Long-lasting pro-inflammatory suppression of microglia by LPS-preconditioning is mediated by RelB-dependent epigenetic silencing.

Microglia, the innate immune cells of the central nervous system (CNS), react to endotoxins like bacterial lipopolysaccharides (LPS) with a pronounced inflammatory response. To avoid excess damage to the CNS, the microglia inflammatory response needs to be tightly regulated. Here we report that a single LPS challenge results in a prolonged blunted pro-inflammatory response to a subsequent LPS stimulation, both in primary microglia cultures (100 ng/ml) and in vivo after intraperitoneal (0.25 and 1mg/kg) or intracerebroventricular (5 µg) LPS administration. Chromatin immunoprecipitation (ChIP) experiments with primary microglia and microglia acutely isolated from mice showed that LPS preconditioning was accompanied by a reduction in active histone modifications AcH3 and H3K4me3 in the promoters of the IL-1ß and TNF-α genes. Furthermore, LPS preconditioning resulted in an increase in the amount of repressive histone modification H3K9me2 in the IL-1ß promoter. ChIP and knock-down experiments showed that NF-κB subunit RelB was bound to the IL-1ß promoter in preconditioned microglia and that RelB is required for the attenuated LPS response. In addition to a suppressed pro-inflammatory response, preconditioned primary microglia displayed enhanced phagocytic activity, increased outward potassium currents and nitric oxide production in response to a second LPS challenge. In vivo, a single i.p. LPS injection resulted in reduced performance in a spatial learning task 4 weeks later, indicating that a single inflammatory episode affected memory formation in these mice. Summarizing, we show that LPS-preconditioned microglia acquire an epigenetically regulated, immune-suppressed phenotype, possibly to prevent excessive damage to the central nervous system in case of recurrent (peripheral) inflammation.

Combined analysis of HLA class I, HLA-E and HLA-G predicts prognosis in colon cancer patients.

BACKGROUND: Evasion of immune surveillance and suppression of the immune system are important hallmarks of tumour development in colon cancer. The goal of this study was to establish a tumour profile based on biomarkers that reflect a tumour's immune susceptibility status and to determine their relation to patient outcome. METHODS: The study population consisted of 285 stage I-IV colon cancer patients of which a tissue micro array (TMA) was available. Sections were immunohistochemically stained for the presence of Foxp3+ cells and tumour expression of HLA Class I (HLA-A, -B, -C) and non-classical HLA-E and HLA-G. All markers were combined for further analyses, resulting in three tumour immune phenotypes: strong immune system tumour recognition, intermediate immune system tumour recognition and poor immune system tumour recognition. RESULTS: Loss of HLA class I expression was significantly related to a better OS (P-value 0.005) and DFS (P-value 0.008). Patients with tumours who showed neither HLA class I nor HLA-E or -G expression (phenotype a) had a significant better OS and DFS (P-value <0.001 and 0.001, respectively) compared with phenotype b (OS HR: 4.7, 95% CI: 1.2-19.0, P=0.001) or c (OS HR: 8.2, 95% CI: 2.0-34.2, P=0.0001). Further, the tumour immune phenotype was an independent predictor for OS and DFS (P-value 0.009 and 0.013, respectively). CONCLUSION: Tumours showing absence of HLA class I, HLA-E and HLA-G expressions were related to a better OS and DFS. By combining the expression status of several immune-related biomarkers, three tumour immune phenotypes were created that related to patient outcome. These immune phenotypes represented significant, independent, clinical prognostic profiles in colon cancer.

Type III bare lymphocyte syndrome associated with a novel RFXAP mutation: a case report.

Type III bare lymphocyte syndrome (BLS) is a severe combined immunodeficiency disease caused by the absence of MHC Class II expression associated with low expression of class I molecules. Here, we report a case with type III BLS who lacked RFXAP (Regulatory factor X-associated protein) expression as a result from a novel mutation introducing a premature stopcodon in DE-region at amino acid 73.

The relationship between region E1a and E1b of human adenoviruses in cell transformation.

