Mahavir Singh

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.

Stimulation of Th1-Polarizing Cytokines, C-C Chemokines, Maturation of Dendritic Cells, and Adjuvant Function by the Peptide Binding Fragment of Heat Shock Protein 70

The peptide binding C-terminal portion of heat shock protein (HSP)70 (aa 359–610) stimulates human monocytes to produce IL-12, TNF-α, NO, and C-C chemokines. The N-terminal, ATPase portion (HSP701–358) failed to stimulate any of these cytokines or chemokines. Both native and the truncated HSP70359–610 stimulation of chemokine production is mediated by the CD40 costimulatory molecule. Maturation of dendritic cells was induced by stimulation with native HSP70, was not seen with the N-terminal HSP701–358, but was enhanced with HSP70359–610, as demonstrated by up-regulation of CD83, CCR7, CD86, CD80, and HLA class II. In vivo studies in macaques showed that immunization with HSP70359–610 enhances the production of IL-12 and RANTES. Immunization with peptide-bound HSP70359–610 in mice induced higher serum IgG2a and IgG3 Abs than the native HSP70-bound peptide. This study suggests that the C-terminal, peptide-binding portion of HSP70 is responsible for stimulating Th1-polarizing cytokines, C-C chemokines, and an adjuvant function.

A Conserved Mycobacterial Heat Shock Protein (hsp) 70 Sequence Prevents Adjuvant Arthritis upon Nasal Administration and Induces IL-10-Producing T Cells That Cross-React with the Mammalian Self-hsp70 Homologue

Immunization with Mycobacterium tuberculosis heat shock protein (hsp) 60 has been shown to protect rats from experimental arthritis. Previously, the protection-inducing capacity was shown to reside in the evolutionary conserved parts of the molecule. Now we have studied the nature of the arthritis suppressive capacity of a distinct, antigenically unrelated protein, M. tuberculosis hsp70. Again, a conserved mycobacterial hsp70 sequence was found to be immunogenic and to induce T cells that cross-reacted with the rat homologue sequence. However, in this case parenteral immunization with the peptide containing the critical cross-reactive T cell epitope did not suppress disease. Upon analysis of cytokines produced by these peptide-specific T cells, high IL-10 production was found, as was the case with T cells responsive to whole hsp70 protein. Nasal administration of this peptide was found to lead to inhibition of subsequent adjuvant arthritis induction. The data presented here shows the intrinsic capacity of conserved bacterial hsp to trigger self-hsp cross-reactive T cells with the potential to down-regulate arthritis via IL-10.

Passive protection with immunoglobulin A antibodies against tuberculous early infection of the lungs

We report on a new approach toward protection against tuberculosis, based on passive inoculation with immunoglobulin A (IgA) antibodies. In a mouse model of tuberculous lung infection, intranasal inoculations of mice with an IgA monoclonal antibody (mAb) against the α‐crystallin antigen of Mycobacterium tuberculosis reduced up to 10‐fold the lung bacterial counts at nine days after either aerosol‐ or intranasal challenge. This effect involved synergism between mAb inoculations shortly before and 3 days after infection. Monomeric IgA reduced the colony‐forming unit counts to the same extent as the polymeric IgA, suggesting antibody targeting to Fcα, rather than poly‐immunoglobulin receptors on infected lung macrophages. The protective effect was of short duration, presumably due to the rapid degradation of the intranasally applied IgA. Our results provide evidence of an alternative approach which could be further developed toward immunoprophylaxis against tuberculosis in immunocompromised subjects.

HSP70 Peptide Binding Mutants Separate Antigen Delivery from Dendritic Cell Stimulation

Microbial heat shock proteins (HSPs) have been implicated in the induction of both the innate and adaptive arms of the immune response. We now show that human dendritic cells (DC) pulsed with peptide-loaded mycobacterial HSP70 complexes generate potent antigen-specific cytotoxic lymphocyte (CTL) responses, which are dependent on an HSP70-stimulated calcium signaling cascade. From the calculated peptide binding affinity of mycobacterial HSP70 (K(D) = 14 microM) we show that 120 pM HSP70 bound peptide is sufficient to generate a peptide-specific CTL response that is up to four orders of magnitude more efficient than peptide alone. The minimal 136 amino acid, mycobacterial HSP70 peptide binding domain can generate CTL responses, and a single amino acid mutant HSP70 designed to prevent peptide binding but retain stimulatory capacity has allowed us to separate antigen delivery from DC immunostimulation.

