HIV‐1 Nef: Taking Control of Protein Trafficking

The Nef protein of the human immunodeficiency virus is a crucial determinant of viral pathogenesis and disease progression. Nef is abundantly expressed early in infection and is thought to optimize the cellular environment for viral replication. Nef controls expression levels of various cell surface molecules that play important roles in immunity and virus life cycle, by directly interfering with the itinerary of these proteins within the endocytic and late secretory pathways. To exert these functions, Nef physically interacts with host proteins that regulate protein trafficking. In recent years, considerable progress was made in identifying host‐cell‐interacting partners for Nef, and the molecular machinery used by Nef to interfere with protein trafficking has started to be unraveled. Here, we briefly review the knowledge gained and discuss new findings regarding the mechanisms by which Nef modifies the intracellular trafficking pathways to prevent antigen presentation, facilitate viral particle release and enhance the infectivity of HIV‐1 virions.      

Function and dysfunction of CD4+ T cells in the immune response to melanoma

A major difficulty for tumor immunotherapy derives from the phenomenon that the encounter of the immune system with an antigen does not necessarily result in activation, but may also be followed by the induction of tolerance either by anergy or physical deletion. It is well established that the immune system becomes alerted only in the face of danger, i.e. upon ligand recognition in the context of increased expression of costimulatory molecules, adhesion molecules, and MHC molecules on antigen-presenting cells (APC). The pivotal role of CD4⁺ T lymphocytes in this process has been established. However, encounter of CD4⁺ T cells with either MHC class II-expressing melanoma cells or certain tumor antigen-presenting APC has been reported to induce antigen-specific tolerance. Thus, as more is learned about the molecular regulation of immune responses and the role of CD4⁺ T cells in particular, additional strategies to block inhibitory pathways of T-cell activation will be developed. Such strategies are likely to be based on a modulation of the context in which antigen is encountered by the immune system, e.g. in situ cytokine therapy, induction of costimulatory molecules or the simulation of `danger' signals. Received: 20 March 1999 / Accepted: 3 May 1999     

Presence of a helix in human CD4 cytoplasmic domain promotes binding to HIV-1 Nef protein

The Nef proteins of simian and human immunodeficiency viruses are known to directly bind and downregulate the CD4 receptor of infected cells. Recent results suggest that residues forming an alpha-helix N-cap in the CD4 cytoplasmic domain play a role in binding of CD4 to human immunodeficiency virus type 1 Nef protein. We determined the dissociation constants between Nef and several CD4 peptides that contain or do not contain the respective alpha-helix N-cap. Further, we compared helical secondary structure content of these CD4 peptide variants by circular dichroism spectroscopy. We conclude that presence of an alpha-helix in CD4 cytoplasmic domain increases CD4 affinity to Nef. In addition, the amino acid sequence of residues forming the helix N-cap influences CD4 affinity to Nef, too. Finally, the structural changes induced in Nef and CD4 upon binding to each other are investigated.     

Complex formation of CD23/surface immunoglobulin and CD23/CD81/MHC class II on an EBV-transformed human B cell line and inferable role of tetraspanin

CD23, a low-affinity IgE receptor, is a type II transmembrane protein having a C-type lectin domain and it associates noncovalently with MHC class II on B cells. The results of our immunoprecipitation analysis suggest that CD23 co-exists with at least two additional molecules, surface immunoglobulin (sIg) and CD81 (and/or CD9), on the cell surface of L-KT9 cells (an Epstein-Barr virus (EBV)-transformed human B cell line). When both CD23 and sIg molecules were stimulated simultaneously by the corresponding antibodies, a large increase in CD81 in the immunoprecipitation was observed as compared with the case of stimulation by only one antibody. Simultaneous stimulation by anti-CD23 and anti-Ig may mimic the situation of B cells stimulated by an antigen/IgE complex. In addition, a large increase in MHC class II in the immunoprecipitation was also observed by cross-linking of CD23 with anti-CD23 and its second antibody as compared with the case of stimulation by anti-CD23 alone. The cross-linking of CD23 with anti-CD23 and its antibody may mimic the situation of B cells stimulated by an IgE/antigen/IgE complex. Therefore, the complex formation among CD23, sIg, MHC class II, and CD81 on the cell surface of L-KT9 cells by the antigen/IgE or IgE/antigen/IgE complex is most likely to be closely related to B cell regulatory events by signaling through sIg or MHC class II. Tetraspanins such as CD81 and CD9 are thought to be involved in the formation and the preservation of various different membrane complexes consisting of several functional proteins.     

