Alloantibody Levels Research Articles

Donor B-Cell Alloantibody Deposition and Germinal Center Formation Are Required for the Development of Murine Chronic Gvhd and Bronchiolitis Obliterans

Abstract 307 Introduction.Chronic GVHD (cGVHD) poses a significant risk for hematopoietic stem cell transplantation (HSCT) patients. Preclinical development of new therapies has been hindered by the dearth of models with pathological findings that simulate the development of human cGVHD. Although the exact causes of cGVHD in HSCT patients are unknown, higher alloantibody levels and the organ systems in which alloantibodies are deposited have not been well-represented in preclinical models. The present studies extend our recent publication on a mouse model of cGVHD and bronchiolitis obliterans (BO), which has been deemed pathognomonic of cGVHD diagnosis in patients according to the NIH consensus criteria. Further, we have elucidated the role that alloantibody deposition plays in cGVHD pathogenesis and have developed a new interventional approach that is highly effective in preventing BO and cGVHD manifestations. Methods/Results. B10.BR (H2k) recipients received myeloablative conditioning with Cytoxan and high dose radiation followed by allogeneic C57BL/6 (H2b) bone marrow (BM) and a low (sublethal) dose of T cells to induce a chronic anti-host immune response. Survival was ≥90% at 2 months in all studies and mice did not lose significant weight or have clinical manifestations of acute GVHD. On day 60 after HSCT, pulmonary function was measured in anesthesized mice by whole body plethysmography using the Flexivent mechanical ventilator. Mice with cGVHD and BO had decreased compliance and increased airway resistance along with histological features of BO, characterized by airway blockade, peri-bronchiolar fibroproliferation and bronchiole obliteration. Tissue manifestations of cGVHD occurred in a wide-spectrum of target organs, including the tongue. Fibrosis was demonstrated in the lung and liver, associated with CD4+ T-cells and B220+ B-cell infiltration and alloantibody deposition. Whereas serum IgM and IgG isotype levels did not differ in cGVHD compared with BM controls, alloantibody deposition (primarily IgG2c, IgG2b) could be found in the lung and liver. Recipients given μMT B cell deficient BM did not develop BO and had reduced cGVHD, implicating donor BM-derived B cell alloantibody production in cGVHD pathogenesis. To distinguish between alloantibody deposition and reduced B cell APC function, we utilized (m+s)IgMxJhD BALB/c donors, capable of generating membrane bound IgM antibody and secreting IgM but secreting ≥100-fold less antigen-specific IgG than similarly immunized controls. Recipients of (m+s)IgMxJhD BM and wildtype splenocytes had significantly reduced BO, indicating that the lack of IgG alloantibody secretion by donor BM-derived B-cells precluded BO. Germinal center (GC) formation is necessary for Ig class switching. cGVHD was associated with robust GC formation, in striking contrast to BM only or non-HSCT controls. Lymphotoxin-beta (LTβ), produced by T- and B- cells and LTβR signaling is required for GC formation. Thus, we sought to determine whether disruption of LTβR signaling by infusion of the known blocking reagent, mouse LTβR-Ig fusion protein, would reduce cGVHD pathology. Mice treated with LTβR-Ig showed disrupted GCs, reduced alloantibody deposition and no BO as assessed by pulmonary function tests. Conclusions. Our studies have identified a requirement for donor B cells and alloantibody deposition in cGVHD pathogenesis. Importantly, these studies have discovered LTβR-Ig as a potential clinical interventional strategy for prevention and therapy of cGVHD. Disclosures:Ranger:Biogen Idec: Employment. Browning:Biogen Idec: Employment.

