Published online before print October 4, 2005
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* Institute for Cellular Therapeutics, Department of Microbiology and Immunology, University of Louisville, Kentucky; and
The Corixa Corporation, Hamilton, Montana
1 Correspondence: Institute for Cellular Therapeutics, Donald Baxter Research Building, 570 S. Preston Street, Room 404C, Louisville, KY 40202. E-mail: tom.mitchell{at}louisville.edu
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following a 21-day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long-term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low-inflammation adjuvants, such as MPL and RC529, are capable of enhancing short-term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long-term retention of antigen-specific T cells.
Key Words: AGP compounds • clonal expansion • T cell survival • mice • Toll-like receptors
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(TNF-), many of which are inflammatory and can cause severe damage or death in sufficient quantities in the host. The extreme toxicity of LPS has led to the search for alternative compounds with similar immunostimulatory properties, which would be better tolerated in humans. Sequential acid and base hydrolysis of LPS produces an immunoactive lipid A fraction, monophosphoryl lipid A (MPL), which lacks the saccharide groups and all but one of the phosphates present in LPS (ref. [4 ], reviewed in ref. [5 ], structure shown in ref. [6 ]). It is important that MPL retained many of the immunostimulatory properties of LPS, even as toxic side effects were lost. MPL was able to mature dendritic cells (DCs) from human donors, causing the up-regulation of human leukocyte antigen-DR, CD80, CD86, CD40, and an activation marker, CD83, in vitro [7 ]. It was also shown that treatment with MPL leads to the production of Th1 and Th2 cytokines [6 , 7 ] and could enhance the production of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) [8 , 9 ]. Even with these immunostimulatory effects, MPL was observed to have low levels of side effects in clinical trials [9 ]. In fact, more than 273,000 doses of MPL have been administered in various clinical trials with a side-effect panel similar to the classical vaccine adjuvant alum. The strong safety profile and immunostimulatory properties of MPL® adjuvant led to the recent European approval of Fendrix®, GlaxoSmithKline Biological’s (Belgium) novel hepatitis B vaccine containing MPL.
Following the successes of MPL, a better understanding of the functional contributions of its acyl side-chains and the desire for a homogeneous synthetic compound led to the synthesis of a family of derivatives with modified acyl chain and backbone structures, which were considerably less toxic than LPS (ref. [10
]; structure shown in ref. [6
]). Similar to MPL, these synthetic mimetics, aminoalkyl glucosaminide 4-phosphates (AGPs), were found to activate cells of the innate immune system, including macrophages, DCs, B cells, and other antigen-presenting cells. One AGP compound, in particular, RC529, has shown great promise and has become a leading candidate vaccine adjuvant for use in humans. Like LPS and MPL, RC529 has been shown to signal through TLR4, resulting in the up-regulation of cell-surface costimulatory molecules and receptors, cytokines, and chemokines [6
7
8
]. The cytokine elaboration of IL-1ß, IL-10, IL-6, IL-8, and TNF- following in vitro stimulation of freshly isolated peripheral blood mononuclear cells was found to be similar with MPL and RC529 [8
]. It is most important that RC529 was shown to be well-tolerated and effective during a clinical trial in which healthy patients were given a vaccine against hepatitis B. The antibody titer in subjects receiving the vaccine containing RC529 showed a significant increase as compared with subjects given the vaccine containing alum [8
]. Thus, the use of less-toxic MPL or its mimetic RC529 has been shown to be safe and effective in clinical vaccinations by taking advantage of the ability to uncouple the useful immunostimulatory properties of a natural bacterial adjuvant (LPS) from its inherent toxicity.
The efficacy of low-toxicity adjuvants such as MPL and RC529 appears to contradict the widely held view that the more inflammatory an agent is, the greater its immunostimulatory properties. MPL, for example, could theoretically have the same spectrum of activities as LPS, while being weaker at producing them, such that adequate immunostimulation is achieved with tolerable levels of inflammation. To determine whether MPL is simply a weaker form of LPS, we compared the effects that LPS, MPL, and RC529 had on the clonal expansion, long-term survival, and differentiation [via interferon- (IFN-) production] of CD4+ T cells in vivo. We found that all three compounds increased peak clonal expansion in vivo and the propensity to survive enforced growth factor withdrawal in ex vivo culture and were correlated with increased serum content of IFN--inducible protein-10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1). At low doses, MPL was as potent as its parent compound LPS and at high doses, was notably more efficacious. We also found that the ability of CD4+ T cells to produce IFN- following a 3-week immunization/recall protocol was similar, whether or not the T cells were activated in the presence of inflammatory (LPS) or less-inflammatory (MPL and RC529) compounds. However, our results suggest that inflammatory signaling enhances the long-term retention of antigen-specific T cells, as many more CD4+ T cells responded to the 3-week challenge if LPS were used as the adjuvant. Therefore, our data show that high levels of inflammation are dispensable for T cell priming but may be needed for maximal persistence of those cells afterwards.