MHC Class I Presentation of an Exogenous Polypeptide Antigen Encoded by the Murine AIDS Defective Virus

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25-mer polypeptide (P12–25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H-2^b) CD8⁺ T cells in vitro. The presentation of P12–25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome-inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA-074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12–25 polypeptide. In fact, when P12–25 was digested with purified cathepsin B in vitro, a highly antigenic 11-mer peptide containing the class I (H-2D^b)-binding motif was obtained. Our results favor the phagosome/macropinosome-to-cytosol-to-endoplasmic reticulum (ER)-to-cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8⁺ T cell immunity in vivo.     

Construction and immunogenicity prediction of Plasmodiumfalciparum CTL epitope minigene vaccine

The minigenes encoding Plasmodium falciparum CTL epitopes restricted to human MHC class I molecular HLA-A2 and HLA-B51, which were both at high frequency among Chinese population, were constructed as mono-epitope CTL vaccines named pcDNA3.1/frand pcDNA3.1/ sh. The minigenes of the two epitopes were then tandem linked to form a dimeric CTL epitope minigene recombinant vaccine. After DNA transfection, the epitope minigenes were expressed respectively in two human cell lines, each bearing one MHC class I molecule named CIR/HLA-A2.1 and K562/HLA-B51. The intraceliular expression of the CTL epitope minigenes not only enhanced the stability of HLA-A2.1 and HLA-B51 molecules but also increased the assemblage of MHC class I molecules on cell surfaces, which testified the specific process and presentation of those endogenous expressed epitopes. For the cells transfected with the dimeric minigene encoding two tandem linked epitopes, the expression and presentation of each epitope were also detected on cell membran     

Sequence, linkage to H2-K, and function of mouse tapasin in MHC class I assembly

 Assembly of major histocompatibility complex (MHC) class I molecules in human cells is dependent on the accessory protein tapasin, which mediates their interaction with the transporters associated with antigen processing (TAP) and thereby ensures efficient peptide binding. Analysis of a mouse tapasin complementary DNA defined a conserved polypeptide sharing sequences diagnostic of a transmembrane protein related to the immunoglobulin superfamily, and an endoplasmic reticulum retention motif. The mouse tapasin gene was mapped about 70 kilobases from H2-K at the centromeric end of the mouse MHC. Expression of mouse tapasin in a tapasin-deficient human mutant cell line restored the normal assembly and expression of class I alleles. Thus, tapasin is a structurally and functionally conserved component of the MHC class I antigen processing pathway. Its genetic linkage to the class I and TAP subunit genes in the MHC may be of significance in the coordinate expression and functional coadaptation of the diverse gene products. Received: 1 February 1998 / Revised: 23 March 1998     

A geometric study of the amino acid sequence of class I HLA molecules

 HLA class I alleles are studied by representing them in a metric space where each dimension corresponds to each one of the amino acid positions. Their similarity in reference to their ability to present peptides to T cells is then evaluated by calculating the correlation matrix between the amino-acid-composition tables (or binding affinity tables) for the sets of peptides presented by each allele. This correlation matrix is considered an empirical similarity matrix between HLA alleles, and is modeled in terms of possible structures defined in the metric space of HLA class I amino acid sequences. These geometric structures are adequate models of the peptide-binding data currently available. The following clusters of HLA class I molecules are identified in reference to their ability to present peptides: Cluster I) HLA-A3/ HLA-A11/ HLA-A31/ HLA-A33/ HLA-A68; Cluster II) HLA-B35/ HLA-B51/ HLA-B53/ HLA-B54/ HLA-B7; and Cluster III) HLA-A29/ HLA-B61/HLA-B44; the last cluster showing possible similarities between alleles from different loci. In modeling these natural clusters, the geometric structures with more predictive power confirm the importance of those positions in the peptide-binding groove, particularly those in the B pocket. In addition, other positions (46, 79, 113, 131, 144, and 177) appeared to bear some relevance in determining which peptides can be presented by which HLA alleles. Received: 20 January 1998 / Revised: 30 March 1998     

