Immunization of mice with the heat shock protein (HSP) gp96 but not control proteins leads to 5- to 7-fold enlargement of draining lymph nodes (LNs) resulting from accumulation of large numbers of mature CD11c+ cells, but not T or B lymphocytes in them. The increase in size and cellularity is time-dependent; the draining LNs reach their peak size between 12 and 24 h after injection and regress to their normal size between 48 and 72 h after injection. The increment is elicited specifically in the draining LN but not in other LNs. This observation uncovers a novel aspect of HSP-APC interaction and adds to the mechanistic explanation for the unusually high immunogenicity of HSP-peptide complexes.

Heat shock proteins (HSPs)3 interact with the immune system in a multiplicity of ways, all of which rely on the interaction of HSPs with APCs (Fig. 1). HSPs chaperone antigenic peptides (1, 2, 3, 4, 5, 6, 7) and interact with APCs through receptors, such as CD91 (for the HSP gp96; Ref. 8). HSPs are also released from cells as a result of necrotic, but not apoptotic, death (9), and when released, interact with APCs to mediate translocation of NF-κB into the nucleus with rapid kinetics (9). This results in elaboration of a number of pro-inflammatory cytokines such as TNF-α, IL-12, GM-CSF, and IL-1β (9, 10). Bacterial HSPs, hsp60 (11), and mammalian HSPs gp96 (9), hsp90 (9), hsp70 (9), and calreticulin (9) have been shown to mediate a number of these effects.

FIGURE 1.

Two modes of HSP-APC interaction. Top panel, The acquisition of HSP-peptide complexes by APCs through an HSP receptor such as CD91 (8 ), followed by representation of HSP-peptide complexes by the MHC I molecules of the APC (15 ). These MHC I-peptide complexes stimulate the T lymphocytes. Bottom panel, The stimulation of APC by HSPs leading to elaboration of cytokines and expression of Ag presenting and costimulatory molecules in an Ag-independent manner (9 ).

FIGURE 1.

Two modes of HSP-APC interaction. Top panel, The acquisition of HSP-peptide complexes by APCs through an HSP receptor such as CD91 (8 ), followed by representation of HSP-peptide complexes by the MHC I molecules of the APC (15 ). These MHC I-peptide complexes stimulate the T lymphocytes. Bottom panel, The stimulation of APC by HSPs leading to elaboration of cytokines and expression of Ag presenting and costimulatory molecules in an Ag-independent manner (9 ).

Close modal

The studies reported here uncover a phenomenon in vivo that reveals a novel aspect of HSP-APC interaction. Immunization of mice with the HSP gp96 but not control proteins leads to enlargement of draining lymph nodes (LNs) resulting from accumulation of large numbers of mature CD11c+ cells, but not other cells in them. This observation suggests that immunization with HSPs results in elaboration of signals that exert a powerful effect on trafficking of APCs, and adds to the mechanistic explanation for the extraordinary immunogenicity of HSP-peptide complexes.

C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were maintained by Center for Laboratory Animal Care at the University of Connecticut Health Center. Anti-CD11c, CD4, CD8, CD80 (B7-1), CD86 (B7-2), ICAM-1, MHC I, MHC II, and B220 Abs used for flow cytometry were purchased from PharMingen. The Fc Block (anti-CD16/32) Ab was used to block binding to Fc receptors on APCs in all the FACScan analysis. Flow cytometry was performed on a FACScan (Becton Dickinson, San Jose, CA).

All inoculations were intradermal and, except in Fig. 2,C, were mid-ventral (at the navel). All material was injected in 100 μl PBS. In the experiment shown in Fig. 2 C, inoculations were biased toward the right or left as shown. To determine the identity of the draining LNs, initial inoculations were performed with patent blue dye (Sigma, St. Louis, MO). LNs staining blue after 10–20 min were determined to be the draining LN. At the site of inoculation chosen, the left and right axillary and inguinal LNs were draining LNs. For ease of measurement, the inguinal LNs were studied in this report.

FIGURE 2.

