Alloimmunity: no toll exemption.
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Humans↗
Biomedical subjects
C P Larsen
Publications and source records attributed to C P Larsen.
Toward transplantation tolerance: a large step on a long road.
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Bone Marrow Transplantation↗
NK cells mediate costimulation blockade-resistant rejection of allogeneic stem cells during nonmyeloablative transplantation.
Although T-cell CD28/CD40 costimulation blockade represents a powerful mechanism to promote immune tolerance during murine allotransplantation, it has not yet been successfully translated to clinical transplantation. We determined the impact of natural killer (NK) cells on costimulation blockade-resistant rejection of donor bone marrow. We found that NK cells represent a potent barrier to engraftment: host NK depletion led to increased donor stem cell survival, increased mixed hematopoietic chimerism and to engraftment of low doses of donor marrow (1 x 10(8)/kg) that were otherwise rejected. To understand the mechanisms of NK alloreactivity, we employed an in vivo NK-specific cytotoxicity assay. We found that an increased proportion of target cells were killed between days 2 and 8 after cell transfer, and that NK killing of parental targets was inducible: NK cells preprimed with allotargets were more efficient at their elimination upon reexposure. Finally, both transplant and in vivo NK-killing models were used to determine the contribution of LFA-1 to NK alloreactivity. Blockade of LFA-1 led to decreased NK-mediated killing, and increased alloengraftment. These results identify NK alloreactivity as an integral component to costimulation blockade-resistant rejection, and suggest that its inhibition may represent an important target in the clinical translation of tolerance-induction transplantation.
Animals↗
A new look at blockade of T-cell costimulation: a therapeutic strategy for long-term maintenance immunosuppression.
Activated T cells orchestrate the immune response that results in graft rejection; therefore, a common goal among current immunosuppressive therapies is to block T-cell activation, proliferation and function. Current immunosuppressive regimens that inhibit T cells and immune cells have greatly reduced the incidence of acute rejection following solid-organ transplant. However, the expected improvements in long-term outcomes have not been realized. This may be related to the non-immune side effects of current maintenance immunosuppressants, which target ubiquitously expressed molecules. The focus in transplantation research is shifting in search of maintenance immunosuppressive regimens that might offer improved long-term outcomes by providing efficacy in prevention of acute rejection combined with reduced toxicities. An emerging therapeutic strategy involves an immunoselective maintenance immunosuppressant that inhibits full T-cell activation by blocking the interaction between costimulatory receptor-ligand pairs. This review describes costimulatory pathways and the development of molecules, which inhibit them in the context of transplantation research. Recent clinical data using the selective costimulation blocker, belatacept (LEA29Y), as a part of a CNI-free maintenance immunosuppressive regimen in renal transplantation is highlighted.
Abatacept↗
Transplant tolerance in non-human primates: progress, current challenges and unmet needs.
Given the significant morbidity associated with current post-transplant immunosuppressive regimens, induction of immune tolerance continues to be an important goal of clinical organ transplantation. While many strategies for inducing tolerance have been successfully applied in murine models, significant barriers are faced when translating these approaches to the clinic. This has necessitated pre-clinical studies in the more closely related model system, the non-human primates (NHP). In this review, we will discuss the four most prominent strategies for inducing transplantation tolerance and highlight their relative success and shortcomings in NHP. These strategies are: (1) T-cell costimulation blockade (2) mixed chimerism induction (3) T-cell depletion and (4) tolerance induction through regulatory T-cells. After discussing the progress that has been made with each of these strategies, we will identify this field's most pressing unmet needs and discuss how we may best overcome the resulting barriers to tolerance induction.
Animals↗
A mouse model for polyomavirus-associated nephropathy of kidney transplants.
