抗独特型抗体与T细胞记忆的关系

记忆T细胞作为人体免疫系统中的一个组成部分,在免疫应答中发挥着至关重要的作用,因此利用抗独特型抗体制备诱导产生记忆T细胞的疫苗是免疫学领域的一个重要方向。抗独特型抗体Fab段具有与特异性抗原相似的抗原决定簇的结构,其作为抗原替代物制备的疫苗所激发机体产生的记忆T细胞具有特异性强和安全性高的特点,成为一种比较理想的疫苗.就抗独特型抗体与T细胞记忆之间的联系及其应用效果作一简要综述。     

Isolation and characterization of a monoclonal antibody that inhibits HIV-1 infection

To identify a cell surface molecule other than CD4 involved in infection of cultured cells with human immunodeficiency virus type 1 (HIV-1), mice were immunized with the CD4-negative Raji human B-cell line in order to isolate a monoclonal antibody (mAb). We isolated mAb 33A, which inhibited the infection of CD4-positive T cells, B cells, human peripheral blood lymphocytes (PBL), and brain-derived cells with HIV-1. Formation of viral DNA was also blocked when CD4-positive Raji cells were treated with 33A after adsorption of HIV-1, but not before its adsorption. mAb 33A had little effect on syncytium formation induced by cocultivation with HIV-1-producing cells. Flow cytometry revealed that 33A reacted with HTLV-I-positive T-cell lines, Burkitt's lymphoma cell lines, phytohemagglutinin (PHA) -stimulated PBL, brain-derived fibroblast-like cells, and some adherent cell lines, but hardly at all with immature T-cell lines. Immunoblotting experiments showed that 33A recognized an antigen with an apparent molecular mass of 32 kDa, but did not recognize chemokine receptors such as CXCR4, CCR5, or CCR3. The distribution characteristic of the antigen recognized by 33A on various cells and its molecular weight suggest that mAb 33A recognizes a new cellular antigen that is necessary for HIV-1 entry.     

Inefficient cytotoxic T lymphocyte-mediated killing of HIV-1-infected cells in vivo

Understanding the role of cytotoxic T lymphocytes (CTLs) in controlling HIV-1 infection is vital for vaccine design. However, it is difficult to assess the importance of CTLs in natural infection. Different human leukocyte antigen (HLA) class I alleles are associated with different rates of progression to AIDS, indicating that CTLs play a protective role. Yet virus clearance rates following antiretroviral therapy are not impaired in individuals with advanced HIV disease, suggesting that weakening of the CTL response is not the major underlying cause of disease progression and that CTLs do not have an important protective role. Here we reconcile these apparently conflicting studies. We estimate the selection pressure exerted by CTL responses that drive the emergence of immune escape variants, thereby directly quantifying the efficiency of HIV-1-specific CTLs in vivo. We estimate that only 2% of productively infected CD4+ cell death is attributable to CTLs recognising a single epitope. We suggest that CTLs kill a large number of infected cells (about 10(7)) per day but are not responsible for the majority of infected cell death.     

Partial escape of HIV-1 from cytotoxic T lymphocytes during chronic infection

Viral mutational escape from CD8(+) cytotoxic T lymphocytes (CTLs) is typically considered to be a dichotomous process and uncommon during chronic HIV-1 infection. Ex vivo passaging of HIV-1 from persons with chronic infection, however, revealed the evolution of many fixed substitutions within and around CTL-targeted regions, with an associated increase in replicative capacity. This indicates an evolution of mutations during chronic HIV-1 infection that trade replicative fitness for incomplete evasion of CTLs, or "partial escape."     