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Preparation of carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells
Spleen and lymph nodes were harvested from untreated DORAG mice, processed into single-cell suspensions, resuspended at 4 x 107/ml in Hanks’ balanced salt solution (HBSS), and incubated with an equal volume of 10 µM CFSE (carboxyfluorescein diacetate SE; Molecular Probes, Eugene, OR) for 10 min at 37ºC. After labeling, the cells and CFSE were diluted by the addition of at least 10 vol HBSS, pelleted, washed again in HBSS, counted, and resuspended in HBSS at a density of 4 x 106 cells per 0.1 ml for injection into recipient B10.D2 mice via tail-vein injection.
Activation of CD4+ transgenic T cells
Mice were injected intravenously (i.v.) with 50 µg Ovap (Peptron Inc., Yuseong-ku, Daejeon, South Korea) or 50 µg Ovap plus varying combinations of the indicated TLR4 agonists: phenol-extracted LPS from Salmonella Minnesota (Sigma-Aldrich, St. Louis, MO) and MPL® adjuvant, RC529, or RC526 (Corixa Corp., Seattle, WA). MPL, 529, and 526 were manufactured under Food and Drug Administration-approved good manufacturing practice conditions and resuspended for injection with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Avanti Polar Lipids, Alabaster, AL) in water-for-injection (Abbott Laboratories, Abbott Park, IL). Stocks of each of the compounds used in this study were 1 mg/ml with the following concentrations of DPPC: MPL, 108 µg/ml; RC525, 216 µg/ml; RC529, 1728 µg/ml; and vehicle control, 1728 µg/ml. Ovap preparations contained less than 0.05 endotoxin units/ml, as measured in the quantitative chromogenic QCL-1000 Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). i.v. injection was used in all cases to avoid inflammatory adjuvant effects, which could be experienced at sites proximal to a site of subcutaneous injection.
Flow cytometric analysis
Cells harvested from target organs were processed by disruption through a 100-µM mesh cup, washed in HBSS, and resuspended in RPMI-1640 media supplemented with 9% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Red blood cells from splenic populations were lysed using standard ammonium chloride lysis buffer (0.16 M NH4Cl, 10 mM KHCO3, 1x10–4M EDTA). Cells were then surface-stained with a clonotypic antibody against DO-11.10 TCR (KJ1-26, Caltag Laboratories, Burlingame, CA) and anti-CD4 (BD PharMingen, San Diego, CA). Flow cytometry was performed using a Becton Dickinson (San Jose, CA) FACSCaliburTM followed by analysis with BD Biosciences (San Jose, CA) CellquestTM software. The analysis for live/gating following ex vivo culture was performed as described previously [2
, 12
].
Calculation of the average number of divisions of CD4+ T cells
The average number of divisions of antigen-specific T cells was determined as described elsewhere [13
]. Briefly, CFSE division profiles were analyzed using successive "bins" corresponding to the division peaks obtained from analysis of the CD4+/DO-11.10+ T cells. The undivided peak was set from the cells, which were harvested from animals that received only transferred CFSE-labeled DORAG cells. The percentages of cells in the peaks were multiplied by the number of divisions represented by that bin, and the average number of divisions for all bins was summed.
Detection of acute toxicity by serum amyloid A (SAA) enzyme-linked immunosorbent assay (ELISA)
B10.D2 mice were injected i.v. with 3 µg LPS, 3 µg MPL, 30 µg RC529, 30 µg RC526, or a vehicle control. Twenty-four hours following the injection, blood was collected from each experimental animal via the tail vein. The blood was allowed to clot for 2 h at room temperature, and then the serum was separated via centrifugation, collected, and stored at –80°C. Thawed serum was assayed for the presence of SAA using a solid-phase sandwich ELISA kit from Biosource International (Camarillo, CA).
Cytokine detection using the Luminex multiplex assay system
Splenocytes and lymph node cells were harvested from DORAG mice, CFSE-labeled, and 4 x 106 cells were transferred via tail-vein injection into B10.D2 mice. Recipient B10.D2 mice were injected i.v. with 50 µg OvaP alone or in combination with 1, 3, 10, and 30 µg LPS; 1, 3, 10, and 30 µg MPL; 1, 3, 10, 30, and 100 µg RC529; or 1, 3, 10, 30, and 100 µg RC526. Two hours following injection, blood was collected from each experimental animal via the tail vein. The blood was allowed to clot for 2 h at 4°C, and then the serum was separated via centrifugation, collected, and stored at –80°C. The serum was then assayed for the following cytokines using a Biosource International Multiplex antibody bead kit and Luminex (Austin, TX) 100 IS instrument: IL-1ß, IP-10, TNF-, IL-6, and MCP-1. Standard curves were run for each cytokine or chemokine and found to be within acceptable ranges before assaying test serum.
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Figure 1. Inflammatory signals are not required for short-term clonal expansion of CD4+ T cells. CFSE-labeled splenocytes from DORAG transgenic mice were transferred into recipient B10.D2 mice. The recipient mice were treated with 50 µg OvaP alone or in combination with increasing doses of MPL, RC529, RC526, or LPS from S. Minnesota. Splenocytes from B10.D2 recipient mice were harvested 72 h following activation and processed into single-cell suspensions. (A) Splenocytes were stained with anti-CD4 and anti-DO-11.10 TCR and analyzed via flow cytometry. The fold increase as compared with treatment with OvaP alone ± SE in the number of CD4+/DO-11.10+ T cells at the peak of expansion is shown for each dose tested. (B) Splenocytes were stained with anti-CD4 and anti-DO-11.10 TCR and analyzed via flow cytometry for their CFSE dye dilution. Representative CFSE profiles obtained from CD4+/DO-11.10+ T cells from mice treated with nothing, OvaP alone, or OvaP plus TLR4 agonist (10 µg MPL) are shown at left. The average number of divisions from activated CD4+/DO-11.10+ T cells following treatment with nothing or 50 µg OvaP alone or in combination with 10 µg LPS, MPL, RC529, or RC526 is shown at right. (C) The splenocytes tested for CFSE division upon harvest in B above were cultured at 37°C in minimal media containing 9% FBS but no added growth factors for 20 h. Before and after culture, the cells were stained for CD4 and DO-11.10 TCR and assayed using the live/dead gating method. At the outset of culture, 97–99% of CD4+/DO-11.10+ T cells were alive (data not shown). After culture, the percent of CD4+/DO-11.10+ T cells that were alive versus dead is shown in histogram form above. Comparison of CFSE fluorescence before and after the culture period showed that the cells did not continue to divide in vitro (data not shown). All data are the average of two to five animals ± SE and are representative of two independent experiments.
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An attribute of TLR agonists is their ability to enhance the survival of activated T cells upon culture ex vivo under conditions of limited growth factor availability (refs. [1 , 2 , 15 ] and unpublished results). To test for the propensity of LPS-, MPL-, and RC529-treated cells to survive growth factor withdrawal, splenocytes from mice following 72 h in vivo activation were cultured for 20 h at 37°C in RPMI with 9% FBS but no added cytokines. Following the culture period, cells were harvested, stained with anti-CD4 and anti-DO-11.10 TCR monoclonal antibodies (mAb), and analyzed via flow cytometry using a live/dead gating technique to determine the proportion of CD4+/DO11.10+ cells that remained alive after culture (Fig. 1C) . Treatment with LPS, MPL, and RC529 resulted in enhanced survival of activated CD4+/DO-11.10+ cells as compared with activation with OvaP alone (Fig. 1C) . In contrast, treatment of activated T cells with RC526 resulted in little protection from growth factor withdrawal during the ex vivo culture period.
MPL and RC529 are not acutely toxic, as judged by reduced production of SAA protein
SAA is an acute-phase protein, which is produced by the liver in response to inflammatory cytokines, such as IL-1, IL-6, and TNF-, and has been used as a marker of inflammation and pulmonary infection [16
]. In mice, SAA has been shown to be the major acute-phase protein, whereas the major acute-phase protein in humans is C-reactive protein [17
]. To confirm the reduced toxicity of MPL and RC529 in our model, we measured the production of SAA via ELISA 24 h after treatment of B10.D2 mice with 3 µg LPS, 3 µg MPL, 30 µg RC529, 30 µg RC526, or a vehicle control. These doses were chosen, as they resulted in a similar peak yield of CD4+ T cells in vivo. Treatment with the vehicle alone was associated with low levels of SAA production (4.1 µg/ml; data not shown). Compared with treatment with the vehicle control, the inclusion of LPS caused a 915-fold increase in the amount of SAA present in the serum at 24 h. MPL and RC529 caused increases in SAA of 176- and 199-fold, respectively, as compared with treatment with the vehicle control (Fig. 2
). Treatment with the AGP control, RC526, resulted in an increase of only eightfold compared with the vehicle control. Normalization of the SAA values to the doses of TLR4 agonist administered indicated that LPS was roughly fivefold more toxic than MPL and 50-fold more toxic than RC529. These results confirm the extensive data supporting the greatly reduced toxicity of MPL and RC529.
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Figure 2. The toxicity of MPL and RC529 is greatly reduced as compared with the parent LPS compound. B10.D2 mice were injected i.v. with 3 µg LPS, 3 µg MPL, 30 µg RC529, 30 µg RC526, or a vehicle control. Blood was collected via the tail vein from each experimental animal 24 h following injection. The blood was allowed to clot for 2 h at room temperature, and then the serum was separated via centrifugation and assayed for the presence of SAA via sandwich ELISA. The data shown are averaged ± SD from triplicate experimental animals. A similar trend of increased toxicity with LPS was observed in three independent experiments. The normalized toxicity, shown above each value, was calculated by dividing each value by that obtained for MPL and multiplying by the difference in dosage relative to MPL (0.1 for RC526 and RC529). *, P < 0.0001, as compared with LPS treatment using a Student’s t-test, assuming equal variance.
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, IL-1ß, and IL-6 (data not shown and ref. [8
]). It is interesting that we found that two chemokines, IP-10 and MCP-1, were up-regulated following treatment with all three TLR4 agonists—LPS, MPL, and RC529—which enhanced T cell clonal expansion (Fig. 3A
and 3B
). In fact, the addition of each of the TLR4 agonists resulted in an increase of roughly 1000–1800 pg/ml IP-10 and 600–1000 pg/ml MCP-1. As expected, the addition of RC526 caused no detectable increase in either chemokine (Fig. 3A
and 3B)
.
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Figure 3. IP-10 and MCP-1 are correlated with adjuvant-enhanced survival of CD4+ T cells. Splenocytes from DORAG transgenic mice were transferred into recipient B10.D2 mice, which were injected i.v. with 50 µg OvaP alone or in combination with increasing doses of LPS, MPL, RC529, or RC526. Blood was collected via the tail vein from each experimental animal 2 h following injection and allowed to clot for 2 h at 4°C. The serum was separated via centrifugation and assayed for the presence of MCP-1 (A) and IP-10 (B) via a multiplex cytokine assay using a Luminex-100 cytometer. The data shown are combined from four to seven experimental animals for each treatment group from two independent experiments.
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-producing Th1 cells following challenge. We adoptively transferred 4 x 106 splenocytes plus lymph node cells from DORAG mice into B10.D2 recipients, and then the DO-11.10 T cells were activated by injection of 50 µg OvaPalone or in combination with 10 µg MPL, 10 µg RC529, 15 µg LPS, or 10 µg RC526 on Day 0 and again on Day 21. Three days following the last treatment, splenocytes were harvested and cultured in the presence of phorbol 12-myristate 13-acetate (PMA)/ionomycin and Brefeldin A for 6 h at 37°C. Following culture, the cells were harvested and surface-stained with anti-CD4 and anti-DO-11.10 TCR mAb, fixed in paraformaldehyde, then stained intracellularly for IFN-, and analyzed via flow cytometry (Fig. 4
). We found that the ability of the transferred DO-11.10+ T cells to produce IFN- was similar following treatment with LPS or MPL, and treatment with RC529 resulted in increased amounts of IFN- as compared with treatment with antigen alone (Fig. 4A)
. Notably, MPL treatment was associated with the same levels of Th1 differentiation as its parent compound, LPS.
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Figure 4. Toxicity is correlated with long-term CD4+ T cell yield but not Th1 differentiation. Splenocytes from DORAG transgenic mice were adoptively transferred into B10.D2 recipients and activated via i.v. injection of 50 µg OvaP alone or in combination with 15 µg LPS, 10 µg MPL, 10 µg RC529, or 10 µg RC526. The mice were then challenged again with the same treatment (OvaP plus various TLR4 agonists) 21 days later via i.v. injection. (A) Following challenge (72 h), splenocytes were harvested and cultured for 6 h in the presence of PMA/ionomycin and Brefeldin A. At the end of the culture period, cells were surface-stained for CD4 and DO-11.10 TCR and fixed in 1% paraformaldehyde. Cells were then permeabilized using 1% Triton X-100 and stained intracellularly for IFN-. The average percentage ± SE of CD4+/DO-11.10+ T cells producing IFN- is shown. *, P < 0.05, as compared with treatment with OvaP alone using a two-tailed Student’s t-test, assuming equal variance. (B) The yield of CD4+/DO-11.10 TCR+ T cells as determined by flow cytometric analysis is shown 72 h following challenge (at the time of harvest). *, P < 0.02, as compared with treatment with OvaP plus LPS using a two-tailed Student’s t-test assuming equal variance. In addition, treatment with OvaP plus LPS, MPL, or RC529 resulted in a significant difference from treatment with OvaP alone (P<0.02). The data shown in A and B are the average ± SE from triplicate animals and are representative of two independent experiments. Analysis of T cell numbers in the spleens of mice given primary immunization but no recall challenge showed that  1.6 x 104 or 25 x 104 CD4+/DO-11.10+ T cells persisted on Day 21 after treatment with Ovap and MPL or Ovap and LPS, respectively (n=7 for each group, P=0.01 by two-tailed Student’s t-test, assuming equal variance).
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Diminished numbers of CD4+/DO-11.10+ cells after recall in the MPL-treated mice indicated that the DO11.10 T cells in MPL-treated mice were less abundant before challenge or proliferated poorly after challenge or both. To determine which was true, adoptively transferred mice, which had been given peptide with LPS or with MPL as described above, were harvested for analysis on Day 21, without receiving challenge injections. In spleens of mice given peptide plus LPS, there were 16-fold the number of DO11.10 T cells as in mice given peptide plus MPL: Ovap+ LPS produced an average of 25 x 104 CD4+DO11.10+ cells on Day 21, and Ovap+ MPL gave 1.6 x 104 CD4+DO11.10+ cells (Fig. 4B
legend). The fold-increase of DO11.10 cells after challenge using LPS was approximately seven (Fig. 4B)
. Therefore, the decreased numbers of DO11.10 cells that were observed after challenge using MPL as adjuvant were probably attributable solely to faster loss of cells sometime after peak expansion rather than a failure to clonally expand upon recall challenge. These findings suggest that differentiation and long-term maintenance of CD4+ T cells can be uncoupled, and high levels of inflammation are dispensable for differentiation but helpful for persistence of antigen-specific T cells after priming.
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-producing Th1 cells yielded mixed results. The ability to differentiate into IFN--producing Th1 cells was the same whether the cells were challenged with highly inflammatory LPS or less-inflammatory MPL. Treatment with RC529 resulted in nearly a twofold increase in the percentage of cells producing IFN- following challenge, confirming the observation that toxicity is not a requirement for Th1 differentiation in this scenario. In contrast, we observed a marked decrease in the number of CD4+ T cells recovered after challenge with less inflammatory TLR4 agonists as compared with treatment with the parent LPS compound. We recovered 62-fold more CD4+ T cells following challenge with LPS as compared with challenge with OvaP alone. We only recovered nine- and twofold more CD4+ T cells following challenge with MPL and RC529, respectively, suggesting that high levels of inflammatory signaling may increase the ability of activated CD4+ T cells to persist longer after priming. Recent work by Pasare and Medzhitov [18
] demonstrated a requirement for the TLR signaling adaptor myeloid differentiation primary-response protein 88 (MyD88) during memory CD4+ T cell formation. Our finding fits well with their observation, suggesting that enhanced long-term survival may depend on cytokines that are signaled downstream of MyD88, such as IL-6 and TNF-. MPL and RC529 stimulate reduced expression of these cytokines compared with LPS; so, this may explain why recovery of CD4+ T cells from these groups was lower following challenge. Current work is aimed at understanding the contributions of inflammatory signaling to long-term CD4+ T cell survival.
