Durable targeting of B-lymphocytes in living mice.

Transfer to and enduring expression of genes in B cells has proved a vexing challenge. We report here a novel method for the specific and durable targeting of B lymphocytes in living mice. The method involves generation of lentiviruses pseudotyped with an anti-CD19 antibody. CD19 targeting viruses injected in the spleen of living mice efficiently transduced B cells and plasma cells detected by flow cytometry analysis of GFP expression. Expression of the reporter gene could be detected in the intact animal by external imaging for more than a year and was enhanced by booster immunization. Our method thus enables the specific delivery, expression and localization by external imaging of exogenous genes to the B cells and plasma cells of living individuals.

Cryptic B cell response to renal transplantation.

Transplantation reliably evokes allo-specific B cell and T cell responses in mice. Yet, human recipients of kidney transplants with normal function usually exhibit little or no antibody specific for the transplant donor during the early weeks and months after transplantation. Indeed, the absence of antidonor antibodies is taken to reflect effective immunosuppressive therapy and to predict a favorable outcome. Whether the absence of donor-specific antibodies reflects absence of a B cell response to the donor, tolerance to the donor or immunity masked by binding of donor-specific antibodies to the graft is not known. To distinguish between these possibilities, we devised a novel ELISPOT, using cultured donor, recipient and third-party fibroblasts as targets. We enumerated donor-specific antibody-secreting cells in the blood of nine renal allograft recipients with normal kidney function before and after transplantation. Although none of the nine subjects had detectable donor-specific antibodies before or after transplantation, all exhibited increases in the frequency of donor-specific antibody-secreting cells eight weeks after transplantation. The responses were directed against the donor HLA-class I antigens. The increase in frequency of donor-specific antibody-secreting cells after renal transplantation indicates that B cells respond specifically to the transplant donor more often than previously thought.

Absence of donor-specific anti-HLA antibodies after ABO-incompatible heart transplantation in infancy: altered immunity or age?

Specific B-cell tolerance toward donor blood group antigens develops in infants after ABO-incompatible heart transplantation, whereas their immune response toward protein antigens such as HLA has not been investigated. We assessed de novo HLA-antibodies in 122 patients after pediatric thoracic transplantation (28 ABO-incompatible) and 36 controls. Median age at transplantation was 1.7 years (1 day to 17.8 year) and samples were collected at median 3.48 years after transplantation. Antibodies were detected against HLA-class I in 21 patients (17.2%), class II in 18 (14.8%) and against both classes in 10 (8.2%). Using single-antigen beads, donor-specific antibodies (DSAs) were identified in six patients (all class II, one additional class I). Patients with DSAs were significantly older at time of transplantation. In patients who had undergone pretransplant cardiac surgeries, class II antibodies were more frequent, although use of homografts or mechanical heart support had no influence. DSAs were absent in ABO-incompatible recipients and class II antibodies were significantly less frequent than in children with ABO-compatible transplants. This difference was present also when comparing only children transplanted below 2 years of age. Therefore, tolerance toward the donor blood group appears to be associated with an altered response to HLA beyond age-related effects.

Two novel assays of alloantibody-secreting cells demonstrating resistance to desensitization with IVIG and rATG.

Donor-specific alloantibody presents a major barrier to the successful transplantation of kidneys and hearts. However, the study of alloantibody production has been hampered by both an inadequate source of antibody-secreting cells (ASCs) and a paucity of assays to determine their function. We describe two new assays that allow for the determination of the frequency and specificities of allo-ASCs in humans using purified HLA as targets. These assays demonstrated allo-ASCs in the CD138(+) fraction of the bone marrow, but not in peripheral blood. Alloantibody specificities in these assays correlated well with those detected in the serum suggesting that bone marrow-derived ASCs are indeed a major source of alloantibody in vivo. However, ASCs for a specific HLA antigen were rare with an estimated frequency of only 1/2 x 10(6) marrow cells. Pretransplant treatment in vivo with multiple plasmaphereses and low-dose IVIG alone or in combination with rATG had no effect on ASC number or alloantibody production. These techniques allow for the study of allospecific ASCs and provide a method to test the potential efficacy of agents on alloantibody production in vivo.

Banff '05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy ('CAN').

The 8th Banff Conference on Allograft Pathology was held in Edmonton, Canada, 15-21 July 2005. Major outcomes included the elimination of the non-specific term "chronic allograft nephropathy" (CAN) from the Banff classification for kidney allograft pathology, and the recognition of the entity of chronic antibody-mediated rejection. Participation of B cells in allograft rejection and genomics markers of rejection were also major subjects addressed by the conference.

