The autoimmune pathogenesis of multiple sclerosis.

One of the major goals of biomedical research is to reveal the pathomechanisms that lead to a disease on a level on which diagnostic criteria and causal therapies can be designed. The understanding and treatment of multiple sclerosis (MS) is still far from this goal, but exciting developments are on the way. MS is thought to be an autoimmune disease that is mediated by brain tissue-reactive lymphocytes, T cells and B cells, but so far these lymphocytes could not be reliably detected. This article highlights recent developments that permit the detection of autoreactive B cells in MS, the implications of this finding for early diagnosis of the disease, monitoring its activity, and eventually for gaining insight into the specific immune pathology that drives MS.

Assessment of the innate and adaptive immune system in proliferative vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) is the leading cause of failure of surgery for rhegmatogenous retinal detachment. Although indirect evidence suggests that this disease might be autoimmune in nature, direct proof for this hypothesis is lacking. The purpose of this study was to determine in a murine model whether PVR can develop in the absence of T- or B-cell immunity. METHODS: Four- to six-week-old Rag-1 gene knockout (KO) and congenic wild-type mice (WT) on the C57.Bl/6 background were studied. PVR was induced by intravitreal injection of 3 µl dispase at the concentration of 0.2 U/µl. PVR development was monitored by electroretinograms, the macroscopic observation of hemorrhage, cataract, retinal folds, and of an uneven iris, as well as the histological detection of epiretinal membranes on haematoxylin-eosin stained tissue. Additionally, immunofluorescence analysis was performed. These manifestations of PVR were assessed 1, 2, 4, 6, and 8 weeks after the intravitreal injection. RESULTS: The data showed that the immune-deficient Rag-1 KO mice developed PVR with similar kinetics and severity as did the fully immune competent congenic WT mice. Carboxyfluorescein diacetate succinimidyl ester-labeled T cells that are specific for ovalbumin were detected in the inflamed vitreous and retina showing that T cells that are not specific for autoantigens present in the eye can migrate to PVR lesions. Therefore, the mere presence of T cells in PVR lesions does not imply an autoimmune pathogenesis. CONCLUSION: This study suggests that T- and B-cell immunity is not essential for the induction of PVR.

Gene-dependent cell death in yeast.

Caspase-dependent apoptotic cell death has been extensively studied in cultured cells and during embryonic development, but the existence of analogous molecular pathways in single-cell species is uncertain. This has reduced enthusiasm for applying the advanced genetic tools available for yeast to study cell death regulation. However, partial characterization in mammals of additional genetically encoded cell death mechanisms, which lead to a range of dying cell morphologies and necrosis, suggests potential applications for yeast genetics. In this light, we revisited the topic of gene-dependent cell death in yeast to determine the prevalence of yeast genes with the capacity to contribute to cell-autonomous death. We developed a rigorous strategy by allowing sufficient time for gene-dependent events to occur, but insufficient time to evolve new populations, and applied this strategy to the Saccharomyces cerevisiae gene knockout collection. Unlike sudden heat shock, a ramped heat stimulus delivered over several minutes with a thermocycler, coupled with assessment of viability by automated counting of microscopic colonies revealed highly reproducible gene-specific survival phenotypes, which typically persist under alternative conditions. Unexpectedly, we identified over 800 yeast knockout strains that exhibit significantly increased survival following insult, implying that these genes can contribute to cell death. Although these death mechanisms are yet uncharacterized, this study facilitates further exploration.

ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators.

Measurements of antibodies in bodily fluids (e.g., by ELISA) have provided robust and reproducible results for decades and such assays have been validated for monitoring of B-cell immunity. In contrast, measuring T-cell immunity has proven to be a challenge due to the need to test live cells in functional assays ex vivo. Several previous efforts looking into the reproducibility of ex vivo T-cell assays between different laboratories, or even within the same laboratory, have provided rather discouraging results. The hypothesis we tested in this study is that those poor results are due to the lack of assay and data analysis standardization, rather than the inherent complexity of T-cell assays. In this study, 11 laboratories across Europe and the United States were provided identical reagents and were asked to follow the same protocol while testing aliquots of the same three cryopreserved peripheral blood mononuclear cells (PBMC) in an interferon-gamma (IFNgamma) ELISPOT assay measuring the antigen-specific T-cell response to a CMV peptide. All individuals performing the assays were ELISPOT novices. At their first attempt, while three of these individuals failed with the basic logistics of the trial, eight detected the peptide-specific CD8+ T-cells in frequencies approximating the values established by the Reference Laboratory. The data show that ELISPOT assays provide reproducible results among different laboratories when the assay procedure and data analysis is standardized. Since ELISPOT assays have been qualified and validated for regulated studies, they are ideal candidates for robust and reproducible monitoring of T-cell activity in vivo.

A T cell clone's avidity is a function of its activation state.

At present it is unclear how Ag dose-dependent T cell functions, such as cytokine production, reflect TCR affinity and how the signal strength afforded by the Ag dose affects the kinetics of cytokine production by the individual T cell. We used a computer-assisted ELISPOT approach to address these issues. IFN-gamma release by a clonal population of CD4 T cells was monitored on a clonal population of APC while titrating the nominal peptide. The frequency of cytokine-producing cells, the net per-cell output of cytokine, and the onset of cytokine production were each found to be functions of the signal strength. Sigmoidal dose-response curves were seen at the clonal population level, but the activation thresholds for the individual T cells followed a Gaussian distribution. Moreover, the overall dose-response curve of the T cell clone revealed cyclic changes, becoming increasingly shifted toward lower Ag concentrations with the duration of time that elapsed since the last restimulation with Ag. Therefore, responsiveness to Ag ("functional avidity") is not a constant parameter of a T cell clone but a function of the T cell's history of last Ag encounter. The implications of such shifting activation thresholds are discussed for autoimmune disease.

Protective anti-Helicobacter immunity is induced with aluminum hydroxide or complete Freund's adjuvant by systemic immunization.

To determine whether systemic immunization against Helicobacter pylori could be achieved with an adjuvant approved for human use, the efficacy of vaccination with Helicobacter antigen in combination with aluminum hydroxide (AlOH) was evaluated in a murine model of Helicobacter infection. Immunization with antigen and AlOH induced interleukin-5-secreting, antigen-specific T cells, and immunization with antigen and complete Freund's adjuvant induced interferon-gamma-secreting, antigen-specific T cells, as determined by ELISPOT assay. Both immune responses conferred protection after challenge with either H. pylori or H. felis, as confirmed by the complete absence of any bacteria, as assessed by both histology and culture of gastric biopsy samples. Protection was antibody independent, as demonstrated with antibody-deficient muMT mice (immunoglobulin-gene knockout mice), and CD4(+) spleen T cells from immunized mice were sufficient to transfer protective immunity to otherwise immunodeficient rag1(-/-) recipients. These results suggest an alternative and potentially more expeditious strategy for development of a human-use H. pylori vaccine.

Frequencies of neuroantigen-specific T cells in the central nervous system versus the immune periphery during the course of experimental allergic encephalomyelitis.

Direct measurements of the frequency and the cytokine signature of the neuroantigen-specific effector cells in experimental allergic encephalomyelitis (EAE) are a continuing challenge. This is true for lymphoid tissues, and more importantly, for the CNS itself. Using enzyme-linked immunospot analysis (ELISPOT) assays, we followed proteolipid protein (PLP) 139--151-specific T cells engaged by active immunization of SJL mice. The total numbers of PLP(139--151)-specific CD4 cells were highest before disease onset. At this time, these cells resided in lymphoid and nonlymphoid tissues, but were not detected in the CNS. While the PLP(139--151)-specific cells reached high frequencies in the CNS during clinical EAE, in absolute numbers, less than 20% of them were present in the target organ, with the majority residing in the periphery throughout all stages of the disease. The numbers of PLP(139--151)-specific cells gradually declined in both compartments with time. While eventually this first wave of effector cells completely disappeared from the CNS, PLP(178--191)-specific cells became engaged, being detected first in the CNS. These data suggest that throughout all stages of EAE, the effector cells in the CNS are recruited from a vast peripheral reservoir, and that the second wave of effector cells is engaged while the first wave undergoes exhaustion.

Comprehensive assessment of determinant specificity, frequency, and cytokine signature of the primed CD8 cell repertoire induced by a minor transplantation antigen.