Immunization with gp96 leads to enlarged draining LNs with a higher cellularity. A, Draining LNs were dissected and photographed 12 h after inoculation with 1 μg gp96, 1 μg albumin, or PBS as indicated (left). Right, A patent blue dye was used to highlight the LN from mice injected with the dye alone, or with gp96 as well as the dye. These experiments were conducted with at least 25 mice in each group and the data are representative of these many experiments. B, Mice were immunized with 1 μg gp96, phosphorylase b, SA, or PBS or were sham-injected with a needle, as indicated. The draining LNs were isolated and measured with a caliper and also used to make cell suspensions at 0, 3, 6, 12, 24, 48, and 72 h after injection. The volumes of the LNs (top graph) and the total number of cells (bottom graph) in the LNs was measured and plotted. For all immunizations except with gp96, only the measurements from the 12-h time point are shown. These experiments were conducted with at least 25 mice in each group and the data are representative of these experiments. C, Gp96 was injected at the mid-ventral position of mice biased toward the right (left panel) or in the middle at the navel (right panel). The former leads to draining in the right nodes, while the latter leads to draining into both left and right nodes. These experiments were conducted with at least 10 mice in each group and the data are representative of these experiments. The error bars are visible on some data and not on others in B and C because of the extremely small variability of the data at some time points.

FIGURE 2.

Immunization with gp96 leads to enlarged draining LNs with a higher cellularity. A, Draining LNs were dissected and photographed 12 h after inoculation with 1 μg gp96, 1 μg albumin, or PBS as indicated (left). Right, A patent blue dye was used to highlight the LN from mice injected with the dye alone, or with gp96 as well as the dye. These experiments were conducted with at least 25 mice in each group and the data are representative of these many experiments. B, Mice were immunized with 1 μg gp96, phosphorylase b, SA, or PBS or were sham-injected with a needle, as indicated. The draining LNs were isolated and measured with a caliper and also used to make cell suspensions at 0, 3, 6, 12, 24, 48, and 72 h after injection. The volumes of the LNs (top graph) and the total number of cells (bottom graph) in the LNs was measured and plotted. For all immunizations except with gp96, only the measurements from the 12-h time point are shown. These experiments were conducted with at least 25 mice in each group and the data are representative of these experiments. C, Gp96 was injected at the mid-ventral position of mice biased toward the right (left panel) or in the middle at the navel (right panel). The former leads to draining in the right nodes, while the latter leads to draining into both left and right nodes. These experiments were conducted with at least 10 mice in each group and the data are representative of these experiments. The error bars are visible on some data and not on others in B and C because of the extremely small variability of the data at some time points.

Close modal

Homogenous preparations of gp96 were obtained by published procedures (7). Albumin and phosphorylase b were purchased from Sigma.

LNs were measured in three dimensions using vernier calipers. The average of the three measurements was used as the radius to calculate the volume of the LN. The number of cells was obtained by counting crushed LNs with a hemocytometer under a light microscope (Telaval 31; Zeiss, Oberkochen, Germany).

CD11c+ cells were purified from the LN of mice injected 12 h earlier with 1 μg EG.7- or EL4-derived gp96. Purification was performed using the magnetic bead system (Miltenyi Biotec, Auburn, CA) and purity of >96% was routinely obtained as determined by FACS analysis. Purified CD11c+ (105) cells were adoptively transferred i.v. into mice via the retro-orbital route. Spleen cells were harvested 1 wk after the transfer and stimulated with irradiated EG.7 cells for 1 wk and a further restimulation for another week. Cytotoxic T cell assays were performed using EG.7 or EL4 cells as targets.

C57BL/6 mice were immunized intradermally, as described in Materials and Methods, with 1 μg of normal liver-derived gp96. All gp96 preparation used in this study had undetectable levels (<0.02 endotoxin units (eu)/ml) of bacterial LPS. The draining inguinal LNs of the mice were observed to be significantly larger in size as compared with the corresponding LNs of unimmunized mice or mice immunized with serum albumin (SA), saline, or a patent blue dye used to highlight the nodes (Fig. 2,A). Normal liver-derived gp96, tumor-derived gp96, or in vitro reconstituted complexes of gp96 with an antigenic peptide (SIINFEKL) elicited the same response, indicating the peptide-independent nature of this response (data not shown). This phenomenon was investigated in more detail in terms of its kinetics and specificity. Groups of mice (two mice per group) were injected intradermally with PBS, or with 1 μg phosphorylase b, SA, or gp96. As an additional control, one group of mice was sham-injected with a needle so as to pierce the skin without administering any substance. The volumes of the draining inguinal LN as well as the total number of cells in the LN were measured 12 h after injection. In addition, the kinetics of these two parameters was measured at 3, 6, 12, 24, 48, and 72 h after injection in case of gp96-injected mice (Fig. 2 B). The following was observed: 1) the increase in size of the draining LN was specific to gp96-injected mice, in that sham injection, or injection with saline or control proteins did not lead to such increase; 2) the increase in size of the LN was reflected in an increase in the number of cells in the LN, and 3) the increase in size and cellularity was time-dependent. The peak size was attained at 12–24 h after injection and the LN returned to their original size 72 h after injection. 4) Three quantities of gp96 at 0.3, 1, and 3.0 μg were tested in this assay. Although the effect was detectable at 0.3 μg as well, the effect was optimal at 1 μg and appeared to have reached a plateau, as 1 and 3 μg showed an identical activity (data not shown).

Specificity of the phenomenon was observed in yet another way. The increase in size of the LN was found to be restricted to the draining LN. Thus, if mice were injected ventrally biased to the right side of the mouse, the right inguinal, but not the left inguinal, LN was found to be increased in size (Fig. 2,C, left panel). In contrast, if the mice were injected in the middle (mid-ventral), which drains into the left and the right sides, inguinal LNs on both sides were observed to be enlarged (Fig. 2 C, right panel).

The observed effects cannot be attributed to contaminating LPS for the following reasons: 1) all gp96 preparation used in this study had undetectable levels (<0.02 eu/ml) of bacterial LPS. The presence of gp96 does not interfere with the LAL assay used to detect LPS. 2) The quantity of LPS required to obtain the effect seen with gp96 is 1 mg (∼400 eu/ml) or several log scales higher than the highest possible level of LPS contamination of the gp96 preparations used here.

The LNs of naive and gp96-immunized mice were examined for their major cellular constituents, the CD11c+ cells, CD4+ and CD8+ T and B lymphocytes. CD11c+ cells were observed to constitute less than 2% of the total LN cells, while CD4+, CD8+, and B lymphocytes constituted 29, 25, and 38% respectively. In contrast to these numbers in the naive mice, and much to our surprise, CD11c+ cells were observed to constitute as much as 30–40% of the draining LN of the gp96-injected mice (Fig. 3,A). There was no increase in the numbers of T or B lymphocytes in the LNs of gp96-immunized mice (Fig. 3,A). Indeed, as expected, the proportion of the CD8+ and CD4+ T lymphocytes and B lymphocytes in the enlarged LNs was considerably lower than that in naive mice (Fig. 3 A). The total absolute number of the CD11c cells (T and B lymphocytes) in naive and gp96-immunized mice was nearly identical (data not shown).

FIGURE 3.

Gp96-elicited increase in LN volume is due to infiltration of mature CD11c+ cells. A, Draining inguinal LNs were removed from mice immunized with saline or with 1 μg gp96. Single-cell suspensions were obtained and stained with Abs to CD11c, CD4, CD8, or B220 molecules, as described in Materials and Methods. B, CD11c+ cells in the LNs of gp96-injected mice were double stained for CD11c and MHC I, MHC II, CD80, CD86, or ICAM-1 expression. CD11c+ cells were gated for (green and red lines). For the bottom panel of this figure, LN cells were stained for MHC II and B220 expression or MHC II and CD11c expression. CD11c+ cells (green and red lines) or B220+ cells (pink line) were gated for. C, The kinetics of accumulation of CD11c+ cells in the draining LN was measured after injection of 1 μg gp96 or SA. In the experiment shown, the CD11c+ cells in the SA-injected mice were monitored only up to 12 h; this experiment has been conducted four times and the CD11c+ cells do not accumulate to higher levels in SA-injected mice even at later time points. D, CD11c+ cells from gp96-injected mice are functionally active. C57BL/6 mice were injected with 1 μg gp96 derived from E.G7 or EL4 cells. Draining LN cells were isolated 12 h later and CD11c+ cells were purified and transferred (105 cells/mouse) adoptively to naive C57BL/6 mice. Seven days later, spleen cells from these mice were placed in mixed lymphocyte tumor cultures with irradiated E.G7 cells as described in Materials and Methods, and were restimulated once. The cultures were tested for their ability to lyze E.G7 and EL4 cells.

FIGURE 3.