The enhancement of short-term clonal expansion of CD4+ T cells following treatment with less inflammatory TLR4 agonists would suggest that MPL and RC529 are able to provide the appropriate signals to DCs during T cell activation. The maturation of DCs following stimulation with MPL or RC529 is known to occur, suggesting that this may be enough to allow CD4+ T cell activation and expansion [6
, 7
]. Treatment with MPL and RC529 has been shown to induce lower levels of inflammatory cytokines, such as IL-1ß, IL-6, and TNF- [8
]. However, there may be a threshold level of inflammatory cytokines required to orchestrate the immune system to function correctly. We hypothesize that these less inflammatory compounds trigger TLR signaling and still allow for the production of low levels of inflammatory cytokines, such that they are not clinically symptomatic but are effective in maturing DCs and stimulating the innate immune system. Indeed, recent work by Sporri and Reis e Sousa [19
] has shown that DC maturation by inflammatory cytokines alone supports CD4+ T cell clonal expansion but not differentiation into IFN--producing Th1 or IL-4-producing Th2 cells. They concluded that TLR signaling is qualitatively different from signaling via inflammatory cytokines, such that both are required for the full activation of DCs. Thus, similar to LPS, MPL and RC529 provide TLR signaling and at least a basal level of cytokine signaling, which promotes DC activation that sustains CD4+ T cell clonal expansion and differentiation into IFN--producing Th1 cells.
In addition to maturation of DCs, other signals have been shown to enhance the activation and clonal expansion of CD4+ T cells. For example, ligation of costimulatory molecules, such as members of the TNF receptor family, including CD40 and OX-40, as well as cytokines such as IL-1, IL-2, and IL-7 can increase the survival of activated CD4+ T cells during clonal expansion [3
, 20
21
22
23
]. Here, we have shown that expression of two chemokines, IP-10 and MCP-1, is correlated with increases in CD4+ T cell clonal expansion and Th1 differentiation. IP-10 (CXC chemokine ligand 10) is an IFN--inducible chemokine, which is involved in stimulation of monocytes, migration of natural killer and T cells, and the maturation of T cells and bone marrow progenitor cells. Recently, IP-10 has been shown to modulate the migration of activated T cells to the site of infection and to have proliferative and protective effects on mesengial cells [24
, 25
]. In addition, the IP-10 promoter has been shown to contain a binding site for a key modulator of the adjuvant effect in T cells, Bcl-3, suggesting a role in mediating the survival of activated T cells [2
, 26
]. MCP-1 (CC chemokine ligand 2) is a potent stimulator of macrophage migration and has been shown to have protective and CTL-enhancing properties during T cell activation [27
]. In that study, MCP-1 was shown to be largely responsible for the infiltration of T cells and macrophages, which occurred in response to an alloantigen. Although the direct effects of IP-10 and MCP-1 on CD4+ T cells in our model remain unknown, we show here that the expression of both chemokines is increased and correlated with enhanced clonal expansion and survival of activated CD4+ T cells.
Overall, our results indicate that less inflammatory TLR4 agonists, such as MPL and RC529, are just as effective in promoting CD4+ T cell activation and initial clonal expansion as the highly inflammatory parent compound, LPS. MPL was capable of promoting the differentiation of CD4+ T cells into IFN--producing Th1 cells to a similar extent as LPS. However, we found that MPL and RC529 did not fully support the long-term persistence of previously primed CD4+ T cells, suggesting that a more complex set of signals is needed during activation. Our results have important implications for vaccine design, as they show that the toxicity of the TLR4 agonists can be uncoupled from their immunostimulatory effects in the dose range where these compounds have adjuvant effects on CD4+ T cells (as shown by our SAA data in Fig. 2
). Short-term clonal expansion of CD4+ T cells does not seem to be impaired by marked decreases in inflammatory signaling; however, long-term retention of the cells was less effective. Our current research is aimed at understanding this disparity in the requirement for inflammatory signaling to enhance long-term survival of clonally expanded CD4+ T cells.
Received March 30, 2005; revised July 10, 2005; accepted August 5, 2005.
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