Ibrutinib suppresses alloantibody responses in a mouse model of allosensitization.

BACKGROUND: Ibrutinib is a Bruton's tyrosine Kinase (BTK) antagonist that inhibits B cell receptor (BCR) signaling. Complete BTK deficiency is associated with absence of B-cells. Ibrutinb is currently approved by FDA for treatment of B-cell malignancies, including Waldenström macroglobulinaemia. We recently carried out studies to determine if ibrutinib could modify alloantibody responses. MATERIALS AND METHODS: A mouse model of allogenic sensitization using a C57BL/6 mouse as the recipient of a skin allograft from an HLA-A2 transgenic mouse was utilized to examine the effects of ibrutinib on alloantibody responses and B cell effector functions. Donor-specific antibody (DSA) levels were measured in a flow-cytometric antibody binding assay. Splenic T and B cell subsets and plasma cells were analyzed in flow cytometry. RESULTS: Control mice developed peak levels of DSA IgM at day 14 PTx while the ibrutinib treated mice had significantly lower levels of DSA IgM (p=0.0047). Control mice developed HLA.A2-specific IgG antibodies at day 14 (230±60 MFI) and reached peak levels at day 21 (426±61 MFI). In contrast, mice in the treatment group had low levels of HLA.A2-specific IgG at day 14 (109±59 MFI, p=0.004) and day 21 (241±86 MFI, p=0.003). FACS analysis found a reduction of B220+ or CD19+ B cell population (p<0.05). In addition, ibrutinib attenuated recall DSA IgG responses to re-sensitization (p<0.05) and reduced CD38+CD138+ plasma cells (p<0.05) in the spleens. CONCLUSIONS: Ibrutinib is effective in suppressing alloantibody responses through blocking BTK-mediated BCR signaling, leading to reduction of B cells and short-lived plasma cells in the spleens. Use of ibrutinib may provide benefits to HLA-sensitized transplant patients for alloantibody suppression.

Immunological characterization of de novo and recall alloantibody suppression by CTLA4Ig in a mouse model of allosensitization.

It is well known that CTLA4Ig inhibits allogenic T-cell activation in transplantation. The immunological features and mechanisms associated with alloantibody suppression by CTLA4Ig, however, are poorly understood. Here, we used a mouse model of allosensitization to evaluate the efficacy of CTLA4Ig (abatacept) in suppression of donor-specific antibody (DSA) during de novo and recall alloantibody responses. We found that abatacept inhibited de novo DSA IgM and IgG responses to HLA-A2 expressing skin grafts. Abatacept administered during primary T cell priming also reduced recall IgG responses induced by re-immunization. Suppression of de novo DSA responses by abatacept is associated with a reduction in splenic expression of the germinal center activation marker GL7 and a reduction of CD4(+)PD1(+)CXCR5(+) follicular T helper (Tfh) subset in splenic lymphocytes detected by flow cytometry. The efficacy of abatacept on recall DSA suppression is moderate. In vitro experiments demonstrated that abatacept inhibited DSA IgG secretion by CD138(+) plasma cells isolated from allograft recipients. Additional experiments using an IgG1 secreting mouse hybridoma cell line showed that abatacept binds to CD80 expressed on these cells with subsequent inhibition of cell proliferation and reduction in IgG ELISpot formation. In conclusion, CTLA4Ig is a potent suppressor of de novo DSA responses and also affects recall responses. The data suggests modification of recall DSA responses is due to a direct suppressive effect on plasma cells.

Anti-interleukin 6 receptor antibodies attenuate antibody recall responses in a mouse model of allosensitization.