Baby rat kidney (BRK) cells were transfected either with intact region E1 DNA of adenovirus type 5 (Ad5) or with mixtures of DNA fragments containing the separated E1a and E1b regions. The results showed that mixtures of regions E1a and E1b transform with a similar efficiency as intact region E1. DNA fragments containing region E1b alone have no detectable transforming activity in primary BRK cells nor in established rat cell lines. When region E1a and Ad5 was combined with region E1b and Ad12 complete transformation was also obtained. Characterization of the cell lines transformed by separated E1a and E1b regions have led to the following conclusions: (1) Expression of region E1b is not dependent on specific linkage to region E1a as it occurs in the intact E1 region. (2) Region E1b is normally expressed into the corresponding major adenovirus T antigens (65,000 and 19,000 Mr with region E1b of Ad5; 60,000 and 19,000 Mr with E1b or AD12). (3) Region E1b of Ad12 can be activated by region E1a of Ad5 indicating that the Ela regions of both serotypes are functionally similar in transformation. (4) Cell lines containing region E1b of Ad5 are weakly oncogenic in nude mice whereas cells containing E1b of Ad12 are highly oncogenic in nude mice, indicating that the degree of oncogenicity is determined by region E1b.

Direct assessment of junctional diversity in rearranged T cell receptor beta chain encoding genes by combined heteroduplex and single strand conformation polymorphism (SSCP) analysis.

In order to define the extent of T cell heterogeneity and clonality, unique DNA sequences in the junctional region in rearranged T cell receptor (TcR) genes can be studied. For this purpose we have adapted a non-denaturing nucleic acid gel electrophoresis procedure to detect TcR junctional diversity. Detection of junctional diversity is based upon electrophoretic separation of single stranded (ss) and double stranded (ds) DNA molecules via mobility shifts due to nucleotide sequence polymorphism. To examine the capacity of this nucleic acid gel electrophoresis procedure to detect nucleotide sequence polymorphism in the CDR 3 region within TcR V beta gene family sequences polymerase chain reaction (PCR) amplified TcR V beta 5.1/5.4 and V beta 14 cDNA sequences were analyzed. The results of this study showed that (1) the single strand conformation polymorphism (SSCP) procedure has a low capacity to discriminate between diverse TcR V beta cDNA sequences due to comigration of the ssDNA molecules, which results in an underestimation of the heterogeneity in a given T cell population; (2) comigrating ssDNA and/or dsDNA (homoduplex) molecules can be separated by the formation of heteroduplex molecules; these heteroduplex molecules provide essential additional information on the degree of nucleotide sequence polymorphism in the CDR 3 region within the TcR V beta cDNA sequences; (3) the double strand conformation polymorphism (DSCP) procedure provides a fast and reliable procedure to detect junctional diversity within the sequences tested. Using DSCP a more detailed assessment of amplified TcR V beta cDNA sequences can be obtained as compared with SSCP analysis only. Data obtained by gel analysis were very similar to those obtained by conventional bacterial cloning and DNA sequencing procedures on the corresponding cDNA clones. In conclusion, this new gel electrophoresis procedure allows a direct assessment of the extent of T cell heterogeneity and clonality by screening junctional diversity in TcR chain encoding sequences in clinical conditions with (oligo)clonal expansion of T lymphocytes.

Functional and molecular characterization of graft-infiltrating T lymphocytes propagated from different biopsies derived from one heart transplant patient.

Alloreactive T lymphocytes play an important role in graft rejection. In the present study, we have analyzed the cytolytic capacity against donor cells of graft infiltrating T lymphocyte cell lines, which were propagated from various endomyocardial biopsies taken at different time points after transplantation, including during a rejection crisis. Also, T cell clones were generated from the rejection biopsy and evaluated for their cytolytic capacity and nucleotide composition of the TCR alpha and beta chains. The results of these studies revealed a strong cytolytic activity against donor cells by T cells derived from the rejection biopsy, whereas from the other biopsies, no cytolytic T cell clones could be established. The T cells that were responsible for this activity, as detected by T cell cloning and TCR gene analysis, could not been identified in earlier biopsies, indicating that these cytolytic cells were recently recruited toward the endomyocardium.

