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Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4 + T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca 2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca 2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4 + T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4 + T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4 + T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.

CD4 + Foxp3 + regulatory T (Treg) cells are central modulators of autoimmune diseases. However, the timing and location of Treg cell-mediated suppression of tissue-specific autoimmunity remain undefined. Here, we addressed these questions by investigating the role of tumor necrosis factor (TNF) receptor 2 (TNFR2) signaling in Treg cells during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We found that TNFR2-expressing Treg cells were critical to suppress EAE at peak disease in the central nervous system but had no impact on T cell priming in lymphoid tissues at disease onset. Mechanistically, TNFR2 signaling maintained functional Treg cells with sustained expression of CTLA-4 and Blimp-1, allowing active suppression of pathogenic T cells in the inflamed central nervous system. This late effect of Treg cells was further confirmed by treating mice with TNF and TNFR2 agonists and antagonists. Our findings show that endogenous Treg cells specifically suppress an autoimmune disease by acting in the target tissue during overt inflammation. Moreover, they bring a mechanistic insight to some of the adverse effects of anti-TNF therapy in patients., Competing Interests: The authors declare no competing interest.

The present study evaluates the impact of immune cell populations on metastatic development in a model of spontaneous melanoma [mice expressing the human RET oncogene under the control of the metallothionein promoter (MT/ret mice)]. In this model, cancer cells disseminate early but remain dormant for several weeks. Then, MT/ret mice develop cutaneous metastases and, finally, distant metastases. A total of 35% of MT/ret mice develop a vitiligo, a skin depigmentation attributable to the lysis of normal melanocytes, associated with a delay in tumor progression. Here, we find that regulatory CD4(+) T cells accumulate in the skin, the spleen, and tumor-draining lymph nodes of MT/ret mice not developing vitiligo. Regulatory T-cell depletion and IL-10 neutralization led to increased occurrence of vitiligo that correlated with a decreased incidence of melanoma metastases. In contrast, inflammatory monocytes/dendritic cells accumulate in the skin of MT/ret mice with active vitiligo. Moreover, they inhibit tumor cell proliferation in vitro through a reactive oxygen species-dependent mechanism, and both their depletion and reactive oxygen species neutralization in vivo increased tumor cell dissemination. Altogether, our data suggest that regulatory CD4(+) T cells favor tumor progression, in part, by inhibiting recruitment and/or differentiation of inflammatory monocytes in the skin.

Understanding the complexity and dynamics of cancer cells in response to effective therapy requires hypothesis-driven, quantitative, and high-throughput measurement of genes and proteins at both spatial and temporal levels. This study was designed to gain insights into molecular networks underlying the clinical synergy between retinoic acid (RA) and arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), which results in a high-quality disease-free survival in most patients after consolidation with conventional chemotherapy. We have applied an approach integrating cDNA microarray, 2D gel electrophoresis with MS, and methods of computational biology to study the effects on APL cell line NB4 treated with RA, ATO, and the combination of the two agents and collected in a time series. Numerous features were revealed that indicated the coordinated regulation of molecular networks from various aspects of granulocytic differentiation and apoptosis at the transcriptome and proteome levels. These features include an array of transcription factors and cofactors, activation of calcium signaling, stimulation of the IFN pathway, activation of the proteasome system, degradation of the PML-RARalpha oncoprotein, restoration of the nuclear body, cell-cycle arrest, and gain of apoptotic potential. Hence, this investigation has provided not only a detailed understanding of the combined therapeutic effects of RA/ATO in APL but also a road map to approach hematopoietic malignancies at the systems level.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.

Gene sequences highly similar to major histocompatibility complex (Mhc) class I and class II genes were recently recognized as mapping to a site in the genome of the chicken separate from the Mhc class I, class II, and B-G genes of the major histocompatibility (B) complex. The present study was undertaken to see whether this complex of Mhc-like genes designated as restriction fragment pattern Y (Rfp-Y) might reside in one of three clusters of cosmid clones contained within the molecular map of chicken Mhc genes, since only two of the three clusters can be assigned to the B system. To determine whether the third cluster (cluster II/IV) might contain Rfp-Y, a subclone (18.1) from within cluster II/IV near a polymorphic lectin gene was used to analyze the DNA of families in which Rfp-Y haplotypes are known to be segregating. The restriction fragment polymorphisms revealed by the 18.1 probe were found to segregate in parallel with the restriction fragment polymorphisms defining the Rfp-Y haplotypes, thus establishing the location of Rfp-Y within cosmid cluster II/IV. Two of six Mhc class I genes and two of five Mhc class II genes map to cosmid cluster II/IV, so a substantial fraction of chicken Mhc genes, including at least one that may be expressed, are located in a chromosomal region separate from the B system. In further linkage analyses, Rfp-Y was found to assort independently from more than 400 markers in the present linkage map of the chicken genome.