The future of organ replacement: needs, potential applications, and obstacles to application.

The supply of organs available for transplantation is already far smaller than the demand, and the demand may grow substantially in the near future. For this reason, it is timely to consider how organ function might be replaced in the future. In this article, we consider new technologies that might be used to replace organ function, the obstacles to applying new technologies, and how those obstacles might be overcome in the development of new strategies for organ replacement.

Cryptic speciation in the cosmopolitan and clonal human pathogenic fungus Aspergillus fumigatus.

Microbes and other organisms smaller than one to a few millimeters in size are hypothesized to have global populations, in contrast to the geographically restricted ranges of larger organisms. However, fungi, which routinely have reproductive propagules no larger than 10 micrometers, challenge the generality of this hypothesis because recent studies have shown that globally distributed morphological species embrace two or more geographically restricted phylogenetic species. We used the concordance of gene genealogies to recognize phylogenetic species in the globally distributed opportunistic human pathogenic fungus, Aspergillus fumigatus. Based on DNA sequence data of five loci for each of 63 individuals collected from five continents, we have delineated two phylogenetic species in this single morphological species. Unlike all other fungi examined to date, both genetically isolated groups showed a global distribution with no evidence of a correlation between genotype and geographic location. Sexual reproduction has never been observed in A. fumigatus, but when the same data were used to explore the association of alleles at the five loci for one of the phylogenetic species, evidence was found to support recombination. The discovery of a cryptic species is medically relevant because different species are likely to differ in virulence or drug resistance. The discovery of a globally distributed A. fumigatus species clade highlights the need for ecological studies of the fungus to either document global dispersal or propose alternative mechanisms by which it persists as single, global phylogenetic population.

New approaches to replacing failing organs.

The foremost cause of death and disability is organ failure. Failure of the heart, lungs, kidney, and liver is typically addressed by organ transplantation; however, the number of human organs available for this purpose is quite limited. For this reason, some new technologies are being advanced for the treatment of organ failure. This communication summarizes potential limitations of these technologies.

B cell-dependent T cell development.

T and B cells are thought to develop independently. While it is widely recognized that T cells help B cells in the production of antibodies to protein antigens, less well understood is whether or how B cells contribute to T cell development and function. Defects in cell-mediated immunity in individuals with B cell deficiency and in B cell-deficient mice suggest that B cells contribute to T cell function. The question of whether T cell development is B cell dependent was revisited using two novel mouse strains: mice with monoclonal T cells (MT) and mice with monoclonal compartments of both B and T cells (MBT). It was found that T cell development and thymocyte selection is modified by the presence of B cells. These results suggest that B cells, or B cell products, contribute to thymocyte selection and T cell development.

Genetic modification of xenografts.

For nearly a century, xenotransplantation has been seen as a potential approach to replacing organs and tissues damaged by disease. Until recently, however, the application of xenotransplantation has seemed only a remote possibility. What has changed this perspective is the advent of genetic engineering of large animals; that is, the ability to add genes to and remove genes from lines of animals that could provide an enduring source of tissues and organs for clinical application. Genetic engineering could address the immunologic, physiologic and infectious barriers to xenotransplantation, and could allow xenotransplantation to provide a source of cells with defined and even controlled expression of exogenous genes. This communication will consider one perspective on the application of genetic engineering in xenotransplantation.

The possibility of B-cell-dependent T-cell development.

The development of T cells is thought to be independent of B cells. However, defects in cell-mediated immunity in individuals with B-cell deficiency suggest the contrary. To test whether B cells affect T-lymphocyte development, we constructed mice with a monoclonal T-cell compartment (MT) and monoclonal B- and T-cell compartments (MBTs). In these mice, the T cells expressed a DO 11.10 transgenic (DO-T) cell receptor restricted to major histocompatibility complex (MHC) class IId. While CD4+ DO-T lymphocytes are rare in transgenic H-2b MT mice, we found that in H-2b MBT mice under the influence of B cells, DO-T lymphocytes mature into large numbers of CD4+ peripheral T cells. H-2b MBT mice have more CD4+ thymocytes than H-2b MT mice. These data are consistent with the view that B cells play some role in thymocyte development.

Deficiency of 5-lipoxygenase accelerates renal allograft rejection in mice.

