Search

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of β2-microglobulin (β2m)-specific shRNAs reduced β2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

There is compelling evidence that self-reactive CD8(+) T cells are a major factor in development and progression of type 1 diabetes in animals and humans. Hence, great effort has been expended to define the specificity of autoimmune CD8(+) T cells and to alter their responses. Much work has focused on tolerization of T cells using proteins or peptides. A weakness in this approach is that residual autoreactive T cells may be activated and exacerbate disease. In this report, we use a novel approach, toxin-coupled MHC class I tetramers. Used for some time to identify Ag-specific cells, in this study, we use that same property to delete the Ag-specific cells. We show that saporin-coupled tetramers can delete islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive T cells in vitro and in vivo. Sequence analysis of TCRbeta-chains of IGRP(+) cells reveals the repertoire complexity in the islets is markedly decreased as NOD mice age and significantly altered in toxic tetramer-treated NOD mice. Further tetramer(+) T cells in the islets are almost completely deleted, and, surprisingly, loss of tetramer(+) T cells in the islets is long lasting. Finally, we show deletion at 8 wk of age of IGRP(+) CD8(+) T cells, but not dystophia myotonica kinase- or insulin B-reactive cells, significantly delays diabetes in NOD mice.

The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. Certain MHC class II-bound self peptides from autoimmune MRL/lpr mice are not found in eluates from class II molecules of MHC-identical C3H mice. Eleven of 16 such peptides elicited lymph node cell and spleen cell T cell proliferation in both MRL/lpr (stimulation index = 2.03-5.01) and C3H mice (stimulation index = 2.03-3.75). IL-2 and IFN-gamma production were detected, but not IL-4. In contrast to what was seen after immunization, four self peptides induced spleen cell proliferation of T cells from naive MRL/lpr, but not from C3H and C57BL/6.H2(k), mice. These peptides were derived from RNA splicing factor SRp20, histone H2A, beta(2)-microglobulin, and MHC class II I-A(k)beta. The first three peptides were isolated from I-E(k) molecules and the last peptide was bound to I-A(k). T cell responses, evident as early as 1 mo of age, depended on MHC class II binding motifs and were inhibited by anti-MHC class II Abs. Thus, although immunization can evoke peripheral self-reactive T cells in normal mice, the presence in MRL/lpr mice of spontaneous T cells reactive to certain MHC-bound self peptides suggests that these T cells actively participate in systemic autoimmunity. Peptides eluted from self MHC class II molecules may yield important clues to T cell epitopes in systemic autoimmunity.

The escape of tumor cells from immune recognition is a central problem in tumor immunology. Here, we examined the functional role of somatic beta 2-microglobulin (beta2m) gene mutations in human lung and breast cancers. Using single-strand conformational polymorphism (SSCP) analysis and DNA sequencing, we found mutations in the beta2m gene in 2 of 110 tested lung, colon and breast tumors and tumor cell lines. No mutations were identified in 63 breast cancer tumors, in B-lymphoblastoid cell lines or normal tissues from these or other patients. In these cell lines, beta2m protein was undetectable by Western blot analysis and there was no MHC class I on their cell surface even after treatment with interferon-gamma. Transfection of these tumor cell lines with the beta2m gene, but not addition of purified beta2m protein restored MHC expression without addition of exogenous pepticles, indicating that endogenous beta2m expression is necessary for proper intracellular MHC assembly and stabilization by endogeneous pepticles. Mutation in beta2m caused cell line H2009 to be resistant to specific lysis by influenza virus-specific CTL from HLA matched donors, and transfection of the beta2m gene restored this killing. A small cell lung cancer cell line with low class I expression and with a normal beta2m genomic sequence nonetheless also demonstrated increased class I expression after transfection of the beta2m expression vector alone, indicating that the availability of beta2m may be rate limiting for MHC assembly in this line. Our results indicate that somatic mutations or selective loss of expression of the beta2m gene in human lung cancer is rare, but can cause defective MHC class I expression and function allowing these cells to escape recognition by cytotoxic T cells.

Increased levels of beta 2-microglobulin (part of the class I major histocompatibility complex molecules) in body fluids are associated with activation of the immune system and pathophysiological processes. Various anorexigenic cytokines, including interferon-gamma and tumor necrosis factor-alpha, induce the expression of class I molecules. Therefore, it is possible that beta 2-microglobulin may participate in the feeding suppression induced by cytokines or may have direct effects on feeding. In the present study, the effects of beta 2-microglobulin on the central regulation of feeding were investigated. Intracerebroventricular (ICV) microinfusion of beta 2-microglobulin (0.01-5.0 micrograms/rat) suppressed the nighttime food intake dose dependently. The most effective dose of beta 2-microglobulin, 5.0 micrograms/rat, decreased nighttime feeding by 38% and total daily food intake by 28%. Computerized analysis of behavioral patterns demonstrated that beta 2-microglobulin decreased meal size and meal frequency during the initial 4-h interval, but decreased only meal size during the second 4-h interval after the ICV microinfusion of 5.0 micrograms beta 2-microglobulin/rat; meal duration was not significantly affected in any interval. For the complete nighttime period, only meal size was significantly decreased. Cerebrospinal fluid-brain and rectal temperatures did not change significantly. ICV microinfusion of heat-treated beta 2-microglobulin or intraperitoneal administration of beta 2-microglobulin, in doses equivalent to those administered centrally, had no effect on food intake. The results suggest that beta 2-microglobulin may act centrally to decrease feeding, and this effect may participate in the anorexia frequently accompanying pathological processes.

