Youping Li

Costimulatory molecule-deficient dendritic cell progenitors (MHC class II+, CD80dim, CD86−) prolong cardiac allograft survival in nonimmunosuppressed recipients

We have shown previously that granulocyte-macrophage colony-stimulating factor-stimulated mouse bone marrow-derived MHC class II+ dendritic cell (DC) progenitors that are deficient in cell surface expression of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) can induce alloantigen-specific T-cell anergy in vitro. To test the in vivo relevance of these findings, 2 x 10(6) B10 (H2b) mouse bone marrow-derived DC progenitors (NLDC 145+, MHC class II+, B7-1dim, B7-2-/dim) that induced T-cell hyporesponsiveness in vitro were injected systemically into normal C3H (H2k) recipients. Seven days later, the mice received heterotopic heart transplants from B10 donors. No immunosuppressive treatment was given. Median graft survival time was prolonged significantly from 9.5 to 22 days. Median graft survival time was also increased, although to a lesser extent (16.5 days), in mice that received third-party (BALB/c; H2d) DC progenitors. Ex vivo analysis of host T-cell responses to donor and third-party alloantigens 7 days after the injection of DC progenitors (the time of heart transplant) revealed minimal anti-donor mixed leukocyte reaction and cytotoxic T lymphocyte reactivity. These responses were reduced substantially compared with those of spleen cells from animals pretreated with mature granulocyte-macrophage colony-stimulating factor + interleukin-4-stimulated DC (MHC class IIbright, B7-1+, B7-2bright), many of which rejected their heart grafts in an accelerated fashion. Among the injected donor MHC class II+ DC progenitors that migrated to recipient secondary lymphoid tissue were cells that appeared to have up-regulated cell surface B7-1 and B7-2 molecule expression. This observation may explain, at least in part, the temporary or unstable nature of the hyporesponsiveness induced by the DC progenitors in nonimmunosuppressed recipients.

Systemic administration of cellular interleukin-10 can exacerbate cardiac allograft rejection in mice

Cellular interleukin-10 (cIL-10) has been shown to inhibit cytokine production by T helper type 1 (Th1) cells by blocking antigen presenting cell function. This activity has suggested that IL-10 might be useful in the treatment of transplant rejection. Stimulatory effects of IL-10 however, have also been observed both on T and B cell differentiation. In this study, we examined the influence of recombinant (r) mouse (m) IL-10 on heterotopic vascularized heart allograft survival in the B10(H2b)-->C3H(H2k) strain combination that crosses both major histocompatibility complex (MHC) and non-MHC-histocompatibility antigen (non-MHC-HA) barriers. The influence of IL-10 was also examined in the B10.BR (H2k)--> C3H combination with disparity at only non-MHC-HA loci. Postoperative intraperitoneal administration of IL-10 (100 microg/d, days 0-6) significantly accelerated heart graft rejection both in the B10-->C3H (mean survival time [MST] 7.8+/-0.2 days; control MST 10.6+/-0.6 days; P<0.05) and the B10.BR-->C3H combination (MST 14.3+/-0.5 days; control MST 77.7+/-14.4 days). Ex vivo IL-10 perfusion of donor hearts for either 15 min or 2 hr did not affect subsequent graft survival. Immunologic monitoring of transplanted mice revealed that IL-10 treatment (100 microg/d, i.p., days 0-6) increased both the circulating complement-dependent cytotoxic (CDC) antibody titer and splenic anti-donor cytotoxic T lymphocyte (CTL) activity measured up to 3 weeks posttransplant. These findings indicate that post transplant systemic administration of cIL-10 can promote vascularized allograft rejection, and that this may reflect stimulation both of B and T cell alloimmune responses.

ISOLATION, PHENOTYPE, AND ALLOSTIMULATORY ACTIVITY OF MOUSE LIVER DENDRITIC CELLS

Donor liver-derived dendritic cells (DC) have recently been identified within various lymphoid and nonlymphoid tissues of organ allograft recipients, including nonimmunosuppressed mice transplanted with and permanently accepting major histocompatibility complex (MHC)-disparate hepatic allografts. These findings have raised questions about the basis of the tolerogenicity of the liver--and, in particular, about the properties of liver-derived DC. To study further the structure, immunophenotype and allostimulatory activity of leukocytes resident in normal mouse (B10.BR;H-2k, I-Ek) liver, a procedure was developed to maximize the yield of viable, nonparenchymal cells (NPC) obtained following collagenase digestion of perfused liver fragments and density centrifugation (Percoll). These cells comprised populations expressing lymphoid and myeloid cell surface antigens. As compared with spleen cells, they proved good allostimulators of naive (B10; H-2b, I-E-) splenic T cells when tested in primary mixed leukocyte reactions (MLR). After overnight (18-hr) incubation of the NPC, enrichment for transiently adherent, low-density (LD) cells on metrizamide gradients permitted the recovery of low numbers of cells (approx. 2-5 x 10(5) per liver), many of which displayed distinct DC morphology. Flow cytometric analysis revealed that these cells were CD3-, CD4-, CD8-, and B220-, but strongly expressed CD45 (leukocyte-common antigen), and mild-to-moderate levels of CD11b, heat-stable antigen, and CD44. The cells also expressed moderate intensity of NLDC 145 but not 33D1, DC restricted markers which have been shown to be differentially expressed on mouse DC isolated from various organs. This DC-enriched population was more strongly MHC class II(I-Ek)+ than NPC, as determined by immunocytochemistry and flow cytometry and exhibited much more potent allostimulatory activity for naive T cells. These findings demonstrate that freshly isolated murine liver NPC, and perhaps their counterparts in situ, exhibit allostimulatory activity that is enhanced in the non-adherent, low-density (DC-enriched) fraction after overnight culture. They further suggest that the maturation of liver DC may play a key role in determining the immunogenicity and or tolerogenicity of hepatic allografts.

In vitro propagation and homing of liver-derived dendritic cell progenitors to lymphoid tissues of allogeneic recipients: Implications for the establishment and maintenance of donor cell chimerism following liver transplantation

Dendritic cell (DC) progenitors were propagated in liquid culture from nonparenchymal cells resident in normal mouse (B10.BR; H-2k, I-E+) liver in response to granulocyte-macrophage colony stimulating factor (GM-CSF). The liver-derived DC progenitors were MHC class II-/dim and did not express counter receptors for CTLA-4, a structural homologue of the T cell activation molecule CD28. Following subcutaneous or intravenous injection, these liver-derived cells migrated to T cell-dependent areas of lymph nodes and spleen of unmodified, allogeneic (B10; H-2b; I-E-) recipients, where they were identified 1-5 days, and 1 and 2 months after injection by their strong surface expression of donor MHC class II (I-Ek) and their dendritic morphology. Maximal numbers of liver-derived DC in the spleen were recorded 5 days after injection. Both clusters of strongly donor MHC class II+ cells--and (more rarely) dividing cells--could also be identified, suggesting cell replication in situ. Using the same techniques employed to generate DC progenitors from normal liver, GM-CSF-stimulated cells were propagated for 10 days from the bone marrow and spleen of nonimmunosuppressed mice sacrificed 14 days after orthotopic liver transplantation (B10;H-2b-->C3H;H-2k). Immunocytochemical staining for recipient and donor MHC class II phenotype revealed the growth both of host cells with DC characteristics, and of cells expressing donor alloantigens (I-Ab). These results are consistent with the growth, in response to GM-CSF, of donor-derived DC from progenitors seeded from the liver allograft to recipient lymphoid tissue. The functional activity of the progenitors of chimeric DC and the possible role of these cells in the establishment and maintenance of donor-specific tolerance following liver transplantation remain to be determined.

Second-set rejection of mouse liver allografts is dependent on radiation-sensitive nonparenchymal cells of graft bone marrow origin.

Livers are accepted spontaneously when transplanted orthotopically from B10 (H2b) or BALB/c (H2d) to C3H (H2k) mice without host immunosuppression. Presensitization to donor can, however, be induced by skin grafting two weeks prior to liver transplantation, resulting in second-set or accelerated liver graft rejection, within 4-5 days. In this study, the role of liver nonparenchymal cells (NPC) in second-set rejection was tested by donor whole-body irradiation and replacement of donor B10 liver NPC with those of either third-party (BALB/c) or recipient strain (C3H) bone marrow. Irradiation alone (9.5 Gy) of normal B10 donors before liver transplantation significantly prolonged graft survival in presensitized C3H recipients. Three months after bone marrow transplantation (BMT), chimeric livers expressing third-party or recipient haplotype, were transplanted orthotopically into unmodified C3H recipients. Graft survival was prolonged significantly compared with livers from normal or syngeneically reconstituted B10 donors. Prolonged survival of chimeric (BALB/c->B10) livers was also evident in C3H mice presensitized to alloantigens expressed on both the liver parenchymal (B10) and third-party NPC (BALB/c) by simultaneous grafting of skin from each donor strain. Determination of graft cytokine mRNA profiles 4 days posttransplant showed that replacement of donor with third-party or recipient strain BM-derived cells was associated with reduced expression of IL-2 and IFN-gamma mRNA compared with that in grafts from syngeneically reconstituted donors. IL-10 transcripts, however, were not significantly affected. The results are consistent with a key role of radiation-sensitive, donor-specific NPC of bone marrow origin in second-set liver rejection in presensitized hosts. In this process, competent donor-strain NPC appear to augment the activity of primed T cells responsible for the second-set rejection.

Interferon-γ induced expression of MHC antigens facilitates identification of donor cells in chimeric transplant recipients

After whole organ transplantation, donor bone marrow-derived cells migrate out of the graft into the recipient, leading to establishment of chimerism, which is the first step towards the subsequent induction of donor-specific tolerance. In routine immunohistochemical staining, monoclonal antibodies specific for heterotopic MHC alleles are used to identify donor and recipient cells. However, it is difficult to detect these cells using this technique in long-term allograft recipients who have a persistently low donor cell population (microchimerism). Because Interferon-gamma (IFN-γ) is known to induce expression of MHC class I and class II cell surface molecules, we used this cytokine 12-48 h before sacrifice, to facilitate the identification of donor and recipient cells in the tissues of animals transplanted with either liver (B10 → C3H) or bone marrow (LEW → BN). In long-term allograft recipients, the use of IFN-γ for as briefly as 12 h prior to sacrifice, results in marked upregulation of class I and class II antigens, leading to easy identification of ubiquitously distributed low numbers of donor cells.

Isolation of Dendritic Leukocytes from Mouse Liver

Dendritic cells (DC) are a minor population of large, bone marrow-derived leukocytes that are distributed ubiquitously throughout the body (1,2). DC resident in the interstitial connective tissue of non-lymphoid organs are believed to be important “passenger” leukocytes that migrate to T-dependent areas of host lymphoid tissue following organ transplantation (3). Recently, it has been suggested that the chimeric cells observed in various organs of recipients of liver or other allografts include, predominantly, cells of DC lineage. These cells may play an important role in modulation of the immunological interaction between host and donor (4,5). Since the liver is the most tolerogenic of all transplanted organs (6–8) and is accepted spontaneously across major histocompatibility complex (MHC) barriers in mice without the need for immunosuppressive therapy (8), studies on mouse liver DC may provide important clues to mechanisms underlying tolerance induction. We have isolated DC-enriched cell populations from normal mouse liver and describe herein their immunophenotype and in vitro allostimulatory activity compared with freshly-isolated liver non-parenchymal cells (NPC).