Polyomavirus-associated nephropathy is an important cause of dysfunction and failure of renal transplants. BK virus is an ubiquitous human polyoma virus that persistently infects the kidney. This otherwise silent infection can reactivate in immunosuppressed individuals, resulting in renal complications. Because polyoma viruses are highly species-specific, we developed a mouse polyoma virus-renal transplant model in order to investigate the pathogenesis of polyomavirus-associated nephropathy. Using this model, we found that polyoma virus preferentially replicates in the allogeneic kidney grafts, accelerating graft failure; thus, this animal model is able to mimic the polyomavirus-associated nephropathy seen in human renal transplant patients. Acute polyoma virus infection of mouse allograft recipients augmented the alloreactive CD8+ T-cell response, while maintaining the anti-viral CD8+ T-cell response. In addition to the known virus-induced cytopathology, these findings demonstrate a potential role for an enhanced anti-donor T-cell response in the pathogenesis of polyomavirus-associated nephropathy.
Animals↗
Six years' experience of using the BacT/ALERT system to screen all platelet concentrates, and additional testing of outdated platelet concentrates to estimate the frequency of false-negative results.
BACKGROUND AND OBJECTIVES: Approximately 1 in every 2000 units of platelets is contaminated with bacteria. The BacT/ALERT automated blood culture system can be used to screen platelet concentrates (PCs) for bacterial contamination. MATERIALS AND METHODS: Data were collected from May 1998 until May 2004. The number of PCs tested during this period was 36 896, most of which were produced from pools of four buffy-coats. On the day following blood collection or platelet apheresis, a 5-10 ml sample of the PC was aseptically transferred to a BacT/ALERT culture bottle for detection of aerobic bacteria. The sample was monitored for bacterial growth during the entire storage period of the PC (6.5 days). When a positive signal was generated, the culture bottle, the PC and the erythrocyte concentrates were tested for bacterial growth. In order to determine the frequency of false-negative BacT/ALERT signals, 1061 outdated PCs were tested during the period from May 2002 to May 2004. RESULTS: Eighty-eight positive signals were detected by the BacT/ALERT system, of which 12 were interpreted as truly positive. Fourteen signals were interpreted as truly false positive. Thirty-three signals were interpreted to be probably false positive. Two of 1061 outdated units tested positive, and Bacillus spp. and Staphylococcus epidermidis, respectively, were isolated from these PCs. CONCLUSIONS: Between 0.03% and 0.12% of the PCs were contaminated with bacteria. BacT/ALERT is an efficient tool for monitoring PCs for bacterial contamination; however, it is important to realize that false-negative results may occur.
Bacillus↗
Ability of donor splenocytes with costimulation blockade to induce mixed hematopoietic chimerism and transplantation tolerance.
We reported stable mixed chimerism and specific tolerance to a fully allogeneic graft after a minimally myelosuppressive regimen including costimulation blockade (CB), donor bone marrow cells (BMC), and busulfan (Bu), a chemotherapeutic conditioning agent that makes niches for engraftment of BMC. For clinical application, the strategy may have the limitation of the number of donor BMC when a deceased donor offers transplants to multiple recipients. Herein, we examined whether donor splenocytes can serve as an alternative source to induce mixed chimerism and tolerance. When a C57BL/6 (H-2b) recipient was treated with CB (CTLA4-Ig and anti-CD154 mAb, on days 0, 2, 4, 6) and donor BALB/c (H-2d) BMC (2 x 10(7) cells on day 0) in the absence of Bu, survival of BALB/c skin graft was remarkably prolonged but not indefinite (median survival time [MST]: 138 days). The recipients never showed durable chimerism. When the recipient was treated with CB and donor splenocytes ([DST] 2 x 10(7) cells on day 0), survival was not indefinite either (MST: 114 days). When the dose of DST was increased to 2 x 10(8) cells, survival was further prolonged; two of six recipients had indefinite survival (MST: 132 days). Moreover, one recipient showed a low level of chimerism. When treated with CB, donor DST (2 x 10(7) cells on day 0) and Bu (20 mg/kg, day -1), six of seven recipients showed a stable, high level of chimerism and enjoyed tolerance of skin allografts. DST combined with CB and Bu may be an alternative source of hematopoietic stem cells to induce mixed chimerism and transplantation tolerance in our model.
Animals↗
Positive BacT/ALERT signal from a platelet concentrate 23 h before the donor was admitted to hospital with Streptococcus pneumoniae septicaemia.
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Bacterial Typing Techniques↗
Role of CD28-B7 interactions in generation and maintenance of CD8 T cell memory.
