Peter J. van den Elsen

Site α Is Crucial for Two Routes of IFNγ-Induced MHC Class I Transactivation: The ISRE-Mediated Route and a Novel Pathway Involving CIITA

Abstract The constitutive and cytokine-induced levels of major histocompatibility (MHC) class I expression are tightly controlled at the transcriptional level. In this study, it is shown that the cis -acting regulatory element site α of the MHC class I promoter is essential for the IFNγ-induced transactivation of MHC class I gene expression through the ISRE. Moreover, it was discovered that the class II transactivator (CIITA), which is itself under the control of the IFNγ induction pathway, strongly transactivates MHC class I gene expression and exerts its activity through site α. Therefore, site α is a crucial regulatory element, mediating the classic route of IFNγ induction via the ISRE as well as a novel route of MHC class I transactivation involving CIITA.

A Novel Autocrine Pathway of Tumor Escape from Immune Recognition: Melanoma Cell Lines Produce a Soluble Protein That Diminishes Expression of the Gene Encoding the Melanocyte Lineage Melan-A/MART-1 Antigen Through Down-Modulation of Its Promoter

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as “Ag silencing,” is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

Regulation of mycobacterial heat-shock protein-reactive T cells by HLA class II molecules: lessons from leprosy.

Major hislocompatibility (MHC) class II molecules are heterodimeric cell surface glycoproteins that are expressed by immunocompetenl cells. MHC class II molecules present processed peptide fragments to T-lymphocyte receptors, and thus can restrict and regulate T-cell responses against specific antigens. Class II molecules are highly polymorphic. This polymorphism is responsible for the genetically controlled differences in T cell-dependent responses and presumably also for genetic differences in disease susceptibility. A human disease where (HLA) class II genes play an important role is leprosy, the chronic infectious disease caused by Mycobacterium leprae. Protective immunity against M. leprae and other mycobacterial species is dependent on antigen-specific helper T lymphocytes. This is elegantly illustrated by the leprosy spectrum where strong Tlympbocyte reactivity to M. leprae is seen in the tuberculoid or high responder form of the disease as well as in healthy exposed individuals, but low or no Tcell responsiveness in the disseminated lepromatous form (Bloom & Godal 1983). HLA class Il-linked genes control the type of leprosy that develops upon infection as well as the type of cell-mediated immunity against M. leprae and related mycobacteria in skin testing (reviewed by Ottenhoff & de Vries 1987). Since leprosy type and skin test responsiveness both strongly correlate with M. lepraespecific T-cell reactivity, HLA class II gene products might regulate these responses by restricting and regulating T-helper cell activity against M. leprae

Definition of a novel complementation group in MHC class II deficiency

In this study we analyzed fibroblasts derived from an MHC class II deficiency patient (type III bare lymphocyte syndrome). Northern blot analysis showed that upon induction with IFN-γ these fibroblasts did not express HLA class II genes and displayed a strongly reduced level of HLA class I gene expression when compared with fibroblasts of a healthy individual. However, when analyzed by RT-polymerase chain reaction (PCR), residual expression could be detected for HLA-DRA, DPB, and DQA, but not for HLA-DRB, DPA, and DQB. The lack of HLA-DRB transcripts in the patient fibroblasts and the high degree of sequence polymorphism of HLA-DRB were exploited in the further analysis of these fibroblasts. Thus far, at least three, and probably four, complementation groups have been defined among patient-derived and experimentally-derived MHC class II-negative cell lines. Transient heterokaryons between the patient fibroblasts and representative B-lymphoblastoid cell lines from each of the complementation groups were analyzed by RT-PCR and Southern blotting, using HLA-DRB-specific primers and biotin-labeled sequence specific oligonucleotides, respectively. These analyses showed that the fibroblasts of this particular patient belonged to a novel complementation group in MHC class II deficiency.

Oxidative Phosphorylation Induces De Novo Expression of the MHC Class I in Tumor Cells through the ERK5 Pathway

Most cancer cells use anaerobic-like glycolysis to generate energy instead of oxidative phosphorylation. They also avoid recognition by CTLs, which occurs primarily through decreasing the level of MHC class I (MHC-I) at the cell surface. We find that the two phenomena are linked; culture conditions that force respiration in leukemia cells upregulate MHC-I transcription and protein levels at the cell surface, whereas these decrease in cells forced to perform fermentation as well as in leukemia cells lacking a functional mitochondrial respiratory chain. Forced respiration leads to increased expression of the MAPK ERK5, which activates MHC-I gene promoters, and ERK5 accumulation in mitochondria. Respiration-induced MHC-I upregulation is reversed upon short hairpin RNA-mediated ERK5 downregulation and by inactive mutants of ERK5. Moreover, short hairpin RNA for ERK5 leukemia cells do not tolerate forced respiration. Thus, the expression of ERK5 and MHC-I is linked to cell metabolism and notably diminished by the metabolic adaptations found in tumor cells.

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.

The RAG cell line defines a new complementation group of MHC class II deficiency.