The low-affinity interactions underlying antigen recognition by T-cell receptors (TCRs) are thought to involve antigen-independent adhesion mechanisms. Using a hydrodynamic approach, we found that antigen-independent adhesion occurred between human B cells and resting T cells in a transient and temperature-dependent fashion. The mean cell-cell adhesion force was 0.32 x 10(-9) N and was generated by similar contributions (0.16 x 10(-9) N) of the LFA-1- and CD2-dependent adhesion pathways. After T-cell stimulation with a phorbol ester, the force contributed by LFA-1 was drastically increased, while that of CD2 was unaffected. We propose that weak receptor-mediated adhesion initiates antigen-independent intercellular contacts required for antigen recognition by the TCR and is upregulated following TCR engagement. The method used permits adhesion forces between living cells to be resolved at the molecular level and should prove valuable for the rapid assessment of interaction forces between various types of cells and cell-sized particles.

CD4, a cell surface glycoprotein expressed primarily by T lymphocytes and monocytes, interacts with HLA class II antigens to regulate the immune response. In AIDS, CD4 is the receptor for the human immunodeficiency virus, which binds to CD4 through envelope glycoprotein gp120. Delineation of the ligand-binding sites of CD4 is necessary for the development of immunomodulators and antiviral agents. Although the gp120 binding site has been characterized in detail, much less is known about the class II binding site, and it is as yet uncertain whether they partially or fully overlap. To investigate CD4 binding sites, a cellular adhesion assay between COS cells transiently transfected with CD4 and B lymphocytes expressing HLA class II antigens has been developed that is strictly dependent on the CD4--class II interaction, quantitative, and highly reproducible. Mutants of CD4 expressing amino acids with distinct physicochemical properties at positions Arg-54, Ala-55, Asp-56, and Ser-57 in V1, the first extracellular immunoglobulin-like domain, have been generated and studied qualitatively and quantitatively for interaction with HLA class II antigens, for membrane expression, for the integrity of CD4 epitopes recognized by a panel of monoclonal antibodies, and for gp120 binding. The results obtained show that the mutations in this tetrapeptide, which forms the core of a synthetic peptide previously shown to have immunosuppressive properties, affect the two binding functions of CD4 similarly, lending support to the hypothesis that the human immunodeficiency virus mimicks HLA class II binding to CD4.

The structure of a chicken CD3 chain has been determined by isolating a cDNA clone (T11.15) that encodes a 175-amino-acid-long protein, including the NH2-terminal signal peptide. In Northern blot experiments, the earliest expression of the T11.15 transcript was detected in the thymus at embryonic day 10 (i.e., 1 day after cytoplasmic expression of a CD3 epitope recognized by a specific monoclonal antibody [CT3; Chen, C.L.H., Ager, L.L., Gartland, G.L. & Cooper, M.D. (1986) J. Exp. Med. 164, 375-380], but 2 days before the appearance of clonotypic components of the T-cell antigen receptor). Sequence similarity of this chicken protein sequence compared with that of the known mammalian CD3 gamma and delta polypeptides was 36-39% and 39-40%, respectively. Amino acid sequence alignments between avian and mammalian CD3 revealed maximum conservation in the transmembrane and cytoplasmic domains as well as in the regions flanking the cysteine residues in the extracellular domain, underlining their functional importance. The difficulty of unambiguously assigning this chain to a single mammalian CD3 subunit on the basis of sequence comparison raises the possibility that this polypeptide represents a derivative from an ancestral form of the gamma and delta chains. It is thus possible that a single chain may play the role of both CD3 gamma and delta subunits in the chicken CD3 complex or, alternatively, that gene duplications occurred independently in the avian and mammalian lineages.