Terminal Complement Inhibition Decreases Antibody-Mediated Rejection in Sensitized Renal Transplant Recipients

Sensitized renal transplant recipients with high levels of donor-specific alloantibody (DSA) commonly develop antibody-mediated rejection (AMR), which may cause acute graft loss or shorten allograft survival. We examined the efficacy of terminal complement inhibition with the humanized anti-C5 antibody, eculizumab, in the prevention AMR in renal transplant recipients with a positive crossmatch against their living donor. The incidence of biopsy-proven AMR in the first 3 months posttransplant in 26 highly sensitized recipients of living donor renal transplants who received eculizumab posttransplant was compared to a historical control group of 51 sensitized patients treated with a similar plasma exchange (PE)-based protocol without eculizumab. The incidence of AMR was 7.7% (2/26) in the eculizumab group compared to 41.2% (21/51) in the control group (p = 0.0031). Eculizumab also decreased AMR in patients who developed high levels of DSA early after transplantation that caused proximal complement activation. With eculizumab, AMR episodes were easily treated with PE reducing the need for splenectomy. On 1-year protocol biopsy, transplant glomerulopathy was found to be present in 6.7% (1/15) eculizumab-treated recipients and in 35.7% (15/42) of control patients (p = 0.044). Inhibition of terminal complement activation with eculizumab decreases the incidence of early AMR in sensitized renal transplant recipients (ClincalTrials.gov number NCT006707).

B-Cell-Dependent Memory T Cells Impede Nonmyeloablative Mixed Chimerism Induction in Presensitized Mice

Presensitization to HLA antigens limits the success of organ transplantation. The achievement of donor-specific tolerance via mixed chimerism could improve outcomes of transplantation in presensitized patients. In presensitized B-cell-deficient μMT B6 mice, we developed nonmyeloablative bone marrow transplantation (BMT) regimens that successfully tolerized presensitized T cells, achieving long-term (LT) multilineage chimerism and tolerance to donor-type skin. To apply these regimens in wild-type (WT) animals while avoiding antibody-mediated destruction of donor bone marrow cells, presensitized WT B6 mice were rested >2 years to allow alloantibody clearance. However, chimerism and tolerance were not reliably achieved in LT presensitized WT B6 mice in which alloantibody had declined to minimal or undetectable levels before BMT. Strong antidonor memory T-cell responses were detected in LT presensitized WT B6 mice after rejection of donor bone marrow (BM) occurred, whereas levels of alloantibody remained consistently low. In contrast, presensitized μMT B6 mice had diminished memory T-cell responses compared to WT B6 mice. These data implicate T-cell memory, but not alloantibody, in rejection of donor BM in LT presensitized WT mice.

Algorithm to Manage Highly Sensitized Kidney Transplant Recipients in Poland

Objective Programs for immunized transplant recipients are essential to achieve graft survivals comparable to those of non-immunized recipients. The threshold in Poland is a PRA by the complement-dependent cytotoxicity (CDC) method greater than 50%, which includes approximately 3.8% of the patients. At the same time the United Network for Organ Sharing there recipients represent approximately 16% of the waiting list in the United Network for Organ Sharing (UNOS). The underestimation of the immunized group in Poland may be due to differences in laboratory techniques to assess alloantibodies. Materials and methods This study investigated 55 potential recipients with a PRA by CDC > 50%. We used the following algorithm to assess their immunization: Luminex screening test for an HLA antibody; specificity assessed with Luminex Single Antigen, vPRA (evaluation of immunization of the patient); and analysis of acceptable HLA incompatibilities (HLAMatchmaker). Results All recipients were positive class I anti-HLA antibodies and 94.5% were positive for class II. For the groups of subjects with PRA-CDC from 50% to 79% versus those greater than 80%, the average values of PRA-CDC were 62.2% and 89.5%, respectively. The virtual PRA results for these groups were 95.7% and 97.2%, respectively. In addition, anti-HLA-Cw, anti-DQ and anti-DP antibodies were detected in 77%, 84%, and 51% of recipients, respectively. Immunized recipients reported to the next transplant were characterized by the antibodies against mismatch only in 68%. For all potential recipients, additional acceptable non-compliance was determined with HLAMatchmaker: 152 specificity for locus A and 252 for locus B. Conclusions Evaluation of immunization status of recipient candidates should be routinely performed using tests to assess class and specificity as well as level of alloantibodies to enable determination of a safe potential donor. As a routine test, PRA-CDC underestimates the number of highly immunized patients. Exclusion from the list of patients with repeated non-compliance is a simplification, which reduces their chance for transplantation.