Although the role of CD28-B7 interaction in the activation of naive T cells is well established, its importance in the generation and maintenance of T cell memory is not well understood. In this study, we examined the requirement for CD28-B7 interactions in primary T cell activation and immune memory. Ag-specific CD8 T cell responses were compared between wild-type (+/+) and CD28-deficient (CD28(-/-)) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). During the primary response, there was a substantial activation and expansion of LCMV-specific CD8 T cells in both +/+ and CD28(-/-) mice. However, the magnitude of the primary CD8 T cell response to both dominant and subdominant LCMV CTL epitopes was approximately 2- to 3-fold lower in CD28(-/-) mice compared with +/+ mice; the lack of CD28-mediated costimulation did not lead to preferential suppression of CD8 T cell responses to the weaker subdominant epitopes. As seen in CD28(-/-) mice, blockade of B7-mediated costimulation by CTLA4-Ig treatment of +/+ mice also resulted in a 2-fold reduction in the anti-LCMV CD8 T cell responses. Loss of CD28/B7 interactions did not significantly affect the generation and maintenance of CD8 T cell memory; the magnitude of CD8 T cell memory was approximately 2-fold lower in CD28(-/-) mice as compared with +/+ mice. Further, in CD28(-/-) mice, LCMV-specific memory CD8 T cells showed normal homeostatic proliferation in vivo and also conferred protective immunity. Therefore, CD28 signaling is not necessary for the proliferative renewal and maintenance of memory CD8 T cells.
Animals↗
Characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance.
Simultaneous blockade of the CD28 and CD40 T cell costimulatory pathways has been shown to effectively promote skin allograft survival in mice. Furthermore, blockade of one or both of these pathways has played a central role in the development of strategies to induce mixed hematopoietic chimerism and allospecific tolerance. It has recently been observed that the beneficial effects of CD40 blockade and donor splenocytes in prolonging skin graft survival can be abrogated by some viral infections, including lymphocytic choriomeningitis virus (LCMV). In this study, we show that LCMV infection prevents prolonged allograft survival following CD28/CD40 combined blockade. We further show that LCMV prevents the induction of allospecific tolerance and mixed hematopoietic chimerism, while delay of infection for 3-4 wk posttransplant has no effect on tolerance induction. Because of reports of anti-H-2(d) activity following LCMV infection, we assayed the ability of LCMV-specific T cells to respond to alloantigen at a single cell level. Although we confirm that LCMV infection induces the generation of alloreactive cells, we also demonstrate that LCMV-specific T cells do not divide in response to alloantigen. The alloresponse suppressed by costimulation blockade is restored by LCMV infection and correlates with increased dendritic cell maturation. We hypothesize that the costimulation blockade-resistant rejection mediated by LCMV could be partly attributable to the up-regulation of alternative costimulatory pathways subsequent to LCMV-induced dendritic cell maturation.
Animals↗
Analysis of the CD40 and CD28 pathways on alloimmune responses by CD4+ T cells in vivo.
BACKGROUND: Blockade of the CD40 and CD28 pathways is a powerful strategy to inhibit CD4-mediated alloimmune responses. In this study, we examine the relative roles of the CD40 and CD28 pathways on CD4-mediated allograft rejection responses, and further characterize the role of these pathways on CD4+ T-cell activation, priming for cytokine production, and cell proliferation in response to alloantigen in vivo. METHODS: BALB/c skin allografts were transplanted onto C57BL/6 Rag 1-/- recipients reconstituted with CD4 cells from CD28-/- or CD40L-/- donors. The popliteal lymph node assay was used to study the role of these pathways on CD4-cell activation and priming in vivo. To investigate the role of CD40 and CD28 blockade on CD4-cell proliferation, the fluorescein dye carboxyfluorescein diacetate succinimidyl ester was used. We performed heterotopic cardiac transplantation using CD40-/- mice to evaluate the role of CD40 on donor versus recipient cells in CD4-mediated rejection. RESULTS: B6 Rag 1-/- recipients reconstituted with CD28-/- CD4+ T cells acutely rejected allografts (median survival time 15 days), whereas recipients reconstituted with CD40L-/- CD4+ T cells had significantly prolonged survival of BALB/c skin grafts (MST 71 days). CD40L blockade was equivalent to or inferior to CD28 blockade in inhibition of in vivo CD4-cell activation, priming for cytokine production, and proliferation responses to alloantigen. BALB/c recipients depleted of CD8 cells promptly rejected donor B6 CD40-/- cardiac allografts, whereas B6 CD40-/- recipients depleted of CD8 cells had significantly prolonged survival of BALB/c wild-type cardiac allografts. CONCLUSIONS: The CD40/CD40L pathway, but not the CD28/B7 pathway, is critical for CD4-mediated rejection responses, however, the responsible mechanisms remain unclear.
