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.

Transcriptional control of MHC genes and T cell development in MHC class II deficiency.

MHC class II deficiency has proven to be an excellent model to study transcription regulation of MHC genes and T cell development. Cell lines established from MHC class II deficient patients have been of great value for the identification of proteins necessary for MHC expression and their study has resulted in the identification of a common regulatory pathway for MHC class II and class I genes. The lack of MHC class II expression was found to have a profound effect on the development of the CD4+ T cell lineage, in particular on the composition of the T cell receptor repertoire, revealing aberrant thymic selection processes in these patients. Here, we will discuss several aspects of the transcriptional regulation of MHC genes and the impact of deficient MHC class II expression on T cell development.

Incomplete T-cell immune reconstitution in two major histocompatibility complex class II-deficiency/bare lymphocyte syndrome patients after HLA-identical sibling bone marrow transplantation.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II-deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8(+) T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II-expressing CD4(+) and in the CD8(+) T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.

Molecular analysis of an MHC class II deficiency patient reveals a novel mutation in the RFX5 gene.

Patients suffering from major histocompatibility complex (MHC) class II deficiency, a rare primary immunodeficiency, are characterized by a lack of MHC class II expression which is the result of defects in trans-acting factors. At least four complementation groups, A, B, C, and D, can be discerned. The gene affected in group C patients is known to be RFX5 and encodes one of the subunits of the multimeric phosphoprotein complex, RFX. In the present study we fused fibroblasts of a recently identified MHC class II deficiency patient, OSE, with fibroblasts derived from patients representative of each of the four complementation groups. Transient heterokaryon analysis indicated that OSE belonged to complementation group C. Furthermore, transfection of wild-type RFX5 cDNA into OSE fibroblasts resulted in restoration of the defect. Mutation analysis revealed that the RFX5 mRNA lacked four nucleotides and that this deletion was the consequence of a G to A transition in a splice acceptor site. Genomic oligotyping demonstrated that OSE was homozygous for the splice site mutation.

Regulation of MHC class I and II gene transcription: differences and similarities.

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes.

Introduction of exogenous class II trans-activator in MHC class II-deficient ABI fibroblasts results in incomplete rescue of MHC class II antigen expression.

Previously, we have shown that fibroblasts established from type III bare lymphocyte syndrome patient ABI are characterized by the absence of MHC class II gene expression and a strongly reduced amount of MHC class I transcripts. Complementation analysis has suggested that the gene defective in these ABI fibroblasts is different from that encoding the class II trans-activator (CIITA), which has been attributed an essential role in both constitutive and inducible expression of MHC class II genes. In the present study it is shown by reverse transcriptase-PCR analysis that the amount of CIITA transcripts in ABI fibroblasts is greatly reduced compared with that in fibroblasts derived from a healthy individual. Transient cotransfection of a construct in which CIITA is under the control of a constitutive promoter with an HLA-DRA promoter-luciferase reporter plasmid resulted in enhanced luciferase expression in ABI fibroblasts. Furthermore, ABI fibroblasts stably transfected with CIITA re-express functional HLA-DR Ags, but do not express HLA-DQ and DP Ags at the cell surface. Comparison of these data with those obtained for normal fibroblasts and fibroblasts defective for CIITA indicate that the gene defect and the resulting lack of MHC class II expression in ABI fibroblasts can only partly be corrected by the introduction of CIITA. Furthermore, DNase I hypersensitivity analysis of ABI fibroblasts has revealed a closed chromatin structure in the promoter region of the MHC class II DRA gene. However, CIITA transfection resulted in an open DNA configuration, which suggests a role for CIITA in provoking changes in the chromatin structure of the DRA gene.

Selective usage of defined TCRBV genes in response to filarial antigens.

The characterization of T cell reactivities that are prone to down-modulation by filarial parasites is central to understanding how these nematodes can survive for long periods of time within their human host and to design appropriate immunoprophylactic measures. In the present study, TCRBV gene usage was analyzed in response to filarial antigens by PCR using a panel of TCRBV gene segment family-specific oligonucleotide primers. Analysis of individuals highly responsive to Brugia malayi adult worm antigen (BmA) (n = 4) indicated that following stimulation with BmA a maximum of four TCRBV gene families were over-represented in each subject. Those were TCRBV2, 9, 19 and 23 in subject 1; TCRBV8, 9 and 16 in subject 2; TCRBV2, 8, 9 and 11 in subject 3; and TCRBV13 and 23 in subject 4. The analysis of one subject who was unresponsive to BmA before but regained responsiveness after diethylcarbamazine treatment revealed that there was no overexpression of a particular TCRBV gene family before chemotherapy, whereas after chemotherapy three TCRBV gene families (TCRBV8, 16 and 19) were found to be overexpressed. Complementarity determining region 3 size analysis of a selection of the overexpressed TCRBV genes displayed oligoclonality in some of the observed expansions. Together these observations show that limited T cell subpopulations are clonally amplified in BmA-stimulated peripheral blood mononuclear cells of filarial responder subjects, possibly driven by a restricted number of antigens.

