Inducible heat shock protein 70 reduces T cell responses and stimulatory capacity of monocyte-derived dendritic cells

Heat shock protein 70 (Hsp70) has gained a lot of attention in the past decade due to its potential immunoregulatory functions. Some of the described proinflammatory functions of Hsp70 became controversial as they were based on recombinant Hsp70 proteins specimens, which were later shown to be endotoxin-contaminated. In this study we used low endotoxin inducible Hsp70 (also known as Hsp72, HSPA1A), and we observed that after a 24-h incubation of monocyte-derived immature dendritic cells (mo-iDCs) with 20 μg/ml of low endotoxin Hsp70, their ability to stimulate allogenic T cells was reduced. Interestingly, low endotoxin Hsp70 also significantly reduced T cell responses when they were simulated with either IL-2 or phytohemagglutinin, therefore showing that Hsp70 could alter T cell responses independently from its effect on mo-iDCs. We also reported a greater response of Hsp70 treatment when activated versus nonactivated T cells were used. This effect of Hsp70 was similar for all tested populations of T cells that included CD3(+), CD4(+), or CD8(+). Taken together, our observations strongly suggest that Hsp70 might dampen, rather than provoke, T cell-mediated inflammatory reactions in many clinical conditions where up-regulation of Hsp70 is observed.     

Patterns of in vivo lymphoid responses to tumour specific antigens

A tumour-derived antigen complex injected induced a different cellular immune response in tumour-bearing mice when compared with unprimed controls. A different statistical approach, namely the linear correlation, allowed us to discern significant differences in the response pattern.     

Functional activation of T cells by dendritic cells and macrophages exposed to the intracellular parasite Neospora caninum

Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. We studied how the interaction between murine conventional dendritic cells or macrophages and N. caninum influences the generation of cell-mediated immunity against the parasite. We first explored the invasion and survival ability of N. caninum in dendritic cells and macrophages. We observed that protozoa rapidly invaded and proliferated into these two cell populations. We then investigated how Neospora-exposed macrophages or dendritic cells distinguish between viable and non-viable (heat-killed tachyzoites and antigenic extract) parasites. Viable tachyzoites and antigenic extract, but not killed parasites, altered the phenotype of immature dendritic cells. Dendritic cells infected with viable parasites down-regulated the expression of MHC-II, CD40, CD80 and CD86 whereas dendritic cells exposed to N. caninum antigenic extract up-regulated the expression of MHC-II and CD40 and down-regulated CD80 and CD86 expression. Moreover, only viable tachyzoites and antigenic extract induced IL-12 synthesis by dendritic cells. MHC-II expression was up-regulated and CD86 expression was down-regulated at the surface of macrophages, regardless of the parasitic form was encountered. However, IL-12 secretion by macrophages was only observed under conditions using viable and heat-killed parasite. We then analysed how macrophages and dendritic cells were involved in inducing T-cell responses. T lymphocyte IFN-γ-secretion in correlation with IL-12 production occurred after interactions between T cells and dendritic cells exposed to viable tachyzoites or antigenic extract. By contrast, for macrophages IFN-γ production was IL-12-independent and only occurred after interactions between T cells and macrophages exposed to antigenic extract. Thus, N. caninum-induced activation of murine dendritic cells, but not that of macrophages, was associated with T cell IFN-γ production after IL-12 secretion.     

Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens: evidence from studies of proliferative responses to SB antigens

The T4 molecule has been identified as a marker of human T cell differentiation, but the function of this molecule remains to be defined. We have investigated its possible functional involvement in T cell proliferative responses to class II HLA antigens encoded by the recently described SB locus. The responses of SB-primed cells (specific for each of four different SB antigens) were studied with the use of two proliferation-inducing stimuli, SB antigen or TCGF. The proliferative responses to both stimuli were found to be mediated by T4+, T8- cells. Monoclonal antibodies against some epitopes on the T4 molecule (OKT4A and OKT4B) substantially blocked antigen-stimulated proliferative responses; antibodies against other epitopes of the T4 molecule (OKT4, T4C, T4D) blocked less well. Inhibition of SB-specific proliferation by antibodies to the T4 molecule was maximal only when the antibodies were incubated with the responder cells before the addition of stimulator cells. Proliferative responses of SB-primed cells stimulated with TCGF alone were not inhibited by any of the OKT4-related antibodies, but were completely inhibited by the anti-Tac monoclonal antibody, which reacts with the TCGF receptor. These results lend further support for the hypothesis that the T4 molecule is involved in T cell recognition of and/or activation by class II HLA antigens. We suggest that 1) the T4 molecule binds a nonpolymorphic epitope on class II HLA molecules, and 2) this interaction may facilitate, but not be an obligate requirement for, T cell activation by class II antigens.     