The role of activated cytotoxic T cells in inflammatory bowel disease

The role of cell-mediated cytotoxicity in the pathogenesis of ulcerative colitis and Crohn's disease has been controversial since reports indicating either a decreased, or an increased, activity of cytotoxic T cells in active stages of inflammatory bowel disease exist. Some of these discrepancies may be attributed to the fact that so far mostly peripheral blood lymphocytes rather than intestinal T cells have been examined. To overcome some of these limitations we performed in situ hybridizations for the detection of perforin and granzyme A mRNA expressing cells, i.e. of cytotoxic cells activated in situ, in the affected intestinal mucosa. These studies revealed increased frequencies of activated, cytotoxic T cells in active stages of ulcerative colitis and Crohn's disease. Interestingly, activated perforin mRNA expressing T cells are present both in the CD4 and in the CD8 T cell subsets. In the latter T cell subset up to 60% of the mucosal T cells isolated from the affected sites express perforin mRNA at detectable levels. The elevated frequency of activated cytotoxic cells and their histological distribution also in close proximity to the epithelial cells may thus indicate an important role for cytotoxic cells in the pathogenesis of inflammatory bowel disease since activated cytotoxic T cells may further exacerbate the inflammatory process through the production of pro-inflammatory cytokines such as interferon-gamma or tumor necrosis factor-alpha, but also through the release of pro-inflammatory cytokines and chemokines upon lysis of epithelial cells and the increased influx of luminal antigens at the site of epithelial erosions.     

Dynamics of CD4+ T cells in HIV-1 infection

Mathematical modeling is becoming established in the immunologist's toolbox as a method to gain insight into the dynamics of the immune response and its components. No more so than in the case of the study of human immunodeficiency virus (HIV) infection, where earlier work on the viral dynamics brought significant advances in our understanding of HIV replication and evolution. Here, I review different areas of the study of the dynamics of CD4+ T cells in the setting of HIV, where modeling played important and diverse roles in helping us understand CD4+ T-cell homeostasis and the effect of HIV infection. As the experimental techniques become more accurate and quantitative, modeling should play a more important part in both experimental design and data analysis.     

The HIV-1 Tat protein selectively enhances CXCR4 and inhibits CCR5 expression in megakaryocytic K562 cells

The hematopoietic compartments act as long-term reservoirs for human immunodeficiency virus type-1 (HIV-1). Although hematopoietic progenitor cells (HPCs) are rarely infectable, HPCs committed to the megakaryocytic lineage can be infected and support a productive infection by both the X4 and R5 strains of HIV-1. Indeed, in contrast to the CD34+ progenitors, the lineage-committed HPCs express high levels of the HIV-1 co-receptors, CXCR4 and CCR5. The HIV-1 transactivator (Tat) protein has been shown to alter co-receptor expression in T lymphocytes and macrophages. We hypothesized that Tat may regulate co-receptor expression in lineage-specific HPCs as well. We have monitored the effects of Tat protein on co-receptor expression and on lineage-specific differentiation, using the HPC cell line, K562. Butyric acid (BA)-induced erythroid differentiation in K562 cells was suppressed by 1-100 ng/ml of Tat, as evident from a 70-80% decrease in hemoglobin (Hb) production and a 10-30-fold decrease in glycophorin-A expression. However, Tat treatment enhanced phorbol myristate acetate (PMA)-induced megakaryocytic differentiation, as evident from a 180-210% increase in 3H-serotonin uptake and a 5-12-fold increase in CD61 expression. Tat did not significantly alter co-receptor expression in erythroid cells. However, Tat co-treatment profoundly effected both CXCR4 and CCR5 gene expression and protein levels in megakaryocytic cells. In PMA-stimulated cells, Tat increased CXCR4 and decreased in CCR5 expression, this was potentiated in cells chronically exposed to Tat. In conclusion, Tat protein suppresses erythroid and facilitates megakaryocytic differentiation of K562 cells. In megakaryocytic cells, Tat differentially effected CXCR4 and CCR5 expression. Because megakaryocytes may play a crucial role in HIV-1 infectivity in viral reservoirs, our findings implicate a role for Tat protein in dictating co-receptor usage in lineage-committed HPCs.     

Monoclonal anti-idiotypic antibody mimicking the principal neutralization site in HIV-1 GP120 induces HIV-1 neutralizing antibodies in rabbits

