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Originally published online as doi:10.1189/jlb.0305172 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1273-1280.)
© 2005 by Society for Leukocyte Biology

The low-toxicity versions of LPS, MPL® adjuvant and RC529, are efficient adjuvants for CD4+ T cells

Bruce S. Thompson*, Paula M. Chilton*, Jon R. Ward{dagger}, Jay T. Evans{dagger} and Thomas C. Mitchell*,1

* Institute for Cellular Therapeutics, Department of Microbiology and Immunology, University of Louisville, Kentucky; and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less-toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long-term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon-{gamma} 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is a continuing need for powerful adjuvants that are safe to use. For example, bacterial lipopolysaccharide (LPS) has long been known to have strong immunostimulatory adjuvant properties, but its extreme toxicity has precluded therapeutic use. LPS elicits an immune response through its interaction with Toll-like receptor 4 (TLR4) and has been shown to be a strong T cell adjuvant, enhancing the clonal expansion and the propensity of activated T cells to survive simulated growth factor withdrawal, as well as their ability to differentiate into helper T (Th) cells [1 2 3 ]. The initial response to LPS is marked by the coordinated release of many factors, such as interleukin-1ß (IL-1ß), IL-6, IL-10, IL-12, and tumor necrosis factor {alpha} (TNF-{alpha}), 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-{alpha} 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-{gamma} (IFN-{gamma}) 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-{gamma}-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-{gamma} 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
B10.D2/nSnAi and B10.D2/AiTac-TgN(DO11.1)-Ragtml (DORAG) were purchased from Taconic Farms (Germantown, NY) via the Emerging Models program of National Institute of Allergy and Infectious Diseases (Bethesda, MD). TCR transgenic mice were backcrossed to the B10.D2 subline at least eight times (described in detail at www.taconic.com/emerging/004091.htm). B10.D2 recipients were used to avoid the Th2 bias associated with the BALB/c background originally used to harbor the DO11.10 TCR transgene [11 ], while retaining the H-2d genotype needed for presentation of ovalbumin peptide 323–339 (Ovap) by I-Ad. All mice were housed in a specific pathogen-free facility at the University of Louisville School of Medicine (KY), and the University of Louisville Institutional Animal Care and Use Committee approved all animal protocols.

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-{alpha}, 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LPS and its less-toxic derivatives MPL and RC529 enhance CD4+ T cell clonal expansion
We and others (refs. [1 , 2 ] and unpublished results) have previously shown that various TLR agonists enhance the in vivo clonal expansion and ex vivo survival of activated CD4+ T cells. To test if clonal expansion of CD4+ T cells could occur to similar extents in the presence of inflammatory and less-inflammatory TLR4 agonists, we tested LPS, MPL, and RC529. To assess the clonal expansion of CD4+ T cells, CFSE-labeled splenocytes and lymph node cells from DORAG mice were adoptively transferred into B10.D2 recipient mice and activated with 50 µg OvaP alone or in the presence of increasing amounts of LPS, MPL, RC529, or RC526, which was included as a negative AGP control, as the length of its secondary acyl chain renders it inactive as a TLR4 agonist [14 ]. At the peak of clonal expansion (72 h), splenocytes were harvested, stained with anti-CD4 and anti-DO-11.10 TCR, and analyzed via flow cytometry. The clonal expansion of the CD4+/DO-11.10+ T cells was enhanced to similar extents by the addition of LPS or MPL (Fig. 1 ). Treatment with RC529 resulted in enhanced clonal expansion at higher doses, and the addition of RC526 had little effect on the peak yield. As shown in Figure 1A , the expansion of CD4+/DO-11.10+ T cells increased two- to 15-fold above treatment with antigen alone. The levels of expansion of LPS and MPL were similar at lower doses. It is interesting that the yield of CD4+ T cells at the peak of clonal expansion following treatment with LPS at doses above 3–10 µg was not enhanced, and MPL and RC529 showed enhanced yield at doses up to 30 µg. This raises that possibility that LPS was too toxic for T cell effects above 3–10 µg, such that the reduced toxicity of MPL and RC529 allows them to continue to work at higher doses.