HIV/AIDS患者CD4水平对甲状腺功能影响的比较研究

目的观察HIV/AIDS患者甲状腺功能的变化,了解HIV对内分泌的影响。方法检测168例HIV/AIDS患者TT4、TT3、FT4、FT3、TSH,并分析不同CD4水平对甲状腺功能影响。结果HIV/AIDS患者甲状腺功能可保持正常,但低CD4组的FT3、TSH水平低于高CD4组。结论HIV/AIDS患者甲状腺功能可保持正常,但HIV感染不同阶段会对内分泌系统有一定影响。HIV感染对甲状腺影响的具体机制尚不十分清楚,利用动物模型进行进一步研究非常必要。     

Crystal Structure of Myeloid Cell Activating Receptor Leukocyte Ig-like Receptor A2 (LILRA2/ILT1/LIR-7) Domain Swapped Dimer: Molecular Basis for Its Non-binding to MHC Complexes

The leukocyte Ig-like receptor (LILR/ILT/LIR) family comprises 13 members that are either activating or inhibitory receptors, regulating a broad range of cells in the immune responses. LILRB1 (ILT2), LILRB2 (ILT4) and LILRA1 (LIR6) can recognize MHC (major histocompatibility complex) class I or class I-like molecules, and LILRB1/HLA-A2, LILRB1/UL18 and LILRB2/HLA-G complex (extracellular domains D1D2) structures have been solved recently. The details of binding to MHC have been described. Despite high levels of sequence similarity among LILRA1, LILRA2 (ILT1), LILRA3 (ILT6) and LILRB1/B2, all earlier experiments showed that LILRA2 does not bind to MHC, but the reason is unknown. Here, we report the LILRA2 extracellular D1D2 domain crystal structure at 2.6 Å resolution, which reveals structural shifts of the corresponding MHC-binding amino acid residues in comparison with LILR B1/B2, explaining its non-binding to MHC molecules. We identify some key residues with great influence on the local structure, which exist only in the MHC-binding receptors. Moreover, we show that LILRA2 forms a domain-swapped dimer. Further work with these key swapping residues yields a monomeric form, confirming that the domain-swapping is primarily amino acid sequence-specific. The structure described here supports the dimer conformation in solution observed earlier, and implies a stress-induced regulation by dimerization, consistent with its function as a heat shock promoter.     

Recognition of naturally processed and ovarian cancer reactive CD8<Superscript>+</Superscript> T cell epitopes within a promiscuous HLA class II T-helper region of NY-ESO-1

NY-ESO-1 is frequently expressed in epithelial ovarian cancer (EOC) and elicits spontaneous humoral and cellular immune responses in a proportion of EOC patients. The identification of NY-ESO-1 peptide epitopes with dual HLA-class I and class II specificities might be useful in vaccination strategies for generating cognate CD4+ T cell help to augment CD8+ T cell responses. Here, we describe two novel NY-ESO-1-derived MHC class I epitopes from EOC patients with spontaneous humoral immune response to NY-ESO-1. CD8+ T cells derived from NY-ESO-1 seropositive EOC patients were presensitized with a recombinant adenovirus encoding NY-ESO-1or pooled overlapping peptides. These epitopes, ESO127-136 presented by HLA-A68 molecule, and ESO127-135 restricted by HLA-Cw15 allele, are located within ESO119-143, a promiscuous HLA-class II region containing epitopes that bind to multiple HLA-DR alleles. The novel epitopes were naturally processed by APC or naturally presented by tumor cell lines. In addition, these epitopes induced NY-ESO-1-specific CTL in NY-ESO-1 seropositive EOC patients. Together, the results indicate that ESO119-143 epitope has dual HLA classes I and II specificities, and represents a potential vaccine candidate in a large number of cancer patients.     

