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Keywords:

  • Cardiac;
  • neonate;
  • non-specific;
  • tissue-specific;
  • tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Neonatal tolerance is exclusively donor-specific when assessed by skin allograft survival and in vitro alloreactivity assays. In contrast, we reported previously that acceptance of primarily vascularized cardiac allografts was not donor-specific in C3H/He (C3H, H-2k) mice treated as neonates with BALB/c-derived (BALB, H-2d) lymphohematopoietic cells, but included third-party C57BL/10 (B10, H-2b) allografts. The present study examined whether this unusual pattern is limited to heart grafts in this strain combination, and defined the relative importance of the donor cell H-2d haplotype for third-party cardiac allograft acceptance. C3H neonates were injected with (C3HxBALB)F1 bone marrow and spleen cells. Tolerance was assessed at age 8–10 weeks by transplantation of heart or skin allografts from several donor strains, and by in vitro assays of proliferation and cytotoxicity. Additionally, cells from H-2d and H-2b-expressing strains on BALB or non-BALB minor histocompatibility (miH) antigen backgrounds were tested as tolerizing inocula. Prolonged survival of cardiac grafts from all donor strains was observed in neonatally treated mice, whereas skin grafting and in vitro assays demonstrated donor-specific hyporesponsiveness. Both H-2d haplotype and non-H-2 miH background of graft donor and tolerizing cell donor were important to third-party cardiac allograft acceptance. These results suggest that the functional alteration in alloreactivity induced by neonatal alloantigen exposure depends partly on method of assessment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Landmark experiments carried out in the 1950s by Billingham, Brent and Medawar demonstrated that transplantation tolerance could be induced in immunologically immature mice by neonatal exposure to lymphohematopoietic cells expressing alloantigens of the eventual donor strain (1–5). The ease of tolerance induction was related to the immunogenetic susceptibility of the recipient strain as well as the antigenic disparity between donor and recipient. Early evidence supported central clonal deletion of developing recipient alloreactive T cells as the predominant mechanism of neonatal tolerance (1,4–8), reviewed by Brent and by Streilein (9,10). Subsequently, many investigators have demonstrated evidence for additional mechanisms, including clonal inactivation and active suppression or cytokine immunomodulation, illustrating the immunologic complexity of neonatally induced tolerance (9–29).

In many of these studies, a neonatal tolerizing inoculum composed of semi-allogeneic bone marrow and spleen cells was injected, thereby inducing permanent acceptance of genetically compatible skin allografts, while skin grafts from unrelated third-party strains were rejected promptly. This in vivo phenotype of neonatal tolerance typically was reflected in vitro by donor-specific hyporesponsiveness in alloreactivity assays. (Semi-allogeneic cells were used in the tolerizing inoculum instead of fully allogeneic cells to avoid graft-vs.-host disease, GVHD) (2–4). Our previous studies were the first to use primarily vascularized, heterotopically placed cardiac allografts for assessment of neonatally induced tolerance (30–32). We demonstrated that unresponsiveness to cardiac allografts was not donor-specific, using donor and recipient strains with full major histocompatibility complex (MHC) and multiple minor histocompatibility (miH) antigen disparity. In C3H/He (C3H, H-2k) mice exposed as neonates to BALB/c-derived alloantigens (BALB, H-2d), we observed prolonged survival of cardiac allografts from unrelated third-party C57BL/6 (B6, H-2b) donors, in addition to acceptance of donor-type BALB grafts.

Donor-specific tolerance is often cited as a goal of transplantation tolerance protocols. However, in most clinical transplant situations the identity of a cadaveric organ donor usually cannot be known sufficiently far in advance to allow consideration of donor-specific tolerance induction protocols. This time element is particularly crucial in heart transplantation, where the interval between identification of a potential donor and graft implantation in the recipient necessarily must be brief to minimize effects of brain death and ischemia on post-transplant myocardial function. Thus, an immunologic manipulation leading to acceptance of heart grafts from any potential organ donor, i.e. ‘tissue-specific’ vs. ‘donor-specific’ tolerance, would be invaluable, if this could be accomplished without inducing a state of global immunodeficiency or immunosuppression. In this regard, understanding the mechanisms leading to acceptance of third-party cardiac allografts in the neonatal murine model has important implications for clinically relevant tolerance induction protocols, particularly in relation to infants transplanted during the immunologic immaturity of very early life.

The present study was undertaken to explore in more detail the unexpected pattern of neonatal tolerance discussed in the previous model above, and to define the requirements for acceptance of third-party cardiac allografts. In particular, we wished to determine whether ‘non-donor-specific’ graft acceptance induced in C3H mice by neonatal exposure to BALB-derived alloantigens is limited to B6 cardiac allografts, or includes cardiac allografts from other third-party strains and skin allografts, as well as alloreactivity responses in vitro of cells from neonatally treated mice. We also investigated the relative importance of the H-2 haplotype of the tolerizing cell donor vs. the non-MHC background of multiple miH antigens to the development of this novel phenomenon.