A murine mAb BAT123 (Ab1) directing to the principal neutralization site of human T cell leukemia virus (HTLV)-IIIB gp120 (amino acid residue 308-322) was used to generate syngeneic anti-Id mAb (Ab2). Among the Ab2, a mAb AB19-4 was characterized by both serologic and biologic methods to be paratope-specific (Ab2 beta), bearing the internal image of the neutralization site. AB19-4 was found to bind specifically to BAT123 and also to its mouse-human chimeric form in ELISA. The binding of AB19-4 to BAT123 was specifically inhibited by HTLV-IIIB gp120 and the synthetic epitope peptides of HTLV-IIIB and HTLV-IIIMN defined by BAT123. AB19-4 also inhibited the binding of BAT123 to HTLV-IIIB-infected H9 cells in flow cytometric studies. Polyclonal goat and sheep antisera against HTLV-IIIB gp120 reacted specifically with AB19-4, suggesting that AB19-4 may recognize cross-species idiotopes. Rabbits immunized with purified AB19-4 generated anti-anti-Id antibodies (Ab3) that reacted specifically with HTLV-IIIB gp120 and the BAT123-binding epitope peptides of HTLV-IIIB and HTLV-IIIMN. The Ab3 bound to H9 cells infected by HTLV-IIIB or HTLV-IIIMN and inhibited the infection of CEM cells by HTLV-IIIB or HTLV-IIIMN, whereas BAT123 also bound H9 cells infected by HTLV-IIIB or HTLV-IIIMN but neutralized only HTLV-IIIB. Our data suggest that AB19-4 mimics the neutralization site on HIV-1 gp120 defined by BAT123. The induction of immunity to HIV using internal-image Ab2 to HIV-neutralizing antibodies may provide a viable approach for developing effective vaccines for AIDS.     

Vaginal langerhans cells nonproductively transporting HIV-1 mediate infection of T cells

Although implied by other models, proof that Langerhans cells (LCs) in the human vagina participate in dissemination of infectious human immunodeficiency virus type 1 (HIV-1) has been lacking. Here, we show that LCs migrate from HIV-1-exposed vaginal epithelia and pass infectious virus to CD4+ T cells without being productively infected themselves, and we point to a pathway that might enable HIV-1 to avoid degradation in vaginal LCs. Transport by migratory LCs to local lymphatics in a nonproductive but infectious form may aid HIV-1 in evasion of topical microbicides that target its intracellular productive life cycle.     

Anti-Lyt-1 monoclonal antibody inhibits induction of anti-self-TNP but not of alloreactive cytotoxic T lymphocytes

The role of Lyt-1+ T cells in the induction of anti-self + TNP and anti-allogeneic CTL was investigated by the use of monoclonal antibodies directed against these molecules. When present during the 5 days of in vitro induction of CTL, anti-Lyt-1.1 mAb partially inhibited the induction of anti-self + TNP CTL but did not affect the induction of alloreactive CTL. The inhibitory effect was dose-dependent, Lyt-1 allotype-specific, and directed at the responding cells. The inhibition could be abolished by the addition of interleukin 2-containing supernatants. These results suggest that under some experimental conditions, Lyt-1-specific mAb can be shown to affect a T cell function, probably a helper function. Possible differences in the induction mechanisms of anti-self + TNP and alloreactive CTL are discussed.     

Neutralizing antibody responses to HIV-1 infection

Neutralizing antibodies represent an important component of immune control in many viral infections. In HIV-1 infection, almost all individuals develop antibodies capable of neutralizing autologous viruses in vitro; however, the role of these antibodies in vivo still remains unclear. Their absence during the acute phase of infection, when the viral levels are brought under control, suggests they play a minor role in immune control and that cellular immune responses are more critical during this time. However, during chronic infection these antibodies may be important in preventing cell-to-cell spread and they still represent our best hope of providing sterilizing immunity (i.e., prevention of infection) by vaccination. Significant advances over the last few years in understanding the structure of the envelope glycoproteins have renewed interest in the role of neutralizing antibodies and the possibility that immunogens capable of stimulating a neutralizing antibody response can be developed.     

NKT cells in HIV-1 infection

Natural killer T （NKT） cells are a unique T cell population that have important immunoregulatory functions and have been shown to be involved in host immunity against a range of microorganisms. It also emerges that they might play a role in HIV-1 infection, and therefore be selectively depleted during the early stages of infection. Recent studies are reviewed regarding the dynamics of NKT depletion during HIV-1 infection and their recovery under highly active antiretroviral treatment （HAART）. Possible mechanisms for these changes are proposed based on the recent developments in HIV pathogenesis. Further discussions are focused on HIV＇s disruption of NKT activation by downregulating CDld expression on antigen presentation cells （APC）. HIV-1 protein Nefis found to play the major role by interrupting the intracellular trafficking of nascent and recycling CDld molecules.     

Exposure to apoptotic activated CD4+ T cells induces maturation and APOBEC3G-mediated inhibition of HIV-1 infection in dendritic cells

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4(+) T cells (ApoInf) or apoptotic uninfected activated CD4(+) T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4(+) T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4(+) T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.     

