Biomedical subjects

M D Mannie

Publications and source records attributed to M D Mannie.

At least 37 records · Page 2Linked to original sources

PubMed1996

Anergy-associated T cell antigen presentation. A mechanism of infectious tolerance in experimental autoimmune encephalomyelitis.

CD4+ T cells promote immune responses against foreign Ags while actively suppressing responses against self Ags. To address how CD4+ T cells ensure self-tolerance, we focused on two CD4+ T helper cells specific for myelin basic protein (MBP). GP2.E5/R1 T cells recognized rat MBP (RMBP) as a partial agonist and mediated mild experimental autoimmune encephalomyelitis (EAE), whereas R2 T cells recognized RMBP with full efficacy and mediated severe EAE. GP2.E5/R1 T cells were more susceptible to anergy induction than R2 T cells. Anergic GP2.E5/R1 T cells lacked proliferative reactivity, but expressed both I-A glycoproteins and high levels of radioresistant APC activity. During induction of anergy, these T cells acquired the ability to present MBP. In a separate subsequent culture without further addition of Ag, anergic GP2.E5/R1 T cells elicited full proliferative and IL-2 production responses by R2 T cells. Unlike activations induced via irradiated splenocytes, irradiated anergic T cells elicited anergy in R2 T cells in the form of a postactivational phase of nonresponsiveness. Anergic GP2.E5/R1 T cells not only transferred anergy to pathogenic R2 T cells in vitro, but these anergic T cells also transferred resistance to EAE in Lewis rats subsequently challenged with guinea pig MBP in CFA. Antagonistic signaling by autologous RMBP was more tolerogenic than that of guinea pig MBP in both in vitro and in vivo models of infectious anergy. We conclude that in the presence of tolerogenic mAb, antagonistic signaling by a self protein elicited the coordinate expression of anergy and T cell-mediated APC activity as a mechanism for the genesis and spread of infectious tolerance.

Animals↗

PubMed1996

The post-activation refractory phase: a mechanism to measure antigenic complexity and ensure self-tolerance among mature peripheral T lymphocytes.

This paper outlines a model describing how the post-activation refractory phase of mature T cells may promote adaptive self/nonself-discrimination among mature peripheral T cells in extrathymic tissues. This model is based on the following experimental observations. First, activation of myelin basic protein-specific T-helper cells elicits a single episode of interleukin 2 production followed by a 7-10 day refractory phase during which antigenic restimulation elicits proliferation but without interleukin 2 production or reexpression of encephalitogenic activity. Secondly, T lymphoblasts exhibiting post-activation refractoriness are substantially more susceptible to anergy as compared to resting T cells. Third, myelin basic protein-induced activation at low T-cell densities elicits a refractory phase that is prolonged as compared to that of high T-cell density cultures. These results support a model by which a pioneer T-cell encountering peripheral antigen produces a limited supply of interleukin 2. This T cell will upregulate effector functions or will become anergic, depending on continual immigration of additional antigen-reactive clones, each of which makes a limited supply of interleukin 2 before entering into the post-activation refractory phase. By this model, immune responses will be sustained in areas with high degrees of antigenic complexity (infectious process) but will falter in regions of low antigenic complexity (unaltered self tissues).

Animals↗

PubMed1995

Autologous rat myelin basic protein is a partial agonist that is converted into a full antagonist upon blockade of CD4. Evidence for the integration of efficacious and nonefficacious signals during T cell antigen recognition.

