Macrophages as main inducers of IFN-
in T cells following administration of human and mouse heat shock protein 60
Minka Breloer1,
Solveig H. Moré1,
Anke Osterloh1,
Felix Stelter2,
Robert S. Jack2 and
Arne von Bonin1
1 Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht Strasse 74, 20359 Hamburg, Germany 2 Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, 17489 Greifswald, Germany
Correspondence to: A. von Bonin; E-mail: Arne_von_Bonin{at}magicvillage.de
Transmitting editor: S. H. E. Kaufman
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Abstract
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Human Hsp60 (hHsp60) elicits a potent pro-inflammatory response in cells of the innate immune system. Here we compared the capacity of peritoneal exudate cells (PEC) and bone marrow-derived dendritic cells (DC) to stimulate murine T cells in the presence of Hsp60. Hsp60 induced a specific secretion of high amounts of IFN-
in T cells with PEC as antigen-presenting cells (APC). Although DC are highly efficient APC, they were much less potent as inducers of IFN-
in the presence of Hsp60. The IFN-
-inducing effect of Hsp60 is dependent on co-stimulatory signals provided by B7CD28 interactions. In addition to hHsp60, we used syngenic murine recombinant Hsp60 (mHsp60) and show that mHsp60 also induces IFN-
in TCR transgenic T cells. These results demonstrate that mHsp60 as an endogenous self molecule can induce an inflammatory response. Interestingly, mHsp60, although sharing >98% protein sequence identity with the hHsp60 homologue, does not bind to human CD14 molecules. Taken together, our results indicate a finely tuned activation of cells from the innate and adaptive immune system by self Hsp60 that depends strongly on the type of APC.
Keywords: antigen-presenting cell, danger signal, dendritic cell, Hsp60
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Introduction
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Heat shock proteins (HSP) are thought to protect against cellular stress (1). From an immunological point of view it has been shown that eukaryotic HSP, besides their peptide-specific effects (26), e.g. in generating a cytotoxic T lymphocyte response (7), serve as danger signals (8) to the innate immune system (911). Murine and human macrophages were found to elicit a profound pro-inflammatory response when incubated with recombinant human Hsp60 (hHsp60) (12). The response included the up-regulation of co-stimulatory molecules, and the release of inflammatory cytokines like IL-6 and tumor necrosis factor (TNF)-
. It was shown that CD14, a monocyte receptor for lipopolysaccharide (LPS), is necessary for hHsp60-mediated signaling (13). CD14 is a GPI-linked receptor which has no transmembrane domain. Signaling through CD14 requires the Toll-like receptor (TLR)-4 molecule as a co-receptor. Activation of monocytes and macrophages by hHsp60 depends on the presence of TLR-4 and mice carrying a mutant TLR-4 are non-responsive to Hsp60 (14).
We have recently shown that hHsp60, in addition to its ability to activate professional antigen-presenting cells (APC), also influences the activation of T cells. Addition of hHsp60 to cultures containing peritoneal exudate cells (PEC) and ex vivo purified T cells, expressing transgenic MHC class I- or -II-restricted TCR specific for chicken ovalbumin (OVA)-derived peptides, specifically induce a release of IFN-
(15). In contrast, proliferation and IL-2 secretion were not changed in cultures containing hHsp60.
So far, we have no information as to whether dendritic cells (DC) (16), the most potent APC, are able to respond to Hsp60 by inducing increased IFN-
release from T cells. In addition, little is known as to whether Hsp60 molecules derived from different species influence the activation of the innate immune system. The high degree of sequence identity of mammalian HSP [there is >98% amino acid sequence identity between mouse (mHsp60) and hHsp60] strongly suggests conservation of biological functions. Here we compared different APC and the specific induction of IFN-
in ex vivo purified T cell cultures of the hHsp60 and the mHsp60 molecules.
