Co-expression of HLA-B7 and HLA-B27 alleles is associated with B7-restricted immunodominant responses following influenza infection.

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2-K(-/-)/D(-/-) double-knockout transgenic mice expressing either one or two human MHC-I alleles. We hypothesized that co-expression of different allele combinations figures critically in immunodominance and examined this in influenza-infected, double Tg MHC-I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2-restricted matrix I.58-66, the B7-restricted NP418-426 or the B27-restricted NP383-391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu-infected B7/B27 mice, a significantly reduced level of B27/NP383-restricted CTL response was detected while there was no change in the B7/NP418-restricted CTL response. Flu-specific tetramer studies revealed a partial deletion of Vß8.1(+) NP383/B27-restricted CD8(+) T cells, and a diminished Vß12(+) CD8(+) T-cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co-dominant expression of MHC-I alleles for host immune response to pathogens.

A major costimulatory molecule on antigen-presenting cells, CTLA4 ligand A, is distinct from B7.

CTLA4 ligands are important costimulatory molecules because soluble CTLA4Ig blocks the induction of T cell responses and induces T cell tolerance. As CTLA4 immunoglobulin (CTLA4Ig) binds B7 when the latter is expressed on fibroblasts, it was widely assumed that CTLA4Ig blocks T cell costimulation by blocking the function of B7. Here we show that the major costimulatory ligand bound by CTLA4Ig (which we term CTLA4 ligand A) on antigen-presenting cells are not encoded by the B7 gene. CTLA4 ligand A also differs from B7 in cellular distribution and in the respective levels of expression. Both B7 and CTLA4 ligand A are critically involved in T cell costimulation.

B7 but not intercellular adhesion molecule-1 costimulation prevents the induction of human alloantigen-specific tolerance.

Presentation of antigen by the major histocompatibility complex to T lymphocytes without the requisite costimulatory signals does not induce an immune response but rather results in a state of antigen-specific unresponsiveness, termed anergy. To determine which costimulatory signals are critical for the T cell commitment to activation or anergy, we developed an in vitro model system that isolated the contributions of alloantigen and each candidate costimulatory molecule. Here, we show that transfectants expressing HLA-DR7 and either B7 or intercellular adhesion molecule 1 (ICAM-1) deliver independent costimulatory signals resulting in alloantigen-induced proliferation of CD4-positive T lymphocytes. Although equivalent in their ability to costimulate maximal proliferation of alloreactive T cells, B7 but not ICAM-1 induced detectable interleukin 2 secretion and prevented the induction of alloantigen-specific anergy. These results are consistent with the hypothesis that blockade of the ICAM-1:lymphocyte function-associated 1 pathway results in immunosuppression, whereas blockade of the B7:CD28/CTLA4 pathway results in alloantigen-specific anergy. This approach, using this model system, should facilitate the identification of critical costimulatory pathways which must be inhibited in order to induce alloantigen-specific tolerance before human organ transplantation.

HLA class I heavy-chain gene promoter elements mediating synergy between tumor necrosis factor and interferons.

The cytokines tumor necrosis factor (TNF), beta interferon (IFN-beta), and IFN-gamma increase major histocompatibility complex class I molecule expression. A greater than additive (i.e., synergistic) induction of class I heavy-chain mRNA is observed in HeLa cells treated with TNF in combination with either type of IFN. To define the cis-acting elements mediating cytokine synergy, the promoter of a human major histocompatibility complex class I heavy-chain gene (HLA-B7) was placed in front of a reporter gene and transfected into HeLa cells. Deletion analysis mapped the elements required for synergy to a 40-bp region containing a kappa B-like element, which is necessary for the response to TNF, and an interferon consensus sequence (ICS), which is necessary for the responses to IFNs. When the orientation of these elements was reversed or their normal 20-bp spacing was reduced by 5 or 10 bp, i.e., one half or one full turn of the DNA helix, essentially equivalent responses were obtained, suggesting that these parameters are not critical. In electromobility shift assays, a p50-containing NF-kappa B nuclear factor from TNF-treated cells binds kappa B-containing probes, and ISGF-2 from IFN-gamma-treated cells binds ICS-containing probes. A probe containing both the kappa B and ICS elements (kappa B-ICS) forms a novel complex with nuclear factors isolated from cells treated with both TNF and IFN-gamma; this complex also forms when nuclear factors from individually cytokine-treated cells are mixed in vitro. The natural variant ICS found in HLA-A responds to IFN-gamma and can mediate synergy with TNF. However, the variant kappa B found in HLA-C does not respond to TNF, nor can it mediate synergy between TNF and IFN-gamma. These observations suggest that synergy between TNF and IFNs in the induction of HLA class I gene expression results from the sum of individual interactions of cytokine-activated enhancer-binding factors with the transcription initiation complex.

Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes.

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE.

Functional CD40 ligand is expressed on epidermal Langerhans cells.