We previously described RAG, a mouse adenocarcinoma cell line, as deficient for the induction of major histocompatibility (MHC) class II antigens by IFN-γ, but responding normally for MHC class I antigen stimulation and anti-viral protection. We had established that the fusion of RAG with various human cell lines restored the induction of MHC class II antigens, whenever the human chromosome 16 was present in somatic cell hybrids. Here we show that the RAG cell line does not exhibit any induction by IFN-γ ofDMA, DMB, and theinvariant chain (Ii) mRNAs, and that the induction is restored in somatic cell hybrids containing human chromosome 16. In order to define the gene (designatedF16) affected in the RAG cells, we performed a complementation analysis by fusing RAG with previously described human cell lines defective for MHC class II antigen expression (e.g., BLS cell lines), and which belong to five different complementation groups. Our data show that the resulting somatic cell hybrids present an inducible expression of mouse MHC class II antigens, Ii, DMA, and DMB. Therefore, the RAG cell line represents a yet undescribed cellular mutant affected in the expression of MHC class II antigens. In addition, we demonstrate that MHC class II antigens can be constitutively expressed in the RAG cell line when transfected with the cDNA encoding humanCIITA driven by the RSV LTR promoter. Since the complementation analysis assessed that F16 and CIITA are distinct, our data suggest that F16 is required for the expression of CIITA.

Alterations in cord blood leukocyte subsets of patients with severe hemolytic disease after intrauterine transfusion therapy

OBJECTIVES The aim of this study was to compare, at delivery, the cord blood mononuclear cells of infants with severe hemolytic disease who received intrauterine transfusion (IUT) therapy with the cord blood mononuclear cells of healthy nonimmunized control neonates. STUDY DESIGN The expression of leukocyte markers on CBMNC of 14 IUT-treated and 18 control neonates was analyzed by means of a panel of well-defined monoclonal antibodies and flow cytometry. RESULTS Patients with severe hemolytic disease requiring IUT treatment displayed significant altered expression of some leukocyte markers when compared with control subjects. The circulating CD34+ progenitor cells were significantly increased in comparison with cord blood of nonimmunized neonates. IUT-treated patients also showed a statistically significant decrease in natural killer (NK) cell associated markers (CD16, CD57, and CD69), which correlated with a lower expression of CD56. In these patients an increased expression of CD3/CD45RO and CD3/CD5 was also noted. Although these latter alterations were statistically significant in a single-parameter analysis, the significance disappeared after multi-parameter analysis because of a loss of statistical power. CONCLUSIONS Compared with nonimmunized healthy newborn infants, patients who underwent IUT also exhibited a down-regulation of NK cells and NK cell associated markers, as well as increased numbers of CD34+ progenitor cells.

Therapeutic Regulation of T Cells in Rheumatoid Arthritis

TCR repertoire studies in RA have yielded conflicting data. These studies were initiated on the premise that clonal expression of T cells at the site of inflammation could serve as a target for immune therapies designed on the basis of the option to inactivate or eliminate the presumed pathogenic T cells. These analyses have demonstrated the existence of a highly diverse overall TCR repertoire on the basis of extensive usage of TCR V genes both in synovial fluid and tissue. However, clusters of RA patients can be recognized who share increased usage frequencies of defined TCR V genes among synovial fluid or synovial tissue lymphocytes. Subsequent analysis of the CDR3 regions among diverse overall TCR repertoires have revealed the presence of conserved amino acid sequences in the CDR3 regions of the variable portions of TCRs in T lymphocytes derived from the site of inflammation. These findings suggest that a selective, antigen-driven expansion of T lymphocytes is occurring in the inflamed joints. Parallel to the TCR-repertoire studies, we investigated whether vaccines prepared from synovial T cells could modulate T-cell reactivity. The studies were based on previous work on TCV in animals, revealing that attenuated non-specific T-cell lines could serve as a vaccine. The results obtained in 13 RA patients showed no clear indication for a cellular or humoral immune response. Our experience with TCV in RA patients showed that this technique is feasible and safe. We found some evidence for a modulated T-cell reactivity both in vivo and in vitro. These results show at least some immunomodulatory effect af T-cell vaccination, although the antigen specificity of the effect of this intervention remains to be shown. Because of the convincing studies in animals and MS patients, further studies in RA should focus on the effect of vaccination using vaccines prepared from disease-inducing cells. In this respect, determination of the CDR3 regions of synovial T cells could lead to the identification of those T cells that are relevant for the disease.

Analysis of early fetal T-cell receptor δ chain in humans

In the mouse it has been found that a number of T-cell receptor (Tcr) gd phenotypes are generated during fetal thymic development. To examine whether such “waves” of Tcrgd phenotypes can be found in man, we studied the V-region usage and junctional diversity of the T-cell receptor δ chain in human fetal and post-partum thymocytes and peripheral blood T cells. Using the polymerase chain reaction (PCR)-amplification technique it was found that in fetal thymocytes of 15–17 weeks of gestation the Tcrd-V3 gene segment was mainly employed, whereas in post-partum thymocytes the Tcrd-V1 gene segment was preferentially used. These Tcrd-V3 transcripts contained only a single D element (Dδ3) and a limited random nucleotide insertion. The Dδ3 element was also present in Tcrd-V3-containing transcripts derived from peripheral blood γδ Tcr+ clones. These data suggest that a wave of Tcr γδ might exist early in human fetal development that preferentially use the Tcrd-V3 gene segment.