Methods and Materials

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Part A: Comparison of the specificity of unresponsiveness induced in neonatal C3H mice by exposure to BALB-derived alloantigens using different assessments of tolerance

Experimental design: Within 24 h of birth, neonatal mice were injected with a cellular tolerizing inoculum as described below, derived from semi-allogeneic adult donors. No further treatment was administered. At approximately 8 weeks of age, treated mice and untreated control mice were assessed for tolerance using both in vivo and in vitro methods. No immunosuppressive therapy was administered.

Assessment of unresponsiveness: Treated and naïve control mice were transplanted with gender-matched, fully allogeneic donor-type or third-party heart grafts or skin grafts, or were sacrificed for harvesting of lymph node cells (LNC) for in vitro assessment of alloreactivity in proliferative and cytotoxicity assays.

Immunogenetic combinations: Neonatal recipients were all C3H (expressing the H-2k haplotype). Donors of tolerizing cells were adult (C3HxBALB)F1 mice of H-2k/d haplotype. Cardiac and skin allografts were from fully allogeneic adult BALB donors (H-2d) or unrelated third-party donors of other fully disparate H-2 haplotypes including B6 (H-2b), NZW(H-2z), SJL (H-2s) or adult outbred CD1 mice. Controls for transplant experiments were neonatally treated C3H mice transplanted with syngeneic heart or skin grafts and untreated C3H recipients transplanted with heart or grafts from all of the above donor strains. For in vitro experiments, responder cells were derived from neonatally treated and untreated C3H recipients. Stimulators and target cells were derived from BALB donors, B6 and NZW third-party strains and from syngeneic C3H mice.

Part B: Determination of the contribution of the donor cell H-2d haplotype to the tolerance phenotype induced in H-2k neonates

Experimental design: To determine the relative importance of the donor cell H-2 haplotype vs. the non-MHC genetic background of miH antigens to the development of non-donor-specific cardiac allograft acceptance, C3H neonates were injected with tolerizing inocula from semi-allogeneic adult donors expressing either H-2k/d or H-2k/b on various BALB and non-BALB backgrounds, then transplanted at approximately 8 weeks of age with heart grafts or skin grafts from one of several gender-matched donors as follows: grafts were from either the same allogeneic donor strain represented in the tolerizing inoculum; H-2 haplotype-matched donors on a different miH antigen background; H-2 haplotype-mismatched donors on the same miH antigen background as the tolerizing strain; or unrelated third-party donors or syngeneic donors (Table 1).

Table 1. : Strains of mice used for immunogenetic analysis of neonatally induced cardiac allograft acceptance
Tolerizing cell donors‘Related’ graft donors (related by either H-2 haplotype or miH antigen background)‘Unrelated’ donors
  1. Tolerizing cells were prepared from spleen and bone marrow(1 : 1 proportion) harvested from adult F1 animals, as described in the Methods and Materials section.

(C3HxDBA/2)F1 H-2k/dDBA, BALBB6, NZW
(C3HxB10.D2)F1 H-2k/dB10.D2, DBA, BALB, B6NZW
(C3HxBALB.B)F1 H-2k/bBALB.B, B6, BALBNZW

General methods and materials

Animals: C3H/He (KkIAkIEkDk), C57BL/6 J (KbIAbIEDb), BALB/c (KdIAdIEdDd), and CD1 mice were obtained from Charles River Laboratories (St. Consant, Quebec). NZW(KzIAzIEzDz), BALB.B (KbIAbIEDb), B10.D2 (KdIAdIEdDd), DBA/2 (KdIAdIEdDd) and SJL (KsIAsIEDs) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All housing, treatment protocols and procedures were reviewed and approved by an independent Animal Care Committee in accordance with the current regulations and standards of The Canadian Council of Animal Care.

C3H breeding pairs were set up at staggered times to ensure continuous production of newborn pups to serve as neonatal recipients of tolerizing inocula, and subsequently as recipients of cardiac and skin allografts. Other breeding pairs were set up for production of various F1 donors of semi-allogeneic bone marrow and spleen cells.

Preparation of cellular inocula for neonatal injection: Bone marrow and spleen inocula were prepared from various adult F1 donor mice semi-allogeneic to the neonatal recipients. Bone marrow plugs were harvested from isolated tibia and humeri. Tissues were gently disrupted by mechanical prodding. Pooled single cell suspensions were prepared in PBS containing 2% fetal calf serum (Cansera). Splenocytes were layered over Histopaque-1083 (Sigma) and centrifuged for 20 min at 1800 r.p.m. and 20 °C; the interface containing lymphocytes was removed. Bone marrow cells and splenocytes were washed three times with PBS containing 2% fetal calf serum and counted; viability was assessed by trypan blue exclusion. Cells were resuspended (1 : 1 proportion) to a final concentration of 1.5 × 108/mL in 0.9% saline with 15 U/mL heparin (Leo Laboratories) for injection.

Induction of neonatal tolerance: Neonatal mice less than 24 h old were injected intravenously with 15 × 106 bone marrow and spleen cells into the anterior facial vein using a 30G needle. Injected mice were returned to the nest, received no further treatment and were weaned from mothers at 3 weeks of age. No immunosuppressive therapy was administered.

