Trevor Whittall

CD40 Is a Cellular Receptor Mediating Mycobacterial Heat Shock Protein 70 Stimulation of CC-Chemokines

The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.

Identification of Stimulating and Inhibitory Epitopes within the Heat Shock Protein 70 Molecule That Modulate Cytokine Production and Maturation of Dendritic Cells

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407–426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457–496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457–496 but enhanced by p407–426.

Oral tolerization with peptide 336–351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease

Behcets disease (BD) specific peptide (p336–351) was identified within the human 60 kD heat shock protein (HSP60). Oral p336–351 induced uveitis in rats which was prevented by oral tolerization with the peptide linked to recombinant cholera toxin B subunit (CTB). This strategy was adopted in a phase I/II clinical trial by oral administration of p336–351‐CTB, 3 times weekly, followed by gradual withdrawal of all immunosuppressive drugs used to control the disease in 8 patients with BD. The patients were monitored by clinical and ophthalmological examination, as well as extensive immunological investigations. Oral administration of p336–351‐CTB had no adverse effect and withdrawal of the immunosuppressive drugs showed no relapse of uveitis in 5 of 8 patients or 5 of 6 selected patients who were free of disease activity prior to initiating the tolerization regimen. After tolerization was discontinued, 3 of 5 patients remained free of relapsing uveitis for 10–18 months after cessation of all treatment. Control of uveitis and extra‐ocular manifestations of BD was associated with a lack of peptide‐specific CD4+ T cell proliferation, a decrease in expression of TH1 type cells (CCR5, CXCR3), IFN‐γ and TNF‐α production, CCR7+ T cells and costimulatory molecules (CD40 and CD28), as compared with an increase in these parameters in patients in whom uveitis had relapsed. The efficacy of oral peptide‐CTB tolerization will need to be confirmed in a phase III trial, but this novel strategy in humans might be applicable generally to autoimmune diseases in which specific antigens have been identified.

Effect of heterosexual intercourse on mucosal alloimmunisation and resistance to HIV-1 infection

BACKGROUND Unprotected sexual intercourse between regular heterosexual partners could elicit alloimmune responses that might be associated with inhibition of in-vitro HIV-1 infectivity. We investigated this hypothesis in people practising unprotected sex and those using protection. METHODS We recruited 82 participants from an outpatient genitourinary medicine clinic. 29 monogamous heterosexual couples having unprotected sex; and 15 women and 10 men having condom protected or no sex. We used the mixed leucocyte reaction (MLR), stimulating one partners peripheral blood mononuclear cells (PBMC) with the other partners irradiated PBMC and compared the resulting response with control PBMC. We studied resistance to HIV-1 infection by challenging activated CD4-positive T cells with CCR5-binding and CXCR4-binding HIV-1 strains, and comparing the infectivity in participants having unprotected sex with those practising protected sex. We used the correlation coefficient to establish the significance of the relation between MLR and HIV-1 infectivity. FINDINGS We recorded a significant increase in the stimulation indices in PBMC from women whose cells were stimulated with irradiated PBMC (2%, 10%, or 50%) from their regular partners. The mean with 10% partners cells was 8.6 (SD 7.7), compared with those from unrelated cells (4.7 [3.9], p=0.009). Significant alloimmune responses were also seen in corresponding male partners, but only with 50% stimulating cells (p=0.013). Dose-dependent inhibition of activated CD4-positive T cells to HIV-1 infection with both binding strains was noted in vitro in women practising unprotected intercourse, compared with those having protected sex or having no sex for more than 1 year. Highly significant differences were found for CCR5 (p=0.0001) and for CXCR4 (p=0.001) strains of HIV-1 at all four virus-concentrations. Male partners also showed in-vitro inhibition of HIV-1 but this was less than that in women. INTERPRETATION Unprotected sexual intercourse might result in alloimmunisation stimulated by HLA antigens in seminal or cervicovaginal fluid. Mucosal alloimmunisation may reduce infection by HIV-1, and the role of such immunisation in preventive and therapeutic vaccination should be investigated.