BACKGROUND: Interleukin (IL)-6 is a regulatory cytokine for T helper type 17 (Th17) and Treg cells and a potent stimulus for B/plasma cells. The current study evaluated the effect of IL-6 receptor (IL-6R) blockade with an antiYIL-6R monoclonal (mMR16-1) in alloantibody recall responses. METHODS: A mouse model of human leukocyte antigen (HLA).A2 sensitization was used for studies to evaluate the efficacy of antiYIL-6R on alloantibody recall responses and to examine the impact of IL-6R blockade on Th17, Treg, follicular T helper (Tfh) and plasma cells using multiparameter flow cytometry, flow antibody binding, and enzyme-linked immunospot (ELISpot) assay. RESULTS: Re-exposure of C57BL/6 mice to HLA.A2(+) skin allografts resulted in a surge of donor-specific (antiYHLA.A2) immunoglobulin (Ig)G antibodies. AntiYIL-6R treatment significantly decreased but did not eliminate alloantibody responses (IgG mean fluorescence intensity, 486 T 153 vs. control 792 T 193, P = 0.0076). Flow cytometry analysis showed that antiYIL-6R treatment resulted in reduction of IL-21+CD4+ (Th17) cells (P = 0.006 vs. control) and CXCR5(+)CD4(+) Tfh cells (P = 0.04), but increased foxp3(+)CD4(+) (Treg) cells in the CD4(+) population (P =0.04 vs. control). The IgG ELISpot experiments showed a significant reduction of IgG spots in the bone marrow and the spleen cells from the antiYIL-6RYtreated mice. In vitro treatment of mouse hybridoma (PA2.1) cultures with antiYIL-6R decreased IgG spot formation but had limited effect on cell proliferation. CONCLUSION: The data indicate that antiYIL-6R therapy attenuates alloantibody recall responses by modulating a number of immune regulatory and effector cells, including Th17, Tfh, Treg, and importantly, the long-lived plasma cells in the bone marrow.

Anti-CD3ε induces splenic B220lo B-cell expansion following anti-CD20 treatment in a mouse model of allosensitization.

Antibodies targeting T cells and B cells are increasingly used for immunosuppression in clinical transplantation. However, the impact of T-cell depletion by antibodies on B-cell homeostasis is poorly understood. Using a mouse model of allosensitization with skin allograft, we investigated whether targeting T cells by anti-CD3ε alters peripheral B-cell homeostasis and alloantibody responses following B-cell depletion by anti-CD20. We found that anti-CD3ε induced a discrete B220(lo), but not a conventional B220(hi) subset, in the spleens of the allosensitized mice 14 days after anti-CD20 treatment. The splenic B220(lo) cells were refractory to anti-CD20 depletion. Flow cytometry revealed that the splenic B220(lo) cells were phenotypically similar to the B220(lo) AA4.1(+) CD23(-) sIgM(lo) sIgD(-) developing B cells (pre-B to immature B) normally presented in the bone marrow. Despite the presence of the splenic B220(lo) cells, mice treated with combined anti-CD3ε/CD20 produced limited alloantibodies in response to the primary skin allografts. Alloantibody production increased significantly in the mice following re-immunization by donor-specific splenocytes. We conclude that anti-CD3ε can induce an expansion of B220(lo) B cells in the spleens after B-cell depletion by anti-CD20. These B cells are not producing alloantibodies, but re-immunization of the mice with alloantigen leads to risk of alloantibody response.

Immunologic parameters and viral infections in patients desensitized with intravenous immunoglobulin and rituximab.

Desensitization with IVIG and rituximab followed by transplantation with alemtuzumab or daclizumab induction is an effective clinical protocol. Here, we examined the effects of this protocol on immune cell number, T cell function by Cylex ImmuKnow®, CMV-specific CD8+ T cell (CMV-Tc) activity, total and viral-specific immunoglobulin levels and viral infections. In 17 highly HLA-sensitized (HS) patients who received desensitization, CD19+ cells were undetectable immediately after desensitization, while other immune cells were unchanged. No alteration in Cylex or CMV-Tc levels was seen. In separate 14 HS patients who were desensitized followed by transplantation, T cell numbers were near zero after alemtuzumab, while NK cell reduction was minimal. Early B cell recovery was not a risk for antibody-mediated rejection. Total IgG, IgM, and IgA remained in the normal range up to 12.6 months post-transplant, and CMV IgG level did not change. CMV-Tc activity was eliminated post-transplant in some patients, but recovered by 4 months post-transplant. None of them developed CMV infection. In conclusion, IVIG-rituximab-desensitization does not significantly alter T cell function pre-transplant, or reduce Ig levels below the normal range post-transplant. Although post-transplant induction therapy is associated with a transient depletion of viral-specific CD8+ memory cells, it does not increase risks for viral infections.

Intracellular IFNgamma production in CD3 negative cells exposed to allo-antigens is an indicator of prior sensitization.

