found in the grafts. However, gene expression of β-actin, IL-1β, IL-2, IL-12p40, and TNF-α was increased in both peripheral and regional lymph nodes, whereas that of IFN-γ was upregulated only in the peripheral lymph nodes. On day 3, several ICCs still remained intact. An increased level of transcripts for β-actin was detected in the grafts for the first time and correlated with the infiltration of immunocompetent cells. A few TCR⁺, CD4⁺, and OX-62⁺ cells were seen in the border between the ICC xenografts and the adjacent kidney parenchyma, whereas moderate numbers of ED1⁺ cells were found in the central parts of the grafts. A few CD8⁺ cells were also found in this area of the grafts. Only a few graft-infiltrating cells were found expressing iNOS, and either none or only a few apoptotic cells were detected. Among the cytokines, primarily IFN-γ transcript levels were elevated in the grafts. The gene expression of β-actin peaked on day 3 in both regional and peripheral lymph nodes. Transcripts for IL-10 were increased for the first time at this time point, but as for IFN-γ and TNF-α, there was no difference between peripheral and regional lymph nodes. In contrast, the gene expression of IL-1β, IL-2, and IL-12p40 were markedly higher in the regional lymph nodes compared with the peripheral lymph nodes.

On day 5, the grafts were heavily infiltrated by immunocompetent cells, and only occasional remnants of ICCs remained. The marked increase in infiltrating cells was reflected by a pronounced increase in the number of transcripts for β-actin in the grafts at this time point. Many ED1⁺, ED2⁺, and CD8⁺ cells were evenly distributed, entirely covering the grafts. A moderate number of CD4⁺ cells infiltrated the central part of the grafts, but even more were accumulated in the periphery. Moderate numbers of TCR⁺ cells were found in the peripheral parts of the grafts, and only a few TCR⁺ cells were located in the central area. Few to moderate numbers of NKR-P1⁺ and OX-62⁺ cells were evenly spread within the grafts. Moderate numbers of infiltrating ED1⁺ and ED2⁺ cells expressed iNOS. Only occasional apoptotic cells were detected in the grafts. The number of transcripts for IL-10 and IFN-γ was markedly increased, whereas that of IL-1β and IL-2 was only modestly increased in the grafts at 5 days after transplantation. The transcript levels for IL-1β, IL-2, IL-12p40, and β-actin decreased markedly in the lymph nodes from days 3 to 5, possibly reflecting a relocation of immunocompetent cells from the lymph nodes to the grafts.

On day 12 after transplantation, the ICC xenografts were totally rejected. A massive infiltration of immunocompetent cells still persisted in the grafts. However, a reduction of ∼50% in number of transcripts of β-actin was found when compared with day 5. ED1⁺, ED2⁺, CD4⁺, and CD8⁺ cells, ranging from many to massive numbers, completely covered the grafts. In the central part of the grafts, there was a dense accumulation of cells positively stained for all these antigens. Moderate numbers of these cells expressed iNOS. Only a few apoptotic cells were found, and these were evenly distributed within the graft. TCR⁺, NKR-P1⁺, and OX-62⁺ cells could be found in all parts of the grafts, but the majority of these cells were seen in the periphery. A few eosinophils were found in the grafts for the first time. The transcript levels in the grafts increased slightly for IL-4 but decreased for all of the other cytokines analyzed. Elevated transcript levels in the regional lymph nodes were seen for β-actin, IL-10, and TNF-α. In the peripheral lymph nodes, the mRNA expression of these genes was approximately the same as at day 5, whereas the IL-12p40 transcripts increased to the levels found at day 3. The gene expression of IL-4 increased slightly in the peripheral lymph nodes but was markedly enhanced in the regional lymph nodes. The transcript levels of IL-1β, IL-2, and IFN-γ remained stable when compared with that found on day 5.

All rats had preformed xenoreactive anti-ICC IgM antibodies, and the level of these antibodies did not increase after ICC transplantation. However, a significant increase in the level of anti-ICC IgG was found at 12 days after transplantation. Infiltrating B-cells or depositions of IgM, IgG, or C3 were either not detected or rarely detected within the ICC xenografts.

The present findings clearly demonstrate that not only the cellular infiltration profile but also the cytokine mRNA expression profile in the acute cellular rejection of an ICC xenograft resembles that of a DTH reaction. However, a Th2 response is triggered at the point at which most of the graft has been rejected, corresponding to the infiltration of eosinophils and an increase in anti-ICC IgG titers.