Recruitment of dendritic cells and macrophages during T cell-mediated synovial inflammation

Adoptive transfer of adjuvant-induced arthritis was used in this study to examine local macrophages and dendritic cells (DCs) during T cell-mediated synovial inflammation. We studied the influx of CD11b+CD11c+ putative myeloid DCs and other non-lymphoid CD45+ cells into synovium-rich tissues (SRTs) of the affected hind paws in response to a pulse of autoreactive thoracic duct cells. Cells were prepared from the SRTs using a collagenase perfusion-digestion technique, thus allowing enumeration and phenotypic analysis by flow cytometry. Numbers of CD45+ cells increased during the first 6 days, with increases in CD45+MHC (major histocompatibility complex) II+ monocyte-like cells from as early as day 3 after transfer. In contrast, typical MHC II(-) monocytes, mainly of the CD4(-) subset, did not increase until 12 to 14 days after cell transfer, coinciding with the main influx of polymorphonuclear cells. By day 14, CD45+MHC IIhi cells constituted approximately half of all CD45+ cells in SRT. Most of the MHC IIhi cells expressed CD11c and CD11b and represented putative myeloid DCs, whereas only approximately 20% were CD163+ macrophages. Less than 5% of the MHC IIhi cells in inflamed SRT were CD11b(-), setting a maximum for any influx of plasmacytoid DCs. Of the putative myeloid DCs, a third expressed CD4 and both the CD4+ and the CD4(-) subsets expressed the co-stimulatory molecule CD172a. Early accumulation of MHC IIhiCD11c+ monocyte-like cells during the early phase of T cell-mediated inflammation, relative to typical MHC II(-) blood monocytes, suggests that recruited monocytes differentiate rapidly toward the DC lineage at this stage in the disease process. However, it is possible also that the MHC IIhiCD11c+ cells originate from a specific subset of DC-like circulating mononuclear cells.     

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.     

TLR-dependent activation stimuli associated with Th1 responses confer NK cell stimulatory capacity to mouse dendritic cells

Dendritic cells (DCs) have an important role in the activation of NK cells that exert direct antitumor and antimicrobial effects and can influence the development of adaptive T cell responses. DCs acquire NK cell stimulatory capacity after exposure to various stimuli. In this study we investigated the nature of the stimuli that confer to DCs the NK cell-activating capacity. After exposure of DCs to TLR-dependent and -independent microbial stimuli and to nonmicrobial stimuli, we evaluated the ability of activated DCs to elicit IFN-gamma production from NK cells in vitro and to promote NK cell activation in vivo. We show in this study that only TLR-dependent microbial stimuli typically associated with Th1 responses confer to DCs the ability to activate NK cells, whereas stimuli associated with Th2 responses do not have this property.     

Distribution of Lyt antigens on the surface of thymocytes associated with thymic macrophages and dendritic cells

Summary Thymocyte subpopulations that are associated with macrophages and dendritic cells of the thymus in vivo were isolated from the thymuses of C57Bl/6 mice, and their Lyt phenotypes were analyzed. Electron-microscopic examination of immunogold-labeled cells revealed that the thymic complexes formed by macrophages mainly contained Lyt-2-positive thymocytes, while Lyt-1-positive thymocytes were more frequently associated with dendritic cells. The characteristic distributions of Lyt antigens on the surface of thymocytes in regions of reciprocal contact with macrophages (Lyt-2-positive cells) and dendritic cells (Lyt-1-positive cells) suggest that these antigens play a role in specific interactions between thymocytes and stroma cells.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Cytotoxic and proliferative lymphocyte responses to allogeneic and xenogeneic antigens in vitro.

In vitro lymphoproliferative responses to foreign histocompatibility antigens are phylogenetically restricted. Responses occur most readily to allogeneic or closely related xenogeneic leucocytes, but not to unrelated xenogeneic cells. Specific cytotoxic T cell responses to foreign histocompatibility antigens show the same phylogenetic restriction. This lack of xenoreactivity is not due to a lack of precursor cells for the xenoantigens; guinea-pig lymphocytes, although normally unresponsive to mouse antigens, have a similar precursor frequency for these antigens as do lymphocytes of allogeneic mouse strains. Specific cytotoxic responses of guinea-pig lymphocytes to mouse antigens can be generated if a factor released from con A stimulated guinea-pig spleen cells is added to the culture medium. The factor produced by con A-activated spleen cells (CS) is also phylogenetically restricted in its action; CS must be obtained from animals homologous with the donor of the responding lymphocytes.     

Memory and effector T cells modulate subsequently primed immune responses to unrelated antigens

Memory and effector T cells modulate subsequently primed T cell responses to the same antigen. However, little is known about the impact of pre-existing memory and effector T cell immunity on subsequently primed immune responses to unrelated antigens. Here, we show that an antigen-primed first wave of Th1 and Th2 immunity enhanced or inhibited the subsequently primed T cell immunity to an unrelated antigen, depending on whether the second antigen was administered in the same or opposite type of adjuvant. The regulatory effects of the first wave of T cell immunity on the subsequent T cell responses to an unrelated antigen attenuated over time. Notably, following challenge with the second antigen, there was a mutual cross-regulation between the first and second wave of humoral responses to unrelated antigens. Thus, immunization with one antigen not only primes immune responses to that antigen, but also influences subsequently primed immune responses to unrelated antigens.     

Role of cross-talk between IFN-alpha-induced monocyte-derived dendritic cells and NK cells in priming CD8+ T cell responses against human tumor antigens

In the present study we evaluated the role of IFN-alpha in the generation of dendritic cells (IFN-DCs) with priming activity on CD8(+) T lymphocytes directed against human tumor Ags. A 3-day treatment of monocytes, obtained as adherent PBMCs from HLA-A*0201(+) healthy donors, with IFN-alpha and GM-CSF led to the differentiation of DCs displaying a semimature phenotype, but promptly inducing CD8(+) T cell responses after one in vitro sensitization with peptides derived from melanoma (gp100(209-217) and MART-1/Melan-A(27-35)) and adenocarcinoma (CEA(605-613)) Ags. However, these features were lost when IFN-DCs were generated from immunosorted CD14(+) monocytes. The ability of adherent PBMCs to differentiate into IFN-DCs expressing higher levels of costimulatory molecules and exerting efficient T cell priming capacity was associated with the presence of contaminating NK cells, which underwent phenotypic and functional activation upon IFN-alpha treatment. NK cell boost appeared to be mediated by both direct and indirect (i.e., mediated by IFN-DCs) mechanisms. Experiments performed to prove the role of contaminating NK cells in DC differentiation showed that IFN-DCs generated in the absence of NK were phenotypically less mature and could not efficiently prime antitumor CD8(+) lymphocytes. Reciprocally, IFN-DCs raised from immunosorted CD14(+) monocytes regained their T cell priming activity when NK cells were added to the culture before IFN-alpha and GM-CSF treatment. Together, our data suggest that the ability of IFN-DCs to efficiently prime anti-tumor CD8(+) T lymphocytes relied mostly on the positive cross-talk occurring between DCs and NK cells upon stimulation with IFN-alpha.     

Capacities of migrating CD1b+ lymph dendritic cells to present Salmonella antigens to naive T cells

Dendritic cells (DCs) are well known as professional antigen-presenting cells (APC) able to initiate specific T-cell responses to pathogens in lymph nodes (LN) draining the site of infection. However, the respective contribution of migratory and LN-resident DCs in this process remains unclear. As DC subsets represent important targets for vaccination strategies, more precise knowledge of DC subsets able to present vaccine antigens to T cells efficiently is required. To investigate the capacities of DCs migrating in the lymph (L-DCs) to initiate a specific T-cell response, we used physiologically generated DCs collected from a pseudoafferent lymphatic cannulation model in sheep. The CD1b+ L-DCs were assessed for presenting antigens from the vaccine attenuated strain of Salmonella enterica serovar Abortusovis. CD1b+ L-DCs were able to phagocytose, process and to present efficiently Salmonella antigens to effector/memory T cells in vitro. They were shown to be efficient APC for the priming of allogeneic naive T cells associated with inducing both IFN-γ and IL-4 responses. They were also efficient in presenting Salmonella antigens to autologous naive T cells associated with inducing both IFN-γ and IL-10 responses. The capacities of L-DCs to process and present Salmonella antigens to T cells were investigated in vivo after conjunctival inoculation of Salmonella. The CD1b+ L-DCs collected after inoculation were able to induce the proliferative response of CD4+ T cells suggesting the in vivo capture of Salmonella antigens by the CD1b+ L-DCs, and their potential to present them directly to CD4+ T cells. In this study, CD1b+ L-DCs present potential characteristics of APC to initiate by themselves T cell priming in the LN. They could be used as target cells for driving immune activation in vaccinal strategies.     

A subset of human dendritic cells in the T cell area of mucosa-associated lymphoid tissue with a high potential to produce TNF-alpha

Recently, a new class of human dendritic cell (DC) precursors has been described in the peripheral blood recognized by the mAb M-DC8. These cells represent approximately 1% of PBMC and acquire several characteristics of myeloid DC upon in vitro culture. In this report we show that M-DC8(+) monocytes secrete in response to LPS >10 times the amount of TNF-alpha as M-DC8(-) monocytes, but produce significantly less IL-10. Consistent with a role in inflammatory responses, we found that M-DC8(+) cells localized in the T cell area of inflamed human tonsils and in the subepithelial dome region of Peyer's patches. In patients with active Crohn's disease, abundant M-DC8(+) cells were detectable in inflamed ileal mucosa, which were entirely depleted after systemic steroid treatment. Our results indicate that M-DC8(+) cells are cells of DC phenotype in inflamed mucosa-associated lymphoid tissue that may contribute to the high level of TNF-alpha production in Crohn's disease. We infer that selective elimination of M-DC8(+) cells in inflammatory diseases has therapeutic potential.     

Antibodies to Tp67 and Tp44 augment and sustain proliferative responses of activated T cells

We have shown previously that binding of a monoclonal antibody (MAb) to Tp44 molecules increased the proliferation of anti-CD3-activated T cells by causing enhanced IL 2 receptor expression and IL 2 release. We now show that anti-CD5 (Tp67) antibodies have a similar effect under conditions in which monocytes are suboptimally activated or where monocytes are not present. The activity did not depend on antibody isotype or on the precise CD5 epitope recognized. Functional experiments indicated that both IL 2 production and IL 2 receptor expression were enhanced by antibody binding. Anti-Tp67 and anti-Tp44 appear to augment proliferation through distinct mechanisms, because both antibodies together had greater activity than either antibody alone. In neither system is the Fc portion of the antibody required, because F(ab')2 fragments had activity equivalent to that of the intact antibody and were effective at concentrations as low as 10 ng/ml. Fab fragments of anti-Tp67 were active, but Fab fragments of anti-Tp44 had no effect. Anti-Tp67 and anti-Tp44 were able to sustain continuous proliferation of anti-CD3-Sepharose-stimulated T cells for up to 2.5 wk without exogenous IL 2 or feeder cells. These experiments suggest that Tp67 and Tp44 are receptors that play a critical regulatory role in the control of T cell proliferation.     

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.     

Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells

Signaling through the PGI(2) receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI(2.) In this study, we determined that PGI(2) analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI(2) analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI(2) analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-alpha, IL-1alpha, IL-6) and chemokines (MIP-1alpha, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-kappaB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI(2) signaling through the IP may exert anti-inflammatory effects by acting on DC.     

The cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage

Anti-CD3 (T3) Ab reacting with different proportions of thymocytes (anti-CD3a: UCHT1, anti-CD3b: T10B9, and anti-CD3c: OKT3) were tested for cytoplasmic (cCD3) and membrane (mCD3) expression in the bone marrow, thymus, and blood in man and selected primates. The expression of cCD3a and cCD3c in the perinuclear and Golgi area of large, BrdU-incorporating, strongly TdT+ thymic blasts probably represents one of the earliest signs of T cell commitment, because these blast cells are CD1-, CD4-, CD8-, and mCD3-. The cCD3+, TdT+ cells are normally restricted to the thymus and are absent among the TdT+ cells of bone marrow. The anti-CD3b Ab used, T10B9, co-caps and co-modulates with the other anti-CD3 Ab and is a T cell-specific reagent at a membrane level but does not bind to perinuclear cCD3. Instead, this reagent cross-reacts with a filamentous cytoplasmic network in non-T cells in man and in primates S. oedipus and M. rhesus despite their T cell negativity for mCD3. The characteristics of all T-ALL cases studied: cCD3+, CD7+ along with nuclear TdT+ suggest lineage fidelity to early thymic blasts. As a marked contrast, cCD3 is absent in common ALL and in AML, including cases that concomitantly express CD7 and myeloid antigens. Thus, the cCD3, TdT combination provides a very sensitive assay for residual T-ALL blasts outside the normal thymus.