BACKGROUND: Sensitization to HLA antigens (Ags) is a significant obstacle to kidney transplantation and risk factor for antibody-mediated rejection (AMR). Current screening methods to assess HLA Ag exposure include various antibody assays. However, tools to accurately measure cell-mediated immunity to allo-Ags in a clinical setting are lacking. Here we report on an intracellular cytokine flow cytometry (CFC) assay that detects intracellular gamma-interferon (IFNgamma) production in non-T cell populations (CD3-) that appears to assess sensitization from previous allo-Ag exposure. METHODS: Blood from 106 highly-HLA sensitized (HS) patients (pre-, post-IVIG-treatment [Rx] and/or post-transplant) and 14 3(rd) party normal controls (3(rd)N) were incubated with donor or 3(rd)N peripheral blood mononuclear cells (PBMCs), and IFNgamma+/CD3- cells were enumerated. RESULTS: The percentage of IFNgamma+ cells in CD3- cells without stimulation in pre-IVIG-Rx HS patients was similar to normals, but significantly increased with incubation with donor and/or 3(rd)N PBMCs. Reactivity in normals was minimal. Reactivity was higher in HS females than HS males. Normal females with previous pregnancy (PG) showed significantly higher response than females without PG or non-sensitized normal males. Donor-specific reactivity in the CFC assay better correlated with donor-specific B cell crossmatch than total anti-HLA antibody levels or PRA. HS patients who developed AMR post-transplant showed significantly higher reactivity than those without AMR. CONCLUSIONS: The CFC assay measures IFNgamma production in CD3- cells that may indicate a memory response to allo-Ags. This response is limited to HS patients and normal females with previous PG. Patients undergoing AMR show significantly higher reactivity. This assay may represent a novel approach to measurement of allo-sensitization with clinical utility in predicting those at risk for AMR.

Flow cytometric isolation and phenotypic characterization of two subsets of ED2(+) (CD163) hepatic macrophages in rats.

AIMS: Macrophages in the liver are well known for their functional heterogeneity. However, subpopulations of the hepatic macrophages are not well defined. METHODS: Two subsets of hepatic macrophages isolated from rats via FACS with immunolabeling of ED2 (anti-CD163) antibody were studied for phenotypic and functional characteristics. RESULTS: A subset showed an ED2(high) and autofluorescence(high) (ED2(high)/AF(high)) phenotype, exhibiting characteristics consistent with the description of the Kupffer cells (KC). A second subset, displaying an ED2(dim)/AF(dim) phenotype, was smaller in size, monocyte-like and weak in phagocytosis. Transmission electron microscopy demonstrated that both subsets are phagocytes. Quantitative RT-PCR revealed that in addition to expression of macrophage-related surface markers such as CD14, ED1 (CD68), fucose receptor, and CD163, the ED2(dim)/ AF(dim) cells expressed mRNA encoding for myeloid lineage differentiation markers ERMP12 (PECAM) and ERMP20 (Ly-6C). These two subsets exhibited differential in gene expression of selected cytokines, extracellular matrix proteinases, and Toll-like receptor in normal livers, as well as significantly upregulated expression in cholestatic livers induced by bile duct ligation. CONCLUSION: The data suggest that the ED2(high)/AF(high) population of the liver cells represent the conventional Kupffer cells. The ED2(dim)/AF(dim) cells, however, are small hepatic resident macrophages characteristically different from the conventional Kupffer cells.

Anti-CD20 antibody suppresses anti-HLA antibody formation in a HLA-A2 transgenic mouse model of sensitization.

BACKGROUND: B cell depletion by anti-CD20 antibody is used in desensitization protocols and for treatment of antibody-mediated rejection (AMR). However, little is known about the efficacy and the mechanism(s) of action. METHODS: A mouse model of HLA sensitization was used to study the effectiveness of anti-CD20 treatment on B cell depletion and anti-HLA antibody suppression. RESULTS: Immunization of C57BL/6 mice with skin grafts from a transgenic C57BL.Tg/HLA-A2.1 mouse resulted in robust production of anti-HLA IgM and IgG antibodies, and accelerated rejection of a secondary skin allograft (within 3 days) featured by intragraft IgG and C4d deposition. Both IgM and IgG alloantibodies are specific to HLA-A2 as well as to a panel of class I HLA, including A1, A3, A25, A26, A29, and A30. These alloantibodies were complement-dependently cytotoxic (CDC) against HLA-A2 expressing target cells. Administration of 2 doses of a mouse-anti-mouse CD20 monoclonal antibody significantly reduced the levels of anti-HLA IgG2a antibodies, suppressed serum CDC, and prolonged survival of the secondary skin allografts. Suppression of anti-HLA IgG antibodies was associated with significant depletion of B220(+)/CD5(-) B cells from the blood, the spleen and the bone marrow of the treated animals. CONCLUSION: Anti-CD20 treatment effectively depletes B220(+)/CD5(-) B cells, resulting in potent suppression of anti-HLA IgG and prolongation of skin graft survival. The data are in support for the use of anti-CD20 antibodies in highly-HLA sensitized patients undergoing desensitization and for the treatment of AMR.