Functional domains of HSP70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity

Microbial HSP70 (heat-shock protein 70) consists of three functionally distinct domains: an N-terminal 44 kDa ATPase portion (amino acids 1-358), followed by an 18 kDa peptide-binding domain (amino acids 359-494) and a C-terminal 10 kDa fragment (amino acids 495-609). Immunological functions of these three different domains in stimulating monocytes and dendritic cells have not been fully defined. However, the C-terminal portion (amino acids 359-610) stimulates the production of CC chemokines, IL-12 (interleukin-12), TNFalpha(tumour necrosis factor alpha), NO and maturation of dendritic cells and also functions as an adjuvant in the induction of immune responses. In contrast, the ATPase domain of microbial HSP70 mostly lacks these functions. Since the receptor for HSP70 is CD40, which with its CD40 ligand constitutes a major co-stimulatory pathway in the interaction between antigen-presenting cells and T-cells, HSP70 may function as an alternative ligand to CD40L. HSP70-CD40 interaction has been demonstrated in non-human primates to play a role in HIV infection, in protection against Mycobacterium tuberculosis and in conversion of tolerance to immunity.

Stimulation of Cell Surface CCR5 and CD40 Molecules by Their Ligands or by HSP70 Up-Regulates APOBEC3G Expression in CD4+ T Cells and Dendritic Cells

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like-3G (A3G) is an intracellular innate antiviral factor that deaminates retroviral cytidine to uridine. In an attempt to harness the anti-HIV effect of A3G, we searched for an agent that would up-regulate A3G and identify the receptors involved. Stimulation of cell surface CCR5 with CCL3 and CD40 with CD40L or both molecules with microbial 70-kDa heat shock protein (HSP)70 up-regulated A3G mRNA and protein expression in human CD4+ T cells and monocyte-derived dendritic cells (DC), demonstrated by real-time PCR and Western blots, respectively. The specificity of CCR5 and CD40 stimulation was established by inhibition with TAK 779 and mAb to CD40, as well as using human embryonic kidney 293 cells transfected with CCR5 and CD40, respectively. A dose-dependent increase of A3G in CCL3- or HSP70-stimulated CD4+ T cells was associated with inhibition in HIV-1 infectivity. To differentiate between the inhibitory effect of HSP70-induced CCR5 binding and that of A3G, GFP-labeled pseudovirions were used to infect human embryonic kidney 293 cells, which showed inhibition of pseudovirion uptake, consistent with A3G being responsible for the inhibitory effect. Ligation of cell surface CCR5 receptors by CCL3 or CD40 by CD40L activated the ERK1/2 and p38 MAPK signaling pathways that induced A3G mRNA expression and production of the A3G protein. These in vitro results were corroborated by in vivo studies in rhesus macaques in which A3G was significantly up-regulated following immunization with SIVgp120 and p27 linked to HSP70. This novel preventive approach may in addition to adaptive immunity use the intracellular innate antiviral effect of A3G.

Interaction between the CCR5 chemokine receptors and microbial HSP70

Evidence is presented that the microbial 70‐kD heat shock protein (HSP70) binds to CCR5 chemokine receptors in CCR5‐transfected cell lines and in primary human cells. Significant CCR5‐mediated calcium mobilization was stimulated by HSP70 and inhibited with TAK 779, which is a specific CCR5 antagonist. HSP70‐mediated activation of the p38 MAPK phosphorylation signaling pathway was also demonstrated in CCR5‐transfected HEK 293 cells. Direct binding of three extracellular peptides of CCR5 to HSP70 was demonstrated by surface plasmon resonance. Functional evidence of an interaction between HSP70, CCR5 and CD40 was shown by enhanced production of CCL5 by HEK 293 cells transfected with both CD40 and CCR5. Primary monocyte‐derived immature DC stimulated with HSP70 produced IL‐12 p40, which showed dose‐dependent inhibition of >90% on treatment with both TAK 779 and anti‐CD40 mAb. Stimulation of IL‐12 p40 or TNF‐α by HSP70 was related to the differential cell surface expression of CCR5 in primary human immature and mature DC, and those with the homozygous ▵▵32 CCR5 mutation. These findings may be of significance in the interaction between HSP70 and immune responses of CCR5+ T cells in HIV‐1 infection, as well as in inflammatory bowel disease.

