Heterogeneous yet stable Vδ2(+) T-cell profiles define distinct cytotoxic effector potentials in healthy human individuals.

Human γδ T cells display potent responses to pathogens and malignancies. Of particular interest are those expressing a γδ T-cell receptor (TCR) incorporating TCRδ-chain variable-region-2 [Vδ2(+)], which are activated by pathogen-derived phosphoantigens (pAgs), or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Once activated, Vδ2(+) T cells exhibit multiple effector functions that have made them attractive candidates for immunotherapy. Despite this, clinical trials have reported mixed patient responses, highlighting a need for better understanding of Vδ2(+) T-cell biology. Here, we reveal previously unappreciated functional heterogeneity between the Vδ2(+) T-cell compartments of 63 healthy individuals. In this cohort, we identify distinct "Vδ2 profiles" that are stable over time; that do not correlate with age, gender, or history of phosphoantigen activation; and that develop after leaving the thymus. Multiple analyses suggest these Vδ2 profiles consist of variable proportions of two dominant but contrasting Vδ2(+) T-cell subsets that have divergent transcriptional programs and that display mechanistically distinct cytotoxic potentials. Importantly, an individual's Vδ2 profile predicts defined effector capacities, demonstrated by contrasting mechanisms and efficiencies of killing of a range of tumor cell lines. In short, these data support patient stratification to identify individuals with Vδ2 profiles that have effector mechanisms compatible with tumor killing and suggest that tailored Vδ2-profile-specific activation protocols may maximize the chances of future treatment success.

Why are Behçet's disease patients always exhausted?

OBJECTIVES: Patients with Behçet's disease (BD) constantly complain of fatigue and many have problems with poor sleep. This ultimately has a major impact on all aspects of normal living. To attempt to understand this, Artificial Intelligence (AI) was used to identify potential biomarkers. These were alpha-melanocyte stimulating hormone (α-MSH), vasoactive intestinal peptide (VIP) and some inflammatory cytokines. We assessed the association of fatigue, quality of sleep and disease activity with circulating concentration of α-MSH, VIP and inflammatory cytokines. METHODS: There were 127 participants, 97 BD patients, and 30 healthy controls (HC). All completed the Multi-Dimensional Assessment of Fatigue questionnaire (MAF) and the Pittsburgh Sleep Quality Index (PSQI) on the day of their clinical assessment. Enzyme-linked immunosorbent assays (ELISA) were used to evaluate the serum concentrations of α-MSH, VIP and cytokines (IL-1ß, IL-6, IL-10, and TNF-α). RESULTS: 64% of BD patients experienced high fatigue scores, and 63% had poor quality of sleep. When BD and HC were compared the MAF and PSQI scores as well as the serum concentrations of α-MSH, VIP, and IL-6 were significantly higher in BD (p values were: 0.001, 0.001, 0.001, 0.004 and 0.036, respectively). Both α-MSH and IL-6 had significant impact on MAF and PSQI. Interestingly, VIP had a significant influence on PSQI and disease activity, but not on MAF. CONCLUSIONS: A better understanding of these complex clinical and biochemical interactions between α-MSH, VIP and IL-6 might lead to the development of novel approaches to manage fatigue and sleep disorders as well as disease activity in BD patients.

Immunization with recombinant macaque major histocompatibility complex class I and II and human immunodeficiency virus gp140 inhibits simian-human immunodeficiency virus infection in macaques.