The real-time quantitative RT-PCR technique has not been previously applied in studies of acute cellular xenograft rejection. The technique enables quantification of the specific number of a defined messenger present in the graft/lymph nodes at a certain time point. The present findings, showing T-cell infiltration mainly in the periphery of the grafts and numerous macrophages in the central parts, in parallel with mRNA expression of TNF-α, IFN-γ, and IL-2, indicate that ICC xenograft rejection shares significant characteristics with that of a DTH reaction. The interpretation that acute cellular xenograft rejection is a Th2-associated reaction, which has been put forth in previous studies using RT-PCR to analyze the local cytokine response, is contradictory to the present findings. This discrepancy may be explained either by the much higher resolution that can be gained by using the real-time RT-PCR technique or by differences in species combinations. The notion of cellular xenograft rejection being a Th2-dependent reaction is, however, not supported by previous findings demonstrating that the rejection of an islet xenograft proceeds almost unchanged in IL-4– or IL-5–deficient mice (6). The only effect observed in these animals was an absence of infiltrating eosinophils, strongly suggesting that acute cellular rejection proceeds normally in mice lacking the functional capacity to mount a Th2-associated response. On the other hand, the previous finding of islet xenograft rejection being a highly specific process (19) argues against the view that it shares significant characteristics with those of a DTH reaction. However, it should be noted that the DTH reaction is per definition an immune response observed after previous immunization and generation of memory T-cells, and that almost nothing is known about the immunological mechanisms orchestrating the reaction or its specificity at the time when the antigen is first encountered. Interestingly, both the tempo and strength of the ICC xenograft rejection is increased, whereas the specificity of the rejection is lost in animals previously immunized with pig islets (20). Based on the temporal correlation between immunohistological findings and cytokine profiles in this xenograft model, it is argued that cellular rejection is caused by Th1 CD4⁺ T-cell–mediated activation of macrophages, i.e., a DTH reaction. Nonetheless, ICC xenograft rejection has been demonstrated to persist in IFN-γ–deficient mice (21), even though the rejection process was slightly delayed in these mutant mice compared with normal controls. Other cytokines, such as TNF-β or TNF-α, possibly substitute for the loss of IFN-γ in these mice. In fact, simultaneous but not single inhibition of IFN-γ, TNF-α, and IL-2 could prevent ICC xenograft rejection in some animals (22).

A Th2 response is triggered by IL-4, locally released at the site of antigen presentation to naive Th cells. Subsequently, activated T-cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 (23,24,25). Similar to previous reports of RT-PCR studies of cytokines in islet xenografts (8,9,10), we were able to detect IL-4 and IL-10 transcripts in the rejecting grafts. However, because the IL-4 mRNA levels were still low in the lymph nodes as well as in the grafts the first 5 days after transplantation, it does not seem as if a Th2 response is induced at the earlier observation times. This notion is strongly supported by the present immunohistological observations, as well as the late appearance of a humoral immune response. Hence, it is more likely that the IL-10 transcripts detected at earlier time points, before the increase in IL-4 messenger, are expressed by the highly activated macrophages. Stimulation by lipopolysaccharide (LPS) triggers monocytes to produce proinflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Subsequently, the anti-inflammatory cytokine IL-10, which downregulates proinflammatory cytokines as well as its own synthesis, is secreted. IL-10 and TNF-α production are induced more or less simultaneously by GM-CSF (26,27). This effect is exerted at least at the transcriptional level. Also, in the present study, the transcripts of TNF-α and IL-10 were produced with similar kinetics. Moreover, the release of cytokines from human lymphomononuclear cells in response to allogeneic or xenogeneic islets was recently reported (28). Only xenogeneic islets caused an increased production of IL-10. Importantly, removal of the monocyte-macrophage cells caused a marked reduction of IL-6, IL-10, and TNF-α in response to xenogeneic but not allogeneic islets, suggesting that the source of these cytokines may be different in allotransplantation compared with xenotransplantation.

The graft site contains many chemotactic factors, necrotic debris, and superoxide radicals, representing a significant inflammatory focus. The role of locally produced IL-10 might be to keep the developing ICC xenograft rejection well localized and of minimum duration by reducing bystander damage on adjacent tissues. It may be speculated that this balanced immune response is lost when an animal is rechallenged with the same antigen, leading to a fully mature DTH reaction characterized by the well-known dominance of Th1-associated cytokines and a pronounced bystander effect. Our previous observations regarding the specificity and intensity in rejection of an ICC xenograft in nonimmunized compared with immunized animals support this notion (20).

The first appearance of eosinophils infiltrating the ICC xenografts as well as the increase in anti-ICC IgG titers occurred 12 days after transplantation. This fits well with the observed increase of IL-4 transcripts in the regional lymph nodes and within the ICC xenograft. This mounting of a Th2 response was, however, observed when the vast majority of the ICCs had already been rejected. It may be speculated that this sequential activation of first a Th1 response and then a subsequent Th2 response could be beneficial after “naturally occurring xenotransplantation,” e.g., parasite infections. This would enable the host to minimize bystander destruction of adjacent tissues and at the same time protect itself from the invading microorganisms escaping the local DTH response, e.g., via penetration of a blood vessel. Opsonization of the invading microorganism in the blood with xenoreactive antibodies could then result in enhanced phagocytosis, antibody-mediated complement activation, and antibody-dependent cell cytotoxicity.