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Methods
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Mice, cells and antibodies
Eight- to 10-week-old OT-1 TCR transgenic mice (specific for OVA257264/H2-Kb) (17) and DO11.10 mice (specific for OVA323339/H2-Ad) (18) were maintained in the animal facilities at the BNI. All cells were cultured in RPMI 1640 medium supplemented with 10% FCS, HEPES and L-glutamine. To induce peritoneal macrophages, mice were injected with 500 µl pristane (Sigma, Deisenhofen, Germany) i.p. PEC were harvested 56 days later by rinsing the peritoneum with ice-cold medium. Only freshly prepared PEC were used in the experiments described. FACS analysis of isolated PEC showed a >95% staining for the macrophage surface marker Mac-1. T cells from spleens from TCR transgenic mice were enriched via adherence to nylon wool. The resulting cells were typically >60% for TCR+ cells. Quantification of IFN-
and IL-2 was performed by ELISA (PharMingen, Heidelberg, Germany).
Hsp60
Hsp60s were recombinant mycobacterial (SPP881, lot 006408; Stressgen, Victoria, BC, Canada), human (ESP740, lot 008404) and murine (SPP741, lot 008414; Stressgen and mHsp60 obtained from Dr Ruurd van der Zee, Utrecht, The Netherlands). Quantification of contaminating endotoxins revealed a concentration of LPS in the range of 10150 EU/mg Hsp60 (Limulus amebocyte lysate kit, QCL-1000; Biowhittaker Walkersville, MD). Individual Hsp60 samples were tested by SDSPAGE and silver staining followed by Western blotting (ECL; Amersham-Pharmacia, Little Chalfont, UK) using mAb specific for human Hsp60 (LK-1; Stressgen).
T cell assays
Purified naive T cells (5 x 104/well) from TCR transgenic mice were incubated for the indicated time periods in the presence or absence of Hsp60 with syngenic pristane-induced PEC or bone marrow-derived DC (5 x 104/well) that had or had not been pulsed with OVA peptides (2 h, 37°C). For cytokine quantification, supernatant from the individual cultures was collected and analyzed for IFN-
or IL-2 content, respectively. Remaining cultures were labeled with [3H]thymidine for an additional 18 h to quantify proliferation.
DC
Bone marrow-derived DC were generated as previously described (19). Briefly, bone marrow was collected from tibias and femurs of BALB/c mice and resuspended in complete RPMI-1640 medium. Then 2 x 106 cells were placed in round plastic Petri dishes (no tissue culture surface) in 10 ml complete RPMI 1640 containing 20 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF; Biomol, Hamburg, Germany). On day 3, 10 ml RPMI 1640 was added containing 20 ng/ml GM-CSF. On day 6 and 8, 10 ml of the culture supernatants was exchanged with 10 ml fresh medium (20 ng/ml GM-CSF). DC cultures were
6070% positive for CD11c staining. Less than 10% of all cells analyzed stained for the marker CD14. Polymyxin B (100 U/ml; Sigma, Deisenhofen, Germany) was added in some experiments as an internal LPS control.
CD14 binding assay and FACS analysis
CHO-human CD14 or mock-transfected CHO-DHFR cells (2 x 105) were subsequently incubated with 25 µl FCS, a competing protein or LPS (Escherichia coli strain 055:B5; Sigma, Deisenhofen, Germany) and 0.3 µg FITC-labeled LPS at 4°C. Samples were washed, fixed and analyzed in a FACScan (Becton Dickinson, Heidelberg, Germany). As a control, the competing proteins were heated to 95°C for 15 min prior addition to the transfected CHO cells.
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Results
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Analysis of the different Hsp60 preparations
hHsp60, mHsp60 and mycobacterial Hsp65 (as a control) were obtained from Stressgen, and analyzed for purity by SDSPAGE followed by silver staining. As depicted in Fig. 1(A), all Hsp60 preparations tested showed a single protein band of
60 kDa. Western blotting with a hHsp60-specific mAb confirmed the protein band as the Hsp60 molecule and revealed no detectable degradation products (Fig. 1B). Mycobacterial Hsp65 sequences differ significantly from both hHsp60 and mhsp60, and therefore it does not show up on this Western blot developed with a hHsp60-specific mAb.