Modulation of the T cell compartment by blood transfusion. Effect on cytotoxic and helper T lymphocyte precursor frequencies and T cell receptor Vbeta usage.

Recent data suggest that the favorable effect of pretransplant blood transfusion (BT) on transplant outcome depends on the HLA match. HLA-DR or haplotype shared transfusions lead to transplantation tolerance, and HLA-mismatched BT leads to immunization. The immunological mechanism involved is still unknown. To investigate the effect of HLA compatibility between blood donor and recipient on the T cell compartment, we determined the frequency of cytotoxic and helper T cell precursors specific for blood donor cells (n=20) and the T cell receptor Vbeta (TCRBV) repertoire of the CD4- and CD8-positive peripheral blood mononuclear cells before, at 2 weeks after, and at more than 10 weeks after BT (n=10). Patients had received one transfusion of a nonstored (<24 hr after withdrawal) erythrocyte concentrate without buffy coat containing on average 6x10(8) leukocytes. Eight patients shared an HLA-B and -DR antigen, nine patients shared one HLA-DR antigen, and three patients shared no HLA class II antigens with the blood donor. All patients showed a significant increase in both cytotoxic and helper T cell precursor frequencies against the blood donor 2 weeks after BT. In most patients, the frequencies reached pretransfusion levels again long after BT. In 5 of 10 patients, an expansion of one or more TCRBV families was observed in either the CD4 or CD8 compartment. This study demonstrates that BT, irrespective of the degree of HLA matching, induces activation of the T cell compartment. The degree of sharing of HLA antigens was not correlated with quantitative changes in cytotoxic T lymphocyte precursor or helper T lymphocyte precursor frequencies, or changes induced in the TCRBV repertoire. Cytotoxic and helper T lymphocyte precursor frequencies and TCRBV repertoire determined after BT do not give an indication for a state of tolerance prior to transplantation.

Uveal melanoma: no expression of HLA-G.

PURPOSE: To investigate whether uveal melanoma cells express HLA-G, a nonclassical HLA class I molecule that has been shown to be a critical mediator in the inhibition of natural killer (NK) cell-mediated cytolysis. METHODS: Eleven human uveal melanoma cell lines were analyzed for the expression of HLA-G by flow cytometry, immunocytochemistry, Western blot analysis, and RT-PCR followed by Southern blot analysis. Two HLA-G-specific monoclonal antibodies were used, 87G and MEM-G/1. In addition, HLA-G expression was determined on frozen tissue sections of 17 primary uveal melanomas. RESULTS: With all HLA-G detection methods, no evidence for HLA-G expression by uveal melanoma cells was found. In contrast, the trophoblast cell line JEG-3 clearly expressed HLA-G transcripts and protein in all cases. Furthermore, interferon-gamma did not induce HLA-G expression in the uveal melanoma cell lines. Notably, all cell lines expressed HLA-E, and this expression was significantly enhanced by interferon-gamma. CONCLUSIONS: Because none of the uveal melanoma cell lines nor any of the primary uveal melanomas displayed expression of HLA-G, it is unlikely that HLA-G plays a role, direct or indirect, in the modulation of cellular immunity against uveal melanoma tumors.

Cloning of the MCF1233 murine leukemia virus and identification of sequences involved in viral tropism, oncogenicity and T cell epitope formation.

MCF1233 is an oncogenic C57BL-derived retrovirus of the Murine Leukemia Virus (MuLV) family, that causes T and B lymphomas in an MHC-associated fashion. In this study, we cloned MCF1233, determined its nucleotide sequence and, by comparison with its MuLV relatives, identified the sequences that relate to the leukemogenic character of this virus. MCF1233 was found to have an ecotropic backbone, and carried acquired polytropic sequences in the 3' pol and 5' env region. The gag-region contained six specific nucleotides, determining the viral B-tropism. Short sequences within the U3 LTR shared specific homology with the xenotropic Bxv-1 MuLV, which is the U3 donor for leukemogenic MCF MuLV of AKR origin. These sequences, in combination with specific ecotropic sequences present in env p15E, most likely determine the viral oncogenicity. Currently, the deduced MCF1233 amino sequence is being exploited for T cell epitope analysis, which in this paper is discussed with respect to antigenically distinct Friend/Moloney/Rauscher types of MuLV. Identification of these T cell epitopes will contribute to our understanding of the fundamental aspects of immune control on MCF1233-induced lymphomagenesis. It will help to elucidate the mechanisms that underlie immune escape of T lymphomas, rarely arising in immunoresistant mice, and allow the development of vaccination protocols for tumor therapy.