Alloimmunization to transfused HOD red blood cells is not increased in mice with sickle cell disease

Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease. Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array. Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals. In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease.

Surveillance of alloantibodies after transplantation identifies the risk of chronic rejection

The monitoring of the levels of alloantibodies following transplantation might facilitate early diagnosis of chronic rejection (CR), the leading cause of renal allograft failure. Here, we used serial alloantibody surveillance to monitor patients with preoperative positive flow cytometric crossmatch (FCXM). Sixty-nine of 308 renal transplant patients in our center had preoperative positive FCXM. Blood was collected quarterly during the first postoperative year and tested by FCXM and single antigen bead luminometry, more sensitive techniques than complement-dependent cytotoxic crossmatching. Distinct post-transplant profiles emerged and were associated with different clinical outcomes. Two-thirds of patients showed complete elimination of FCXM and solid-phase assay reactions within 1 year, had few adverse events, and a 95% 3-year graft survival. In contrast, the remaining third failed to eliminate flow FCXM or solid-phase reactions directed against HLA class I or II antibodies. The inferior graft survival (67%) with loss in this latter group was primarily due to CR. Thus, systematic assessment of longitudinal changes in alloantibody levels, either by FCXM or solid-phase assay, can help identify patients at greater risk of developing CR.

Limitations of Rituximab/IVIg desensitization protocol in kidney transplantation; Is this better than a tincture of time?

A plasmapheresis-free protocol for desensitization of donor kidney transplant candidates with high Calculated Panel Reactive Antibody (CPRA) was initiated at our center. The protocol was adopted from previously published work by Vo, Jordan et al. Five patients with CPRA of 94 ± 18%, awaiting kidney transplant from living or deceased donors received rituximab (1 g × 2 doses) and intravenous immunoglobulin (IVIG 2 g/kg × 2 doses) without plasmapheresis. Levels of donor specific antibodies (DSA) and T/B cell crossmatches were followed using solid phase flow cytometry. Three out of 5 patients were sensitized only to Class II HLA antigens. All patients had very high levels of alloantibodies before initiation of the treatment. All of the candidates initially demonstrated reduced levels of HLA antibody, but statistical significance was only obtained in one patient Class II antibody and in another only for Class I. Depletion was transient with observed antibody rebound. Rituximab effectively depleted CD20 cells in peripheral blood. None of the patients were transplanted due to persistently high levels of antibody and strong positive flow cytometry crossmatches. Under this protocol, reduction of HLA antibodies in patients with high levels was insufficient. Highly allo-sensitized patients with a CPRA above 85% may not benefit from a combination of rituximab-IVIG alone. The previously published protocol does not help all patients achieve an acceptable crossmatch. An individualized approach to the treatment of highly sensitized patients is still required.

CD8+ T Cells Negatively Regulate IL-4–Dependent, IgG1-Dominant Posttransplant Alloantibody Production

We have previously reported that CD8(+) T cells significantly influence Ab production based on the observation that posttransplant alloantibody levels in CD8-deficient murine hepatocyte transplant recipients are markedly enhanced. However, the precise mechanisms contributing to enhanced alloantibody production in the absence of CD8(+) T cells is not understood. We hypothesized that alloactivated CD8(+) T cells inhibit Ab production by skewing toward a proinflammatory cytokine profile, whereas when these cells are absent, an anti-inflammatory cytokine profile shifts the alloimmune response toward alloantibody production. To investigate this possibility, alloantibody isotype profiles were examined in CD8-deficient and wild-type hepatocyte recipients. We found that IgG1 (IL-4-dependent isotype) was the dominant alloantibody isotype in wild-type recipients as well as in CD8-deficient recipients, although the amount of alloantibody in the latter group was substantially higher. Utilizing real-time PCR we found that CD4(+) T cells from wild-type recipients significantly upregulated IFN-γ but not IL-4 mRNA. In contrast, in the absence of CD8(+) T cells, CD4(+) T cells switched to significantly upregulate IL-4 mRNA, while IFN-γ was downregulated. IL-4 knockout mice do not produce any posttransplant alloantibody. However, adoptive transfer of wild-type CD4(+) T cells into CD8-depleted IL-4 knockout mice restores high alloantibody levels observed in CD8-depleted wild-type recipients. This suggests that IL-4-producing CD4(+) T cells are critical for posttransplant alloantibody production. Additionally, this CD8-mediated regulation of posttransplant alloantibody production is IFN-γ-dependent. Further elucidation of the mechanisms by which CD8(+) T cells influence Ab production will significantly contribute to development of therapies to manipulate humoral responses to Ag.

Prevalence and qualitative properties of circulating anti–human leukocyte antigen alloantibodies after pregnancy: No association with unexplained recurrent miscarriage

In pregnant women, circulating alloantibodies, triggered by exposure to paternal HLA antigens, are frequently detectable. The finding of lower alloantibody levels in women who experience spontaneous abortion (miscarriage) has led to the speculation that antipaternal antibodies could favor maintenance of pregnancy, whereas their lack poses a risk of miscarriage. Postulating a role of alloantibodies in the pathogenesis of unexplained abortion, we examined whether different categories of recurrent miscarriage (RM) can be distinguished according to prevalence or distinct qualitative properties of anti–human leukocyte antigen (HLA) antibody patterns. Sera obtained from 167 women with RM were assessed for complement- and non–complement-fixing anti-HLA alloreactivity using Luminex-based bead array technology. Women with RM had less often detectable anti-HLA class I and/or II reactivity (19%) compared with a control group of 96 multiparous women without a history of miscarriage (49%). However, analysis of different categories of RM (unknown [ n = 112] versus known cause [ n = 55]; primary [ n = 125] versus secondary RM [ n = 42]) did not reveal any differences regarding antibody prevalence, number of targeted HLA single antigens, antigen specificity, binding density, or complement-fixing ability of detected alloantibodies. Our results do not support a link between anti-HLA antibody formation and RM, and argue against a diagnostic value of alloantibody detection in the diagnostic work-up of women with RM.

Arsenic trioxide combined with co-stimulatory molecule blockade prolongs survival of cardiac allografts in alloantigen-primed mice

Various memory cell populations existing before organ transplantation are believed to be important barriers to extending the graft survival time. Here, we report that arsenic trioxide (As 2O 3), a common component of some Chinese traditional preparations, could obviously reduce the proliferation of splenic T cells in alloantigen-primed mice in vitro. Furthermore, we evaluated As 2O 3 treatment alone or in combination with blocking monoclonal antibodies (mAb) against co-stimulatory molecules (LFA-1 and CD154) in the prevention of heart transplant rejection in alloantigen-primed mice. Alloantigen-primed mice were randomly divided into 4 groups of 6 animals each. The control group was given normal saline; the As 2O 3 group received As 2O 3 (5 mg/kg/d) (i.p.) on days 0–10 post-transplantation; the Ab group received 100 μg anti-LFA-1 mAb + 250 μg anti-CD154 mAb (i.p.) on the day of transplantation and 3 more times every alternate day and the As 2O 3 + Ab group received a combined As 2O 3 and Ab treatment protocol. The survival of the allografts was almost doubled among the As 2O 3 group and Ab group compared with the control group (5, 5.6 vs 3.3 days). A marked prolongation (16 days) of graft survival was achieved by the combination protocol compared with the control group (3.3 days; P < 0.05). None of the treatment group showed side effects. Five days after transplantation, cardiac allografts, splenic T cells and serum were harvested for analysis. Compared with the control group, most of the treatment groups showed: (i) alleviation of allograft rejection in different levels; (ii) IFN-γ expression was reduced and TGF-β expression increased in both peripheral blood and within the grafts; (iii) the proportions of CD4 + or CD8 + memory T cells in the spleen of the recipients were significantly reduced while the expression of regulatory T cells (Tregs) was enhanced; (iv) the alloresponses of memory T cells were inhibited, and levels of alloantibodies in recipients' sera were also decreased. Among all these changes, the As 2O 3 + Ab group showed the most significant changes. Our study highlights an obvious synergistic action of As 2O 3 when combined with co-stimulatory molecules blockade in prolonging the survival of cardiac allografts in alloantigen-primed mice.

Biomarkers in renal transplantation

An important effort is being made among the transplant community investigating novel biological markers at different biological levels that enable transplant clinicians to identify patients at risk for allograft rejection or, conversely, patients in whom immunosuppression could be safely minimized. Here, we review recent important biomarkers that have shown relevant impact on renal graft outcome. Important collaborative studies have allowed the identification of biomarkers or biological signatures indicative of organ tolerance or rejection in different transplant settings (mainly kidney and liver transplantation). This has required novel and highly specific technological assays focused at different biological levels, employing peripheral blood, urine and grafts tissue. Noteworthy, standardization and methodical validation of reliable tests or biomarkers is being a crucial task since the start of this challenge. These endeavours include detailed characterizations of serum alloantibody levels, measurements of individual soluble or cell-surface markers in peripheral blood, urine or both; profiling gene expression, proteome expression patterns or both and evaluations of peripheral cellular alloimmunity, among others. Despite the important advances achieved so far in the identification of several potentially useful biomarkers of tolerance, rejection or both, validation and demonstration of their clinical utility still needs to be tested. Ultimately, in order to facilitate the achievement of such important goal, all efforts should probably be performed in the context of international cooperative networks.

Suppressing memory T cell activation induces islet allograft tolerance in alloantigen-primed mice

Memory T cells are known to play a key role in prevention of allograft tolerance in alloantigen-primed mice. Here, we used an adoptively transferred memory T cell model and an alloantigen-primed model to evaluate the abilities of different combinations of monoclonal antibodies (mAb) to block key signaling pathways involved in activation of effector and memory T cells. In the adoptively transferred model, the use of anti-CD134L mAb effectively prevented activation of CD4(+) memory T cells and significantly prolonged islet survival, similar to the action of anti-CD122 mAb to CD8(+) memory T cells. In the alloantigen-primed model, use of anti-CD134L and anti-CD122 mAbs in addition to co-stimulatory blockade with anti-CD154 and anti-LFA-1 prolonged secondary allograft survival and significantly reduced the proportion of memory T cells; meanwhile, this combination therapy increased the proportion of regulatory T cells (Tregs) in the spleen, inhibited lymphocyte infiltration in the graft, and suppressed alloresponse of recipient splenic T cells. However, we also detected high levels of alloantibodies in the serum which caused high levels of damage to the allogeneic spleen cells. Our results suggest that combination of four mAbs can significantly suppress the function of memory T cells and prolong allograft survival in alloantigen primed animals.

Isatis tinctoria L. combined with co-stimulatory molecules blockade prolongs survival of cardiac allografts in alloantigen-primed mice

Memory T cells present a unique challenge in transplantation. Although memory T cells express robust immune responses to invading pathogens, they may be resistant to the effects of immunosuppressive therapies used to prolong graft survival. In previous studies, we found that compound K, the synthesized analogue of highly unsaturated fatty acids from Isatis tinctoria L., reduced acute cardiac allograft rejection in mice (Wang et al., 2009 [1]). Here, we further investigated the effect of compound K on cardiac allograft rejection in alloantigen-primed mice. We found that compound K significantly inhibited CD4 + and CD8 + memory T cells proliferation in a mixed lymphocyte reaction (MLR). In vivo, compound K combined with anti-CD154 and anti-LFA-1 monoclonal antibodies (mAbs) significantly extended the survival time of heart grafts in alloantigen-primed mice with no obvious toxic side effects. Furthermore, our data suggests that compound K works by reducing the expression of both IL-2 and IFN-γ within the graft rather than enhancing expression of regulatory T cells (Tregs). Compound K can also inhibit the alloresponses of memory T cells, while increasing the proportion of CD4 + memory T cells in the spleen of the recipients and significantly reducing the level of alloantibodies in the serum. Our study highlights the unique immune effects of compound K that may be further explored for clinical use in extending the survival of transplant grafts.

Proteomic Portrayal of Transplant Pathologies

Despite recent improvements in 1-yr outcomes for kidney transplantation, long-term allograft survival has not changed significantly in the past several decades.1 The survival half-life for kidneys from deceased donors is approximately 11 yr, and the pathogenesis of these late graft losses is multifactorial.2 Analyses of graft histology reflected in the revised Banff criteria indicate chronic allograft injury can be subcategorized, in part, on the basis of evidence of local inflammation and the presence or absence of interstitial fibrosis and tubular atrophy (IF/TA).3 Although specific inciting factors are difficult to define in each situation, distinct histopathologic entities often correlate with likely causes. For example, calcineurin inhibitor toxicity frequently manifests as IF/TA without inflammation; ongoing cellular alloimmunity presents histologically with tubulitis with or without IF/TA; C4d staining suggests transplant glomerulopathy with or without IF/TA; and detectable, donor-specific serum antibodies underlie antibody-mediated allograft injury.3 Because only a subset of patients develop chronic injury and because at present physicians do not have the ability to reverse chronic fibrotic kidney damage, it is essential that the transplant community develop reliable and noninvasive approaches to predict which patients are most likely to develop graft failure so that appropriate interventions can be instituted …

Value of Crossmatch Tests for Verifying the Compatibility in Dog Blood Transfusion Therapy

In case of pets, dogs and cats transfusions are already a current practice, beeing an intensive therapy procedure. In a group of dogs that underwent intensive therapy hole blood transfusion was used based on major and minor Crossmatch test results in order to establishing blood compatibility. The slide and tube techniques used were original and taken from different authors. Using 15 potential donors, 5 patients were transfused, one of them diagnosed with osteosarcoma. In 4 of the patients, all donor tested were compatible and they recovered after blood transfusion. Incompatibility problems appeared in the patient with osteosarcoma, for which 11 potential donors were tested and among them only 2 were compatible, the others showed positive agglutination reaction in major crossmatch In patients with positive reactions of incompatibility, the results were relevant in both variants (slide, tube), meaning the high level of plasmatic alloantibodies. After the evaluation of blood compatibility using major and minor crossmatch tests it was decided to opt for blood transfusion which made possible the recovery of 4 out of 5 dogs undergoing intensive therapy. The development was fatal for the patient with osteosarcoma because of the severe complications caused by the cancer. The highly positive major crossmatch results obtained in the patient with osteosarcoma and 9 potential donors, who never received a transfusion, suggest the possibility of producing alloantibodies to some foreign cancer induced proteins, cross-reactant with common erythrocyte antigens.

Alloantibody Levels and Acute Humoral Rejection Early After Positive Crossmatch Kidney Transplantation

We examined the course of donor-specific alloantibody (DSA) levels early after transplant and their relationship with acute humoral rejection (AHR) in two groups of positive crossmatch (+XM) kidney transplant recipients: High DSA group-41 recipients with a baseline T- or B-cell flow crossmatch (TFXM, BFXM) channel shift >or=300 (molecules of equivalent soluble fluorochrome units (MESF) of approximately 19 300) who underwent pretransplant plasmapheresis (PP), and Low DSA group-29 recipients with a baseline channel shift <300 who did not undergo PP. The incidence of AHR was 39% (16/41) in the High DSA group and 31% (9/29) in the Low DSA group. Overall, mean DSA levels decreased by day 4 posttransplant and remained low in patients who did not develop AHR. By day 10, DSA levels increased in patients developing AHR with 92% (23/25) of patients with a BFXM >359 (MESF of approximately 34 000) developing AHR. The BFXM and the total DSA measured by single antigen beads correlated well across a wide spectrum suggesting that either could be used for monitoring. We conclude that AHR is associated with the development of High DSA levels posttransplant and protocols aimed at maintaining DSA at lower levels may decrease the incidence of AHR.