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Fig. 1. Biochemical analysis of the various Hsp60 preparations. Purity and identity of hHsp60 (100 ng), mHsp60 (100 ng) and mycobacterial Hsp60 (100 ng) was analyzed by SDSPAGE followed by silver staining (A) and Western blotting using a Hsp60-specific antibody (B). Numbers on the side indicate relative molecular masses. (C) LPS content of each Hsp60 preparation was determined as described in Methods. Measurement was repeated twice with triplicate values. Error bars indicate SD.
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Since LPS has very similar effects to Hsp60 in our T cell assays (20), we quantified the endotoxin content of the individual Hsp60 preparations. Figure 1(C) shows that hHsp60 and mHsp60 contained a comparable low level of LPS contamination, with slightly higher amounts measurable in the mHsp60 preparation, whereas in the mycobacterial Hsp60 sample no LPS could be detected. The LPS contents of the human and murine preparations are 1020 times lower than the required amounts of purified E. coli or Staphylococcus abortus-derived LPS required to obtain specific IFN-
secretion in the T cell assays (data not shown).
Macrophages and DC differ in their capacity to induce IFN-
in T cells
Recent data revealed that Hsp60 stimulates murine and human APC to release cytokines (12,13). A direct activation of professional APC with HSP, thus, may directly contribute to the activation of T cells in vitro and in vivo. We have recently shown that professional APC like PEC and bone marrow-derived DC secrete pro-inflammatory cytokines and NO when hHsp60 is added to the cultures (20). To analyze how DC and PEC stimulate IFN-
secretion in T cells when incubated with Hsp60, we first compared the release of IL-12, which is one of the most important regulatory cytokines of IFN-
induction in NK and T cells. As shown in Fig. 2(A), PEC and DC released IL-12 in the presence of hHsp60. The induction of IL-12 in PEC and DC measured in independent experiments is comparable, although in the experiment shown in Fig. 2 DC actually secreted slightly higher amounts of IL-12 in response to hHsp60. Next, purified DO11.10 T cells (expressing a transgenic TCR for OVA323339/H2-Ad) were co-cultured with antigen-pulsed DC or PEC. As shown in Fig. 2(B), OVA peptide-pulsed DC are much better APC with respect to induced T cell proliferation and IL-2 (data not shown) and IFN-
production. However, addition of hHsp60 to the cultures (Fig. 2C) resulted in a significant increase in IFN-
when PEC (and bone marrow-derived macrophages, not shown) were used as APC, whereas DC in the presence of hHsp60 were reproducibly less potent to induce IFN-
in T cells. The induction of IFN-
was completely blocked when the Hsp60 preparations were boiled prior to addition to the cultures, arguing against LPS contamination as a potential source for the observed effects [data not shown and (15)]. Thus, hHsp60-activated PEC and DC respond similarly in their release of soluble factors, but clearly differ in their ability to stimulate IFN-
release.

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Fig. 2. DC and PEC differ in their response to Hsp60. (A) Bone marrow-derived DC or pristane-induced PEC were incubated for 24 h in medium or in medium containing 10 µg/ml hHsp60. IL-12 was determined in the supernatants using a IL-12p40-specific ELISA. (B) Different numbers of OVA323339 (5 µg/ml)-pulsed BALB/c PEC (squares) or DC (circles) (x-axis) were incubated with 5 x 104 purified OVA323339-specific TCR transgenic DO11.10 T cells. (C) Titrated numbers of pulsed PEC (squares) and DC (circles) were incubated together with purified DO11.10 T cells in the absence of Hsp60 (open symbols) or in the presence (closed symbols) of hHsp60 (10 µg/ml) for 24 h. Shown is the amount of IFN- in the supernatants determined with specific ELISAs. Results are presented as means ± SD of triplicates. One out of two independent experiments is shown. Comparable results were obtained in an independent experimental set-up using MHC class I-restricted OVA257264-specific OT-1 T cells.