The emerging role of innate immunity in protection against HIV-1 infection.

Preventive immunization against HIV-1 infection requires a rapid immune response that does not rely exclusively on B or T cell memory. Innate immunity may fulfill this function as it may be activated directly at the time of HIV-1 transmission, inhibit early HIV-1 replication, stimulate adaptive immunity and enable specific antibodies followed by CD8(+) T cells to deal with the virus effectively. The three components of innate immunity - cellular, extracellular and intracellular - are presented, with an example given for each of these components; gammadelta T cells, CC chemokines and APOBEC3G. This brief account is presented to highlight the immuno-virological concept of coordinating activated innate immunity with adaptive antibody and T cell responses in preventive vaccination against HIV-1 infection.

Immunogenicity of the Extracellular Domains of C-C Chemokine Receptor 5 and the In Vitro Effects on Simian Immunodeficiency Virus or HIV Infectivity

The C-C chemokine receptor CCR5 serves an important function in chemotaxis of lymphocytes, monocytes, and dendritic cells. CCR5 is also the major coreceptor in most macrophage-tropic HIV-1 infections. Immunization of rhesus macaques with a baculovirus-generated CCR5 construct or peptides derived from the sequences of the four extracellular domains of CCR5 elicited IgG and IgA Abs, inhibition of SIV replication, and CD4+ T cell proliferative responses to three of the extracellular domains of CCR5. The immune sera reacted with cell surface CCR5 expressed on HEK 293 cells. T and B cell epitope mapping revealed major and minor T and B cell epitopes in the N-terminal, first, and second loops of CCR5. The three C-C chemokines, RANTES, macrophage-inflammatory protein-1α, and macrophage-inflammatory protein-1β, were up-regulated by immunization with the CCR5-derived peptides, and the cell surface expression of CCR5 was decreased. The CCR5 Abs were complementary to the C-C chemokines in inhibiting HIV replication in vitro. Immunization with the four extracellular domains of CCR5 suggests that three of them are immunogenic, with maximal T cell responses being elicited by the second loop peptide. However, maximal Abs to the cell surface CCR5 or viral inhibitory Abs in vitro were induced by the N-terminal peptide. Up-regulation of the three C-C chemokines and down-modulation of cell surface CCR5 were elicited by the second loop, N-terminal, and first loop peptides. The data suggest that a dual mechanism of C-C chemokines and specific Abs may engage and down-modulate the CCR5 coreceptors and prevent in vitro HIV or SIV replication.

Stress-activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory.

Evidence is presented that thermal or oxidizing stress‐activated DC interact with CD4+ T cells to induce and maintain a TCR‐independent homeostatic memory circuit. Stress‐activated DC expressed endogenous intra‐cellular and cell surface HSP70. The NF‐κB signalling pathway was activated and led to the expression of membrane‐associated IL‐15 molecules. These interacted with the IL‐15 receptor complex on CD4+ T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T‐cell proliferation and IFN‐γ production. CD40 ligand on CD4+ T cells in turn re‐activated CD40 molecules on DC, inducing DC maturation and IL‐15 expression thereby maintaining the feedback circuit. The proliferating CD4+ T cells were characterized as CD45RA− CD62L+ central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC‐class‐II‐TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC‐class II antibodies. These findings suggest that stress can activate a DC‐CD4+ T‐cell interacting circuit, which may be responsible for maintaining a homeostatic antigen‐independent memory.