T cell immunity is often focused on one peptide segment of a complex protein Ag, with other epitopes inducing weaker, low frequency responses or no responses at all. Such determinant hierarchy has been well characterized for MHC class II-restricted CD4 cell immunity, but is less well understood for class I-restricted CD8 cell responses. We studied class I determinant recognition in a skin transplant model with beta-galactosidase (beta-gal) as a minor transplantation Ag. CD8 T cells from C57BL/6 mice that rejected congenic C57BL/6 beta-gal transgenic skin were tested in enzyme-linked immunospot assays for recall responses to single-step, overlapping, 9-mer peptides that spanned a 94-aa region of the beta-gal sequence. This approach provided every possible class I-restricted peptide for CD8 cell recognition, allowing us to define the in vivo frequency of CD8 cells specific for each of the 86 individual peptides. While four peptides were predicted to bind to the Kb or Db molecules, only one (beta-gal96-103) actually induced an immune response. No peptides outside of the motifs were recognized. Tolerization to beta-gal96-103 significantly prolonged beta-gal transgenic skin graft survival, confirming its immune dominance. Therefore, single-determinant dominance characterized this CD8 cell response. The data demonstrate the feasibility of large-scale, comprehensive, class I determinant mapping, an approach that should be indispensable in measuring CD8 cell immunity in humans.

Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production.

Identifying and quantifying autoaggressive responses in multiple sclerosis (MS) has been difficult in the past due to the low frequency of autoantigen-specific T cells, the high number of putative determinants on the autoantigens, and the different cytokine signatures of the autoreactive T cells. We used single-cell resolution enzyme-linked immunospot (ELISPOT) assays to study, directly ex vivo, proteolipid protein (PLP)-specific memory cell reactivity from MS patients and controls. Overlapping 9-aa-long peptides, spanning the entire PLP molecule in single amino acid steps, were used to determine the frequency and fine specificity of PLP-specific lymphocytes as measured by their IFN-gamma and IL-5 production. MS patients (n = 22) responded to 4 times as many PLP peptides as did healthy controls (n = 22). The epitopes recognized in individual patients, up to 22 peptides, were scattered throughout the PLP molecule, showing considerable heterogeneity among MS patients. Frequency measurements showed that the number of PLP peptide-specific IFN-gamma-producing cells averaged 11 times higher in MS patients than in controls. PLP peptide-induced IL-5-producing T cells occurred in very low frequencies in both MS patients and controls. This first comprehensive assessment of the anti-PLP-Th1/Th2 response in MS shows a greatly increased Th1 effector cell mass in MS patients. Moreover, the highly IFN-gamma-polarized, IL-5-negative cytokine profile of the PLP-reactive T cells suggests that these cells are committed Th1 cells. The essential absence of uncommitted Th0 cells producing both cytokines may explain why therapeutic strategies that aim at the induction of immune deviation show little efficacy in the established disease.

Revisiting tolerance induced by autoantigen in incomplete Freund's adjuvant.

Injection of autoantigens in IFA has been one of the most effective ways of preventing experimental, T cell-mediated, autoimmune disease in mice. The mechanism that underlies this protection has, however, remained controversial, with clonal deletion, induction of suppressor cells or of type 2 immunity being implicated at one time or another. Using high resolution enzyme-linked immunospot (ELISPOT) analysis, we have revisited this paradigm. As models of autoimmunity against sequestered and readily accessible autoantigens, we studied experimental allergic encephalomyelitis, induced by myelin oligodendrocyte glycoprotein, proteolipid protein, myelin basic protein, and renal tubular Ag-induced interstitial nephritis. We showed that the injection of each of these Ags in IFA was immunogenic and CD4 memory cells producing IL-2, IL-4, and IL-5, but essentially no IFN-gamma. IgG1, but not IgG2a, autoantibodies were produced. The engaged T cells were not classic Th2 cells in that IL-4 and IL-5 were produced by different cells. The IFA-induced violation of self tolerance, including the deposition of specific autoantibodies in the respective target organs, occurred in the absence of detectable pathology. Exhaustion of the pool of naive precursor cells was shown to be one mechanism of the IFA-induced tolerance. In addition, while the IFA-primed T cells acted as suppressor cells, in that they adoptively transferred disease protection, they did not interfere with the emergence of a type 1 T cell response in the adoptive host. Both active and passive tolerance mechanisms, therefore, contribute to autoantigen:IFA-induced protection from autoimmune disease.

Direct visualization of cytokine-producing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients.

We have used computer-assisted cytokine ELISA spot analysis to measure the frequencies, the type of cytokine, and the amount of cytokine produced by individual recall Ag-specific CD4 memory cells in freshly isolated blood. We studied the memory cells specific for tetanus toxoid and purified protein derivative in 18 healthy individuals and in 22 HIV-infected patients on highly active antiretroviral therapy (HAART). In healthy individuals, the frequency, cytokine signature, and cytokine production per cell of these memory cells were stable over time. Although it is presently unclear whether the maintenance of the memory T cell pool depends upon Ag persistence, cross-reactive Ag stimulation, or cytokine-driven bystander stimulations and expansions, our data strongly argue for a stable memory cell pool in healthy individuals. In HIV patients, however, the frequency of these memory cells was a function of the viral load. The decreased numbers of functional memory cells in patients with high viral loads might provide one mechanism behind the immunodeficient state. Although the cytokine output per cell was unaffected in most patients (20 of 24), in some patients (4 of 24) it was >100-fold reduced, which might provide an additional mechanism to account for the reduced immunocompetence of these patients. The ability to visualize directly and quantify the cytokine produced by the low frequency memory cells in freshly isolated blood that have been physiologically stimulated by Ag should aid comprehensive studies of the Ag-specific memory cell pool in vivo, in health and disease.

Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN-gamma to be rare (0-10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Th1 or Th2 cells but by single-cytokine-producing memory cells.

Prevention of murine EAE by oral hydrolytic enzyme treatment.

Clinical trials that test the efficacy of Phlogenzym (consisting of the hydrolytic enzymes bromelain and trypsin and the anti-oxidant rutosid) as a treatment for T cell-mediated autoimmune diseases including multiple sclerosis (MS), type 1 diabetes and rheumatoid arthritis are presently ongoing. We tested the effects of Phlogenzym treatment in the murine model for MS, experimental allergic encephalomyelitis (EAE), a disease induced in SJL mice by immunization with proteolipid protein (PLP) peptide 139-151. Oral administration of Phlogenzym resulted in complete protection from EAE. In Phlogenzym-treated mice, the dose response curve of the PLP:139-151-specific T cell response was shifted to the right, that is, the primed T cells required higher peptide concentrations to become activated. Additionally, the T cell response to this peptide was shifted towards the T helper 2 cytokine profile. Both effects are consistent with an increased T cell activation threshold. In support of this interpretation, we found that the accessory molecules CD4, CD44, and B7-1 (all of which are involved in T cell co-stimulation) were cleaved by Phlogenzym, while CD3 and MHC class II molecules (which are involved in the recognition of antigens by T cells) and LFA-1 were unaffected. These data show the efficacy of oral Phlogenzym treatment in an animal model of T cell-mediated autoimmune disease and suggest that the protective effect might be the result of an increase in the activation threshold of the autoreactive T lymphocytes brought about by the cleavage of accessory molecules involved in the interaction of T cells and antigen presenting cells.

Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response.

Traditionally, protein Ags have been injected in CFA (oil with inactivated mycobacteria) to induce immunity and with IFA (oil alone) to induce tolerance. We report here that injection of hen eggwhite lysozyme, a prototypic Ag, in CFA-induced and IFA-induced pools of hen eggwhite lysozyme-specific memory T cells of comparable fine specificity, clonal size, and avidity spectrum, but with type-1 and type-2 cytokine signatures, respectively. This adjuvant-guided induction of virtually unipolar type-1 and type-2 immunity was observed with seven protein Ags and in a total of six mouse strains. Highly polarized type-1 and type-2 immunity are thus readily achievable through the choice of adjuvant, irrespective of the genetic bias of the host and of the nature of the protein Ag. This finding should have far-reaching implications for the development of vaccines against infectious and autoimmune diseases. Furthermore, our demonstration that Ag injected with IFA is as strongly immunogenic for T cells as it is with CFA shows that the presence of the mycobacteria determines not the priming of naive T cells through the second-signal link but the path of downstream differentiation toward CD4 memory cells that express either type-1 or type-2 cytokines.

Heat shock proteins and experimental autoimmune encephalomyelitis. II: environmental infection and extra-neuraxial inflammation alter the course of chronic relapsing encephalomyelitis.

We wished to study how infections might trigger relapses of autoimmune diseases such as multiple sclerosis (MS) and encephalomyelitis (EAE). We hypothesized that immune responses to heat shock proteins (hsp) induced by an infection could modulate responses to autoantigens. We induced extra-neuraxial inflammation in SJL mice housed either in specific-pathogen free (SPF) or conventional facilities. Mice in conventional housing are continuously exposed to large numbers of infectious agents. Spleen cell proliferative responses to human HSP60 and bacterial HSP65 were measured as were numbers of cells secreting IFN-gamma or IL-5. Proliferative responses to HSP60 were increased in conventionally housed mice compared to SPF mice and this was associated with skewing of secreted cytokines toward a Th2 pattern. Skewing toward a Th1 pattern was noted in SPF mice. Acute and relapsing EAE was induced in both groups of mice. Acute EAE was, in general, equivalent in all groups. However, SPF mice had more severe relapses than did conventionally housed animals and these differences were amplified by extra-neuraxial inflammation. Immunocytochemical analyses of brains from mice with relapsing EAE showed that increased numbers of brain gamma/delta cells were associated with disease remission. Our data suggest that frequent exposure to infectious agents leads to a relative Th2 skewing of immune responses to hsp and that this is associated with milder, less frequent relapses of EAE. They also support the concept that immune responses to hsp are of potential importance in exacerbating and perpetuating organ-restricted autoimmune diseases.

Shifting T-cell activation thresholds in autoimmunity and determinant spreading.

The best-characterized autoimmune T-cell response is that to myelin basic protein (MBP). MBP has classically been regarded as a sequestered antigen that does not cause negative selection. This view has been fostered by the observation that T-cell receptor-transgenic T cells that are specific for the "immunodominant determinant" on the molecule, MBP:Ac1-11, persist as naive cells in MBP-expressing H-2u mice. The same T cells, however, can cause autoimmune pathology once they have been primed by environmental stimulation to become memory cells. Once the autoimmune response to Ac1-11 has been engaged, determinant spreading occurs and second-wave T-cell responses that are specific for weaker, "cryptic" determinants like MBP:121-140 develop. Although the nature of these cryptic determinants has been enigmatic, recent studies using MBP-/- mice have provided new insights. These studies showed that MBP is not a sequestered antigen, but one that causes negative selection; as MBP:121-140 is actually the immunodominant determinant in MBP-/- mice, it tolerizes high avidity clones in MBP+/+ mice, making it appear cryptic. Based on this new information, we attempt here to redefine the MBP-specific repertoire within the theoretical framework of the threshold model for negative selection, and we propose a model of shifting T-cell activation thresholds to explain how ignorant/naive T cells can become effector cells of autoimmune pathology and why this effector cell repertoire spreads.

Endogenous myelin basic protein inactivates the high avidity T cell repertoire.

To study the contribution of endogenous myelin basic protein (MBP) to the positive and/or negative selection of the MBP-specific T cell repertoire, we studied the T cell response to MBP in MBP-deficient shiverer and MBP-expressing congenic C3H mice. Immunization with MBP induced a vigorous T cell response in shiverer mice directed against a single I-Ak- restricted immunodominant determinant, the core of which is peptide MBP:79-87 (DENPVVHFF). Injection of this peptide induced a high avidity T cell repertoire in shiverer mice that primarily consisted of clones capable of recognizing the native MBP protein in addition to the peptide itself. These data show that endogenous MBP is not required for the positive selection of an MBP-specific T cell repertoire. C3H mice, in contrast, were selectively unresponsive to the MBP protein and injection of MBP:79-87 peptide induced a low avidity repertoire that could be stimulated only by the peptide, not by the protein. Therefore, endogenous MBP induced profound inactivation of high avidity clones specific for the immunodominant determinant making that determinant appear cryptic.

High-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice.

BACKGROUND: Whether alloreactive T cells in a naive host derive from naive or memory T cells remains unclear. It is also unclear whether graft rejection alters the phenotype of these T cells. Proliferation assays and cytokine enzyme-linked immunosorbent assays performed on culture supernatants do not differentiate primary T-cell alloreactivity from recall responses in allograft-primed mice, suggesting that these methods are inadequate measures of the alloreactive immune repertoire. METHODS: To better characterize alloreactivity in naive and skin allograft-primed mice, we used a modified, high-resolution cytokine enzyme-linked immunosorbent spot assay capable of detecting cytokine production over short time periods. RESULTS: Twenty-four-hour analysis of alloreactivity in mice that rejected fully MHC-disparate skin allografts revealed a high frequency of interferon (IFN)-gamma- and interleukin (IL)-4-producing, L-selectin-negative T cells, consistent with a memory phenotype. In contrast, 24-hr allostimulation of T cells from naive mice resulted in IL-2 production with minimal secretion of IFN-gamma or IL-4. The frequency of IL-2 producers was low and their phenotype was L-selectin positive, suggesting that they were naive and not memory T cells. When maintained in culture for 48 hr, however, the T cells from the primary mixed lymphocyte reaction began producing IFN-gamma, consistent with in vitro priming. CONCLUSIONS: The primary alloresponse does not seem to involve clones that have been preprimed by environmental antigens, but instead behaves similarly to self-MHC-restricted immunity directed toward prototypic protein antigens: T cells with a naive phenotype are specifically induced to differentiate into high-frequency memory populations. These findings may have important implications for therapeutic induction of allograft tolerance.