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mHsp60 induces IFN-
in ex vivo purified T cells
Addition of hHsp60 to cultures containing purified T cells expressing a transgenic TCR and antigen-pulsed PEC leads to a rapid and high induction of IFN-
in the T cells (Fig. 2) (15,20). As shown in Fig. 3, the addition of mHsp60 also leads to the secretion of IFN-
into the culture supernatants, but with delayed kinetics when compared to the hHsp60 homologue. Following the kinetics of IFN-
in the cultures, mHsp60 and hHsp60 induced comparable amounts of IFN-
at day 2 of culture, and this comparable level of IFN-
was maintained until day 5 (data not shown). Thus, we show that not only xenogenic but also an endogenous, self heat shock protein can activate the immune system.
The IFN-
-inducing effect of hHsp60 and mHsp60 requires co-stimulation
We have previously shown that the enhanced secretion of IFN-
in T cells in the presence of Hsp60 requires IL-12 produced by the APC (15,20). Since a complete activation of T cells requires two signals, signal 1 provided by TCRMHC interactions and signal 2 given by co-stimulatory molecules on the T cell, we tried to block the interactions of B7 and CD28 molecules on APC and T cells using CTLA-4Ig fusion proteins. As shown in Fig. 4(A), titration of CTLA-4Ig into cultures containing hHsp60 or mHsp60 blocked the enhanced secretion of IFN-
completely. As shown in earlier reports (20), the production of IL-2 is not positively affected by the presence of Hsp60 in the T cell assays (Fig. 4A, lower panel). Using the CTLA-4Ig fusion proteins, thus, reveals the requirement for co-stimulatory signals of APC and T cell in the presence of Hsp60 to permit an enhanced synthesis of IFN-
. As shown in Fig. 4(B), hHsp60 and mHsp60 clearly differ in their ability to induce pro-inflammatory cytokines like IL-1 and IL-6. These lower levels of IL-1 and IL-6 may contribute to the weaker stimulation potential of mHsp60.
mHsp60 does not bind to CHO cells transfected with the human CD14 molecule
Since it was shown that the presence of the CD14 molecule plays a crucial role in Hsp60-mediated APC activation (13), we compared the binding of hHsp60, hamster (haHsp60) and mHsp60 to CHO cells transfected with the human CD14 (hCD14) molecule. To detect specific binding of Hsp60, we used a competition assay in which we tried to compete FITC-labeled LPS (FITCLPS) with hHsp60, haHsp60 or mHsp60. This way of performing the assay has the advantage that Hsp60 is not modified by labeling procedures and therefore can bind hCD14 in the native form. As shown in Fig. 5(A), FITCLPS bound to hCD14-transfected CHO cells, but not to mock-transfected CHO-DHFR cells (Fig. 5C). The binding of FITCLPS could be prevented by preincubation with titrated amounts of unlabeled LPS. In a similar way, hHsp60 and haHsp60 competed for binding to hCD14 (Fig. 5B). mHsp60, like the control protein BSA, in contrast, showed no specific binding to CD14 transfected CHO cells, reflected by the unchanged fluorescence intensity of FITCLPS-labeled hCD14-CHO cells (Fig. 5A and B). Even at 3 times higher concentrations (compared to hHsp60), mHsp60 showed only a negligible binding to CD14-transfected CHO cells. hHsp60 and mHsp60, thus, clearly differ in their binding to the hCD14 molecule.

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Fig. 5. mHsp60 does not bind to hCD14. (A) LPSFITC-treated (300 ng) CD14 transfected (CHO-CD14) were incubated without additional reagents (thick line) or with a 10-fold molar excess of unlabeled LPS, hHsp60 or mHsp60 (thin line). The LPS panel also shows the inhibition of a 3-fold molar excess of unlabeled LPS (dotted line). (B) CHO-CD14 and CHO mock-transfected (CHO-DHFR, C) were analyzed in the absence of additional reagents or were analyzed following incubation with the indicated competing reagents (Komp.). Numbers in brackets indicate molar excess to the LPSFITC input. A typical result from three independent experiments is shown.