Epidermal Langerhans cells (LC) are bone-marrow-derived major histocompatibility complex (MHC) class II antigen-expressing antigen-presenting cells (APC) that comprise 1-3% of total epidermal cells (EC). LC express high levels of MHC class II antigen and augment costimulatory molecules such as B7-1, B7-2 during culture. In a previous report, using purified murine LC, we showed that freshly prepared LC (fLC) do not express CD40, whereas cLC express CD40. Tumor necrosis factor alpha (TNF-alpha) enhanced CD40 expression on LC during culture. We examined the expression of CD40L on LC and found that both fLC and cLC expressed mRNA for CD40L. FACS analysis revealed that cLC cultured for 36 h expressed CD40L but fLC did not. When we examined the cytoplasmic CD40L, however, both fLC and cLC expressed cytoplasmic CD40L. TNF-alpha, which up-regulated CD40 expression on LC during culture, did not modulate CD40L. Co-culture of purified LC ith anti-CD40L markedly inhibited the up-regulation of B7-1 expression on LC and caused partial inhibition of B7-2 expression during culture. These results indicate that CD40L is expressed on cLC, and that CD40L on LC modulates the expression of costimulatory molecules such as B7-1 and B7-2 on LC.

Differential enhancement of HLA-B27 by interferon.

To further examine the role of the HLA-B27 antigen in the pathogenesis of disorders such as anterior uveitis and ankylosing spondylitis, we have measured the level of enhancement of this antigen on peripheral blood lymphocytes and compared it with that of the class I HLA antigens HLA-A2 and HLA-B7 following interferon treatment. We found that the level of enhancement of HLA-B7 was greater than that of HLA-A2 or -B7 following treatment by alpha or gamma interferon (p less than 0.002). Similarly, the level of enhancement of HLA-B7 was greater than that of HLA-A2 (p less than 0.002) (Mann Whitney U-test). A differential enhancement of HLA-B27 by lymphokines may be important in the pathogenesis of HLA-B27-related disorders.

Intracellular cysteine residues in the tail of MHC class I proteins are crucial for extracellular recognition by leukocyte Ig-like receptor 1.

The activity of NK cells is regulated by activating receptors that recognize mainly stress-induced ligands and by inhibitory receptors that recognize mostly MHC class I proteins on target cells. Comparing the cytoplasmic tail sequences of various MHC class I proteins revealed the presence of unique cysteine residues in some of the MHC class I molecules which are absent in others. To study the role of these unique cysteines, we performed site specific mutagenesis, generating MHC class I molecules lacking these cysteines, and demonstrated that their expression on the cell surface was impaired. Surprisingly, we demonstrated that these cysteines are crucial for the surface binding of the leukocyte Ig-like receptor 1 inhibitory receptor to the MHC class I proteins, but not for the binding of the KIR2DL1 inhibitory receptor. In addition, we demonstrated that the cysteine residues in the cytoplasmic tail of MHC class I proteins are crucial for their egress from the endoplasmic reticulum and for their palmitoylation, thus probably affecting their expression on the cell surface. Finally, we show that the cysteine residues are important for proper extracellular conformation. Thus, although the interaction between leukocyte Ig-like receptor 1 and MHC class I proteins is formed between two extracellular surfaces, the intracellular components of MHC class I proteins play a crucial role in this recognition.

Differential expression of major histocompatibility complex class II variants in cells infiltrating the meth A sarcoma.

CD4+ T cells are involved in immune responses against the Meth A sarcoma and infiltrate tumours arising from Meth A cells inoculated intradermally in (BALB/c x C57BL/6)F1 (H-2d/b) mice. In order to investigate whether the prerequisites for tumour antigen presentation to CD4+ T cells are fulfilled within the malignant lesion, expression of class II major histocompatibility complex (MHC) molecules and the costimulatory ligand B7 were examined. The I-Ab and I-Ed allomorphs were abundantly expressed by virtually all B cells and 50% of macrophages infiltrating the tumours. In striking contrast, the I-Ad variant was hardly detectable. This pattern of class II expression appeared to be unique for the tumour microenvironment. Thus the proportion of I-Ad+ spleen B cells and peritoneal macrophages did not significantly differ from the proportion expressing I-Ab and I-Ed, and these extratumoral I-Ad+ cells stained as brightly as did cells from healthy mice. Expression of B7 was weak by tumour-infiltrating cells. The profound capacity of the Meth A sarcoma to confer low local I-Ad and B7 expression might preclude T-cell-dependent anti-tumour immune responses and thus promote survival of malignant cells. Whereas MHC class II genes are generally found to be co-ordinately transcribed, this study demonstrates that the expression of MHC class II allelic variants can be differentially regulated in vivo.

Alloreactive cytotoxic T-lymphocyte-defined HLA-B7 subtypes differ in peptide antigen presentation.