Heart transplantation: Mice were anesthetized with 3.6% chloral hydrate (Wiler). Primarily vascularized heterotopic heart transplantation into an abdominal position was performed by a modified technique as originally described by Corry et al. (33), in which the donor aortic root is anastomosed end-to-side to the recipient abdominal aorta and the donor pulmonary artery trunk to the inferior vena cava. Grafts were monitored by direct abdominal palpation of the pulsating graft in the awake animal. A qualitative score of 4 (strongest) to 0 (absence of pulsation) was assigned; rejection day was designated when the graft ceased functioning. Functioning grafts were monitored for 60 days; graft function beyond 60 days was considered ‘indefinite acceptance’. Results are given as median survival time (MST) and as percent of recipients with indefinite graft acceptance

Skin transplantation: Mice were anesthetized with 3.6% chloral hydrate (Wiler); a circumferential band was shaved around thorax and abdomen from the level of the shoulder joints to the level of the hip joints. Skin grafts were carried out using a modified technique originally described by Billingham et al. (34) by laying a full-thickness tail-skin graft into a prepared dermal bed in a dorsal thoracic position. Gender-matched allogeneic test grafts and syngeneic control grafts were placed on all animals on either side of the dorsal thorax with at least 2 cm of undisturbed recipient skin between grafts. Collodion (BDH) was applied at the junction of the recipient skin and donor skin to secure the grafts in place. Graft sites were first covered with vaseline gauze, then bandaged circumferentially with elastic bandage. Bandages were removed at 7 days and grafts inspected visually. Rejection day was assigned when no further viable skin was visible; grafts surviving longer than 60 days were considered to have achieved indefinite survival.

In vitro assessments: At 6–10 weeks of age, in vitro proliferation of lymph node cells (LNC) from treated and untreated mice as indicated by 3H-thymidine incorporation was assessed in mixed lymphocyte reaction (MLR), and effector function was assessed by chromium-release in cell-mediated lympholysis assay (CML) with stimulation by spleen cells from C3H, BALB, B6 and NZW mice, as described by Coligan (35).

Responder LNC (3 × 103−3 × 105 cells/well) were obtained from treated and naïve C3H mice. Stimulator cells(3 × 105 cells/well) were irradiated splenocytes(2000 rads) from naïve C3H and various test strain mice. The responder and stimulator cells were cultured in 96 roundbottom microtitre plates (Nunc) at 37 °C and 5% CO2 in DMEM (Gibco/BRL) with 10% fetal calf serum (Cansera), 10 mm hepes (Gibco/BRL), 5 × 10−6 M 2-mercapto-ethanol (Sigma) and penicillin/streptomycin (Gibco/BRL).

MLR assay: After 72 h culture, 3H-thymidine (Amersham) was added at a concentration of 1 μΧι/well and incubation continued for another 24 h. Cells were harvested with a cell harvester (Skatron), then counted in a beta-counter (Wallac). The stimulation index was calculated as the mean cpm of triplicate cultures of responder cells stimulated with allogeneic cells divided by the mean cpm of responder cells stimulated with syngeneic cells.

CML assay: On day 3, target cells were prepared from splenocytes of appropriate mice. Spleens were harvested and pressed though a sieve using sterile technique; samples were washed three times and counted. Splenocytes (1 × 107) were cultured in a vertical T-25 flask (Falcon) with 10 mL media and 2 μg/mL Concanavalin A (Con A) (Pharmacia) for 48–72 h. On day 5, the Con A stimulated blast target cells were washed and run through Lympholyte M (Cedarlane). The interface was removed, washed and counted. Con A lymphoblasts(1 × 106) were transferred into 15 mL conical tubes, centrifuged; the pellet was resuspended in 100 μΧι Na51CrO4(NEN) with fetal calf serum comprising 66% of the final volume. Targets were incubated in a lead case at 37 °C, 5% CO2 for 2 h with occasional shaking, then washed three times and resuspended in 5 mL media.

After 5 days of in vitro stimulation, 100 mL of the culture supernatant was removed from each well and 3 ×10351Cr-labeled target cells were added to each well. At the same time, spontaneous release (only stimulator and target cells) and maximum release (target cells only and 1% acetic acid) were set up. The plates were then centrifuged at 1000 r.p.m. for 1.5 min and incubated behind a lead shield at 37 °C for 4 h, then centrifuged at 1500 r.p.m. for 5 min. Subsequently, 100 mL of supernatant was removed from each well and counted with a gamma counter (Wallac). Specific lysis was calculated as [(experimental − spontaneous release)/(maximal − spontaneous release)]× 100%.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Part A

Third-party heart, but not skin, allograft acceptance is induced by neonatal injection of (C3HxBALB)F1 lymphohematopoietic cells: Injection of C3H neonates with (C3HxBALB)F1 bone marrow and spleen cells, the same tolerance induction protocol used in previous studies by many investigators, induced prolonged survival of donor-type BALB primarily vascularized cardiac allografts (MST > 60 days; Table 2 and Figure 1a) and skin allografts (MST > 60 days; Table 2 and Figure 1b) and, with 89% of BALB heart grafts and 67% of BALB skin grafts surviving long-term (defined as > 60 days). Untreated age-matched C3H mice rejected BALB heart grafts within 7–15 days, MST 8 days (Figure 1c), and skin grafts within 10–13 days, MST 12 days (Figure 1d).

Table 2. : Survival of donor-type and third-party heart grafts and skin grafts in C3H mice treated as neonates with (C3HxBALB/c)F1 bone marrow and spleen cellsThumbnail image of
image

Figure 1. Survival of donor-type and third-party cardiac allografts was prolonged in neonatally treated C3H mice, whereas prolonged survival of skin grafts was donor-specific. Heart (A) and skin (B) allografts from several donor strains were transplanted to C3H mice injected as neonates with 15 × 106 (C3HxBALB)F1 bone marrow and spleen cells. Controls were untreated C3H mice transplanted with heart (C) or skin allografts (D).

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In order to test the strain specificity of graft prolongation, additional heart and skin allografts were transplanted from several third-party donor strains. Third-party skin grafts from all unrelated allogeneic strains tested were rejected promptly in all treated and untreated animals (Table 2 and Figure 1b,d), confirming not only that skin graft acceptance induced in C3H neonates by exposure to BALB alloantigens was donor-specific, but also that the animals were not rendered significantly immunodeficient by the neonatal tolerizing protocol.

In contrast to these results for third-party skin allografts, neonatally treated animals exhibited prolonged acceptance of third-party cardiac allografts from all strains tested (Table 2 and Figure 1a), while untreated animals rejected cardiac allografts promptly (Table 2 and Figure 1c). The extent of third-party heart graft survival varied amongst allograft donor strains, ranging from MST 19 days (SJL donor grafts) to > 60 days (B6 donor grafts), with indefinite acceptance of B6 grafts in 75% of recipients. Survival of heart grafts, but not skin grafts, from outbred CD1 donors was also prolonged in neonatally treated recipients: MST 66 days with 50% of grafts surviving indefinitely.

Syngeneic heart grafts and skin grafts were accepted long-term in all treated and untreated animals (Table 2 and Figure 1), demonstrating that mechanical, ischemic or inflammatory effects of the operative procedures do not contribute to altered patterns of heart graft or skin graft survival.

Alloreactivity to third-party alloantigens is demonstrable in vitro after neonatal treatment that induces third-party cardiac allograft acceptance: In vitro assessments of alloreactivity were performed to assess the proliferative capacity and cytotoxic effector function of cells from naïve and from neonatally treated animals in response to stimulation by syngeneic (C3H), donor-type (BALB) and third-party (B6 and NZW) alloantigens. In MLR assays, when compared to LNC from naïve animals, proliferation as indicated by 3H-thymidine incorporation of responder LNC from neonatally treated animals was diminished in response to stimulation by irradiated donor-type BALB splenocytes but not by either third-party B6 or NZW splenocyte stimulation (Figure 2). Likewise, compared to naïve animals, cytotoxic effector function of LNC from neonatally treated animals as indicated by chromium release in CML assays was intact for both B6 and NZW third-party targets (in response to B6 and NZW stimulators, respectively), and diminished in only a donor-specific pattern, with decreased lysis of BALB targets in response to stimulation by BALB splenocytes. This donor-specific pattern of in vitro hyporesponsiveness was most pronounced in assays using responder LNC from C3H mice injected as neonates with a higher dose of H-2d-expressing cells (dose 30 × 106 cells/neonate): proliferation and cytotoxic function was intact in response to both B6 and NZW third-party alloantigen stimulation, but diminished to donor-type BALB stimulation.

image

Figure 2. Neonatal injection of (C3HxBALB)F1 bone marrow and spleen cells induces donor-specific hyporesponsiveness when assessed by in vitro assays. MLR (A, B, E, F) and CML (C, D, G, H) assays were carried out using lymph node cells from C3H mice injected as neonates with 15 × 106 (C3HxBALB)F1 bone marrow and spleen cells (B, D) or 30 × 106 (C3HxBALB)F1 bone marrow and spleen cells (F, H) or untreated control mice (A, C, E, G). Data are shown as averaged data collected from three separate single-animal experiments each.

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Part B

Donor-type cardiac and skin grafts (MHC and miH antigen-matched to tolerizing cells) are accepted in neonatally treated recipients: Injection of C3H neonates with 15 × 106 bone marrow and spleen cells from all semi-allogeneic adult donor combinations expressing either theH-2k/d haplotype or the H-2k/b haplotype induced a high level of unresponsiveness for all donor-type allografts (e.g. donor organs matched to tolerizing cell donors for both MHC and miH antigens; Table 3 and Figures 3–5, groups A1, B1 and C1). This was true for both heart allografts (MST > 60 days in all groups, with indefinite graft survival in 83–100% of recipients) and, with somewhat less consistency, for skin allografts (MST > 60 days in all groups, with indefinite graft survival in 50–83% of recipients). Syngeneic heart and skin allografts survived indefinitely in all treated and untreated control animals (groups A5, B6, C5). Untreated control animals rejected all allogeneic heart and skin grafts promptly (Table 3, groups D1, D2, D3).