Macaques immunized with uninfected human cells have been shown to be protected from challenge with simian immunodeficiency virus (SIV) propagated in human cells. To identify the potential antigens involved in this protection, macaques were immunized with uninfected human cells, sucrose density gradient-purified culture fluid from uninfected human cells (mock virus), beta-2 microglobulin (beta 2M), immunoaffinity-purified HLA class I and class II proteins from these human cells, and adjuvant. Although all macaques immunized with beta 2M and HLA class I developed high antibody titers to beta 2M, these animals were not protected from a subsequent challenge with infectious SIV grown in human cells. In contrast, the macaques immunized with class II protein (HLA-DR) and mock virus developed antibodies to class II protein and were protected from the intravenous infectious virus challenge. The class II protein- and mock virus-immunized animals which were protected from challenge were given boosters of the appropriate antigen and challenged with the same SIV propagated in macaque cells. All animals became infected, indicating that the protection seen with human class II protein did not extend to protection from infection with SIV containing macaque class II proteins. Since the virus released from SIV-infected macaque cells would contain macaque class II proteins, our results suggest that the initial SIV infected was completely prevented. In addition, the lack of protection from challenge with SIV propagated in macaque cells provided strong evidence that the protection was due to an immune response to the cellular proteins and not to epitopes cross-reactive between class II proteins and the viral proteins, since the identical virus proteins were present in both challenge stocks. These results are the first demonstration that immunization with a purified cellular protein can protect from virus infection.

beta 2-Microglobulin (beta 2-M) deposits have been found in the destructive bone lesions associated with dialysis-related amyloidosis. To examine whether beta 2-M can cause bone resorption in vivo, doses of beta 2-M alone were compared with parathyroid hormone (PTH), aluminum, and vehicle alone. Eleven injections of 10 micrograms each were made over a period of 56 hours into the subcutaneous tissue overlying the occipital region of mice. Using a computerized image analysis system we measured (1) periosteal and inner bone length, (2) bone marrow interface length, and (3) the extent of resorption along these surfaces expressed as percentage of total length. Injections of either beta 2-M or PTH were associated with 22% +/- 4% and 25% +/- 4% resorption of periosteal surface, respectively, and 15.9% +/- 2% and 19.9% +/- 5% resorption of marrow bone surfaces, respectively, compared with control. In contrast, aluminum did not increase bone resorption over controls. The simultaneous injection of calcitonin, an osteoclast inhibitor, with beta 2-M or PTH did not increase periosteal resorption over controls. The resorption of inner bone surface was similar in all groups. These studies show that beta 2-M and PTH cause bone resorption in the bone surfaces proximate to the site of injection. This suggests that beta 2-M may contribute to the development of the bone cysts in dialysis-related amyloidosis.

The renal extraction of beta 2-microglobulin (beta 2M) was investigated under steady-state conditions achieved by constant infusion of human beta 2M. Fifteen animal experiments were conducted. Beta 2 microglobulin was infused at rates ranging from 51 o 269 micrograms/min. The renal arterial and venous blood levels remained constant throughout the study period. The data showed that renal extraction of beta 2M exceeded the rate of filtration at all levels of beta 2M delivered to the kidney. The tubular uptake of filtered beta 2M increased linearly as did the extraglomerular extraction throughout the range investigated. There was no evidence of beta 2M (FE beta 2M) increased linearly with the fractional excretion of filtered water (FE H2O). The results are interpreted to indicate that beta 2M is extracted from renal blood by glomerular filtration and, in addition, by a mechanism independent of glomerular filtration rate (GFR). Under the conditions existing in these experimental animals, the linear relationship between FE beta 2M and FE H2O is evidence to suggest that factors affecting proximal tubular water reabsorption also affect beta 2M reabsorption.

Highly purified rat beta2-microglobulin (beta2m) as well as cytochrome c and lysozyme were radiolabeled and their catabolism studied in the rat. More than 90 percent of these low molecular weight proteins were removed from the serum within an hour and excreted into the urine by 24 hours. Except for the kidney in which the concentration of these protein is ten- to twentyfold greater than in the serum, there is little evidence that rat tissues are concentrating these proteins. The stomach was found to concentrate radioiodine. The catabolism of rat beta2m differed from that of cytochrome c and lysozyme in that the kidney contained twice as much labeled rat beta2m. In addition, the rat excretes 10 to 15 percent of the injected rat beta2m but only 1 to 5 percent of the cytochrome c or lysozyme. These studies established a basis for turnover studies of beta2m complexed with other cell membrane proteins, for example, HL-A or H-2 peptides.

beta 2-microglobulin (beta 2M) is a protein of 11,800 daltons which occurs in the plasma of normal individuals at concentrations of approximately 2 microgram/ml. It is presumed to be relatively freely filterable. More than 99% of the filtered beta 2M is taken up by an active reabsorptive mechanism and catabolized by the renal tubule. The data presented here demonstrate that renal extraction is only slightly diminished by complete ureteral obstruction. The renal extraction of beta 2M is greater than can be accounted for by filtration alone. These data indicate that some uptake of beta 2M occurs from the peritubular capillary circulation. The loading of animals with beta 2M is associated with a marked tubular proteinuria suggesting that this protein may play a part in inducing tubular injury.