Human T cell repertoire generation in the absence of MHC class II expression results in a circulating CD4+CD8- population with altered physicochemical properties of complementarity-determining region 3.

In this study, we have investigated the impact of deficient MHC class II expression on the use of TCRBV6 and TCRBJ gene elements, and on the pattern of amino acid incorporation exhibited in the N1-D-N2 segments of the third complementarity-determining region (CDR3) of these TCRBV6 rearrangements. To this end, we have analyzed circulating T cells from three, nonrelated MHC class II-deficient (bare lymphocyte syndrome (BLS)) patients and three MHC class II-expressing family members. The patients and healthy controls exhibited similar, nonrandom usage profiles of TCRBV6 and TCRBJ gene elements in both the CD4+CD8- and the CD4-CD8+ subsets of peripheral blood T cells. No statistically significant differences between patients and controls were detected in the length of CDR3, or in the amount of non-germline modification at the sites of recombination. However, detailed analysis of the TCRBV6 rearrangements derived from the CD4+CD8- subsets from the BLS patients revealed patterns of amino acid incorporation into the N1-D-N2 region of CDR3 that resulted in altered charge and hydropathicity properties of the presumed Ag binding site. In this way, we have been able to demonstrate that human T cell repertoire development in the absence of MHC class II expression results in a circulating CD4+CD8- T cell population bearing TCRs with altered CDR3 profiles. Such altered profiles are likely to be a direct reflection of the lack of MHC class II-mediated selection processes in these BLS patients.

T cell development in a major histocompatibility complex class II-deficient patient.

In this report we show that the major histocompatibility complex (MHC) class II-negative thymus of a bare lymphocyte syndrome (BLS) patient contains a reduced CD4+ CD8- T cell population when compared to thymocytes derived from a MHC class II-expressing thymus. Of these CD4+ CD8- BLS thymocytes, approximately only one third co-expressed the CD3 antigen, moreover at a lower expression level when compared to control thymocytes. This suggests a partial maturation of the CD4+ CD8- T cells in the absence of MHC class II expression. Among the BLS thymocytes, CD4+ CD8+ thymocytes could easily be detected. Noteworthy, the number of CD4- CD8+ thymocytes was significantly increased. CD4+ CD8- T cells could also be found among the BLS peripheral blood mononuclear cells, albeit at reduced numbers. Despite the absence of peripheral MHC class II expression, the majority of these CD4+ CD8- T cells co-expressed the CD45RO marker. In the BLS patient, thymocytes as well as peripheral CD4+ CD8- T cells were not restricted in the use of the available T cell receptor (TcR) V gene family pool. However, the lack of detectable levels of thymic and peripheral MHC class II antigen expression in the BLS patient had altered the CD4-skewing patterns of TcR V gene families which were present in normal individuals. In conclusion, the lack of MHC class II expression in the BLS patient does not completely inhibit the CD4+ CD8- T cell development.

The MHC class II deficiency syndrome: heterogeneity at the level of the response to 5-azadeoxycytidine.

The involvement of DNA methylation in aberrant MHC class II gene expression in EBV-transformed B-cell lines from 2 patients (THF and DGN) with the MHC class II deficiency syndrome (bare lymphocyte syndrome) was investigated. Incubation of the cells in the presence of various concentrations of 5-azadeoxycytidine resulted in the induction of expression of HLA DR genes in the DGN cell line, whereas, in the THF cell line, no effect of 5-azadeoxycytidine treatment on the expression of the HLA DR genes could be detected. Subsequent Southern blot analysis using methylation-sensitive restriction enzymes (ApyI, EcoRII and HhaI) and 5-azadeoxcycytidine-treated and untreated genomic DNA, indicated that the lack of HLA DR-A expression in the DGN cell line is not caused by hypermethylation of the 5' region of the HLA DR-A gene. These results indicate that 5-azadeoxycytidine treatment of the DGN cell line leads to activation of a methylation-sensitive factor that is involved in the regulation of transcription of the DR-A gene. In cell line THF, however, demethylation does not restore the activity of this factor. The lack of MHC class II expression in this cell line is caused by some other defect. The results of our analysis indicate that at least two different factors are involved in regulation of MHC class II gene expression.

Unambiguous typing for HLA-DQ TA10 and 2B3 specificities using specific oligonucleotide probes.