Differential beta-adrenoceptor expression induced by nerve growth factor infusion into the canine right and left stellate ganglia.

BACKGROUND: Nerve growth factor (NGF) infusion into the right stellate ganglion (RSG) is antiarrhythmic, while NGF infusion into the left stellate ganglion (LSG) is proarrhythmic in dogs with myocardial infarction (MI) and complete atrioventricular block (CAVB). This functional asymmetry suggests differential neural remodeling. OBJECTIVES: To test the hypothesis that NGF infusion into the RSG and the LSG can lead to differential beta-adrenoceptor (beta-AR) expression in dogs with MI and CAVB. METHODS AND RESULTS: We performed immunostaining to quantify beta(1)-AR and beta(3)-AR immunoreactivity in six dogs with MI and CAVB, nine dogs with MI, CAVB, and NGF infusion to the LSG, six dogs with MI, CAVB, and NGF infusion to the RSG, and six normal dogs. There was significantly increased beta(3)-AR immunoreactivity in dogs with NGF infusion into the LSG and significantly decreased beta(3)-AR immunoreactivity in dogs with NGF infusion into the RSG compared with controls and with the MI and CAVB group. There were no significant differences in beta(1)-AR immunoreactivity among these four groups. To determine protein and mRNA expression of beta-ARs, we created MI and CAVB and infused NGF into the LSG in six additional dogs. The noninfarcted left ventricle free wall was harvested 1 week later. The protein level and receptor density of beta(3)-AR (but not beta(1)- or beta(2)-AR) significantly increased in these six dogs compared with normal controls. CONCLUSIONS: We conclude that NGF infusion into the RSG and the LSG in dogs with MI and CAVB induced differential beta(3)-AR expression in the left ventricular myocardium.

Proteinase and growth factor alterations revealed by gene microarray analysis of human diabetic corneas.

PURPOSE: To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I. METHODS: RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agilent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10. RESULTS: More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin alpha4 chain, and thymosin beta(4) genes were downregulated. The data were corroborated by QPCR and immunohistochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin alpha(3)beta(1), whereas in normal corneas MMP-10 decreased laminin-10 and integrin alpha(3)beta(1) expression. CONCLUSIONS: Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.

Increased expression of membrane type 3-matrix metalloproteinase in human atherosclerotic plaque: role of activated macrophages and inflammatory cytokines.

BACKGROUND: Matrix metalloproteinases (MMPs) are thought to play a prominent role in atherogenesis and destabilization of plaque. Pericellularly localized membrane-type (MT)-MMPs activate secreted MMPs. We investigated the hypothesis that MT3-MMP is expressed in human atherosclerotic plaques and is regulated by locally produced inflammatory cytokines and oxidized low-density lipoprotein (Ox-LDL). METHODS AND RESULTS: Expression and cellular localization of MT3-MMP in normal and atherosclerotic human coronary arteries were examined using specific antibodies. Abundant MT3-MMP expression was noted in medial smooth muscle cells (SMCs) of normal arteries. In atherosclerotic arteries, MT3-MMP expression was observed within complex plaques and colocalized with SMCs and macrophages (Mphi). Cultured human monocyte-derived Mphi constitutively expressed MT3-MMP mRNA and proteolytically active protein, as demonstrated by mRNA analyses, immunoblotting, and gelatin zymography, respectively. Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor caused dose- and time-dependent increases in steady-state levels of MT3-MMP mRNA in cultured Mphi. This correlated with a 2- to 4-fold increase in levels of MT3-MMP immunoreactive protein and enzymatic activity in Mphi membranes. Confocal microscopy and flow cytometry confirmed induction and spatial distribution of MT3-MMP protein from intracellular domains to the Mphi plasma membrane by Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor. CONCLUSIONS: MT3-MMP is expressed by SMCs and Mphi in human atherosclerotic plaques. Proinflammatory molecules cause a progressive increase in the expression of MT3-MMP in cultured Mphi. Our results suggest a mechanism by which inflammatory molecules could promote Mphi-mediated degradation of extracellular matrix and thereby contribute to plaque destabilization.