Gp96-elicited increase in LN volume is due to infiltration of mature CD11c+ cells. A, Draining inguinal LNs were removed from mice immunized with saline or with 1 μg gp96. Single-cell suspensions were obtained and stained with Abs to CD11c, CD4, CD8, or B220 molecules, as described in Materials and Methods. B, CD11c+ cells in the LNs of gp96-injected mice were double stained for CD11c and MHC I, MHC II, CD80, CD86, or ICAM-1 expression. CD11c+ cells were gated for (green and red lines). For the bottom panel of this figure, LN cells were stained for MHC II and B220 expression or MHC II and CD11c expression. CD11c+ cells (green and red lines) or B220+ cells (pink line) were gated for. C, The kinetics of accumulation of CD11c+ cells in the draining LN was measured after injection of 1 μg gp96 or SA. In the experiment shown, the CD11c+ cells in the SA-injected mice were monitored only up to 12 h; this experiment has been conducted four times and the CD11c+ cells do not accumulate to higher levels in SA-injected mice even at later time points. D, CD11c+ cells from gp96-injected mice are functionally active. C57BL/6 mice were injected with 1 μg gp96 derived from E.G7 or EL4 cells. Draining LN cells were isolated 12 h later and CD11c+ cells were purified and transferred (105 cells/mouse) adoptively to naive C57BL/6 mice. Seven days later, spleen cells from these mice were placed in mixed lymphocyte tumor cultures with irradiated E.G7 cells as described in Materials and Methods, and were restimulated once. The cultures were tested for their ability to lyze E.G7 and EL4 cells.

Close modal

The CD11c+ cells in the LNs of gp96-immunized mice were observed to have high expression of MHC II, CD80, CD86, MHC I, and ICAM-1 molecules indicating a mature phenotype (Fig. 3,B). To provide a reference point for the level of MHC II expression on the CD11c+ cells, the MHC II expression was monitored on B cells and CD11c+ cells from the LN. The CD11c+ cells expressed similar or higher levels of MHC II molecules than the B cells, which express high MHC II levels to begin with (Fig. 3 B). The gp96 receptor CD91 is expressed constitutively at high levels on CD11c+ cells and no variations in its expression were detected (data not shown).

The kinetics of accumulation of CD11c+ cells in the draining LN, following immunization with gp96, was monitored. It was observed that similar to the kinetics of increase in size and cellularity of the draining LN observed in Fig. 2,B, the CD11c+ cells continued to accumulate up to about 12 h, and remained in the LN at the peak numbers for at least another 12 h. The fate of the CD11c+ cells after this period was not followed in this study. Immunization with control protein such as albumin did not lead to the increase in the numbers of CD11c+ cells in the draining LN (Fig. 3 C).

Gp96 molecules have been shown previously to have the unique ability to target the peptides chaperoned by them into the endogenous presentation pathway of APCs despite exogenous delivery (15). The gp96-elicited CD11c+ cells were tested in vivo for their ability to stimulate naive T lymphocytes specific for the peptides chaperoned by them. Mice were injected with gp96 derived from E.G7 cells, which express OVA, or the parental EL4 cells, which do not. The draining LNs were dissected and CD11c+ cells from them were purified as described in Materials and Methods. Naive C57BL/6 mice were injected with the each type of CD11c+ cells (105 cells/mouse injected i.v. via the retro-orbital route); 1 wk later, spleen cells from the mice were placed in mixed lymphocyte tumor cultures with irradiated E.G7 cells and were restimulated once with the same. The T lymphocytes were tested for their ability to lyze E.G7 cells and as controls, EL4 cells. It was observed (Fig. 3 D) that T lymphocytes derived from spleens of mice that had received CD11c+ cells from E.G7-gp96 injected mice could lyze E.G7 cells but not EL4 cells, while T lymphocytes derived from spleens of mice that had received CD11c+ cells from EL4-gp96 injected mice could not lyze E.G7 nor EL4 cells. The observation indicates that the CD11c+ cells obtained from the gp96-enlarged LNs were functionally active in vivo in stimulating Ag-specific T lymphocytes.