A central question of TCR function is based on the observation that some MHC ligands may bind TCR without stimulating biologic activity. To address the role of CD4 in this mechanism, we studied the interactions of the anti-CD4 mAb W3/25 with an encephalitogenic line of T helper cells. Proliferative responses to guinea pig (GP) myelin basic protein (GPMBP) were mediated by distinct W3/25-sensitive and W3/25-insensitive mechanisms whereas responses to autologous rat (R) MBP (RMBP) were exclusively mediated by W3/25-sensitive pathways. In assays of IL-2 production, RMBP was a partial agonist that stimulated an intermediate level of IL-2 production but antagonized high levels of GPMBP-stimulated IL-2 production to that intermediate level. In the presence of W3/25, RMBP lacked stimulatory activity but instead exhibited inhibitory activity that completely blocked GPMBP-stimulated proliferative responses. The inhibitory mechanism did not involve antigenic competition for MHC glycoproteins, a blockade of mitogenic signaling, or induction of high zone tolerance. Rather, the mechanism involved specific occupancy of TCR with antagonistic MHC ligands derived from the 72-86 region of RMBP. In proliferative assays, GPMBP was approximately 10-fold more active than RMBP. In the presence of W3/25 however, GPMBP-induced agonism and RMBP-induced antagonism exhibited overlapping dose-response curves. RMBP and W3/25 not only fully inhibited GPMBP-stimulated proliferation, this synergistic combination also elicited an extended phase of T cell energy. In conclusion, RMBP-derived MHC ligands occupy TCR to exhibit full efficacy for CD4-dependent signaling pathways while simultaneously lacking efficacy for W3/25-resistant signaling pathways. These data support an "integrative" model of T cell Ag recognition whereby MHC glycoproteins actively guide specific clustering of MHC-ligated TCR to enable quantitative comparisons of efficacious (nonself) and nonefficacious (self) signals by T cells.

Amino Acid Sequence↗

PubMed1995

Prostaglandin E2 promotes the induction of anergy during T helper cell recognition of myelin basic protein.

Remission of experimental autoimmune encephalomyelitis (EAE) in Lewis rats may involve mediators such as prostaglandins (PG) that are produced within demyelinating lesions and are known to potently inhibit T cell responses. In support, this study shows that PGE2 inhibited myelin basic protein (MBP)-specific responses of proliferation and IL-2 production by continuously propagated lines of T-helper cells. Simultaneous exposure to PGE2 and immunogenic MBP rendered T cells profoundly anergic. Even after several weeks of propagation in IL-2-containing medium, anergic T cells exhibited marked reductions in MBP-stimulated proliferation and IL-2 production responses when restimulated with optimal concentrations of MBP and irradiated splenocytes (SPL). PGE2 did not block other measures of MBP-dependent activation, including induction of postactivation refractoriness in IL-2 production pathways, activation-dependent decreases in MBP reactivity, and activation-dependent increases in PGE2 sensitivity. Proliferative responses by anergic T cells were reduced in magnitude but were not altered in their sensitivity to MBP. PGE2-mediated anergy was manifest as an intrinsic deficit rather than an acquired suppressive activity and was associated with reduced mitogenic responsiveness and a block in IL-2 production pathways. Anergic T cells were responsive to IL-2 and eventually regained full antigenic reactivity after extended propagation in IL-2-supplemented medium. In summary, a limited exposure to PG had long-lasting inhibitory effects on subsequent T cell responsiveness to the target autoantigen MBP. These findings support the hypothesis that PG may promote disease remission by inducing anergy in helper T cells.

Animals↗

PubMed1994

T-helper lymphocytes specific for myelin basic protein: activation-induced refractoriness of IL-2 production pathways augments an anti-CD4-mediated proliferative deficit.