Acute renal allograft rejection is associated with alterations in renal arachidonic acid metabolism, including enhanced synthesis of leukotrienes (LTs). LTs, the products of the 5-lipoxygenase (5-LO) pathway, are potent lipid mediators with a broad range of biologic activities. Previous studies, using pharmacological agents to inhibit LT synthesis or activity, have implicated these eicosanoids in transplant rejection. To further investigate the role of LTs in acute graft rejection, we transplanted kidneys from CByD2F1 mice into fully allogeneic 129 mice that carry a targeted mutation in the 5lo gene. Unexpectedly, allograft rejection was significantly accelerated in 5-LO-deficient mice compared with wild-type animals. Despite the marked reduction in graft survival, the 5lo mutation had no effect on the hemodynamics or morphology of the allografts. Although LTB4 levels were reduced, renal thromboxane B2 production and cytokine expression were not altered in 5-LO-deficient allograft recipients. These findings suggest that, along with their proinflammatory actions, metabolites of 5-LO can act to enhance allograft survival.

Platelet-mediated activation of endothelial cells: implications for the pathogenesis of transplant rejection.

BACKGROUND: Platelets exert their normal functions at sites of endothelial disruption by plugging discontinuities in blood vessels and secreting products that promote thrombosis, inflammation, and the healing of wounds. Whether platelets might induce these changes in xenograft blood vessels, leading to development of acute vascular rejection, has been uncertain. METHODS: To examine the role of human platelets in modulation of xenograft endothelium, pig endothelial cells were treated with human platelets. RESULTS: Treatment of quiescent porcine endothelial cells with human platelets modulated the endothelial cells. Whereas resting human platelets caused little change in normal porcine endothelial cells, platelets activated with small amounts of thrombin induced striking changes in the endothelial cells, including the induction of tissue factor activity, the expression of E-selectin, and the secretion of endothelin-1. These changes were induced, at least in part, by interleukin-1 (IL-1) associated with the platelet surface and were modified by the secretion of transforming growth factor-beta (TGF-beta). CONCLUSION: These findings may explain how the activation of platelets at an early point in the rejection of vascularized organ xenografts or in chronic diseases might contribute to thrombotic, ischemic, and inflammatory changes characteristic of an organ xenograft undergoing rejection.

Unexpected anti-alpha GalNAc antibodies in alpha-galactosyl transferase-deficient mice: complex relationship between genotype and the natural antibody repertoire.

Mice lacking the alpha-galactosyl transferase gene (GalT(-/-) mice) have been used extensively as a model for xenotransplantation. Unlike wild type (WT) mice, GalT(-/-) mice do not produce Gal alpha 1-3Gal and are known to produce natural IgM specific for Gal alpha 1-3Gal, as do humans and higher primates. In addition to natural anti-Gal alpha 1-3Gal IgM in GalT(-/-) mice, we identified natural IgM which bound alpha-N-acetylgalactosamine (alpha GalNAc) but not Gal alpha 1-3Gal or blood group A. Although unexpected, these antibodies were expressed at 10-fold greater concentrations in GalT(-/-) mice than in WT mice. One explanation for this unexpected observation is that the production of natural antibodies is affected by self-antigen(s) that are similar but not identical to targets recognized by the natural antibody. Thus, the natural humoral immune system may be unresponsive to "near-self" antigens even though the individual is not tolerant to those antigens. Another explanation for the unexpected results is that there may be unanticipated and uncharacterized differences between GalT(-/-) mice and WT mice. These studies underscore the need to extensively characterize phenotypes in KO mice and indicate that the relationship between genotype and the natural immune repertoire can be complex.

Human xenospecific T suppressor cells inhibit T helper cell proliferation to porcine aortic endothelial cells, and NF-kappaB activity in porcine APC.

Human T suppressor cells (Ts), capable of preventing autologous T helper cells (Th) from reacting against xenogeneic pig endothelial cells and pig APC can be generated in vitro. Ts derive from a population of CD3(+)CD8(+)CD28(-) T lymphocytes and specifically recognize the MHC class I antigens of the APC used for in vitro immunization. To study the mechanism that underlies suppression, we investigated whether Ts inhibit the expression of costimulatory molecules in xenogeneic professional and semiprofessional APC. We found that Ts down-regulate Th-induced expression of CD86 in pig APC, and that this effect occurs at the level of transcription, as indicated by nuclear run-on and Northern blot assays. EMSA results revealed that inhibition of CD86 expression is mediated by inactivation of transcription factor NF-kappaB. Furthermore, transfection of pig APC with a vector expressing NF-kappaB p65 partially rescued Th-induced expression of the CD86 molecule. These results strongly support the concept that xenospecific Ts inhibit the APC function of xenogeneic cells by preventing activation of NF-kappaB.