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Discussion
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Recent studies from different groups have shown that HSP stimulate the production of cytokines from mononuclear cells (913). Our group has extended these studies by showing that human Hsp60 can act as molecular link between the innate immune response and the adaptive T cell-dependent response, by specifically inducing IFN-
in T cells. DC are highly efficient APC and are able to activate naive T cells (16). In the present study we compared the capacity of DC and PEC to induce IFN-
in T cells following the addition of Hsp60. Interestingly, Hsp60 exhibits a high potential to specifically stimulate IFN-
in T cells when PEC are present as APC. Although DC also induce a measurable release of IFN-
in T cells when cultured together with Hsp60, this effect is much less pronounced. The reason for this discrepancy between DC effectiveness as APC and their relative weak induction of IFN-
in T cells is not completely understood. Both DC and PEC respond comparably to Hsp60 (and LPS) with respect to release of soluble factors like IL-1, IL-12, TNF-
and NO as well as with an up-regulation of co-stimulatory molecules like B7-2 in the absence of T cells (20). Presumably, Hsp60 induces a (so-far unidentified) soluble factor or membrane-bound receptor more efficiently on macrophages than on DC, which in turn leads to higher IFN-
secretion in T cells.
Most studies investigating a potential immunological function of Hsp60 employed recombinant hHsp60. Given the availability of recombinant hHsp60, and the very high homology between the mouse and human proteins, this seems initially a very reasonable approach. Our data show that mHsp60 and hHsp60 have comparable potentials to modulate an immune response. Our data indicate a similar IFN-
-inducing capacity between these two members of the Hsp60 family with differences in the kinetics of IFN-
release. mHsp60 induces lower amounts of IFN-
and reaches the plateau at later time points. Again, these differences might relate to different quantities of soluble factors, produced by the APC or might be due to batch variations of different hHsp60 and mHsp60 preparations. However, the observed differences could be repeated with independently generated Hsp60 preparations. Incubation of purified professional APC with human and mHsp60 revealed that mHsp60 induced lower amounts of IL-1, IL-6 and IL-12 (Fig. 4 and data not shown), which might explain the delayed secretion of IFN-
in T cell assays containing mHsp60.
Most intriguingly, mHsp60 revealed no detectable binding to hCD14 molecules. The region for LPS binding on the hCD14 molecule has been characterized and is located in the region of amino acids 3944 (21). Whether mHsp60 and hHsp60 bind to the same epitope remains to be shown. The lack of binding of mHsp60 to the hCD14 molecule might indicate intrinsic differences of the highly conserved Hsp60 family members. Experiments are ongoing to analyze biochemical properties of Hsp60, e.g. binding to the CD14 receptor, in more detail.
Taken together, our data show that the source of APC in an inflammatory microenvironment has direct consequences for the stimulation of antigen-specific T cells. Moreover, subtle differences in the primary structure in individual members of the Hsp60 family exist, which may have direct consequences for the stimulation of cells from the innate and adaptive immune system.
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Abbreviations
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APCantigen-presenting cell
DCdendritic cell
GM-CSFgranulocyte macrophage colony stimulating factor
hhuman
hahamster
HSPheat shock protein
LPSlipopolysaccharide
mmurine
OVAovalbumin
PECperitoneal exudate cells
TLRToll-like receptor
TNFtumor necrosis factor
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References
|
---|
- Hartl, F. U. 1996. Molecular chaperones in cellular protein folding. Nature 381:571.[ISI][Medline]
- Fourie, A. M., Sambrooks, J. F. and Gething, M. J. H. 1994. Common and divergent peptide binding specificities of hsp70 molecular chaperones. J. Biol. Chem. 269:30470.[Abstract/Free Full Text]
- Li, Z. and Srivastava, P. K. 1994. A critical contemplation on the role of heat shock proteins in transfer of antigenic peptides during antigen presentation. Behring Inst. Mitt. 94:37.[Medline]
- Arnold, D., Faath, S., Rammensee, H. G. and Schild, H. 1995. Cross-priming of minor histocompatibility antigen-specific cytotoxic T cells upon immunization with the heat shock protein gp96. J. Exp. Med. 182:885.