Table 3. : Survival of donor-type and third-party heart grafts and skin grafts in C3H mice treated as neonates with either H-2 k/day or H-2 k/b-expressing cells
Neonatal treatmentGraft donor strainHeart graftsSkin grafts
  nMST% indefinitenMST% indefinite
  1. Neonatal C3H mice were injected intravenously with 15 × 106 F1 bone marrow and spleen cells; treated and untreated mice were transplanted with heart grafts or skin grafts at 8 weeks of age.

Group A
(C3HxDBA)F1A1 DBA (H-2d) 7> 60 d100% 643 d 50%
(H-2k/d)A2 BALB (H-2d) 6> 60 d100% 619.5 d 33%
A3 B6 (H-2b) 9> 60 d 78% 610 d  0
A4 NZW (H-2z) 7> 60 d 57% 6 9.5 d  0
A5 C3H (H-2k) 7> 60 d100%24> 60 d100%
Group B
(C3HxB10.D2)F1B1 B10.D2 (H-2d) 8> 60 d100% 6> 60 d 83%
(H-2k/d)B2 DBA (H-2d) 9> 60 d100% 617.5 d 33%
B3 BALB (H-2d) 8> 60 d100% 618.5 d  0
B4 B6 (H-2b) 9> 60 d 67% 613 d  0
B5 NZW (H-2z)10 8 d  0 616.5 d  0
B6 C3H (H-2k) 7> 60 d100%30> 60 d100%
Group C
(C3HxBALB.B)F1C1 BALB.B (H-2b) 7> 60 d 86% 7> 60 d 57%
(H-2k/b)C2 B6 (H-2b) 6> 60 d100% 514 d  0
C3 BALB/c (H-2d) 735 d 29% 513 d  0
C4 NZW (H-2z) 814.5 d  0 713 d  0
C5 C3H (H-2k) 7> 60 d100%24> 60 d100%
Group D
NoneD1 DBA (H-2d) 7 8 d  0 513 d  0
D2 B10.D2 (H-2d)10 7.5 d  0 611.5 d  0
D3 BALB.B (H-2b) 9 8 d  0 914 d  0
image

Figure 3. Survival of heart and skin allografts in C3H mice injected as neonates with 15 × 106 (C3HxDBA)F1 bone marrow and spleen cells.

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image

Figure 4. Survival of heart and skin allografts in C3H mice injected as neonates with 15 × 106 (C3HxB10.D2)F1 bone marrow and spleen cells.

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image

Figure 5. Survival of heart and skin allografts in C3H mice injected as neonates with 15 × 106 (C3HxBALB.B)F1 bone marrow and spleen cells.

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Cardiac but not skin allografts that are MHC-matched and miH-disparate to tolerizing cells are accepted in neonatally treated recipients: Acceptance of cardiac allografts from donors that were MHC-matched, miH antigen-disparate to the cell donors was induced by both H-2k/d and H-2k/b-expressing cells, with MST > 60 days and 100% indefinite graft acceptance in all groups (Table 3 and Figures 35, heart grafts: groups A2, B2, B3 and C2). Survival of skin grafts from the same donors was prolonged only when the tolerizing haplotype was H-2k/d but not H-2k/b, and to a far lesser extent than heart grafts, with MST ranging from 17.5 days to > 60 days with indefinite graft survival in 0–50% of recipients (Table 3 and Figures 35, skin grafts: groups A2, B2, B3 but not C2).

Cardiac but not skin allografts that are miH-matched and MHC-disparate to tolerizing cells are variably accepted in neonatally treated recipients: Acceptance of cardiac grafts from donors that were miH antigen-matched, MHC-disparate to tolerizing cell donors (Table 3 and Figures 4,5, heart grafts: groups B4 and C3) was prolonged, but to a much greater extent when the tolerizing haplotype wasH-2k/d (group B4: MST > 60 days with 67% indefinite graft acceptance) than H-2k/b (group C3: MST 35 days, with 29% indefinite graft acceptance). In contrast, skin graft survival from miH-matched, MHC-disparate donors was not prolonged (skin grafts: groups B4 and C3).

Cardiac but not skin allografts that are both MHC-disparate and miH-disparate to tolerizing cells are accepted in neonatally treated recipients of H-2k/d-expressing cells: In specificity experiments with transplantation of unrelated third-party cardiac allografts differing from tolerizing cell donors at both MHC and miH antigens, evidence was obtained for a differential effect of these two factors. Acceptance of third-party heart allografts was induced predominantly in neonatally injected recipients of H-2k/d-expressing cells, of (C3HxBALB/c)F1 or (C3HxDBA)F1 origin, but not those of (C3HxB10.D2)F1 origin. Thus compare in Table 2 and Figure 1, heart grafts: groups 2, 3 and 4, and in Table 3 and Figures 3 and 4, heart grafts: groups A3 and A4, but not group B5. No acceptance of MHC and miH antigen mismatched grafts was induced in recipients of H-2k/b-expressing cells, for example, NZW allografts in recipients of (C3HxBALB.B)F1 cells (Table 3 and Figure 5, heart grafts: group C4).