A cDNA library has been constructed from a B cell mRNA fraction enriched for HLA-DR sequences, and cDNA clones corresponding to sequences specifically expressed in B lymphocytes have been isolated by a differential screening procedure. Analysis of these clones with probes specific for the HLA-DR heavy chain gene allowed the characterization of HLA-DR heavy chain-related sequences. One clone, pDCH1, was demonstrated to encode the DC1 heavy chain because the amino acid sequence predicted from its nucleotide sequence matches eight out of nine residues available for comparison in the amino-terminal sequence of the DC1 heavy chain. The heavy chain of the DC1 alloantigen is composed of 232 amino acids and can be divided into two external domains, alpha 1 (amino acids 1-87) and alpha 2 (amino acids 88-181), a connecting peptide (amino acids 182-194), a hydrophobic transmembrane region (amino acids 195-217), and an intracytoplasmic region (amino acids 218-232). Comparison with the HLA-DR heavy chain reveals strong sequence homology in the second external Ig-like domain (alpha 2) and the transmembrane region. In contrast, the first external domain, the connecting peptide, and the intracytoplasmic region are less conserved.

A homozygous a2/a2 and b4/b4 rabbit has been hyperimmunized with Micrococcus lysodeikticus. Poly(A)-containing RNA has been isolated from the spleen and translated in vitro, and translation products have been analyzed by NaDodSO4/polyacrylamide gel electrophoresis. Double-stranded cDNA has been synthesized from poly(A)-containing RNA template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC).oligo(dG) tailing procedure. Tetracycline-resistant ampicillin-sensitive clones containing cDNA complementary to a kappa light chain mRNA have been selected by differential screening and their ability to hybridize to a spleen mRNA having the same size as a mouse kappa light chain mRNA. Two clones, pRk-15 and pRk-32, have been selected to determine the nucleotide sequence of the constant and 3' untranslated regions of kappa light chain mRNA, by the Maxam and Gilbert partial degradation method. Comparison of homologous regions of mouse kappa chain mRNA and b4 rabbit kappa chain mRNA reveals 61% homology in the constant region and 59% homology in the 3' untranslated region.

A class II antigen beta-chain cDNA clone was isolated from a human B-cell cDNA library by using as a probe the murine I-A beta gene. This cDNA clone, pHA beta, was shown to be distinct from the DC beta- and DR beta-related loci by DNA sequence analysis, thus suggesting that it might correspond to a third polymorphic human class II locus, SB, which encodes secondary B-cell antigens. Genetic mapping of this beta-chain cDNA clone to the SB region was performed by the blot hybridization procedure. We showed that (i) within panels of HLA-DR homozygous human B-cell lines and of unrelated individuals who have been typed for HLA antigens, differential mobility of DNA fragments segregated with distinct SB genotypes; (ii) gamma-ray-induced deletion mutants that have lost the expression of DR or DC/MT antigens but maintain SB expression preserved a pattern consistent with (a) their SB phenotype and (b) the genetic independence of the SB locus with respect to DR and DC/MT; and (iii) within an informative family, two siblings differing only for one allele at the SB locus (because of the occurrence of an internal recombination between DR and GLO) and otherwise HLA identical exhibited a restriction enzyme polymorphism linked to the SB locus. Therefore, all available data are compatible with identity between HA beta and SB beta.

Cellular DNAs from individuals of 23 families were digested with five restriction endonucleases (Pvu II, EcoRI, HindIII, BamHI, and EcoRV) and then probed with a DX alpha-chain gene probe. Seventeen allogenotopes were observed, each of which could be assigned to a serologically defined haplotype by noting its segregation in families. Six sets of allogenotopes forming allelic series were noted. In comparison with restriction maps of the DQ alpha and the DX alpha regions, each of these series has been assigned to the DQ alpha or the DX alpha locus. Allogenotopes of the four DQ alpha series constitute three clusters correlating with the supertypic groups of class II histocompatibility antigens DQw1 (DR1, DR2, and DRw6), DRw53 (DR4, DR7, and DR9), and DR3 plus DR5 plus DR8. These 13 DQ alpha fragments constitute 22 different patterns. The two DX alpha series constitute two clusters, one of which is not found to be correlated strongly with DR specificities, whereas the other is correlated loosely (r = 0.45) with DR5 and DR7. This absence of strong linkage disequilibrium between the DX alpha series and the DR series contrasts with the DQ alpha series and suggests a recombination point between DQ alpha and DX alpha loci.