At first sight, the great variation in expression between the different cytokines might seem surprising. However, it should be remembered that this does not necessarily reflect the importance of the different cytokines. Differences in mRNA degradation, translation efficiency, receptor affinity, and receptor expression for all cytokines assayed are not fully characterized. Furthermore, the mRNA for the cytokines could be reverse-transcribed with different efficiencies in the applied cDNA synthesis method. Consequently, variations in mRNA levels of each respective cytokine should be considered rather than comparing the absolute levels between the different cytokines.

The β-actin primer-probe set was, similarly to those for the cytokines, designed to amplify rat but not pig cDNA, allowing an estimate of the number of infiltrating cells within the grafts or of the recruitment of cells to the lymph nodes. Comparisons of the kinetics of the β-actin gene expression in the grafts (Fig. 1) and the histological data (Table 3) correlate well. Surprisingly, there was already an equally strong recruitment of leukocytes in both peripheral and regional lymph nodes at 1 day after transplantation. Notably, analyses of cytokine transcripts indicate specific activation (IL-2 and IL-12p40) preferentially in the regional lymph nodes. The level of β-actin transcripts indicate that the regional lymph nodes displayed a biphasic dynamic recruitment of leukocytes, with a first peak at 3 days after transplantation and a second at day 12. The decrease in cell number within the lymph nodes that was seen on day 5 could reflect the stimulated leukocytes at this time point being directed toward the transplants.

ICC xenotransplantation produced minimal cytokine upregulation in athymic rats, indicating that the surgical trauma caused only a minimal inflammatory reaction. However, the rapid increase in the mRNA expression of TNF-α, IL-1β, IL-10, and IL-12 in both regional and peripheral lymph nodes, in parallel with a marked influx of cells (an increase in β-actin transcript levels) in immunocompetent rats, indicates that xenografts induce a strong systemic T-cell–mediated inflammatory response. This type of systemic immune activation has so far been mainly associated with injection of bacterial components, e.g., LPS. The expression of species-specific carbohydrates within the xenograft may trigger such an inflammatory reaction, providing optimal conditions for the subsequent induced immune response. The first step in this reaction would be migration to the regional lymph nodes of cells with antigen-presenting capacity that have encountered xenoantigens at the site of implantation. Dendritic cells, but not macrophages, have been reported to be the main transporters of antigen from infected tissue sites to the T-cell areas of draining lymph nodes (29). TNF-α is believed to be the major cytokine implicated in the migration of dendritic cells to lymph nodes (30). Previous studies have demonstrated that dendritic cells injected subcutaneously migrate to the draining lymph nodes. After day 1, these cells could be found in the sinus, and by day 3, they entered T-cell–dependent areas. The formation of T-cell–dendritic cell clusters coincided with IL-2 production by the antigen-specific T-cells, followed by proliferation and differentiation into DTH T-cells (31). This temporal migration and subsequent interaction/activation of naive T-cells correlates well with the dynamic mRNA expression of the T-cell–associated cytokines IL-2, IL-12, and IFN-γ in the present study. The principal role for macrophages in this scenario would be to restimulate already-activated T-cells entering the ICC xenograft. These restimulated T-cells would then provide activation signals necessary for macrophages to carry out effector functions (32).

It has been suggested that only myeloid dendritic cells have the capacity to phagocytose an antigen in peripheral tissues, carry it to regional lymph nodes, and efficiently stimulate naive Th cells, whereas one or more of these abilities would be missing in other antigen-presenting cells (33,34). The elevated levels of IL-12p40 transcripts in the regional lymph nodes are in accordance with the presence of activated dendritic cells (antigen-presenting cells). Interestingly, in relation to our previous study of ICC xenograft rejection in IFN-γ–deficient mice (21), the expression of IL-12p40 by dendritic cells does not require IFN-γ (32). Naive Th cells that are stimulated by IL-12 at antigen presentation differentiate into Th1 cells. The mRNA expression of IL-12 has not been studied in xenogeneic islet transplantation before, nor has the presence of cytokine transcripts in the lymph nodes been studied. This might explain why earlier studies of the cytokine response to islet xenografts have not been able to see important Th1 components and have instead emphasized the role of a Th2-associated immune response.

We thank Selina Bari, Ulrika Johansson, and Berit Sundberg for excellent technical assistance. This study was supported by grants from the Swedish Medical Research Council (06P-11813, 16X-12219, and K1999-73X-012232-03A), the Åke Wiberg Foundation, the Nordic Insulin Fund, the Torsten and Ragnar Söderbergs Foundation, the Ernfors Family Fund, the Barn Diabetes Fonden, the Göran Gustafsson Foundation, the Swedish Diabetes Association, the Karolinska Institute, the Reine Westerholm Foundation, the Swedish Medical Society, the Juvenile Diabetes Foundation International, and the Knut and Alice Wallenberg foundation.