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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.

 
Our previous studies have recently indicated that many TLR agonists, including LPS, can promote large increases in CD4+ T cell yield without inducing large increases in the number of cellular divisions undergone by that population of T cells (unpublished results). To test if the same was true for MPL and RC529, we calculated the average number of divisions undergone by the clonally expanded CD4+ T cells 72 h following activation as described in Materials and Methods. We found that the average number of divisions increased at most one to 1.5 divisions following treatment with 50 µg OvaP plus 10 µg LPS, MPL, or RC529, as compared with treatment with OvaP alone (Fig. 1B) , although the yield was increased four- to 12-fold at these doses. As expected, treatment with 10 µg RC526 resulted in, at most, a less than one division increase in the average number of divisions, which corresponded to its small (one- to twofold) increase in T cell yield. As the small increases in division associated with LPS, MPL, or RC529 treatment could only result in a two- to threefold increase in the yield of CD4+ T cells, LPS and its less toxic derivatives MPL and RC529 likely enhanced clonal expansion through similar cellular mechanisms. Previously, we found that enhanced cycling could only explain 15–25% of the net increase in CD4+ T cell yield following treatment with various TLR agonists. The balance, 75–85% of the increase in yield, was attributable to enhanced cellular survival and persistence during clonal expansion (unpublished results). Our calculations indicate that LPS, MPL, and RC529 have similar effects on T cell survival during clonal expansion in this study (data not shown).

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-{alpha}, 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.

 
MCP-1 and IP-10 are correlated with adjuvant activity
To further characterize the cytokines produced following treatment with LPS, MPL, RC529, or RC526, we performed a multiplex cytokine array analysis using a Luminex-100 cytometer on serum from B10D2 mice. We tested a range of doses similar to those tested for their effects on CD4+ T cell clonal expansion. As has been shown previously, treatment with MPL and RC529 resulted in a lessened production of the inflammatory cytokines TNF-{alpha}, 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.

 
LPS, MPL, and RC529 promote the differentiation of CD4+ T cells into IFN-{gamma}-producing Th1 cells
A critical function of adjuvants is to promote the production of long-lived memory cells, which are able to respond quickly to reinfection. To test if MPL and RC529 were as effective as LPS in promoting the long-term survival and differentiation of CD4+ T cells, we assessed the ability of DO-11.10+ T cells for production of IFN-{gamma} 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-{gamma}, and analyzed via flow cytometry (Fig. 4 ). We found that the ability of the transferred DO-11.10+ T cells to produce IFN-{gamma} was similar following treatment with LPS or MPL, and treatment with RC529 resulted in increased amounts of IFN-{gamma} 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-{gamma}. The average percentage ± SE of CD4+/DO-11.10+ T cells producing IFN-{gamma} 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).

 
Comparison of long-term yields, however, showed strikingly more cell recovery after treatment with Ovap and LPS than with Ovap and MPL or RC529 (Fig. 4B) . Treatment with LPS resulted in a robust recall response, marked by a 62-fold increase in the number of CD4+/DO-11.10+ T cells following challenge as compared with challenge with antigen alone, and treatment with MPL and RC529 resulted in only nine- and twofold increases, respectively. As expected, RC526 did not result in any increase in the number of antigen-specific cells following challenge.

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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have compared the effects of inflammatory and less-inflammatory TLR4 agonists on the clonal expansion, long-term survival, and differentiation of CD4+ T cells in vivo. We found that MPL and RC529 share many of the CD4+ T cell immunostimulatory effects of their parent compound, LPS, although they demonstrate dramatically less toxicity. Our results showed that treatment with increasing doses of LPS, MPL, or RC529 resulted in similar enhancements in clonal expansion, cellular division, and ex vivo survival. These attributes have been seen previously only with highly inflammatory TLR agonists, such as LPS, poly(I:C), and immunostimulatory CpG DNA (unpublished results). Thus, it is interesting that the less inflammatory compounds, MPL and RC529, have similar effects on the early stages of CD4+ T cell clonal expansion. Our analysis of the ability of CD4+ T cells to respond to challenge and differentiate into IFN-{gamma}-producing Th1 cells yielded mixed results. The ability to differentiate into IFN-{gamma}-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-{gamma} 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-{alpha}. 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-{alpha} [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-{gamma}-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-{gamma}-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-{gamma}-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-{gamma}-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.


   ACKNOWLEDGEMENTS
 
This work was supported by U.S. Public Health Service Grants AI51377 and AI059023, the Commonwealth of Kentucky Research Challenge Trust Fund, the Kentucky Lung Cancer Research Program, and the Jewish Hospital Foundation. The authors thank Drs. Carolyn Casella and Christopher Cluff for helpful discussions.

Received March 30, 2005; revised July 10, 2005; accepted August 5, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Vella, A. T., McCormack, J. E., Linsley, P. S., Kappler, J. W., Marrack, P. (1995) Lipopolysaccharide interferes with the induction of peripheral T cell death Immunity 2,261-270[CrossRef][Medline]
  2. Mitchell, T. C., Hildeman, D., Kedl, R. M., Teague, T. K., Schaefer, B. C., White, J., Zhu, Y., Kappler, J., Marrack, P. (2001) Immunological adjuvants promote activated T cell survival via induction of Bcl-3 Nat. Immunol. 2,397-402[Medline]
  3. Maxwell, J. R., Ruby, C., Kerkvliet, N. I., Vella, A. T. (2002) Contrasting the roles of costimulation and the natural adjuvant lipopolysaccharide during the induction of T cell immunity J. Immunol. 168,4372-4381[Abstract/Free Full Text]
  4. Ribi, E., Parker, R., Strain, S. M., Mizuno, Y., Nowotny, A., von Eschen, K. B., Cantrell, J. L., McLaughlin, C. A., Hwang, K. M., Goren, M. B. (1979) Peptides as requirement for immunotherapy of the guinea-pig line-10 tumor with endotoxins Cancer Immunol. Immunother. 7,43-58
  5. Ulrich, J. T., Myers, K. R. (1995) Monophosphoryl lipid A as an adjuvant. Past experiences and new directions Pharm. Biotechnol. 6,495-524[Medline]
  6. De Becker, G., Moulin, V., Pajak, B., Bruck, C., Francotte, M., Thiriart, C., Urbain, J., Moser, M. (2000) The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells Int. Immunol. 12,807-815[Abstract/Free Full Text]
  7. Ismaili, J., Rennesson, J., Aksoy, E., Vekemans, J., Vincart, B., Amraoui, Z., Van, L. F., Goldman, M., Dubois, P. M. (2002) Monophosphoryl lipid A activates both human dendritic cells and T cells J. Immunol. 168,926-932[Abstract/Free Full Text]
  8. Evans, J. T., Cluff, C. W., Johnson, D. A., Lacy, M. J., Persing, D. H., Baldridge, J. R. (2003) Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529 Expert Rev. Vaccines 2,219-229[CrossRef][Medline]
  9. Baldridge, J. R., McGowan, P., Evans, J. T., Cluff, C., Mossman, S., Johnson, D., Persing, D. (2004) Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents Expert Opin. Biol. Ther. 4,1129-1138[CrossRef][Medline]
  10. Johnson, D. A., Sowell, C. G., Johnson, C. L., Livesay, M. T., Keegan, D. S., Rhodes, M. J., Ulrich, J. T., Ward, J. R., Cantrell, J. L., Brookshire, V. G. (1999) Synthesis and biological evaluation of a new class of vaccine adjuvants: aminoalkyl glucosaminide 4-phosphates (AGPs) Bioorg. Med. Chem. Lett. 9,2273-2278[CrossRef][Medline]
  11. Murphy, K. M., Heimberger, A. B., Loh, D. Y. (1990) Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo Science 250,1720-1723[Abstract/Free Full Text]
  12. Gomez-Angelats, M., Bortner, C. D., Cidlowski, J. A. (2000) Cell volume regulation in immune cell apoptosis Cell Tissue Res. 301,33-42[CrossRef][Medline]