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.

Early depletion of Mycobacterium tuberculosis-specific T helper 1 cell responses after HIV-1 infection

BACKGROUND The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of acquired immunodeficiency syndrome in patients infected with human immunodeficiency virus (HIV). This study was conducted to better understand the mechanism underlying M. tuberculosis-specific pathogenicity early after onset of HIV infection. METHODS M. tuberculosis-specific T helper 1 (Th1) cells were studied in HIV negative (n=114) and chronically HIV infected (n=68) Tanzanian subjects by using early secreted antigenic target 6 (ESAT6) protein or tuberculin (purified protein derivative) with interferon-gamma ELISPOT and intracellular cytokine staining. In a longitudinal study, the effect of acute HIV infection on M. tuberculosis-specific Th1 cells was determined by polychromatic flow cytometric analysis in 5 subjects with latent M. tuberculosis infection who became infected with HIV. RESULTS In tuberculosis (TB)-asymptomatic subjects (i.e., subjects with unknown TB status who did not show clinical signs suggestive of TB), chronic HIV infection was associated with a decreased percentage of subjects with detectable M. tuberculosis-specific Th1 cells (P< .001) a decrease which was not observed among subjects with active TB. Acute HIV infection induced a rapid depletion of M. tuberculosis-specific Th1 cells in 4 subjects remained TB asymptomatic, whereas the population of these cells remained stable in subjects who remained HIV negative (P< .01). CONCLUSIONS Taken together, these data suggest a mechanism of rapid M. tuberculosis-specific Th1 cell depletion that may contribute to the early onset of TB in individuals with latent M. tuberculosis infection who become HIV infected.

Mycobacterium tuberculosis chaperonin 60.1 is a more potent cytokine stimulator than chaperonin 60.2 (Hsp 65) and contains a CD14-binding domain.

ABSTRACT Much attention has focused on the Mycobacterium tuberculosis molecular chaperone chaperonin (Cpn) 60.2 (Hsp 65) in the pathology of tuberculosis because of its immunogenicity and ability to directly activate human monocytes and vascular endothelial cells. However, M. tuberculosis is one of a small group of bacteria that contain multiple genes encoding Cpn 60 proteins. We have now cloned and expressed both M.tuberculosis proteins and report that the novel chaperonin 60, Cpn 60.1, is a more potent inducer of cytokine synthesis than is Cpn 60.2. This is in spite of 76% amino acid sequence similarity between the two mycobacterial chaperonins. TheM. tuberculosis Cpn 60.2 protein activates human peripheral blood mononuclear cells by a CD14-independent mechanism, whereas Cpn 60.1 is partially CD14 dependent and contains a peptide sequence whose actions are blocked by anti-CD14 monoclonal antibodies. The cytokine-inducing activity of both chaperonins is extremely resistant to heat. Cpn 60.1 may be an important virulence factor in tuberculosis, able to activate cells by diverse receptor-driven mechanisms.

Heat shock protein 70 is a potent activator of the human complement system

Abstract According to new hypotheses, extracellular heat shock proteins (Hsps) may represent an ancestral danger signal of cellular death or lysis-activating innate immunity. Recent studies demonstrating a dual role for Hsp70 as both a chaperone and cytokine, inducing potent proinflammatory response in human monocytes, provided support for the hypothesis that extracellular Hsp is a messenger of stress. Our previous work focused on the complement-activating ability of human Hsp60. We demonstrated that Hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here, we show that another chaperone molecule also possesses complement-activating ability. Solid-phase enzyme-linked immunosorbent assay was applied for the experiments. Human Hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human Hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement-like molecules and primitive immune cells appeared together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.

Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states.

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.