Coreceptor function of CD4 in response to the MHC class I molecule

The specificity of the T-cell receptor (TCR) and its interaction with coreceptors play a crucial role in T-cell passing through developmental checkpoints and, eventually, determine the efficiency of adaptive immunity. The genes for the α and β chains of TCR were cloned from T-cell hybridoma 1D1, which was obtained by fusion of BWZ.36CD8α cells with CD8⁺ memory cells specific for the H-2K^b MHC class I molecule. Retroviral transduction of the 1D1 TCR genes and the CD4 and CD8 coreceptor genes was used to obtain 4G4 thymoma variants that exposed the CD3/TCR complex together with CD4, CD8, or both of the coreceptors on their surface. Although the main function of CD4 is to stabilize the interaction of TCR with MHC class II molecules, CD4 was found to mediate the activation of transfected cells via TCR specific for the H-2K^b MHC class I molecule. Moreover, CD4 proved to dominate over CD8, since the response of CD4⁺CD8⁺ transfectants was suppressed by antibodies against CD4 and the A^b MHC class II molecule but not to CD8. The response of CD4⁺ transfectants was not due to a cross-reaction of 1D1 TCR with MHC class II molecules, because the transfectants did not respond to splenocytes of H-2^b knockout mice, which were defective in the assembly of the MHC class I molecule/β2 microglobulin/peptide complex and did not expose the complex on cell surface. The domination was not due to sequestration of p56^lck kinase, since CD4 devoid of the kinase-binding site was functional in 4G4 thymoma cells. The results were used to explain some features of intrathymic cell selection and assumed to provide an experimental basis for developing new methods of anticancer gene therapy.     

Transfer of GRP94(Gp96)-associated peptides onto endosomal MHC class I molecules

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the K^b-SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I.     

Receptor-mediated ER export of human MHC class I molecules is regulated by the C-terminal single amino acid

Major histocompatibility complex class I (MHC-I) molecules bind antigens in the endoplasmic reticulum (ER) and deliver them to the cell surface for immune surveillance of viruses and tumors. Whereas key steps of MHC-I assembly and its acquisition of peptides in the ER are relatively well defined, little is known about how MHC-I molecules leave the ER for cell surface expression. Here, we show that ER export of human classical MHC-I molecules (HLA-A/-B/-C) is regulated by their C-terminal single amino acid, valine or alanine. These amino acids, conserved in nearly all known human MHC-I alleles, serve as the ER export signal by binding to the Sec23/24 complex, a structural component of coat protein complex II (COPII) vesicles that mediate ER-to-Golgi trafficking. Together, our results strongly suggest that ER export of human classical MHC-I molecules can occur via a receptor-mediated process dictated by a highly conserved ER export signal.     

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4⁺ and/or CD8⁺ T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4⁺ T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8⁺ T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4⁺ T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8⁺ T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4⁺ T cell-mediated antitumor activity in vivo.     

Effect of invariant chain on major histocompatibility complex class I molecule expression and stability on human breast tumor cell lines

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.     

Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation

Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were implicated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting properties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic alloresponses refractory to current immunosuppression.     

Down-regulation of HLA class II and costimulatory CD86/B7-2 on circulating monocytes from melanoma patients

Antigen-presenting cells are crucial for the induction of an antigen-specific antitumoral immune response. Deteriorations in the expression pattern of cell surface molecules important for the presentation of antigens might therefore be indicative of an impaired immune response status in cancer patients. In the present study we investigated the expression of MHC class I and class II molecules, of the costimulatory molecules CD80/B7-1 and CD86/B7-2, of the adhesion molecule CD11c, and of the marker of activation CD71 on CD14⁺ peripheral blood monocytes (PBMs) from 144 melanoma patients in different stages of disease and 43 healthy controls, by flow cytometric analysis. We found a decreased expression of HLA-DR (p<0.0005), HLA-DQ (p=0.006), HLA-DP (p<0.0005), and CD86/B7-2 (p=0.001) on PBMs from melanoma patients compared with healthy controls, whereas no significant difference could be detected in the expression of HLA class I antigens and CD80/B7-1. This down-regulated expression was associated with disease progression. In contrast, CD71 expression was stage-dependently increased on PBMs from melanoma patients compared with healthy controls (p=0.024). No correlation was found between the PBM surface expression pattern and age, gender, tumor load, and current mode of therapy of the patients. The observed down-regulation of HLA class II and CD86/B7-2 on melanoma patients PBMs might reflect an ineffective antigen-presenting function contributing to an impaired antigen-specific immune response in these patients.Both authors S. Ugurel and D. Uhlig contributed equally to this work