Transcriptional regulation of the MHC class Ib genes HLA-E, HLA-F, and HLA-G.

The restricted tissue expression of the MHC class Ib molecules HLA-E, HLA-F, and HLA-G has suggested specialized functions and tight transcriptional control of their genes. Transactivation of classical MHC class I genes is mediated by two groups of juxtaposed cis-acting elements, which can be viewed as regulatory modules. The most upstream module consists of the enhancer A and ISRE, and mediates constitutive and cytokine induced expression, whereas the SXY module is important for the constitutive and CIITA-mediated transactivation of MHC class I genes. Nucleotide sequence divergence in these regulatory elements in the promoters of HLA-E, HLA-F, or HLA-G determines their differential responsiveness to NF-kappaB, IRF1, and CIITA-mediated induction. HLA-E is not inducible by NF-kappaB or IRF1, but is responsive to IFN-gamma through an upstream STAT1 binding site. Furthermore, HLA-E is inducible by CIITA through the SXY regulatory module. HLA-F is inducible by NF-kappaB through the kappaB1 site of enhancer A, is responsive to IFN-gamma through the ISRE, and is inducible by CIITA. Both regulatory modules are divergent in HLA-G rendering this gene unresponsive to NF-kappaB, IRF1, and CIITA-mediated induction. This implies a unique regulation of HLA-G transcription amongst the MHC class Ib genes.

Lymphokine gene transcription in CD4+CD8- T cells of a type III bare lymphocyte syndrome patient.

In this study we analyzed the impact of a MHC class-II-deficient environment on the differentiation of CD4+CD8- T lymphocytes into functional defined subsets of lymphokine-producing T-helper cells. To this end a CD4+CD8- T-cell line and CD4+CD8- T-cell clones, isolated from PBMCs of a type III BLS patient, were stimulated in vitro with anti-CD3 and PMA and assessed for lymphokine transcription patterns. The results of these analyses show that CD4+CD8- T cells that have matured in a MHC class-II-deficient environment display lymphokine transcription patterns that resemble those of MHC class-II-expressing family control-derived CD4+CD8- T cells.

Cellular immune recognition of HLA-A*0201 following gene transfer into a human embryonal carcinoma cell line.

Previously, we have established that transcription and cell surface expression of MHC class I and beta 2m genes in undifferentiated Tera-2 stem cells, a teratocarcinoma-derived cell line, was extremely low. In this study, we have transfected an HLA-A*0201-encoding cDNA driven by the CMV-promoter into Tera-2 cells. Prior to IFN gamma treatment, membrane expression of HLA-A*0201 by these Tera-2 transfectants was nearly lacking. Consequently, the HLA-A*0201 Tera-2 transfectants were not recognized by the allo-HLA-A*0201-specific CTL clone 3E7. Following IFN gamma treatment, which resulted in upregulation of HLA-A*0201, Tera-2 cells were lysed by CTL clone 3E7. In contrast, loading of HLA-A*0201 with the influenza-A-matrix peptide 58-66 resulted in partial lysis of Tera-2 cells by the influenza-A-matrix protein-specific CTL clone Q66-9, and nearly complete lysis was observed following IFN gamma treatment. These results suggest that the HLA-A*0201 transgenes in Tera-2 cells can be loaded with peptides and used as targets for peptide-specific CTLs.

A novel T-cell-defined HLA-DR polymorphism not predicted from the linear amino acid sequence.