Once again, these effects of the donor inoculum were observed only for cardiac allografts. Unrelated third-party skin allografts were rejected promptly by all neonatally treated recipients of cells expressing either H-2k/d or H-2k/b alloantigens (Table 2 and Figure 1, skin grafts: groups 2, 3 and 4; Table 3 and Figures 3–5, skin grafts: groups A3, A4, B5, C4).

In summary, taken from the viewpoint of the graft, cardiac allografts, but not skin allografts, from B6 donors were accepted in all animals injected as neonates with tolerizing cells expressing either H-2k/d or H-2k/b alloantigens, either matched or disparate to the graft for miH antigens (i.e. this is the ‘easiest’ graft to which tolerance is induced in the neonatal C3H recipient). In contrast, cardiac grafts, but not skin grafts, transplanted from NZW third-party donors unrelated to tolerizing cell donors at either MHC or miH antigens, were also accepted, but only in animals injected as neonates with tolerizing cells from H-2k/d-expressing mice, and only on DBA and BALB but not BL strain miH antigen background.

Taken from the perspective of the tolerizing inoculum, injection of cells from either (C3HxBALB)F1 or (C3HxDBA)F1 donors induced acceptance of cardiac grafts from all donor strains tested, including those disparate for both MHC and multiple miH antigens as well as grafts from outbred donor animals (i.e. these are the most ‘widely’ tolerizing donor inocula to the neonatal C3H recipient).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

These experiments demonstrate that the state of immunologic unresponsiveness induced by alloantigen exposure during immaturity is not exclusively specific to the donor MHC haplotype, but rather that the specificity pattern is at least partially dependent on the method of tolerance assessment. Moreover, immunogenetic factors related to both the tolerizing cell donor and the graft donor are important to the development of third-party heart-graft acceptance, and involve miH antigen background as well as MHC haplotype. In vitro assessment of alloreactivity did not predict in vivo cardiac graft survival in this model. Third-party skin grafts were rejected by neonatally injected animals, and third-party-directed alloreactivity was demonstrable in vitro. Thus, the inability to reject third-party cardiac grafts is not due to generalized immunodeficiency or immunosuppression.

This unusual specificity pattern of cardiac graft acceptance was observed with several strain combinations of tolerizing inocula donors and graft donors, all completely disparate to the neonatal recipient across MHC and multiple miH antigen differences. One factor, or more likely a combination of several factors, considered below might account for these differences in heart and skin graft survival, including: expression of cardiac-restricted and/or skin-restricted antigens; tissue variability in the expression of MHC and miH antigens; the load of ‘passenger’ antigen-presenting cells carried with skin grafts vs. heart grafts; different physiologic dispersal of alloantigens to the recipient immune system; or a variable influence of recipient cells which mediate rejection/tolerance of heart allografts vs. skin allografts. In vitro assessment of alloreactivity, which typically measures responses of lymphoid cell populations to direct stimulation by isolated populations of other lymphoid cells (e.g. splenocytes), likely represents a different immune interaction than afforded by in vivo transplantation of either primarily vascularized heart grafts or secondarily vascularized skin grafts. This proviso, as noted by previous investigators, cautions against relying solely on in vitro assessments of alloreactivity when considering the complex in vivo scenario of organ transplantation (18,36–38). Indeed, some investigators have demonstrated that diminished delayed-type hypersensitivity responses may be more applicable than in vitro assays as a correlate of prolonged graft survival (39,40).

Graft and tissue-related factors

Our experiments clearly demonstrate that the organ graft itself is important to the pattern of unresponsiveness induced by a neonatal tolerizing inoculum. Many experimental regimens have been described in which ‘tolerance’ can be induced to heart and other organ allografts but not to skin grafts, which are generally considered more ‘immunogenic’ grafts (37,41–45). Transplantation of skin grafts typically is performed without direct vascular anastomoses (34). Vascular and lymphatic connections occur secondarily, resulting in prolonged ischemia and dispersal of alloantigens to regional lymph nodes. In contrast, transplantation of primarily vascularized allografts with direct vascular anastomoses results in minimal ischemia and dispersal of alloantigens to central lymphoid organs (e.g. spleen) by direct hematogenous dissemination (33,37,41,46,47). Donor-derived cells capable of initiating an immune response, such as dendritic cells, are plentiful in skin but sparse in cardiac tissue (37). Furthermore, the constitutive expression of MHC class I molecules in cardiac myocytes and cardiac endothelium is low, and class II expression is undetectable (47). In contrast, expression of both class I and class II antigens is much greater in a variety of cells present in skin grafts. The increased ischemia of a secondarily vascularized graft compared to a primarily vascularized graft may play an additional role in increasing the vulnerability of skin grafts, as well as augmenting their immunogenicity by causing up-regulation of MHC class II antigens and altered chemokine expression (47,48).

In addition to these substantial differences in the immunological interactions inherent in transplantation of skin vs. cardiac allografts, it has been suggested that local immune events and tissue-restricted or tissue-related antigens in skin and other organs may play a role in differential in vivo allograft survival (38,49–52). Thus, one could speculate that either:

  • a. Heart grafts express antigenic epitopes (i.e. ‘cardiac’ antigens) that are shared with the tolerizing cells, and/or

  • b. Skin grafts express additional unique antigenic structures (i.e. skin-specific or skin-restricted antigens) not shared with the cells of the tolerizing inoculum. Tolerance to these antigens thus would not be induced by the donor cells.

An overlap in tolerance repertoires could be the result of ‘molecular mimicry’. A degree of amino acid sequence homology or shared polymorphic sequences between certain antigens present on lymphohematopoietic cells of the tolerizing inoculum and tissue-specific antigens on cardiac allografts could result in ‘cross-tolerized’ cells which would affect cardiac grafts but not skin grafts. Although not described at the graft level in organ transplantation models, serologic cross-reactivity to alloantigens was described in the early literature in this field (38,47,48), and evidence suggesting immune cross-reactivity or ‘epitope spreading’ has been reported in models of autoimmune diseases (53,54).

In vivo studies using donor-specific transfusion for tolerance induction in adult models have reported a phenomenon known as ‘linked epitope suppression’ (55,56), based on earlier in vitro experiments (57). In these studies using tolerizing cells disparate for only a MHC single class I antigen, prolonged survival was induced of cardiac and skin allografts expressing not only the tolerizing alloantigen but additional alloantigens of the same H-2 haplotype or miH antigens. Allografts that expressed additional alloantigens were accepted provided they also expressed the single mismatched class I tolerizing antigen, which was the common link between tolerizing cell donor and graft donor. In our experiments, there is no obvious common link between tolerizing cell donor and third-party cardiac graft donor; thus it is likely that our results are not due to the same process, or that the linked antigen is not a classical ‘transplantation antigen’.

Donor immunogenetic factors

By varying the strain combinations used as tolerizing cell donors and graft donors for C3H recipients, our experiments demonstrate that the following immunogenetic factors contribute to third-party cardiac graft acceptance in neonatally injected mice:

  • a. MHC antigen haplotype of the tolerizing cell donor (H-2d more effective than H-2b)

  • b. miH (non-MHC) antigen background of the tolerizing cell donor (BALB and DBA more effective than BL)

  • c. MHC antigen haplotype of graft donor (H-2b easier than H-2z; H-2z easier than H-2d or H-2 s). Note: lack of I-E expression is not the reason for B6 graft acceptance, as survival of SJL grafts, also lacking I-E expression, was not as prolonged as B6 graft survival. (Of further note, unlike the studies by Streilein (10) and by McCarthy (18) using donor and recipient combinations with isolated class I or class II antigen disparities, these experiments used only strain combinations with complete MHC disparity between donor and recipient and thus were not designed to distinguish class I vs. class II differences in tolerance induction.)

In general, when H-2d-donor derived cells were used in the neonatal tolerizing inoculum, induction of third-party graft acceptance was more often achieved than when H-2b-expressing cells were injected. In contrast, when H-2d-expressing strains were used as cardiac graft donors, third-party graft acceptance was more difficult to attain (i.e. a more ‘immunogenic’ graft) than acceptance of H-2b-expressing third-party cardiac grafts. Thus the more immunogenic graft donor source was also the most tolerogenic to the neonate. This pattern is consistent with Burnet's early theories of tolerance mechanisms, in which it was suggested that in order for an antigen to induce tolerance, it must be inherently immunogenic to the recipient (58). Thus, under conditions of susceptibility such as recipient immunologic immaturity, a potent immunogen may prove to be a more powerful tolerogen. Strain-related differences in antigenicity may be related to the number of MHC loci expressed by the BALB strain (i.e. several D locus genes), to a qualitative difference in specific allele(s), and/or to uncharacterized non-MHC genetic factors (59,60).

Factors related to recipient immaturity

Based on a number of developmental issues, one could speculate on those neonatal recipient factors that may contribute to third-party cardiac graft acceptance. Development of T-cell receptor (TCR) N-region diversity is limited in the neonatal mouse, with N-region addition beginning only around day 8 of life (61,62). This results in a more limited receptor repertoire than in the adult mouse with only approximately 105 TCR molecules available of an eventual 1012−15, which has important implications for positive and negative selection of immature T cells. T cells would be more promiscuous in terms of the peptide/MHC they recognize, and therefore administration of alloantigens to neonatal mice may inactivate, by whatever mechanism, a larger proportion of developing T cells, giving rise to broader degree of unresponsiveness than when the same tolerizing cells are used in the adult. Tolerizing cells derived from a very immunogenic strain may create a wider ‘hole’ in the developing repertoire of responding T cells when injected into immature (neonatal) animals than cells derived from a less immunogenic strain. Thus H-2d-expressing cells may be more widely tolerogenic to the C3H recipient than H-2b-expressing cells, and may alter the emerging T-cell repertoire in such a way as to allow survival of cardiac allografts from any less immunogenic strain.

