Alex Karlsson-Parra

The main infiltrating cell in xenograft rejection is a CD4+ macrophage and not a T lymphocyte.

Porcine fetal islet-like cell clusters (ICC) or isolated rat islets were implanted under the kidney capsule of normoglycemic rats. The animals were sacrificed 1, 3, 6, 12, or 24 days after transplantation, and a detailed morphological and phenotypic characterization of the different cellular subtypes infiltrating the xenograft was performed and compared with the rejection of allogeneic islets. In xenograft rejection a progressive infiltration of large, polygonal, macrophage-like cells, which with time became the dominating cellular subtype, occurred. These cells expressed the CD4 antigen and the macrophage-specific ED1 antigen. From day 6 and onward, a majority of the macrophage-like cells also expressed the CD8 antigen and the macrophage-specific differentiation antigen ED2. T lymphocytes, defined by their TCR alpha/beta or CD2 expression, were found in low numbers and mainly in the periphery of the graft. At the later stages of xenorejection a substantial number of eosinophilic granulocytes were also found. The allograft rejection, on the contrary, was characterized by a progressive infiltration of T lymphocytes, which with time became the dominating cellular subtype. No clear immunoglobulin or complement deposition was seen in the transplants before day 12, when IgG deposition was found in central necrotic areas of the xenograft. Previous experiments in rodents have underlined the crucial importance of CD4 positive cells in the xenograft rejection process. However, in none of these studies it was conclusively demonstrated that the CD4-expressing cells were T lymphocytes. The presence of CD4-expressing macrophages heavily infiltrating the porcine xenograft seen in our study may thus be in agreement with previous studies in which the anti-CD4 reactive cells were erroneously designated T lymphocytes. Interestingly, the findings in xenograft rejection in the present study have striking similarities with the defense mechanisms active against infections by large parasites such as helminths.

Xenograft rejection of porcine islet-like cell clusters in normal and natural killer cell-depleted mice

Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen were in minority and mainly located in the peripheral parts of the ICC xenograft. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 double-positive cells were found to be larger and more granular than the CD4 single-positive cells. No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (approximately 15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive beta 2-microglobulin-deficient mutant, C4.4-25-, lymphoma cell line EL-4 served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but, readily rejected the NK cell-sensitive lymphoma cell line. Taken together, ICC xenograft rejection in mice seems to be T cell dependent, as evidenced in the nude mice model, while the main effector cell appears to be a macrophage with a unique phenotype.

Recruitment and activation of natural killer cells in vitro by a human dendritic cell vaccine.

Recruitment of circulating natural killer (NK) cells into inflamed lymph nodes is known to provide a potent, IFN-gamma-dependent boost for Th1-polarized immune responses in mouse models. Such NK cell recruitment into draining lymph nodes is induced by certain s.c. injected adjuvants, including mature vaccine dendritic cells (DC), and is mediated by a CXCR3-dependent pathway. Here, we show that monocyte-derived immature human DCs stimulated with polyinosinic acid:polycytidylic acid, IFN-alpha, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IFN-gamma, alpha-type 1-polarized DC (alpha DC1), secrete profuse amounts of the CXCR3 ligand CXCL9/MIG and substantial amounts of CXCL10/IP-10 and CXCL11/I-TAC after withdrawal of maturation stimuli. In sharp contrast, no measurable production of these chemokines was found in DCs after maturation with the current gold standard maturation cocktail for human DC-based cancer vaccines consisting of TNF-alpha, IL-1 beta, IL-6, and prostaglandin-E(2) (PGE(2)-DC). PGE(2)-DCs preferentially produced the Th2 and regulatory T-cell-attracting chemokines CCL17/TARC and CCL22/MDC, whereas only marginal levels of these chemokines were produced by alpha DC1s. Functional studies in vitro showed that supernatants from mature alpha DC1s actively recruited CD3(-)CD56(+) NK cells and that adding anti-CXCL9/MIG antibodies to the alpha DC1 supernatant substantially reduced this recruitment. Finally, alpha DC1s were able to induce IFN-gamma production when cocultured with resting autologous NK cells, but only if concurrent CD40 ligation was provided. These novel findings indicate that injected human alpha DC1-based vaccines have the potential to recruit and activate NK cells during their arrival to draining lymph nodes and that this feature may be of relevance for efficient priming of Th1 cells and CTLs.

Xenograft rejection of porcine islet-like cell clusters in immunoglobulin or Fc-receptor g-deficient mice.