Several genes were found closely associated with major histocompatibility class I and class II beta-chain genes in chicken genomic DNA clusters by hybridizing tissue-specific cDNA probes to cosmid clones. A cDNA probe for one of these genes, probe C12.3 isolated from a chicken liver cDNA library, was used to clone the homologous sequence H12.3 from a human B-lymphoblastoid cell line cDNA library. C12.3 and H12.3 encode exactly the same 317-residue-long protein. The sequence of 12.3 shows significant homology with the two known guanine nucleotide-binding protein beta subunits (GP beta 1 and GP beta 2) and other proteins that all share the same segmented structure with seven internal homologous repeats about 45 residues in length. Unlike the chicken gene, the human H12.3 gene and its mouse counterpart are not located on the same chromosome as the major histocompatibility complex. A possible involvement of the C12.3 gene product in major histocompatibility complex-linked control of lymphocyte proliferation in chickens is discussed.

The DC and DX subregions of the human major histocompatibility complex (MHC) have been cloned from a cosmid library made from a human B-cell line, Priess. The DC subregion, 48 kilobases, includes the DC alpha and DC beta genes. A second DC-like region, the DX subregion, 35 kilobases, contains the DX alpha gene and a newly found beta gene termed DX beta. Since the DC and DX genes are highly homologous in nucleotide sequence, gene size, exon-intron organization, and direction of transcription, the DC and DX subregions were presumably generated by duplication of an ancestral alpha-beta gene pair. Nucleotide sequencing indicates that all four genes have intact coding sequences and promoter regions. Homology between the upstream promoter sequences of these four genes and seven other class II genes at nucleotides -69 to -78 and -98 to -110 highlights these previously described conserved elements. Moreover, a striking conservation of flanking alpha-gene-specific and beta-gene-specific sequences has been observed. Comparison of Southern blots of Priess DNA with DC alpha and DC beta cDNA probes with isolated cosmid clones showed that (i) the human chromosome encodes only two DC alpha-related and two DC beta-related genes, namely, DC alpha, DX alpha, DC beta, and DX beta, and (ii) the DC and DX subregions are homozygous in Priess cells.

A 23-kilobase EcoRI DNA fragment coding for the BALB/c immunoglobulin gamma 2a chain was cloned from mouse embryo DNA in the cosmid pJC74, and a nucleotide sequence of 1904 bases was determined for the entire constant region (CH1, CH2, and CH3), the three intervening sequences (IVS 1, IVS 2, and IVS 3) and the 5' and 3' flanking sequences. When the gamma 2a chain nucleotide sequence was compared with the gamma 2b chain nucleotide sequence, the percent homology of corresponding segments (excluding deletion and insertion) was 82% for the 5' flanking sequence, 87% for CH1, 84% for IVS 1, 96% for the hinge, 95% for IVS 2, 94.6% for CH2, 86% for IVS 3, 74% for CH3, 89% for the 3' untranslated region, and 92% for the 3' flanking region. These findings show that different domains of gamma 2a and gamma 2b have independent rates of evolution and that some of the noncoding segments of the gene are more conserved than are adjacent coding segments. Hypotheses on the possible role of IVS is gene evolution and expression are discussed.

The amino acid sequence of the heavy chain of HLA-DR antigens has been elucidated from the analysis of a genomic clone coding for this protein. A 3.2-kilobase EcoRI fragment includes four exons containing 227 amino acids out of 229 in the mature HLA-DR heavy chain. One exon (alpha 2) encodes a domain of 94 amino acids with strong sequence homology both to Ig constant region domains and to Ig-like domains in HLA-B7, beta 2-microglobulin, and the HLA-DR light chain. These results support a structure for the HLA-DR antigen heterodimer consisting of four extracellular domains, two of which are Ig-like [one in the heavy chain (alpha 2) and one in the light chain (beta 2)]. The third is the amino-terminal polymorphic domain in the light chain (beta 1), and the fourth is an invariant domain in the heavy chain (alpha 1).