The unusually high and specific immunogenicity of tumor-derived gp96 preparations began to be observed nearly 20 years ago (12, 13). A number of observations have helped explain that phenomenon. These include the demonstration that HSPs are associated with antigenic peptides among self peptides (14), that HSP-peptide complexes of exogenous origin are taken up by APCs and the HSP-chaperoned peptides are presented by the MHC I molecules of the APCs (15), that the uptake of HSPs occurs through specific receptors such as CD91 (8), and finally that in addition to these specific events, HSP-APC interaction leads to stimulation to APCs so as to make them release cytokines and mature (9, 10). The studies reported here add an important component to that picture, specifically to the innate, Ag nonspecific component of that picture. They show that immunization with the HSP gp96 (and hsp90, data not shown) leads to accumulation of mature CD11c+ cells in the draining LNs of the mice and that these gp96-elicited mature CD11c+ cells are functionally active in re-presenting exogenous gp96-chaperoned peptides through the endogenous pathway and in stimulating naive T cells in vivo. Together with the previous reports (see 16), this phenomenon suggests (Fig. 4) that injection of gp96 leads to gp96-Langerhans cell interaction resulting in the production of cytokines (9), chemokines (N. Panjwani and P. Srivastava, unpublished observations) and the maturation of Langerhans cells (9). Maturation of Langerhans cells leads to their trafficking to the draining LNs and their accumulation in the LNs. This chain of events would imply that the gp96 injection site should become temporarily depleted of Langerhans cells; a phenomenon akin to this has been reported for another agent whose administration also leads to trafficking of Langerhans cells to the LNs (17).

FIGURE 4.

Schematic representation of the response of CD11c+ cells to an intradermal immunization of gp96. Immature CD11c+ cells in the skin (Langerhans cells; 1) acquire any antigenic peptides if chaperoned by gp96 (2) via the CD91-depepndent pathway and mature by the nonspecific signals provided by gp96 (3). Mature CD11c+ cells migrate to the LNs (4), where they stimulate naive T cells (5).

FIGURE 4.

Schematic representation of the response of CD11c+ cells to an intradermal immunization of gp96. Immature CD11c+ cells in the skin (Langerhans cells; 1) acquire any antigenic peptides if chaperoned by gp96 (2) via the CD91-depepndent pathway and mature by the nonspecific signals provided by gp96 (3). Mature CD11c+ cells migrate to the LNs (4), where they stimulate naive T cells (5).

Close modal

A number of agents such as IL-1 and TNF-α have been shown previously to promote the migration of dendritic cells just as other cytokines such as IL-10 have been shown to inhibit such migration (see Ref. 18 for review). However, cytokines are elaborated only in the context of an ongoing immune response and may not be considered the primary motivators of migration of dendritic cells. Our results suggest that HSPs, specifically gp96, can be primary motivators of migration of dendritic cells. This is particularly significant in light of our recent demonstration (9) that necrotic but not apoptotic cell death leads to liberation of HSPs, and that HSPs stimulate APCs to secrete cytokines including TNF-α and IL-1 and deliver a maturation signal to APCs (9, 10). Therefore, the observations reported here may be extrapolated to certain physiological situations. Events such as infection with lytic viruses and physical trauma resulting from tissue damage can be safely expected to lead to necrotic cell death and liberation of HSPs at very high local concentrations. We show here that as little as 1 μg gp96 can elicit the migration and maturation of CD11c+ cells in vivo. This quantity of gp96 can be released from the lysis of less than 106 cells; indeed as cell lysis shall result in release of other HSPs as well, necrotic death of as little 50,000 cells will be sufficient to elicit the phenomenon shown here. The magnitude of the phenomenon (i.e., the sheer numbers of CD11c+ cells recruited into the draining LNs as a result of exposure to gp96) as well as the rapidity with which it returns to steady state, are truly impressive and provide a sense of the heightened state of readiness of the innate immune system, as well as of its ability to disengage rapidly as needed. These characteristics would be expected of a rapid deployment force. We propose that such events inherently lead to trafficking of the relevant APCs to the draining LNs and concomitant uptake of antigenic peptides by the APCs through the locally generated HSP-peptide complexes. The APCs, re-presenting the HSP-chaperoned peptides on their MHC I molecules, stimulate the naive CD8+ T lymphocytes in the LNs, as also shown here. We also propose that the liberation of IL-1β as a consequence of the HSP-APC interaction is responsible for the fever often associated with trauma, stroke, myocardial infarcts and other such tissue-lytic events.

We thank Drs. Toyoshi Matsutake and Thiru Ramalingham for critically reading this manuscript, and Maria Bausero for her contributions with the gp96 preparations.

1

This work was supported by National Institutes of Health Grant CA84479 and a research agreement with Antigenics, Inc., in which one of us (P.K.S.) has a significant financial interest.

2

Address correspondence and reprint requests to Dr. Pramod K. Srivastava, University of Connecticut School of Medicine, MC1601, Farmington, CT 06030-1920. E-mail address: srivastava@nso2.uchc.edu

3

Abbreviations used in this paper: HSP, heat shock protein; LN, lymph node; SA, serum albumin; eu, endotoxin units.

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