C4d deposition and cellular infiltrates as markers of acute rejection in rat models of orthotopic lung transplantation.

C4d is a useful marker of antibody-mediated rejection in cardiac and renal transplants, but clinical studies examining correlations between circulating alloantibodies, C4d deposition, and rejection in lung transplants have yielded conflicting results. We studied circulating alloantibody levels and C4d deposition in two rat models of lung transplantation: Brown Norway (BN) to Wistar-Kyoto (WKY) and PVG.R8 to PVG.1U lung allografts. The availability of C6 deficient (C6-) and C6 sufficient (C6+) PVG 1U rats allowed evaluation of the effects of the terminal complement components on graft injury and C4d deposition. The lung allografts had histologic features resembling human posttransplant capillaritis, characterized by neutrophilic infiltration of alveoli, edema, and hemorrhage. Immunoperoxidase stains on cross sections of allografts showed intense, diffuse, C4d deposition in a continuous linear pattern on the vascular endothelium. C4d deposits were found in both BN to WKY and PVG R8 to 1U allografts, whereas no staining was detectable in WKY to WKY isografts or native lungs. Complement deposition was associated with vascular disruption in C6+, but not in C6- recipients. The presence of circulating donor-specific alloantibodies was verified by flow cytometry. Cell-specific staining revealed perivascular accumulation of macrophages and T lymphocytes whereas neutrophils were sequestered in the intravascular and alveolar capillary compartments. The deposition of C4d on vascular endothelium as well as the coincident presence of alloantibodies is consistent with previous findings in antibody-mediated rejection of renal and cardiac transplants. Furthermore, the histological features of our allografts support the concept that posttransplant capillaritis is a form of humoral rejection.

Critical Role of Effector Macrophages in Mediating CD4-Dependent Alloimmune Injury of Transplanted Liver Parenchymal Cells

Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4(+) T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4(+) T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.

Recipient Immune Repertoire and Engraftment Site Influence the Immune Pathway Effecting Acute Hepatocellular Allograft Rejection

As novel acute allograft rejection mechanisms are being discovered, determining the conditions that promote or subvert these distinct rejection pathways is important to interpret the clinical relevance of these pathways for specific recipient groups as well as specific tissue and organ transplants. We have employed a versatile hepatocellular allograft model to analyze how the host immune repertoire and immune locale influences the phenotype of the rejection pathway. In addition, we investigated how peripheral monitoring of cellular and humoral immune parameters correlates with the activity of a specific rejection pathway. Complete MHC mismatched hepatocellular allografts were transplanted into immune competent CD4-deficient, CD8-deficient, or C57BL/6 hosts to focus on CD8-dependent, CD4-dependent, or combined CD4 and CD8-dependent alloimmunity, respectively. Hepatocellular allografts were transplanted to the liver or kidney subcapsular space to investigate the influence of the immune locale on each rejection pathway. The generation of donor-reactive DTH, alloantibody, and allospecific cytotoxicity was measured to assess both cellular and humoral immunity. Graft-infiltrating lymphocytes were phenotyped and enumerated in each recipient group. In the presence of CD8+ T cells, cytolytic cellular activity is the dominant mechanism of graft destruction and is amplified in the presence of CD4+ T cells. The absence of CD8+ T cells (CD8 KO) results in potent humoral immunity as reflected by high levels of cytotoxic alloantibody and graft rejection with similar kinetics. Transplant to the liver compared to the kidney site is distinguished by more rapid kinetics of rejection and alloimmunity, which is predominately cell mediated rather than a mix of both humoral and cell-mediated immunity. These studies define several rejection mechanisms occurring in distinct immune conditions, highlighting the plasticity of acute allograft rejection responses and the need to design specific monitoring strategies for these pathways to allow dynamic immune assessment of clinical transplant recipients and targeted immunotherapies.

Cancer in patients on dialysis and after renal transplantation

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