Recent investigations have shown that alloreactive T cells are capable of responding to structures defined by specific linear amino acid sequences on class II molecules. In the present study we show that also a polymorphism can be recognized that is not defined by such linear amino acid sequences. Two human T-cell clones, sensitized to DRw13 haplotypes, are described. The description of clone c50 serves to exemplify the first model. This DRB1-specific clone responds to stimulator cells that carry DR molecules, different in their DRB1 first and second hypervariable regions (HV1 and HV2) but identical in their HV3 regions (i.e., DRw13,Dw18; DRw13,Dw19; DR4,Dw10; and DRw11,LDVII). The second clone, c1443, behaves nonconventionally. It responds to DRw13,Dw18; DRw13,Dw19; and DR4,Dw4 stimulator cells, although no specific amino acid sequence is shared between these specificities. The latter pattern of reactivity suggests the existence of a novel polymorphism recognized by alloreactive T cells. This particular polymorphism may also be biologically significant.

Long-term T cell immune reconstitution in 2 SCID patients after BMT.

To evaluate the long-term reconstitution of the T cell immune repertoire in recipients of an allogeneic Bone Marrow Transplantation (allo-BMT), we have analyzed the T cell receptor (TCR) repertoire in the periphery and the T cell response against tetanus toxoid in two T- B+ Severe Combined Immunodeficiency Disease (SCID) patients more than 11 years after HLA haplo-identical allo-BMT. Our studies demonstrate that in the periphery of allo-BMT recipients, on the basis of TCR V-gene segment usage, the T cell immune repertoire long after allo-BMT is diverse, as is that of the donor. However, when donor and allo-BMT recipient were compared, differences were noted in the TCR Complementarity Determining Region 3 (CDR3) size distributions and in the T cell response against tetanus toxoid. In particular, the tetanus toxoid specific T cell clones differed in their use of HLA restriction elements, and expressed different T cell receptors. Moreover, we have uncovered donor-type tetanus toxoid specific T cell clones which were established from allo-BMT recipient derived peripheral blood lymphocytes and were found to be restricted by the non-shared recipient allele. This observation suggests a role for recipient-mediated T cell selection processes, in the thymus or at extra-thymic sites.

Detection and characterization of HLA class I molecules in the supernatant of an hepatocarcinoma cell line and of EBV-transformed B cell lines.

Human leukocyte antigens (HLA) class I molecules can be detected in "soluble" form in the supernatant of cultured cell lines and in serum and plasma of humans. These "soluble" HLA class I molecules are assumed to play a role in liver transplantation. In order to define the nature and composition of HLA class I molecules found in solution, we studied the HLA class I production of an hepatoma carcinoma cell line (HepG2) and of EBV-transformed B-cell lines. Based on molecular weight (MW) analysis, it was demonstrated that different forms of HLA class I molecules were produced by HepG2 cells and EBV B-cells. Monoclonal antibodies (mAbs) specific for HLA class I alleles were able to recognize the mature 45 kDa form, but failed to interact with the 42 kDa and 39 kDa MW forms of HLA class I. Of these different MW forms of HLA class I molecules the mature 45 kDa product was found predominantly to be associated with subcellular vesicles whereas the alternative MW forms of 42 kDa and 39 kDa exist as truly free entities in supernatants.

Inflamed joints of patients with rheumatoid arthritis contain T cells that display in vitro proliferation to antigens present in autologous synovial fluid. Functional analysis on the basis of synovial-fluid-reactive T-cell clones and lines.

The immunopathology of inflamed joints in patients with RA is thought to result from an antigen-driven T-cell response. The antigen(s) responsible for the activation of synovial T cells, however, are as yet unidentified. In this study, we tested SF as a potential source of (auto)antigen(s). Five of 15 IL-2-expanded T-cell lines generated from SF cells of RA patients displayed a proliferative response to autologous SF. Five CD4+CD8-alpha beta TCR+SF-reactive T-cell clones obtained from responder T-cell lines were studied in more detail. Three T-cell clones from one RA patient were found to recognize epitopes in autologous SF in the context of DR4(Dw4), and two T-cell clones of another RA patient responded to autologous SF in the context of the HLA-DPB1*0401 gene product. The two DP-restricted clones and one of the DR-restricted clones did not proliferate to 50 SF samples of other RA patients, whereas the remaining DR-restricted clones responded to one allogeneic sample. Sequence analysis demonstrated that the latter clones expressed identical V beta 6.9 + TCR beta chains. This was also found for the (V beta 19+) DP-restricted clones. Proliferation of SF-reactive T cells was not only obtained with SF of the joint that had contained the T cells, but also with autologous SF of other affected joints. Together, these findings indicate that epitopes able to stimulate synovial T cells differ among RA patients, but may be similar within multiple joints of an individual patient. The presence of T cells able to respond to SF antigens in inflamed joints suggests that these T cells play an active role in the pathogenesis of RA.