Note that neonatal injection of BALB-derived tolerizing cells abrogated all elements of anti-BALB alloreactivity, resulting in acceptance of BALB heart grafts and skin grafts in vivo, and loss of in vitro alloreactivity in proliferative and functional capacity against BALB alloantigen stimulation. In contrast, the only apparent alteration in anti-B6 alloreactivity is one allowing heart graft acceptance. Thus, in mice receiving neonatal injections of BALB cells, the mechanism(s) responsible for the acceptance of donor-type BALB heart grafts and third-party B6 heart grafts may be quite different, even though operationally the net outcome is the same (cardiac graft acceptance).

Delineating the requirements for acceptance of unrelated cardiac grafts

As we report herein, induction of graft acceptance specific to the tissue type, i.e. ‘organ-specific’ tolerance, may be possible without inducing tolerance to individual donor MHC/miH antigens, a feature with potential implications for outbred human populations. It is likely that a combination of factors contributes to this outcome, including the immaturity (in terms of the immune system) of the recipient; the nature of the graft (a feature which could be explored using other primarily vascularized grafts, i.e. kidney, liver); and the source and nature of the donor tolerizing inoculum. As an example of the latter, from the perspective of the C3H recipient, cells from the DBA donor, with MHC plus miH antigen disparity, represent a universal tolerizing stimulus allowing acceptance of cardiac grafts from any donor strain, in contrast to MHC or miH antigen disparate cells only (see Table 3).

However, other factors clearly influence the outcome of graft survival. Thus, animals made neonatally tolerant to H-2d on any miH antigen background do not reject B6 cardiac allografts, although intact immune responsiveness can be demonstrated to H-2b alloantigens in vitro by proliferative and cytotoxicity assays, and in vivo by rejection of B6 skin allografts. This universal acceptance of B6 heart but not skin grafts, whether in complete disparity with tolerizing cells or in isolated disparity with tolerizing cells at MHC or at miH, is indicative of a complex immunologic process. In contrast to B6 grafts, for NZW grafts, acceptance of heart, but not skin, was observed only when tolerizing cells expressed H-2d haplotype and only in the combination with miH of the BALB and DBA, but not B6, backgrounds (Tables 2 and 3). The potential contribution of other immunologic factors, such as the respective chemokines and/or cytokines induced in these combinations, as well as differential cell trafficking (i.e. of CD4/CD8 T cell subsets), remains to be explored. Moreover, the applicability of these findings to recipients of non-C3H strains has not yet been explored in detail; however, our initial experiments using outbred CD1 neonatal recipients indicate that the pattern of nonspecific cardiac allograft acceptance is not limited to C3H recipients (data not shown).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
  • 1
    The ‘tolerant’ state induced by neonatal exposure to alloantigens is not exclusively donor-specific; rather, specificity varies with the grafted organ. This adds to several other previously noted anatomic, physiologic and immunologic differences in transplantation of skin grafts vs. other organ allografts and may be partially related to tissue-specific factors.
  • 2
    As noted by previous investigators, in vitro assessments of alloreactivity cannot reliably predict outcomes of the more complex in vivo scenario of organ graft transplantation.
  • 3
    In addition to variables related to the grafted organ and the mode of transplantation, third-party cardiac graft acceptance is related to immunogenetic factors of both the donor of the tolerizing inoculum and the heart-graft donor, and these involve miH as well as MHC antigens.
  • 4
    It is often noted that a state of true ‘tolerance’ is present only when all elements of alloreactivity have been abrogated, as indicated by unresponsiveness in the stringent tests of skin grafts and in vitro assays. Nonetheless, complete abolition of alloreactivity appears to be unnecessary for survival of cardiac allografts. In clinical situations of solid organ transplantation, in which the organ donor cannot be identified in advance of transplantation, induction of a state of ‘organ-specific’ tolerance or altered functional alloreactivity leading to acceptance of a particular organ graft (i.e. heart) from any available donor would be extremely helpful, despite continued presence of demonstrable alloreactivity to skin grafts and LNC from the same donor. Although our experiments have not yet elucidated a mechanism to explain nonspecific cardiac allograft acceptance, this investigative pathway is important for the development of this approach as a possible therapeutic tool.
  • 5
    Finally, it should be noted that to date our experiments have not focused on more specifically recipient-related factors. As an example, we do not yet know how restricted these phenomena are to tolerance induced during immunologic immaturity. This takes on particular relevance for fetal and neonatal patients with lethal congenital heart disease requiring transplantation early in life.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

This work was supported by an operating grant from The Heart and Stroke Foundation of Ontario and by funding from The Hospital for Sick Children Research Institute.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Materials
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
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