Oligonucleotide probes specific for the serologically defined TA10 and 2B3 specificities were selected based on a comparison of the available HLA-DQ beta sequences. Panel and family segregation studies confirm a complete correlation between the reactivities of the selected probes and the TA10/IIB3 antibodies. The Glu residue at position 45 of the HLA-DQ beta chain is specific for the TA10 determinants, and a DQ beta Gly-Val-Tyr sequence is found at position 45-47 for all 2B3-positive DQ beta chains.

A family with an apparent HLA-DR triplet: evidence for exchange of functional HLA-DR beta-genes between different haplotypes.

Serological and protein analysis showed that HLA-DR1 and DR2 short (s) segregated together as one haplotype, resulting in a HLA-DR triplet as found in a family study. Both DR1 and DR2 molecules were coordinately expressed and were shown to function as restriction elements in antigen presentation assays. This unique HLA-DR1, 2s haplotype was further studied by Southern blot analysis. Based upon well-known restriction fragment length polymorphisms for the involved gene sets, i.e. DR1 and DR2 along with the DR type from the other haplotype, the genes as identified by restriction fragment length polymorphism of the triplet could be established. Pseudogenes, which are included in the previously described gene sets of HLA-DR1 and DR2 are apparently lacking in the triplet. We therefore postulate that during an unequal crossing-over event the DR beta-pseudogene of DR2 could be exchanged by a functional DR1 beta-gene.

An HLA-DQ alpha allele identified at DNA and protein level is strongly associated with celiac disease.

An HLA-DQ alpha cDNA probe showed upon hybridization a highly significant discrepancy between the RFLP of celiac disease patients and healthy controls. The 4.0-kb Bgl II restriction fragment was present in 97% of celiac disease patients (n = 30), compared to 56% in a healthy control population (n = 72) (RR = 14.9; p less than 0.0005). At the product level all celiac disease patients tested to date have one DQ alpha chain in common, designated HLA-DQ alpha 2.3, which is associated with the 4.0-kb Bgl II fragment. This HLA-DQ alpha allele identified at the DNA level and product level seems to be a better marker for genetic susceptibility to develop celiac disease than those available to date.

RFLP of the HLA-DQ alpha region: a diallelic DX alpha polymorphism, not linked to DR and DQ specificities.

HLA-DQ alpha DNA polymorphism was studied by Southern blot analysis in a panel of 117 individuals, consisting of 56 randomly selected and 61 HLA-DR homozygous individuals. Hybridizing fragments representing DQ alpha genes correlate with DR specificities owing to linkage disequilibrium between DQ and DR. Two fragments representing DX alpha genes were identified with the restriction enzymes PvuII and TaqI and a DQ alpha cDNA probe. The two fragments of PvuII-DQ alpha hybridization (a 7200 and a 7000 basepair fragment) and the two of TaqI-DQ alpha (2200 and 1900 basepairs) have an identical distribution in the panel, and reveal gene frequencies of 49.1% and 50.9%; they behave as alleles of the putative DX alpha locus. The panel study shows that the DX alpha fragments are not linked with the DR or DQ specificities but segregate along with HLA as shown in family studies.

HLA class II DNA analysis by RFLP reveals novel class II polymorphism.

Polymorphism of the HLA-D/DR region has been defined by serologic and cellular methods. Additionally, protein and DNA analyses not only confirmed and refined the definition of the established polymorphisms but also revealed further polymorphisms for which no serologic or cellular correlate are known (yet). To study these in more detail, we analyzed the banding patterns obtained from Southern blot hybridizations with DR beta and DQ alpha cDNA probes. Specific fragments reflecting already defined polymorphisms could be identified. A refined HLA-D/DR definition based upon the presence of DNA subtypes could be introduced. Fragments have also been identified that are associated with the DR/Dw specificities. Moreover, individuals with different DR types may also share fragments in hybridization assays. These shared hybridizing fragments (SHFs) are, for instance, found in individuals typed as DR4, DR5, DR7, and DRw8. In total, 20 SHFs were found using two restriction enzymes and the DR beta and DQ alpha cDNA probes. Some of these SHFs correlate with antigenic determinants defined by broad reacting alloantisera, such as DRw52, but for 14 of these SHFs, no serologic equivalent has been found so far. Thus, SHFs reflect a conservation at the DNA level of the HLA class II region, which suggests that the polymorphic class II genes may be more conserved than previously thought. The possible biologic implications of conserved sequences in the HLA class II genes will be discussed.

An overview of the restriction fragment length polymorphism of the HLA-D region: its application to individual D-, DR- typing by computerized analyses.

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.

Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals.

In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR beta, -DQ alpha, and -DQ beta cDNA probes in Southern blot analysis. Three subtypes are found using the DR beta cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, Dw10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ alpha cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ beta detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ alpha and DQ beta cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ alpha probe, probably due to DX polymorphism. DR beta RFLP divides the LB14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases.