T-cell apoptosis in inflammatory brain lesions: destruction of T cells does not depend on antigen recognition

Elimination of inflammatory T cells by apoptosis appears to play an important role in the down-regulation of inflammation in the central nervous system. Here we report that apoptosis of T lymphocytes occurs to a similar extent in different models of autoimmune encephalomyelitis. Apoptosis is restricted to cells located in the neuroectodermal parenchyma, thereby leaving T cells present in the brain's connective tissue compartments unharmed. Death of T cells in the parenchyma does not depend on antigen presentation by resident microglial cells or astrocytes. Adoptive transfer experiments with T lymphocytes carrying a specific genetic marker revealed that in the central nervous system these cells are destroyed regardless of their antigen specificity or state of activation. Although many of both antigen-dependent and -independent mechanisms in the induction of T-cell apoptosis may act simultaneously, our results suggest that the nervous system harbors a specific, currently undefined, mechanism that effectively eliminates infiltrating T lymphocytes.     

Inflammatory infiltrates in sural nerve biopsies in Guillain-Barre syndrome and chronic inflammatory demyelinating neuropathy

Prompted by observations in experimental autoimmune neuritis we reanalyzed immunohistochemically the inflammatory infiltrates in sural nerve biopsies of 22 cases with Guillain-Barre syndrome (GBS) and 13 cases with chronic inflammatory demyelinating polyneuropathy (CIDP). Endoneurial infiltration of CD3+ T cells was found in 20 cases of GBS (median 5.5 cells/mm(2)) and in 10 cases of CIDP (5 cells). Epineurial T cells were present in all GBS cases (19.5 cells) and in 11 CIDP cases (21 cells). CD68+ macrophages were abundant in these neuropathies and often occurred in endoneurial perivascular clusters. In GBS subgroups the number of endoneurial T cells was significantly higher in patients with hypoesthesia and abnormal electrophysiological findings in the sural nerve. In CIDP hypoesthesia was associated with significantly higher numbers of macrophages. Our study also indicates that other factors including the time point of biopsy or previous corticosteroid treatment may influence the inflammatory cell profile. Quantifying cell infiltration may aid in establishing the diagnosis of an immunoneuropathy in patients with mild and noncharacteristic pathology.     

cDNA cloning and characterization of A3i, an alternatively spliced rat A3 adenosine receptor variant

Programmed cell death of antigen specific T cells (apoptosis) may be an important process in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Fas antigen was recognized as an apoptosis-related antigen. To investigate Fas antigen in multiple sclerosis (MS), we measured the expression rate of Fas antigen and activation markers on T cells in peripheral blood (PB) of 19 MS patients, 18 controls and in cerebrospinal fluid (CSF) of nine MS patients by flow cytometry. The positive rate of Fas antigen in MS patients was higher than that of healthy controls in PB. In patients with MS, the expression rates of Fas antigen and activation markers were higher in CSF than in PB. The above findings suggest that there is acceleration or impairment of apoptosis on activated T cells in MS.     

Expression and characterization of recombinant soluble peptide: I-A complexes associated with murine experimental autoimmune diseases

Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.     

Cytokine production and the pathogenesis of experimental autoimmune neuritis and Guillain-Barré syndrome

Acute inflammatory demyelinating polyradiculoneuropathy (Guillain-Barré syndrome, GBS) and its animal model experimental autoimmune neuritis (EAN) are prototypes of T cell-mediated autoimmune diseases affecting the peripheral nervous system (PNS). Perivascular accumulation of macrophages and T lymphocytes in the PNS, and high levels systemically of PNS myelin antigen-reactive T cells are characteristic features of both diseases, thereby suggesting a pathogenic role for immunoregulatory cytokines. Here we summarise recent studies that have clearly documented that Th1/Th2/Th3 cytokines are differently upregulated during various clinical phases of EAN and GBS. The observations indicate that the role of cytokines in immune regulation and autoimmune disease is more complex than a simple Th1-Th2 dichotomy would suggest. New treatments may be searched for that counteract this complex cytokine imbalance. Treatments with antibodies that selectively target certain pro-inflammatory cytokines, as well as with immunomodulatory preparations that promote cytokines that beneficially influence the disease course should be in focus of future therapeutic trials.     

Attenuation of experimental autoimmune neuritis in the Lewis rat by treatment with an antibody to L-selectin

The leukocyte adhesion molecule L-selectin plays a key role in the initial steps of transendothelial migration of T cells and monocytes. In this study we investigated the role of L-selectin in the pathogenesis of experimental autoimmune neuritis (EAN) an animal model of the Guillain-Barré syndrome. EAN was induced in Lewis rats by sensitization with peripheral nerve myelin. Treatment with HRL3, a monoclonal antibody to L-selectin, efficiently suppressed clinical signs of EAN. Histological examination of the peripheral nervous system (PNS) revealed a marked reduction of inflammatory infiltrates and demyelination during treatment with HRL3. We conclude that L-selectin-dependent mechanisms are of pathophysiological relevance in EAN. Modulation of L-selectin in vivo could be a novel therapeutic approach to autoimmune diseases of the PNS.     

Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals

We compared peripheral and mucosal primary CD8 T cell responses to inflammatory and noninflammatory forms of antigen in a T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover, distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand, B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator, B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inflammatory mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent. The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because of distinct costimulatory and inflammatory signals in the mucosa.     

Identification of a homozygous deletion at 8p12-21 in a human prostate cancer xenograft

Endothelial-monocyte activating polypeptide II (EMAP II) and allograft-inflammatory factor-1 (AIF-1) are two proteins produced by activated monocytes and microglial cells. We now report expression of these factors during experimental therapy of rat neuroautoimmune diseases. Comparative analysis of two therapeutic strategies, treatment with high doses of recombinant autoantigens or with dexamethasone, revealed unexpected differences. High doses of autoantigen were most effective in experimental autoimmune encephalomyelitis and neuritis (EAE and EAN), but less effective in experimental autoimmune uveitis (EAU). Low and high doses of dexamethasone treatment greatly reduced the severity of EAE, EAN and EAU at day 11, but a relapse was observed between days 21 and 26. Only rather limited expression of EMAP II and AIF-1 is seen in the normal central nervous system (CNS). This constitutive expression is not abolished by dexamethasone treatment. In inflammatory autoimmune lesions of the rat CNS, prominent AIF-1 and EMAP II staining was seen with macrophages and monocytes. In particular, parenchymal microglial cells were now activated to express AIF-1 and EMAP II. In accordance with prevention of neurological signs, histological observations revealed that accumulation of activated monocytes expressing EMAP II and AIF-1 in the CNS or peripheral nervous system and the massive expression of these factors by parenchymal microglial cells is inhibited by high doses of autoantigen. Dexamethasone prevented or abolished local expression of EMAP II and AIF-1 at days 10-16. However, an acute and severe relapse occurred in encephalomyelitis between days 20-26. In these cases, a smoldering expression of EMAP II and AIF-1 persisting long after cessation of neurological signs was observed. Thus, expression of EMAP II and AIF-1 by infiltrating activated macrophages is a marker of disease activity and expression of these factors could be used to demonstrate 'silent' lesions in the CNS and prolonged microglial cell activation. Apparently, AIF-1 and EMAP II immunoreactivity are tools to stage activation of monocytes and microglial cells in inflammatory lesions.     

Caspase-1 is not involved in CD95/Fas-induced apoptosis in Jurkat T cells

It is now well established that the caspases, a family of cysteine proteases, play a key role in apoptosis. Although overexpressing each of the caspases in cells triggered apoptosis, the precise role and contribution of individual caspases are still unclear. Caspase-1, the first caspase discovered, was initially implicated in mammalian apoptosis because of its similarity to the gene product ced-3. Using whole cells as well as an in vitro system to study apoptosis, the role of caspase-1 in Fas-mediated apoptosis in Jurkat T cells was examined in greater detail. Using various peptide-based caspase inhibitors, our results showed that N-acetyl-Tyr-Val-Ala-Asp chloromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone efficiently blocked Fas-mediated apoptosis in Jurkat T cells, whereas N-acetyl-Tyr-Val-Ala-Asp aldehyde, which is more specific for caspase-1, had little effect. Cell lysates derived from anti-Fas-stimulated cells, which readily induced apoptotic nuclei morphology and DNA fragmentation in isolated thymocyte nuclei, had no caspase-1 activity using proIL-1beta as a substrate. Time-course studies showed no caspase-1 activity during the activation of apoptosis in Jurkat cells by agonistic Fas antibodies. Furthermore, no pro-caspase-1 protein nor activated form of the protein was detected in normal or apoptotic Jurkat cells. In contrast, both caspase-2 and caspase-3 were readily detected as proenzymes in control cells and their activated forms were detected in apoptotic cells. Incubation of recombinant active caspase-1 with control cell lysates did not activate the apoptotic cascade as shown by the lack of detectable apoptotic nuclei promoting activity using isolated nuclei as substrate. However, under similar conditions proIL-1beta was readily processed into the mature cytokine, indicating that the recombinant caspase-1 remained active in the presence of control cell lysates. Taken together our results demonstrate that caspase-1 is not required for the induction of apoptosis in Jurkat T cells mediated by the Fas antigen.     

Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.     

Neutralizing antibodies to IFN-gamma-inducing factor prevent experimental autoimmune encephalomyelitis

Specific oligonucleotide primers were used to identify and isolate IFN-gamma-inducing factor (IGIF) from the brain of rats with developing experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as a model for multiple sclerosis. IGIF was highly transcribed in the brain at the onset and during the course of active EAE. PCR products encoding rat IGIF were used to generate the recombinant protein that was used to induce anti-IGIF neutralizing Abs. These Abs significantly reduced the production of IFN-gamma by primed T cells proliferating in response to their target myelin basic protein epitope and by Con A-activated T cells from naive donors. When administered to rats during the development of either active or transferred EAE, these Abs significantly blocked the development of disease. Splenic T cells from protected rats were cultured with the encephalitogenic myelin basic protein epitope and evaluated for production of IL-4 and IFN-gamma. These cells, which proliferated, exhibited a profound increase in IL-4 production that was accompanied by a significant decrease in IFN-gamma and TNF-alpha production. Thus, we suggest that perturbation of the Th1/Th2 balance toward Th2 cells is the mechanism underlying EAE blockade by anti-IGIF immunotherapy.     

Microglia are more susceptible than macrophages to apoptosis in the central nervous system in experimental autoimmune encephalomyelitis through a mechanism not involving Fas (CD95)

Morphological studies have shown that macrophages and microglia undergo apoptosis in the central nervous system (CNS) in acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. To assess the relative levels of macrophage and microglial apoptosis, and the molecular mechanisms involved in this process, we used three-colour flow cytometry to identify CD45lowCD11b/c+ microglial cells and CD45highCD11b/c+ macrophages in the inflammatory cells isolated from the spinal cords of Lewis rats 13 days after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Simultaneously, we analyzed the DNA content of these cell populations to assess the proportions of cells undergoing apoptosis and in different stages of the cell cycle or examined their expression of three apoptosis-regulating proteins, i.e. Fas (CD95), Fas ligand (FasL) and Bcl-2. Microglia were highly vulnerable to apoptosis and were over-represented in the apoptotic population. Macrophages were less susceptible to apoptosis than microglia and underwent mitosis more frequently than microglia. The different susceptibilities of microglia and macrophages to apoptosis did not appear to be due to variations in Fas, FasL or Bcl-2 expression, as the proportions of microglia and macrophages expressing these proteins were similar, and were relatively high. Furthermore, in contrast to T cell apoptosis, apoptosis of microglia/macrophages did not occur more frequently in cells expressing Fas or FasL, or less frequently in cells expressing Bcl-2. These results indicate that the apoptosis of microglia and CNS macrophages in EAE is not mediated through the Fas pathway, and that Bcl-2 expression does not protect them from apoptosis. Expression of FasL by macrophages and microglia may contribute to the pathogenesis and immunoregulation of EAE through interactions with Fas+ oligodendrocytes and Fas+ T cells. The high level of microglial apoptosis in EAE indicates that microglial apoptosis may be an important homeostatic mechanism for controlling the number of microglia in the CNS following microglial activation and proliferation.     

Bee and wasp venom allergy in Turkey

Astrocytes and derived factors maintain the morphologic, phenotypic, and physiological properties of the blood-brain barrier. Astroglial cells may also modulate endothelial cell properties associated with the entry of inflammatory cells into the brain. The study of mechanisms of lymphocyte migration through the blood-brain barrier is critical to understanding the pathophysiology of autoimmune (multiple sclerosis) and virus-induced central nervous system diseases (HIV-induced dementia). In this context the contribution of astrocyte derived factors in regulating the interactions between inflammatory cells and endothelial cells of the blood-brain barrier was studied. The treatment of endothelial cells derived from brain or peripheral sources (hepatic) with astrocyte conditioned medium resulted in a dose dependent enhancement of adhesion of T cells to endothelium. The antigen specificity of the T cells did not influence the findings. Identical results were obtained with fresh Concanavalin A activated T cells and T cell hybridomas generated using myelin basic protein or chicken ovalbumin as immunogens. Further studies are in progress to define the active components in astrocyte conditioned medium and endothelial cell adhesion molecules that are regulated in order to gain a better understanding of mechanisms of inflammatory cell entry into the central nervous system.     

Spatiotemporal relationship of apoptotic cell death to lymphomonocytic infiltration in photochemically induced focal ischemia of the rat cerebral cortex

In this study we examined the time course of apoptotic cell death after photochemically induced focal ischemia of the rat cerebral cortex. For unequivocal differentiation between apoptosis and necrosis two criteria of programmed cell death were used: terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) and morphological evidence of fragmentation and marginalization of nuclei. After photothrombosis, many TUNEL-positive cells were found within the infarct region from 12 h to 3 days. By day 6 they were preferentially located in the boundary zone of the infarct, and by day 14 they had disappeared. A high proportion of TUNEL-positive cells displayed fragmentation or marginalization of their nuclei, indicating apoptosis. Neurons, but not T cells and macrophages, were apoptotic. Inflammatory infiltrates were in close contact to apoptotic neurons throughout the infarct areas at day 1 and in the boundary zone between days 2 and 6 after photothrombosis. In summary, our study shows that neuronal apoptosis after cerebral ischemia is a prolonged process to which leukocyte-derived cytokines may contribute. In contrast to autoimmune diseases of the nervous system, termination of the local inflammatory response after cerebral ischemia does not involve apoptosis.     

Certain norditerpenoid alkaloids and their cardiovascular action

Several investigators have reported experimental evidence of epitope spreading in experimental autoimmune encephalomyelitis (EAE). The role of epitope spreading in the pathogenesis of relapsing or chronic autoimmune disease is not established and the in vivo function of the T cells specific for new epitopes which appear during an autoimmune response is unclear. We recently demonstrated that mice which have recovered from an episode of EAE suffer a relapse shortly after reinjection with the original encephalitogen. The reinduced disease occurs in a reproducible fashion with an accelerated onset. This may be due to persistence of an expanded population of previously activated encephalitogenic cells which are rapidly reactivated when re-exposed to antigen. We reasoned that if epitope spread produces a significant number of encephalitogenic cells specific for a new epitope, then reinjection with that epitope should also cause the rapid onset of an episode of EAE. We tested this hypothesis using the known encephalitogenic epitopes in SJL mice. After recovery from EAE induced with the proteolipid protein peptide PLP139-151, five of 16 mice had an accelerated relapse of EAE when reinjected with a second encephalitogenic peptide, PLP178-191. All of the 10 mice reinjected with the original PLP139-151 peptide relapsed. We conclude that epitope spread may produce encephalitogenic cells specific for new epitopes, but that the response to new epitopes is minor compared to the response to the initial epitope.     

Purified truncated recombinant HLA-B7 molecules abrogate cell function in alloreactive cytotoxic T lymphocytes by apoptosis induction

BACKGROUND: Soluble MHC class I molecules are ubiquitous in human body fluids, including serum, urine, sweat, and cerebrospinal fluid. However, their biological function has remained unresolved. Membrane-derived human soluble MHC molecules (soluble human leukocyte antigen; sHLA) have been shown to induce apoptosis in alloreactive cytotoxic T lymphocytes (CTL). Here we report the efficacy of recombinant soluble HLA-B7 (rsHLA-B7) to modulate T-cell function. METHODS: Primers of HLA-B7 were designed to allow amplification of a cDNA lacking the transmembrane and cytoplasmic domains yielding a truncated gene. rsHLA-B7 molecules were expressed in the human myeloma cell line 721.221 and purified by affinity chromatography using the BB7.7 mouse monoclonal antibody. CTL were generated from peripheral blood lymphocytes derived from healthy blood donors by stimulation with irradiated Epstein Barr virus-transformed HLA-B7-positive B cells. CTL were preincubated with rsHLA-B7, and cytotoxicity and apoptosis were tested according to standard procedure. RESULTS: A total of 2 x 10(6) cells/ml secreted 10 microg/ml rsHLA-B7 as determined by a conformation-dependent ELISA, suggesting that rsHLA-B7 do not require the transmembrane and cytoplasmic regions for proper folding. After purification by affinity chromatography, rsHLA-B7 induced apoptosis in anti-HLA-B7 CTL, but not in anti-HLA-A2-specific, CTL. As a consequence, allorecognition of target cells by the CTL was significantly blocked. CONCLUSION: Recombinant sHLA are sufficient binding cues for T cells, which efficiently induce apoptosis and block allorecognition of target cells by CTL. Thus, recombinant sHLA molecules may become a valuable new modality for specific immunological therapeutic intervention.     

MAP kinases and vascular smooth muscle function

Integrins are a subclass of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. Integrins influence transendothelial migration of lymphocytes and monocytes and are suitable targets for experimental immunotherapy. They are critically involved in the pathogenesis of autoimmune neuritis and abnormally expressed in human neuropathies. Also, the role of integrins in myelination, neurite outgrowth, and nerve regeneration suggests that they could be involved in the recovery phase of immune-mediated neuropathies. We investigated by immunohistochemistry the expression of a number of integrin subunits during the course of experimental autoimmune neuritis (EAN). Results were compared with the human immune neuropathy Guillain-Barre syndrome (GBS) and extended in vitro. Inflammation and demyelination in both EAN and GBS induced the down-regulation of beta4 integrin in Schwann cells (SCs), whereas loss of alpha2 was noted only in EAN. When axonal loss was present, SCs displayed alpha5 integrin, in both EAN and GBS. In vitro, basal lamina and inflammatory cytokines modulated the expression of beta4 in SCs, but they did not influence alpha2 and alpha5 expression. Finally, integrins were differentially expressed in blood vessels during EAN. In conclusion, the spatiotemporal changes in integrin expression may be used to characterize, stage, and better understand the pathogenesis and evolution of inflammation during GBS and EAN. This may help to establish useful, novel therapy for immune-mediated neuropathies.     

Induction of cross-reactive antibodies against a self protein by immunization with a modified self protein containing a foreign T helper epitope

Self proteins are handled in the same way as foreign proteins by antigen presenting cells, but because of T-cell tolerance the presentation of self peptides does not normally lead to T cell activation. By providing physically linked T-cell help it is possible to overcome the B cell non-responsiveness toward self antigens. We have shown previously that a very potent antibody response, cross-reactive with a self protein, can be rapidly induced by immunizing with a recombinant immunogen consisting of the self protein with a foreign immunodominant T helper epitope inserted into its sequence (Dalum, I., Jensen, M. R., Hindersson, P., Elsner, H. I. and Mouritsen, S. (1996) J. Immnunol. 157, 4796). In this study we compare this approach for inducing autoantibodies against a self protein with the traditional method of conjugating the self antigen to a foreign carrier protein. The highly conserved self protein ubiquitin with an inserted epitope from ovalbumin (UbiOVA) is used as a model protein and compared to two traditionally conjugated immunogens consisting of ubiquitin chemically conjugated to a peptidic T helper epitope or to ovalbumin. The traditionally conjugated immunogens induce much slower and low titered ubiquitin specific antibody responses than the recombinant construct which also is capable of inducing antibodies directed against a much broader range of potential ubiquitin B cell determinants than the chemically conjugated immunogens. All three constructs are processed by antigen presenting cells and ovalbumin derived T cell epitopes are presented to T helper cells. From these observations it seems likely that the presence of non-shielded autologous B cell determinants on the immunogen is critical for the ability to induce a strong autoantibody response with a diverse fine specificity. Furthermore, the ubiquitin specific antibodies induced by UbiOVA contain higher levels of IgG2a/b relative to IgG1 compared to the conjugates. We therefore speculate that the insertion of a T cell epitope directly into the self antigen could possibly induce an immune response with a different Th1/Th2 balance than a response induced with traditional conjugates.