- Nieland, T. J. F., Tan, M. C. A., Monnee-van Muijen, M., Koning, F., Kruisbeek, A. M. and van Bleek, G. M. 1996. Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94. Proc. Natl Acad. Sci. USA 93:6135.[Abstract/Free Full Text]
- Lammert, E., Arnold, D., Nijenhuis, M., Momburg, F., Hämmerling, G. J., Brunner, J., Stevanovic, S., Rammensee, H. G. and Schild, H. 1997. The endoplasmic reticulum-resident stress protein gp96 binds peptides translocated by TAP. Eur. J. Immunol. 27:923.
- Srivastava, P. K., Udono, H., Blachere, N. E. and Li, Z. 1994. Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 39:93.[ISI][Medline]
- Matzinger, P. 1994. Tolerance, danger and the extended family. Annu. Rev. Immunol. 12:991.[ISI][Medline]
- Asea, A., Kraeft, S. K., Kurt-Jones, E. A., Stevenson, M. A., Chen, L. B., Finberg, R. W., Koo, G. C. and Calderwood, S. K. 2000. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat. Med. 6:435.[ISI][Medline]
- Singh-Jasuja, H., Scherer, H. U., Hilf, N., Arnold-Schild, D., Rammensee, H. G., Toes, R. E. and Schild, H. 2000. The heat shock protein gp96 induces maturation of dendritic cells and down-regulation of its receptor. Eur. J. Immunol. 30:2211.[ISI][Medline]
- Binder, R. J., Anderson, K. M., Basu, S. and Srivastava, P. K. 2000. Heat shock protein gp96 induces maturation and migration of CD11c+ cells in vivo. J. Immunol. 165:6029.[Abstract/Free Full Text]
- Ohashi, K., Burkart, V., Flohe, S. and Kolb, H. 2000. Heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J. Immunol. 164:558.[Abstract/Free Full Text]
- Kol, A., Lichtman, A. H., Finberg, R. W., Libby, P. and Kurt-Jones, E. A. 2000. Heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J. Immunol. 164:13.[Abstract/Free Full Text]
- Vabulas, R. M., Ahmad-Nejad, P., da Costa, C., Miethke, T., Kirschning, C. J., Hacker, H. and Wagner, H. 2001. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J. Biol. Chem. 276:31332.[Abstract/Free Full Text]
- Breloer, M., Dorner, B., More, S. H., Roderian, T., Fleischer, B. and von Bonin, A. 2001. Heat shock proteins as danger signals: eukaryotic Hsp60 enhances and accelerates antigen-specific IFN-gamma production in T cells. Eur. J. Immunol. 31:2051.[ISI][Medline]
- Steinman, R. M. and Banchereau, J. 1998. Dendritic cells and the control of immunity. Nature 392:245.[ISI][Medline]
- Hogquist, K. A., Jameson, S. C., Heath, W. R., Howard, J. L., Bevan, M. J. and Carbone, F. R. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76:17.[ISI][Medline]
- Murphy, K. M., Heimberger, A. B. and Loh, D. Y. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250:1720.[ISI][Medline]
- Lutz, M. B., Kukutsch, N. A., Menges, M., Rossner, S. and Schuler, G. 2000. Culture of bone marrow cells in GM-CSF plus high doses of lipopolysaccharide generates exclusively immature dendritic cells which induce alloantigen-specific CD4 T cell anergy in vitro. Eur. J. Immunol. 30:1048.
- Moré, S. H., Breloer, M. and von Bonin, A. 2001. Eukaryotic heat shock proteins as molecular links in innate and adaptive immune responses: Hsp60-mediated activation of cytotoxic T cells. Int. Immunol. 13:1121.
- Stelter, F., Bernheiden, M., Menzel, R., Jack, R. S., Witt, S., Fan, X., Pfister, M. and Schutt, C. 1997. Mutation of amino acids 3944 of human CD14 abrogates binding of lipopolysaccharide and Escherichia coli. Eur. J. Biochem. 243:100.[Abstract]