Nuclear factor of activated T cells is a driving force for preferential productive HIV-1 infection of CD45RO-expressing CD4+ T cells

Human immunodeficiency virus type-1 (HIV-1) preferentially replicates in CD4-expressing T cells bearing a "memory" (CD45RO+) rather than a "naive" (CD45RA+/CD62L+) phenotype. Yet the basis for the higher susceptibility of these cells to HIV-1 infection remains unclear. Because the nature of the CD45 isoform itself can affect biochemical events in T cells, we set out to determine whether these isoforms could differently modulate HIV-1 long terminal repeat (LTR) activity and thereby replication. Through the use of CD4+ Jurkat T cells specifically expressing distinct CD45 isoforms (i.e. CD45RABC or CD45RO), we demonstrated that a difference in CD45 isoform expression conferred preferential replication of HIV-1 to CD45RO-expressing T cell clones following a physiological CD3/CD28 stimulation. Closer analysis indicated that higher HIV-1 LTR activation levels were consistently observed in CD45RO-positive cells, which was paralleled by more pronounced nuclear factor of activated T cells (NFAT) activation in these same cells. Specific involvement of NFAT1 was revealed in studied Jurkat clones by mobility shift analyses. In addition, preferential activation of the LTR and viral replication in CD45RO T cells was FK506- and cyclosporin A-sensitive. These results underscore the importance of NFAT in HIV-1 regulation and for the first time identify the role of the CD45 isoform in limiting productive HIV-1 replication to the human CD4 memory T cell subset.     

High level expression of a promising anti-idiotypic antibody fragment vaccine against HIV-1 in Pichia pastoris

We have expressed the anti-idiotypic antibody 3H6 Fab directed against the HIV-1 broadly neutralising antibody 2F5 in methylotrophic yeast Pichia pastoris. The chimeric human/mouse Fab fragment was expressed under control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Bioreactor experiments showed the ability of the recombinant P. pastoris clone to secrete up to 260 mg/L Fab fragment in the culture supernatant during a five days cultivation time. Codon optimisation of the Fab expression cassette gave no further improvement of specific productivity when comparing 12 clones of each construct. The subsequent purification of Fab containing supernatants was done by anion exchange and size-exclusion chromatography with a recovery resulting in 70% of the recombinant protein. For verification of the suitability of the expression system we characterised the expressed protein with respect to both, its specificity and binding affinity and could not detect any significant difference between products from yeast derived and the hybridoma derived product. Finally we tested the implicit requirement of the carbohydrate moiety in the H2 loop of the original 3H6 antibody by introducing an asparagine to alanine replacement and, in a second experiment, inhibition of N-glycosylation by tunicamycin treatment. Biochemical analysis confirmed that the N-glycosylation does not contribute to the binding properties of 3H6.     

Human neutrophil alpha-defensin 4 inhibits HIV-1 infection in vitro

Human neutrophil alpha-defensin 4 (HNP4) is more effective than HNP1-3 in protecting human peripheral blood mononuclear cells from infection by both X4 and R5 HIV-1 strains. HNP4 binds to both CD4 and gp120 approximately two orders of magnitude weaker than does HNP1, and is less effectively sequestered by glycosylated serum proteins than HNP1. These results suggest that the HIV-1 inhibition by HNP4 stems at least partially from a unique and lectin-independent property of HNP4 with CD4 and/or gp120. Our finding identifies an anti-HIV-1 property of HNP4 and may have implications in the development of new antiviral agents for AIDS therapy.     

HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages

Immature dendritic cells are among the first cells infected by retroviruses after mucosal exposure. We explored the effects of human immunodeficiency virus-1 (HIV-1) and its Tat transactivator on these primary antigen-presenting cells using DNA microarray analysis and functional assays. We found that HIV-1 infection or Tat expression induces interferon (IFN)-responsive gene expression in immature human dendritic cells without inducing maturation. Among the induced gene products are chemokines that recruit activated T cells and macrophages, the ultimate target cells for the virus. Dendritic cells in the lymph nodes of macaques infected with simian immunodeficiency virus (SIV) have elevated levels of monocyte chemoattractant protein 2 (MCP-2), demonstrating that chemokine induction also occurs during retroviral infection in vivo. These results show that HIV-1 Tat reprograms host dendritic cell gene expression to facilitate expansion of HIV-1 infection.