Animals↗
Gene expression analysis in human renal allograft biopsy samples using high-density oligoarray technology.
BACKGROUND: High-density oligoarray technology is a novel method for screening the expression of thousands of genes in a small tissue sample. Oligoarray analysis of genes expressed during human renal allograft rejection has not been reported previously. METHODS: Seven human renal allograft biopsies with histologic evidence of acute cellular rejection and three renal allograft biopsies without evidence of rejection (control) were analyzed for the expression of 6800 human genes using high-density oligoarrays (GeneChip, Affymetrix, Santa Clara, CA). Quantitative expression of gene transcripts was determined and a comparison analysis between acute rejection and control biopsy samples was performed. Up-regulation of a specific gene transcript during acute rejection was considered to be significant if transcript abundance increased fourfold or more relative to control biopsy samples. RESULTS: Comparison analysis revealed that between 32 and 219 gene transcripts are up-regulated (>fourfold) during acute rejection. Of these transcripts, only four (human monokine induced by interferon-gamma, T-cell receptor active beta-chain protein, interleukin-2 stimulated phosphoprotein, and RING4 (a transporter involved in antigen presentation)) were consistently up-regulated in each acute rejection sample relative to at least two of three control biopsy samples. Six other genes were up-regulated in six of seven acute rejection samples. These were interferon-stimulated growth factor-3, complement factor 3, nicotinamide N-methyltransferase, macrophage inflammatory protein-3beta, myeloid differentiation protein, and CD18. Only two gene transcripts were down-regulated in five of seven acute rejection samples. Significant up-regulation of cytotoxic T-cell effector molecules, previously reported as markers of acute renal rejection in humans, was not detected. CONCLUSIONS: High-density oligoarray technology is useful for screening gene expression in transplanted tissues undergoing acute rejection. Because this method does not rely on a priori knowledge of which genes are involved in acute rejection, it is likely to yield novel insights into the mechanisms and diagnosis of rejection.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.
Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.
Animals↗
Conventional immunosuppression and co-stimulation blockade.
Organ transplantation has become an accepted and successful therapeutic intervention for many patients with end-stage organ disease. Current conventional immunosuppressive regimens achieve very good short-term allograft survival but long-term outcomes are less than adequate. Furthermore, non-specific immunosuppression has its attendant side-effects including increased risks of malignancy and infection as well as drug-specific sequellae. With recent advances in the field of immunology, promising new therapies have arisen that could potentially eliminate lifelong drug therapy and promote indefinite acceptance of the donor tissue. Identification of co-stimulatory signals essential for T-cell activation has provided exciting new possibilities for controlling the alloimmune response. The compatibility of these new agents with proven conventional therapeutics has yielded mixed results. When used in combination, their immunosuppressive properties appear synergistic. However, if the goal of therapy is sustained, specific T-cell hyporesponsiveness, many conventional agents antagonize the effects of co-stimulatory blockade in several immune tolerance models.
Abatacept↗
CD8 T cell-mediated rejection of intestinal allografts is resistant to inhibition of the CD40/CD154 costimulatory pathway.
BACKGROUND: Disruption of the CD40/CD154 pathway inhibits rejection in numerous models. The importance of this pathway on intestinal allograft rejection was examined in this study. METHODS: Intestinal grafts from B6C3F1 mice transplanted into C57BL/6 recipients were assessed histologically for rejection. RESULTS: The monoclonal antibody to CD154, MR1, failed to inhibit rejection in wild-type mice. Similarly, CD154-/- recipient mice rejected intestinal allografts. MR1 did inhibit early rejection in CD8-/- mice, but had no effect in CD4-/- recipients. All MR1-treated CD8-/- recipients eventually developed rejection. No benefit was observed when blockade of the CD40/CD154 pathway by MR1 was combined with blockade of the CD28/B7 pathway by mCTLA4Ig. CONCLUSIONS: These data suggest that CD4+ T cells mediating intestinal allograft rejection may be more dependent upon the CD40/CD154 pathway than CD8+ T cells. This finding highlights the importance of identifying agents that suppress CD8+ T cell-mediated rejection.
Animals↗
FasL is important in costimulation blockade-resistant skin graft rejection.
BACKGROUND: Simultaneous blockade of the CD40 and CD28 costimulatory pathways is effective in prolonging allograft survival in murine and primate models. Recent data suggest that intact apoptotic pathways are crucial for the induction of hyporesponsiveness by costimulation blockade. We have studied the impact of fas/fasL signaling, an important T cell apoptotic pathway, on the effects of costimulation blockade. Methods. Wild type, lpr (fas deficient), and gld (fasL deficient), mice were used as donors and recipients in the murine skin graft model. Allograft survival was compared in untreated and costimulation blockade (500 microg anti-CD40L and 500 microg CTLA4-Ig, days 0, 2, 4, 6) treated recipients. In some recipients, CD4+ T cells were depleted using rat anti-murine CD4 (100 microg day -3, -2, -1, and weekly). RESULTS: gld mice treated with costimulation blockade enjoy a significantly greater increase in skin allograft survival than do wild-type mice. This effect is not replicated using lpr donors or recipients. Experiments in which CD4+ cells were depleted demonstrate that fasL is not necessary for CD8-mediated allograft rejection, and that depletion of CD4+ cells eliminates some of the survival advantage induced by costimulation blockade. CONCLUSIONS: FasL is not required for the establishment of costimulation blockade induced hyporesponsiveness, but rather appears to be required for normal costimulation blockade resistant rejection. Fas expression is not critical for costimulation blockade resistant rejection, suggesting that fasL may be interacting with other receptors. Further, it appears that CD4+ cells are important in the maintenance of allograft protection induced by costimulation blockade in this model.
Animals↗
Expression of the melanocortin 5 receptor on rat lymphocytes.
The expression of melanocortin-5 receptor (MC5-R) mRNA and protein was characterized from isolated rat lymphocytes. The presence of MC5-R mRNA in spleen and thymus tissues was demonstrated by RT-PCR. The RT-PCR product was sequenced to confirm the identification of MC5-R. Tissues from lachrymal glands, adipose, adrenals, thymus, pancreas, and isolated splenic lymphocytes were detergent solubilized. The crude proteins were resolved by SDS-PAGE, transblotted to a nitrocellulose membrane, and probed for MC5-R using anti-receptor rabbit antisera. Two different types of polyclonal rabbit antisera were raised against synthetic peptides representing epitopes found at the amino (alphaN-MC5-R) and the carboxyl termini (alphaC-MC5-R) on the MC5-R. A prominent band at 77,000 (p77) was detected in all tissues except the pancreas. Preimmune sera did not detect p77 by Western analysis and the addition of peptide antigen neutralized the detection of p77 by the specific antisera. The receptor protein was purified from spleen and thymic lymphocytes using protein A agarose that precipitated material complexed to alphaN-MC5-R. The purified MC5-R was detected by Western analysis using alphaC-MC5-R. Both anti-receptor antisera, alphaN-MC5-R and alphaC-MC5-R, detected the p77. The p77 was treated with protein endoglycosidase F to produce a smaller protein band between 34-38,000 (p35); the inferred size is 37,000 based on the cDNA sequence. The data suggest that Asn-linked carbohydrate groups account for much of the p77 mass of the MC5-R. The data also demonstrate the expression of MC5-R protein on rat lymphocytes, thus, supporting the hypothesis that MC5-R is the ACTH receptor on lymphocytes.
Adipose Tissue↗