We investigated T-cell-defined HLA-B7 subtypes using cDNA sequencing, analysis of bound peptides, and reactivity with a panel of alloreactive cytotoxic T-lymphocyte (CTL) clones. Three subtypes (HLA-B*0702, HLA-B*0703, and HLA-B*0705) differ in nucleotide and predicted amino acid sequence. CTL reactivity and pooled peptide sequencing show that these three HLA-B7 subtypes bind distinct but overlapping sets of peptides. In particular B*0702 expresses D pocket residue Asp 114 and binds peptides with P3 Arg, whereas B*0705 expresses D pocket residue Asn 114 and binds peptides with P3 Ala, Leu, and Met. Consistent with different peptide-binding specificities, three alloreactive CTL differentiate between cells expressing B*0702, B*0703, and B*0705 by detecting specific peptide/HLA-B7 complexes. In contrast, three other T-cell-defined HLA-B7 subtypes are identical to HLA-B*0702. The B*0702-expressing cell lines are differentiated by two of ten CTL clones. One CTL clone differentiates B*0702-expressing cells by their ability to present peptide antigen. Thus differences in peptide presentation can explain differential CTL recognition of cell lines expressing structurally identical and variant HLA-B7.

Regulation of meiotic chromatin loop size by chromosomal position.

At meiotic prophase, chromatin loops around a proteinaceous core, with the sizes of these loops varying between species. Comparison of the morphology of sequence-related inserts at different sites in transgenic mice demonstrates that loop size also varies with chromosomal geography. Similarly, chromatin loop lengths differ dramatically for interstitially and terminally located hamster telomeric sequences. Sequences, telomeric or otherwise, located at chromosome termini, closely associate with the meiotic proteinaceous core, forming shorter loops than identical interstitial sequences. Thus, we present evidence that different chromatin packaging mechanisms exist for interstitial versus terminal chromosomal regions, which act separately from those operating at the level of the DNA sequence. Chromosomal position plays the dominant role in chromatin packaging.

Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon gamma receptor: evidence for multiple accessory factors.

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes.

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA-liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis.

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.

HLA-B27 modulates the survival of Salmonella enteritidis in transfected L cells, possibly by impaired nitric oxide production.

Reactive arthritis is triggered by certain microbes that cause primary infections mainly on the gastrointestinal or urogenital mucosa. The disease is strongly associated with HLA-B27. Long persistence of causative microbes or their structures in the body has been thought to have an important role in the pathogenesis of reactive arthritis. This suggests that the elimination of the microbes causing reactive arthritis is ineffective or disturbed in HLA-B27-positive individuals developing this complication. We examined the role of the HLA-B27 antigen in microbe-host interaction in vitro by monitoring the invasion and intracellular survival of Salmonella enteritidis in mouse fibroblasts transfected with HLA-B27, HLA-B7, or beta2-microglobulin only. S. enteritidis invaded into all the three transfectants with the same efficiency. However, at 6 and 10 days after incubation, there were more living intracellular Salmonella organisms in HLA-B27 transfectants than in the other transfected cell lines (P < 0.05), suggesting that the bactericidal effect is impaired in these cells. Impaired NO production in HLA-B27-transfected cells was indicated as a possible mechanism, since the amount of nitrite in the supernatants of the Salmonella-infected HLA-B27-transfected cells was smaller than that in the supernatants of the Salmonella-infected HLA-B7- or beta2-microglobulin-transfected cells (P < 0.001). The inhibition of NO synthesis by N-monomethyl-L-arginine resulted in impaired elimination of Salmonella also in HLA-B7and beta2-microglobulin-transfected cells. The inverse correlation between intracellular survival of Salmonella and the amount of nitrite detected in culture supernatants supports the hypothesis that the L-arginine-dependent NO pathway plays an important role in the murine fibroblast response against Salmonella. We suggest that a major histocompatibility complex class I antigen, HLA-B27, may contribute to the intracellular persistence of Salmonella by a mechanism which involves the NO pathway.

Induction of HLA class I mRNA by cytokines in human fibroblasts: comparison of TNF, IL-1 and IFN-beta.

Expression of HLA class I antigens is known to be regulated by various cytokines at both the mRNA and protein levels. We have examined the induction of HLA-B7 by tumor necrosis factor alpha (TNF), interleukin 1 alpha (IL-1) and interferon beta (IFN-beta) in normal human diploid FS-4 fibroblasts. Optimal induction of HLA-B7 by TNF at 24 h was shown to require a continuous presence of TNF. Since TNF also induces IFN-beta in these cells and the latter cytokine itself has the capacity to upregulate HLA class I expression, we investigated the role of autocrine IFN-beta in the induction of HLA-B7 by TNF. Experiments with neutralizing polyclonal antibodies to recombinant IFN-beta showed that the induction of HLA-B7 mRNA by TNF was partially dependent on autocrine IFN-beta. However, TNF and IFN-beta induced HLA-B7 mRNA with similar kinetics and treatment with saturating concentrations of both TNF and IFN-beta resulted in an additive or possibly synergistic response. The latter findings support the idea that induction of HLA class I by TNF is not mediated solely by autocrine IFN-beta produced in response to TNF. In addition, experiments with the protein synthesis inhibitor cycloheximide suggested that the induction of mRNAs for both the heavy and light (beta 2-microglobulin) chains of the HLA class I antigen by TNF did not require de novo protein synthesis. IL-1 was also shown to increase steady-state mRNA levels of HLA-B7 with kinetics similar to those of TNF and IFN-beta in FS-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)