The aim of the present study was to evaluate the role of xenoreactive antibodies in islet-like cell cluster (ICC) xenograft rejection. For this purpose, normal mice, mice with a targeted disruption of the Fc-receptor (FcR) gamma-chain, or the membrane exon of the immunoglobulin mu-chain gene, were transplanted with fetal porcine ICC under the kidney capsule. Mice lacking the FcR gamma have no functional FcR for IgG or IgE. Mice with disruption of the immunoglobulin mu-chain cannot produce antibodies, because B cell development is arrested at the stage of preB cells. All animals, irrespective of recipient group, readily rejected the ICC xenograft. Analyses of the pattern of cellular infiltration revealed only minor dissimilarities between the different experimental groups. Xenograft destruction was evident on day 6 after transplantation, and a large number of mononuclear cells were found to be evenly distributed throughout the ICC graft. The majority of the infiltrating cells were large, macrophage-like cells expressing the macrophage-specific phenotype marker F4/80. CD3-positive T lymphocytes were found to be mainly accumulated in the peripheral parts of the ICC xenograft. This study has demonstrated that xenoreactive antibodies are not crucial to ICC xenograft rejection in the pig-to-mouse model.

A simple immunoenzyme batch staining method for the enumeration of peripheral human T lymphocyte subsets

A simple batch method was developed for enumeration of peripheral human T lymphocyte subsets by immunoenzyme staining of stored prefixed preparations of peripheral mononuclear cells on multiple well microscope slides. By this method the monoclonal antibodies Leu 1, Leu 2a and Leu 3a stained the same number of cells as by indirect immunofluorescence on suspended cells.

Immunological Characteristics of Islet Cell Xenotransplantation in Humans and Rodents

If ethical, physiological and immunological problems can be solved, it is conceivable that the pig will be a future donor species for human xenotransplantation (Cooper et al. 1991). The pig is a discordant species which implies that all humans have natural antibodies reactive against determinants present on most porcine cells. These determinants are present mainly on glycolipids and glycoproteins present on both nucleated cells and on erythrocytes. Most probably, these determinants are oligosaccharide residues, with structures similar to ABO-blood group substances (Szymanowski et al. 1926, Good et al. 1992, Holgersson et al. 1992, Koren et al. 1993). Porcine cells are sensitive to human complement, hence, porcine tissues can be immediately destroyed by antibody-mediated reactions, such as direct cytotoxicity. Natural antibodies react with determinants present on blood leukocytes, endothelial cells and other cells in a xenogeneic graft. Therefore, discordant vascularized grafts are hyperacutely rejected. However, not all cells are sensitive to the cytotoxic effect of antibodies. Sensitivity is in part determined

T‐Lymphocyte Subsets and HLA‐DR‐Expressing Cells in Rejected Human Kidney Grafts

This communication describes an immunohistochemical analysis of rejected human renal allografts. T‐lymphocyte subsets were identified in frozen tissue sections, by Leu 1 (anti‐ ‘pan’ T lymphocytes). Leu 2a (anti‐ ‘cytotoxic/suppressor’ T cells), and Leu 3a (anti‐ ‘helper/inducer’ T cells) monoclonal antibodies. In addition, HLA‐DR‐positive cells were identified by simultaneous labelling with heterologous anti‐HLA‐DR antibodies. T cells dominated the cellular infiltrates in acute cellular rejection. Leu‐3a‐positive cells were more numerous than Leu‐2a‐positive cells. The Leu‐3a‐positive cells usually appeared in clusters, whereas the Leu‐2a‐positive cells appeared scattered in the tissue. HLA‐DR‐positive non‐T cells were found within clusters of T ‘helper/inducer’ cells. The cell pattern shares many features with the findings in detayed‐type hypersensitivity reactions.

Class II Transplantation Antigens: Distribution in Tissues and Involvement in Disease

The primary reeognition by the immune system of microbial antigens and autoantigens is generally thought to involve eells that are part of the structural framework of a particular organ and migratory cells that are called upon by various stimuli. The immunological response obtained can either afford protection against infections or lead to autoimmune disease in susceptible individuals. It may also be a minor inconsequential part of the normal homeostasis. In these reactions, there is evidence that lymphoid cells, particularly T lymphocytes, control the magnitude of the final response by recognizing microbiai antigens or autoantigens in connection with cell surfaee-bound class II transplantation antigens |5]. Most knowledge of the involvement of class II antigens in T-lymphocyte activation comes from in vitro studies of lymphoid eells. in which mainly T-cell interactions with macrophages and dendritic cells of the spleen and epidermal Langerhans cells have been investigated [45, 46. 48. 52. 54|. These studies indicate that class Il-expressing cells have a major role in T-cell activation both because of their antigen-presenting capabilities and because of their production of lymphokines. More recently, however, immunomorphological studies on tissue seetions have revealed that class II transplantation antigens are not only expressed on cells belonging to the immune system but can also be found on the cell surface of non-lymphoid cells such as endothchal cells and different epithelial cells (11. 15. 35. 56]. In the well-organized structure of an organ various both residential and migratory class II transplantation antigen-expressing cells will thus be encountered, and a precise mapping of the phenotype. origin, and location of the various class Il-cxpressing cells should be fundamental for the understanding of many local immune reactions. Considering the role of class II antigens in local immune reactions, it must, however, be taken into account that expression of these molecules can also be induced on eells that do not express class II antigen constitutively. Thus, in vitro, studies of various cells—among lymphoid cells, mainly B cells and macrophages—have demonstrated that class II antigen expression is not constant but varies with the functional capacities of the ceils in question [18, 52]. Particularly in macrophages. the density of class II antigens on the eell surface appears to correlate well with the antigen-presenting capacity of the cell [52], Studies of class II antigen expression in various tissues must therefore consider not only the cells that always express class II molecules but also various cell types that occur in the organ and change phenotypes as the immune response proceeds through various phases.

Viral triggering of anti-NMDA receptor encephalitis in a child - an important cause for disease relapse.

Herpes simplex encephalitis (HSE) in children is a potentially devastating condition which is occasionally complicated by a clinical relapse. An autoimmune component has long been suspected in these relapses and recent findings suggest that antibodies against N-methyl-D-aspartate receptors (NMDARs) may be part of this mechanism. We here report an 11 months old girl with acute HSE and with negative NMDAR antibody serology at presentation who after an initial response to antiviral treatment deteriorated with seizures, abnormal movements, focal neurologic deficits and psychiatric symptoms. We show that this relapse occurred as production of NMDAR antibodies developed and that clinical improvement followed immunotherapy with a concomitant decrease in NMDAR antibody titers in CSF. She also developed a characteristic 15-20 Hz activity over both hemispheres which has been previously described as an electroencephalographic presentation of anti-NMDAR encephalitis. We conclude that relapse or persisting symptoms in HSE in children may represent an immune-mediated mechanism rather than a viral reactivation and that NMDAR antibodies should be analyzed as this may be of importance for the choice of therapy.

Role of antibody synthesis and complement activation in concordant xenograft retransplantation

A mouse-to-rat heart retransplantation model was used to study the effects of complement depletion and antibody production with regard to graft survival and anti-donor antibody specificity. Retransplantation was performed 3 weeks after the first transplantation in the presence of absence of 15-deoxyspergualin (DSG) immunosuppression. Untreated animals rejected their first graft after 3 days and retransplantation resulted in a hyperacute rejection within 2 min. A low titer of preformed anti-mouse lymphocytotoxic antibodies of the IgM subclass was found in serum collected from the unoperated rat. The rejection gave rise to a synthesis of IgG antidonor antibodies reacting with both graft endothelium and sarcolemma. Immunofluorescent staining of the rejected first heart graft showed moderate IgM and IgG antibody deposits on the graft vascular endothelium, while only IgG was found in the second graft. There was no C3 deposition found in the first mouse graft, as was the case in the second mouse graft. Anti-mouse antibodies cross-reacted with hamster antigens and a hyperacute rejection of a hamster heart graft occurred in a mouse-sensitized rat. Immunofluorescent staining revealed that the antibodies did not bind to hamster heart endothelium, as was expected, but, instead, to graft sarcolemma. DSG treatment prolonged the survival of the first graft by a median of 8 days. Continuous treatment until retransplantation resulted in a prolongation to 30 (20-127) min of the survival of the second graft and no increase in antibody titers against mouse antigens was observed. However, immunofluorescent staining revealed a weak binding of anti-mouse antibodies of the IgM subclass in the rejected mouse heart graft. Additional complement depletion with cobra venom factor in DSG-treated animals resulted in a prolongation of the median graft survival to 48 hr (6-96). No sign or minimal signs of antibody deposition were found in these grafts, but histology revealed massive mononuclear infiltration. In conclusion, xenograft transplantation in a concordant situation results in a shift of antidonor antibody Ig synthesis from IgM to IgG. If daily DSG treatment is administered from the day of transplantation, this reduces the synthesis of antidonor antibodies, and if complement is also depleted, the survival of the second graft is prolonged. The significance of the mononuclear infiltration remains to be established.