Genetic, epidemiological and experimental evidence suggest that the major histocompatibility complex (MHC) is critical in controlling human immunodeficiency virus (HIV) infection. The objectives of this study were to determine whether novel recombinant Mamu MHC constructs would elicit protection against rectal challenge with heterologous simian-human immunodeficiency virus (SHIV) strain SF162.P4 in rhesus macaques. Mamu class I and II gene products were linked together with HIV gp140, simian immunodeficiency virus (SIV) p27 and heat-shock protein 70 to dextran. The vaccine was administered to two groups, each consisting of nine macaques, either subcutaneously (SC), or rectally and boosted by SC immunization. The controls were untreated or adjuvant-treated animals. Repetitive rectal challenges with up to ten doses of SHIV SF162.P4 showed a significant decrease in the peak and sequential viral RNA concentrations, and three macaques remained uninfected, in the nine SC-immunized animals, compared with infection in all nine controls. Macaques immunized rectally followed by SC boosters showed a less significant decrease in both sequential and peak viral loads compared with the SC-immunized animals, and all were infected following rectal challenge with SHIV SF162.P4. Plasma and mucosal IgG and IgA antibodies to Mamu class I alleles and HIV gp120, as well as to RANTES (regulated upon activation, normal T-cell expressed, and secreted; CCR5) were increased, and showed significant inverse correlations with the peak viral load. These results suggested that allo-immunization with recombinant MHC constructs linked to HIV-SIV antigens merits further investigation in preventing HIV-1 infection.

Immunization with recombinant HLA classes I and II, HIV-1 gp140, and SIV p27 elicits protection against heterologous SHIV infection in rhesus macaques.

Major histocompatibility complex (MHC) molecules expressed on the surface of human immunodeficiency virus (HIV) are potential targets for neutralizing antibodies. Since MHC molecules are polymorphic, nonself MHC can also be immunogenic. We have used combinations of novel recombinant HLA class I and II and HIV/simian immunodeficiency virus (SIV) antigens, all linked to dextran, to investigate whether they can elicit protective immunity against heterologous simian/human immunodeficiency virus (SHIV) challenge in rhesus macaques. Three groups of animals were immunized with HLA (group 1, n = 8), trimeric YU2 HIV type 1 (HIV-1) gp140 and SIV p27 (HIV/SIV antigens; group 2, n = 8), or HLA plus HIV/SIV antigens (group 3, n = 8), all with Hsp70 and TiterMax Gold adjuvant. Another group (group 4, n = 6) received the same vaccine as group 3 without TiterMax Gold. Two of eight macaques in group 3 were completely protected against intravenous challenge with 18 50% animal infective doses (AID(50)) of SHIV-SF162P4/C grown in human cells expressing HLA class I and II lineages represented in the vaccine, while the remaining six macaques showed decreased viral loads compared to those in unimmunized animals. Complement-dependent neutralizing activity in serum and high levels of anti-HLA antibodies were elicited in groups 1 and 3, and both were inversely correlated with the plasma viral load at 2 weeks postchallenge. Antibody-mediated protection was strongly supported by the fact that transfer of pooled serum from the two challenged but uninfected animals protected two naïve animals against repeated low-dose challenge with the same SHIV stock. This study demonstrates that immunization with recombinant HLA in combination with HIV-1 antigens might be developed into an alternative strategy for a future AIDS vaccine.

The impact of multifactorial factors on the Quality of Life of Behçet's patients over 10 years.

Objective: This study analyses the 2020 survey and reviews the 2009, 2014 surveys to ascertain which Behçet's symptoms, personal and family status, patients' lifestyle, and work-related outcomes impacted on Health-Related Quality of Life (HRQoL). Methods: Four hundred and fifty-nine Behçet's patients submitted an online survey/questionnaire. Patients provided information on socio-demographic characteristics, disease duration, historical and current symptoms, systemic and topical medication, health related lifestyle, work-related outcomes regarding employment status and claiming benefits and Quality of Life (QoL) measured by EQ-5D index. Results: Four hundred and nineteen patients met the inclusion criteria, and 371 who had full data (Males: Females: Others = 84:285:2, mean-age = 41.1 ± 23.3:38 ± 13.2:40 ± 5). The main symptoms associated with patients seeking medical care were mouth ulcers 30% and genital ulcers 23%, joint 14%, and eye problems 9%. The EQ-5D index for 2009, 2014, 2020 was (mean ± SD); 0.47 ± 0.38, 0.42 ± 0.37, 0.34 ± 0.40, respectively, p < 0.05. 2020 patients had the worst values of the five domains compared to 2014 and 2009. Interestingly, mobility value was the same over the 10 years of monitoring patients. Behçet's syndrome (BS) symptoms that had significant negative impact on QoL were; 2009 (arthropathy, neurological problems, pathergy reaction, and stomach/bowel symptoms), 2014 (arthropathy, headache, neurological problems, pathergy reaction, and skin lesions), 2020 (arthropathy, neurological problems, and stomach/bowel symptoms). The 2014 and 2020 surveys reported the QoL is significantly better in patients on immunosuppressant, who did sport, continued in employment and not receiving benefits. Conclusion: Joints and neurological symptoms are the main symptoms which had negative impact on BS patients over the 10 years, sociodemographic (gender, age, marital, and education status), lifestyle (medication, cannabis, drinking wine, and regular exercise), employment status (employee and no career change), and accessing benefits (never claim benefit) had significant influence on patients' HRQoL.

Cytoskeletal proteins bound to heat-shock protein 70 may elicit resistance to simian immunodeficiency virus infection of CD4(+) T cells.

This study is based on the evidence that immunization of macaques with human CD4(+) T cells elicits prevention of simian immunodeficiency virus (SIV) infection. We hypothesized that heat-shock protein 70 (HSP70) isolated from CD4(+) T cells may act as a chaperone and carry the protective host proteins. Two moieties of HSP70 were affinity-purified from human CD4(+) T cells; an ADP preparation with HSP70-bound proteins (ADP-HSP) and an ATP control preparation. Immunization of rhesus macaques with these preparations showed significant inhibition of SIVmac251 infectivity ex vivo in CD4(+) T cells only with the ADP-HSP (P = 0.01). Proteomic analysis identified three cytoskeletal elements, cofilin, profilin and gamma-actin, exclusively in the ADP-HSP preparation. Investigation of the mechanism of prevention of SIV replication suggests that antibodies to the cytoskeletal proteins may inhibit actin depolymerization and facilitate viral degradation by the innate antiviral APOBEC3G. As cytoskeletal proteins are critical in the formation of virological and immunological synapses, finding specific antibodies and anti-SIV/human immunodeficiency virus (HIV) factors suggests a novel insight into HIV-1 immunopathogenesis.

The oral mucosal and salivary microbial community of Behçet's syndrome and recurrent aphthous stomatitis.

BACKGROUND: Behçet's syndrome (BS) is a multisystem immune-related disease of unknown etiology. Recurrent aphthous stomatitis (RAS) is characterized by the presence of idiopathic oral ulceration without extraoral manifestation. The interplay between the oral microbial communities and the immune response could play an important role in the etiology and pathogenesis of both BS and RAS. OBJECTIVE: To investigate the salivary and oral mucosal microbial communities in BS and RAS. METHODS: Purified microbial DNA isolated from saliva samples (54 BS, 25 healthy controls [HC], and 8 RAS) were examined by the human oral microbe identification microarray. Cultivable salivary and oral mucosal microbial communities from ulcer and non-ulcer sites were identified by matrix-assisted laser desorption/ionization time-of-flight analysis. Mycobacterium spp. were detected in saliva and in ulcer and non-ulcer oral mucosal brush biopsies following culture on Lowenstein-Jensen slopes and Mycobacterial Growth Indicator Tubes. RESULTS: There was increased colonization with Rothia denticariosa of the non-ulcer sites of BS and RAS patients (p<0.05). Ulcer sites in BS were highly colonized with Streptococcus salivarius compared to those of RAS (p<0.05), and with Streptococcus sanguinis compared to HC (p<0.0001). Oral mucosa of HC were more highly colonized with Neisseria and Veillonella compared to all studied groups (p<0.0001). CONCLUSIONS: Despite the uncertainty whether the reported differences in the oral mucosal microbial community of BS and RAS are of causative or reactive nature, it is envisaged that restoring the balance of the oral microbial community of the ulcer sites may be used in the future as a new treatment modality for oral ulceration.

The seroprevalence and salivary shedding of herpesviruses in Behçet's syndrome and recurrent aphthous stomatitis.

BACKGROUND: Behçet's syndrome (BS) is one of the multisystemic diseases that presents with oral ulceration and several other systemic manifestations including genital ulceration, folliculitis, erythema nodosum-like lesions, uveitis, and arthropathy. Ocular manifestation, central nervous system involvement, and gastrointestinal manifestation account for most of the complications of this disease, whereas orogenital ulceration and dermatological involvement affects the quality of life. The cause of the disease is not fully elucidated; however, herpesviruses have long been thought to play a pivotal role in the disease pathogenesis. OBJECTIVE: To investigate the seroprevalence and salivary shedding of herpesviruses in BS. METHOD: The levels of specific immunoglobulin G in six different herpesviruses in serum samples collected from 54 BS, 28 healthy controls (HC), and 7 recurrent aphthous stomatitis (RAS) patients were investigated. Salivary viral load was also quantified for these viruses in matched saliva samples using quantitative real-time polymerase chain reaction. RESULTS: The BS had lower cytomegalovirus (CMV) IgG level in comparison to HC (p=0.0226) and RAS (p=0.0450). There was statistically significant higher salivary shedding of Epstein-Barr virus (EBV) in BS in comparison to HC (p=0.0052), but not RAS (p=0.3318). CONCLUSIONS: A high EBV shedding was observed in both BS and RAS and a lower level of CMV IgG was observed in BS only. The reason for the observed lower level of CMV IgG in BS is not clear. However, one explanation might be a defect in the cross-talk between innate and adaptive immune responses which was suggested by a previously described defect in the toll-like receptor 1 and 2 heterodimer formation and function, this being the initial receptor sensing of CMV.

A novel HIV-CCR5 receptor vaccine strategy in the control of mucosal SIV/HIV infection.

OBJECTIVE: To develop a novel SIV-CCR5 receptor vaccine strategy that will protect macaques from SHIV infection by the vaginal mucosal route. DESIGN: The rationale for this strategy is that humans who express the homozygous delta32 CCR5 mutation and the associated upregulation of CC chemokines, the down-modulation of cell-surface expression of CCR5 and antibodies to CCR5 are protected against HIV infection. METHODS: A vaccine was prepared consisting of three extracellular peptides of CCR5, an N-terminal HIV gp120 fragment generated in transgenic plants and recombinant SIV p27. These were linked to the 70 000 Mr microbial heat shock protein (HSP70) carrier. The vaccine was administered (x3) either by the vaginal mucosal route or by targeting the proximity of the draining iliac lymph nodes. RESULTS: Serum and vaginal fluid IgG and IgA antibodies, IL-2 and IFN-gamma-producing cells, and macrophage-inflammatory protein (MIP) 1beta and MIP-1alpha (CCL4 and CCL3) were significantly raised in immunized macaques (P = 0.01-0.05). Vaginal challenge with SHIV(89.6P) infected all macaques, but sequential analysis over 24 weeks showed a significant variation in viral loads between the animals (P = 0.05). Whereas SHIV(89.6P) persisted in the four unimmunized macaques, in five of the eight immunized macaques the virus was cleared or became undetectable by reverse transcriptase-polymerase chain reaction. The CD4 cell counts in the immunized macaques were significantly higher than those in unimmunized animals (P < 0.05). CONCLUSION: An immunization strategy that targets both the virus and its CCR5 receptor has significantly inhibited SHIV(89.6P) infection and may serve as a novel strategy in the prevention of HIV transmission.

A comparative investigation of CC chemokines and SIV suppressor factors generated by CD8+ and CD4+ T cells and CD14+ monocytes.

The capacity of CD8+ and CD4+ T cells and CD14+ monocytes to generate the CC chemokines, RANTES, MIP-1alpha and MIP-1beta, and SIV suppressor factors were studied using cells separated from PBMC of macaques immunized with the 70-kDa heat shock protein (HSP70). Unimmunized macaques showed low levels of the three CC chemokines and SIV-SF, and they showed little variation between PBMC and the two subsets of T cells stimulated with PHA. Immunization with HSP70 elicited an increase in the in vitro concentration of each of the three CC chemokines and SF. This was found with PBMC, CD4+ and CD8+ T cells and to a lesser extent with monocytes, when conventionally separated enriched cell subsets were examined from the same PBMC. However, the concentrations of the three CC chemokines derived from highly purified cell-sorted populations (>95%) were greatly increased, as compared with the enriched cell subsets. The concentration of each of the three chemokines was highest for CD8+ T cells, decreased with CD4+ T cells and was lowest with the CD14+ monocytes, but the latter were not stimulated. Neutralization assays with antibodies to the three CC chemokines showed that the antiviral activity generated by the four populations of cells could be largely accounted for by the three CC chemokines. The results of this comparative study suggests that CD8+ as well as CD4+ T cells and CD14+ monocytes generate the three CC chemokines and SIV-SF when stimulated with a mitogen, and that the baseline innate level can be upregulated by adaptive immune responses to a specific antigen.

The role of TLR2 and 4 in Behçet's disease pathogenesis.

TLRs are PRRs that play a pivotal role in sensing exogenous pathogens and endogenous danger signals. Their role in the pathogenesis of inflammatory and immune-related diseases is gradually being unravelled. TLR2 and TLR4 are capable of sensing the oral microbial community, which is considered a potential trigger for Behçet's disease (BD). This study aimed to investigate the expression and function of TLR2 and TLR4 in the oral mucosa of BD. A total of 87 patients was included: 55 BD, 24 healthy controls and eight recurrent aphthous stomatitis. Total RNA was purified from non-lesional oral mucosal brush biopsies and analysed for the presence of TLR2 and TLR4 mRNA, along with their splice variants. The response of peripheral blood mononuclear cells to classical TLR2 and TLR4 agonists was also investigated. TLR2b, TLR2d, TLR2e, TLR4.3 and TLR4.4 were significantly elevated in relapsed BD. A significant defect in the response to cognate agonists of TLR1/2 heterodimer and TLR4 was also observed in BD. The expression of unusual splice variants of TLR2 and TLR4 might explain the observed defect in these receptors' function in BD.

The role of innate APOBEC3G and adaptive AID immune responses in HLA-HIV/SIV immunized SHIV infected macaques.

The AID/APOBEC family (activation induced deaminase/apolipoprotein B mRNA editing cytokine deaminase) in B cells play important roles in adaptive and innate immunity. Whereas APOBEC3G has been studied in CD4+ T cells and myeloid cells its functional potential in B cells has received little attention. AID combines two critical functions of antibodies, class switching and affinity maturation and may serve as a functional surrogate of protection. These functions were studied following systemic immunization of rhesus macaques with recombinant HLA constructs, linked with HIV and SIV antigens and HSP70 to dextran. The results showed significant upregulation of AID in CD20+ B cells, APOBEC 3G in CD27+ memory B cells and CD4+ effector memory T cells. After immunization the upregulated APOBEC 3G and AID were directly correlated in B cells (p<0.0001). Following challenge with SHIV SF162.P4 the viral load was inversely correlated with AID in B cells and APOBEC 3G in B and T cells, suggesting that both deaminases may have protective functions. Investigation of major interactions between DC, T cells and B cells showed significant increase in membrane associated IL-15 in DC and CD40L in CD4+ T cells. IL-15 binds the IL-15 receptor complex in CD4+ T and B cells, which may reactivate the DC, T and B cell interactions. The overall results are consistent with AID inhibiting pre-entry SHIV by eliciting IgG and IgA antibodies, whereas APOBEC 3G may contribute to the post-entry control of SHIV replication and cellular spread.

Mucosal immunization in macaques upregulates the innate APOBEC 3G anti-viral factor in CD4(+) memory T cells.

APOBEC3G is an innate intracellular anti-viral factor which deaminates retroviral cytidine to uridine. In vivo studies of APOBEC3G (A3G) were carried out in rhesus macaques, following mucosal immunization with SIV antigens and CCR5 peptides, linked to the 70kDa heat shock protein. A progressive increase in A3G mRNA was elicited in PBMC after each immunization (p<0.0002 to p< or =0.02), which was maintained for at least 17 weeks. Analysis of memory T cells showed a significant increase in A3G mRNA and protein in CD4(+)CCR5(+) memory T cells in circulating (p=0.0001), splenic (p=0.0001), iliac lymph nodes (p=0.002) and rectal (p=0.01) cells of the immunized compared with unimmunized macaques. Mucosal challenge with SIVmac 251 showed a significant increase in A3G mRNA in the CD4(+)CCR5(+) circulating cells (p<0.01) and the draining iliac lymph node cells (p<0.05) in the immunized uninfected macaques, consistent with a protective effect exerted by A3G. The results suggest that mucosal immunization in a non-human primate can induce features of a memory response to an innate anti-viral factor in CCR5(+)CD4(+) memory and CD4(+)CD95(+)CCR7(-) effector memory T cells.

Combining human antisera to human leukocyte antigens, HIVgp120 and 70 kDa heat shock protein results in broadly neutralizing activity to HIV-1.

OBJECTIVE: To elicit broadly neutralizing antibody activity by combining polyclonal human serum IgG antibodies with HIVgp120, human leukocyte antigen (HLA) class I or class II and 70 kDa heat shock protein. DESIGN: : In addition to HIV antigens, HIV-1 virions express HLA class I, HLA class II and 70 kDa heat shock protein molecules, which have quantitative and functional significance. The complementary effect of combining human polyclonal IgG antibodies with these antigens may result in effective broad spectrum neutralizing activity. METHODS: Polyclonal human sera with IgG antibodies and monoclonal antibody to HLA class I or class II, HIVgp120 and 70 kDa heat shock protein were selected and used in single, double or triple combinations. Dose-dependent inhibition studies of HIV-1 clades A, B, C and D were carried out using human CD4 T cells treated with the combinations of human sera and with monoclonal antibodies for clade B. The results are presented as half maximal (IC50) inhibitory concentration and maximum inhibition by these sera. RESULTS: The half maximal (IC50) inhibitory concentration of clade B HIV-1 infection with single or a combination of two antisera was higher than those with three antisera, which also showed maximum inhibition of HIV-1. Further investigations of human sera with HIV-1 clades C and D also showed lower half maximal (IC50) inhibitory concentrations and higher maximum inhibition with combinations of the three antisera, but this was not seen with clade A. CONCLUSION: A novel vaccination strategy eliciting broadly neutralizing antibody activity to the CCR5-using HIV-1 clades B, C and D has been demonstrated by the trimolecular complex of human antisera with HLA class II or class I, HIVgp120 and 70 kDa heat shock protein.

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.

Mucosal alloimmunization elicits T-cell proliferation, CC chemokines, CCR5 antibodies and inhibition of simian immunodeficiency virus infectivity.

The hypothesis was tested that mucosal stimulation with unmatched mononuclear cells would induce systemic alloimmune responses. Rectal or vaginal mucosal administration of 10(4)-10(7) unmatched mononuclear cells induced significant dose-dependent T-cell proliferation stimulated by the allogeneic cells in rhesus macaques. This was associated with a significant upregulation of CD8(+) T-cell-derived suppressor factor, as well as the CC chemokines CCL3, CCL4 and CCL5. In addition, there was a dose-dependent increase in antibodies to CCR5. These responses were associated with decreased in vitro simian immunodeficiency virus (SIV) infectivity of CD4(+) T cells. A further investigation of SIV infectivity of CD4(+) T cells separated from multiparous macaques also showed significant inhibition compared with male macaques. It is suggested that vaginal or rectal exposure to allogeneic stimulation by a partner's HLA antigens in seminal fluid, as occurs during sexual intercourse, or immunization by semi-allogeneic fetuses in multiparous females may elicit protection against SIV or human immunodeficiency virus infection.

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.

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-alpha, NO, and C-C chemokines. The N-terminal, ATPase portion (HSP70(1-358)) failed to stimulate any of these cytokines or chemokines. Both native and the truncated HSP70(359-610) 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 HSP70(1-358), but was enhanced with HSP70(359-610), as demonstrated by up-regulation of CD83, CCR7, CD86, CD80, and HLA class II. In vivo studies in macaques showed that immunization with HSP70(359-610) enhances the production of IL-12 and RANTES. Immunization with peptide-bound HSP70(359-610) 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.