  13. Thompson, B. S., Mitchell, T. C. (2004) Measurement of daughter cell accumulation during lymphocyte proliferation in vivo J. Immunol. Methods 295,79-87[CrossRef][Medline]
  14. Stover, A. G., Da Silva, C. J., Evans, J. T., Cluff, C. W., Elliott, M. W., Jeffery, E. W., Johnson, D. A., Lacy, M. J., Baldridge, J. R., Probst, P., Ulevitch, R. J., Persing, D. H., Hershberg, R. M. (2004) Structure-activity relationship of synthetic Toll-like receptor 4 agonists J. Biol. Chem. 279,4440-4449[Abstract/Free Full Text]
  15. Mitchell, T. C., Teague, T. K., Hildeman, D. A., Bender, J., Rees, W. A., Kedl, R. M., Swanson, B., Kappler, J. W., Marrack, P. (2002) Stronger correlation of bcl-3 than bcl-2, bcl-xL, costimulation, or antioxidants with adjuvant-induced T cell survival Ann. N. Y. Acad. Sci. 975,114-131[Abstract/Free Full Text]
  16. Marhaug, G., Permin, H., Husby, G. (1983) Amyloid-related serum protein (SAA) as an indicator of lung infection in cystic fibrosis Acta Paediatr. Scand. 72,861-866[Medline]
  17. Ochrietor, J. D., Harrison, K. A., Zahedi, K., Mortensen, R. F. (2000) Role of STAT3 and C/EBP in cytokine-dependent expression of the mouse serum amyloid P-component (SAP) and C-reactive protein (CRP) genes Cytokine 12,888-899[CrossRef][Medline]
  18. Pasare, C., Medzhitov, R. (2004) Toll-dependent control mechanisms of CD4 T cell activation Immunity 21,733-741[CrossRef][Medline]
  19. Sporri, R., Reis e Sousa, C. (2005) Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function Nat. Immunol. 6,163-170[CrossRef][Medline]
  20. Croft, M. (2003) Co-stimulatory members of the TNFR family: keys to effective T-cell immunity? Nat. Rev. Immunol. 3,609-620[CrossRef][Medline]
  21. Khoruts, A., Osness, R. E., Jenkins, M. K. (2004) IL-1 acts on antigen-presenting cells to enhance the in vivo proliferation of antigen-stimulated naive CD4 T cells via a CD28-dependent mechanism that does not involve increased expression of CD28 ligands Eur. J. Immunol. 34,1085-1090[CrossRef][Medline]
  22. Dooms, H., Kahn, E., Knoechel, B., Abbas, A. K. (2004) IL-2 induces a competitive survival advantage in T lymphocytes J. Immunol. 172,5973-5979[Abstract/Free Full Text]
  23. Li, J., Huston, G., Swain, S. L. (2003) IL-7 promotes the transition of CD4 effectors to persistent memory cells J. Exp. Med. 198,1807-1815[Abstract/Free Full Text]
  24. Khan, I. A., MacLean, J. A., Lee, F. S., Casciotti, L., DeHaan, E., Schwartzman, J. D., Luster, A. D. (2000) IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection Immunity 12,483-494[CrossRef][Medline]
  25. Wornle, M., Schmid, H., Merkle, M., Banas, B. (2004) Effects of chemokines on proliferation and apoptosis of human mesangial cells BMC Nephrol. 5,8[CrossRef][Medline]
  26. Leung, T. H., Hoffmann, A., Baltimore, D. (2004) One nucleotide in a {kappa}B site can determine cofactor specificity for NF-{kappa}B dimers Cell 118,453-464[CrossRef][Medline]
  27. Tylaska, L. A., Boring, L., Weng, W., Aiello, R., Charo, I. F., Rollins, B. J., Gladue, R. P. (2002) CCR2 regulates the level of MCP-1/CCL2 in vitro and at inflammatory sites and controls T cell activation in response to alloantigen Cytokine 18,184-190[CrossRef][Medline]



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