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-gamma inducible promoter (PIV) of CIITA.

Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-gamma (IFN-gamma)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-gamma-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.

T cell immune reconstitution after allogeneic bone marrow transplantation in bare lymphocyte syndrome.

To study the impact of an MHC class II-negative environment on T cell immune reconstitution, we have analyzed the phenotypical and functional characteristics of FACS-sorted cultured CD4(+) and CD8(+) T cells in two Bare Lymphocyte Syndrome (BLS) patients before and after allo-BMT. A similar analysis was performed in two MHC class II expressing pediatric leukemia patients after treatment with an allo-BMT who were included in our study as control. It was observed that CD4(+) T cells displayed cytolytic alloreactivity in both BLS patients prior to and within the first year after allo-BMT, whereas such cells were absent at a later time-point, in the donors and pediatric leukemia controls. In addition, reduced MHC class II expression was observed in CD8(+) T cells of both recipients early after allo-BMT, irrespective of the T cell chimerism pattern. Lack of endogenous MHC class II expression in BLS patients, therefore, results in aberrant T cell selection within the first year after allo-BMT, analogous to T cell selection before transplantation. These T cell selection processes seem to be normalized at a later time point after allo-BMT probably due to migration and integration of graft-derived MHC class II-positive antigen presenting cells to sites of T cell selection.

Clonal dominance and selection for similar complementarity determining region 3 motifs among T lymphocytes responding to the HLA-DR3-associated Mycobacterium leprae heat shock protein 65-kd peptide 3-13.

In order to establish whether specific MHC class II-peptide complexes are capable of selecting TCR V regions, we investigated in detail the TCR beta chain used in the recognition of HLA-DR3 restricted hsp65 peptide 3-13 in a tuberculoid leprosy patient. Using RT-PCR, a clear dominance of the TCRBV5 gene family was observed in a hsp65 peptide 3-13-specific T-cell line; however, not in fresh, unstimulated PBMCs, PHA-stimulated PBMCs, or a T-cell line specific for tetanus toxoid. DNA sequence analysis of the TCR V regions, comprising TCRBV5 genes, derived from the hsp65 peptide 3-13-specific T-cell line revealed the exclusive usage of the TCRBV55S1 gene segment and a predominance of one V-D-J gene rearrangement, which is indicative of clonal expansion of these T lymphocytes. Additional highly similar V-D-J gene rearrangements were detected at a low level in this hsp65 peptide 3-13 specific T-cell line. These conserved junctional regions (CDR3 regions) could not be detected within the TCRBV5 gene family of fresh PBMCs, PHA-stimulated PBMCs, hsp65, and tetanus-toxoid-specific T-cell lines from this patient. The observations in this tuberculoid leprosy patient reveal that an HLA class-II-restricted T-cell response results in selection of TCRBV regions which are highly similar in amino acid composition to the CDR3 region within the expanding TCRBV regions.

T-cell receptor V-gene usage in synovial fluid lymphocytes of patients with chronic arthritis.

In this study we analyzed the usage frequencies of the TCR V-gene segments by alpha beta+ T cells present in synovial fluid of 17 patients with chronic arthritis, including rheumatoid arthritis. The results of this study, obtained from semiquantitative PCR analyses, showed that in all patients most of the TCR V alpha- and V beta-gene segments could be detected both in fresh PBMCs and in fresh SFMCs. The relative frequencies of use of these V-region genes were variable between the different patients. Although there was some skewing of increased usage frequencies of particular TCR V alpha and V beta genes among SFMC-derived TCRs when compared with PBMCs, we could not correlate such increased TCR V-gene usage with the inflammation in the joints as a disease-specific marker.