Cloned and uncloned lines of encephalitogenic rat T cells produce IL-2 when activated with myelin basic protein (MBP) in the presence of irradiated splenocytes (SPL). Although these T cells use IL-2 as a primary mediator of autocrine growth, regulatory mechanisms controlling production of IL-2 have yet to be fully defined. This study shows that T cells reactivated within approximately 7 days of a prior activation were refractory to the reinduction of MBP-stimulated IL-2 production. In contrast, T cells rested for > 7 days regained the ability to produce optimal levels of IL-2 during activation with MBP. Cultures containing both activated and resting T cells responded to MBP by producing levels of IL-2 that were similar to those obtained from control cultures of resting T cells. The lack of IL-2 production during this refractory phase was associated with lowered responsiveness to MBP in proliferative assays as evidenced by right-shifted dose-response curves. However, this refractory phase did not affect the magnitude of responses elicited by optimal concentrations of MBP. The dissociation of proliferation from IL-2 production suggested parallel pathways of autocrine growth. Indeed, anti-MBP-proliferative responses were mediated by two distinct mechanisms distinguished by differential susceptibility to the anti-CD4 mAb W3/25. The W3/25-sensitive proliferation was desensitized in chronically activated T cells as well as in T cells activated once in the presence of the anti-CD4 mAb W3/25. Conversely, MBP responsiveness of W3/25-insensitive proliferation was unchanged by both chronic activation and by a prior activation in the presence of W3/25. In cultures of T cells recently activated by MBP in the presence of W3/25, the use of nonirradiated SPL rather than irradiated SPL reversed W3/25-mediated tolerance but did not restore MBP-stimulated IL-2 production. In summary, this study reveals mechanisms whereby the engagement of TcR and CD4 negatively regulates subsequent responsiveness of IL-2 production pathways and thereby impairs restimulation of IL-2-dependent proliferation by MBP-specific T-helper cells.

Animals↗

PubMed1994

Inhibition of activation-induced death in T cell hybridomas by thiol antioxidants: oxidative stress as a mediator of apoptosis.

N-Acetylcysteine (NAC) is a well established thiol antioxidant which, after uptake, deacylation and conversion to glutathione functions as both a redox buffer and a reactive oxygen intermediate scavenger. We report here that NAC completely blocks activation induced death and associated DNA fragmentation of myelin basic protein (MBP) specific T cell hybridomas. Conversely, NAC had very little effect on the antigen driven proliferation of a MBP specific T cell line similar to that from which the hybridomas were derived. NAC displayed an analogous absolute inhibition of mitogen mediated activation induced death, even if added up to 3 h post activation. Although glutathione was as efficient as NAC at blocking activation induced death, dithiothreitol displayed minimal inhibition while L-cysteine had no effect at all. The observation that certain thiol antioxidants such as NAC and glutathione can completely block the activation induced death of T cell hybridomas implicates redox regulation in this process.

Acetylcysteine↗

PubMed1994

Parallel costimulatory pathways promote myelin basic protein-stimulated proliferation of encephalitogenic rat T cells.

Activation pathways responsible for myelin basic protein (MBP)-stimulated proliferation of encephalitogenic T cells were studied by derivation of new monoclonal antibodies against rat T cell surface proteins. These monoclonal antibodies were derived by immunization of Balb/c mice with THYB-2 T cell hybrids or with the GP2.E5 line of encephalitogenic T-helper cells. The LRTC1 mAb inhibited MBP-stimulated IL-2 production by THYB-2 hybrids but not by THYB-1 hybrids and did not inhibit the alternative response of MBP-induced growth inhibition by either hybrid subset. Although LRTC1 labeled virtually all rat leukocytes, it selectively inhibited proliferative responses to T cell mitogens but not to B cell mitogens. LRTC1 inhibited MBP-stimulated IL-2 production by GP2.E5 T cells but did not effectively block MBP-stimulated proliferation. Rather, LRTC1 acted in synergy with a second mAb (LRTC2) to effectively inhibit MBP-stimulated proliferation by GP2.E5 T cells. The observation that LRTC1 did not exhibit synergy with a third biologically active mAb (LRTC3) supported the hypothesis that LRTC1 and LRTC2 represented a specific combination of synergistic mAb. In contrast to the inhibitory activity on GP2.E5 T cells, LRTC1 and LRTC2 synergistically stimulated antigen-independent IL-2 production by the THYB-1 hybrid LSS-A1. These results support the hypothesis that GP2.E5 T cells respond to parallel costimulatory pathways that are respectively inhibited by LRTC1 and LRTC2 mAb. Furthermore, these synergistic mAb exhibited inhibitory or stimulatory activities that may be diagnostic of distinct T-helper cell subsets. These novel mAb may thereby facilitate studies of costimulatory pathways and T-helper cell subsets in the pathogenesis of autoimmune disease.

Animals↗

PubMed1993

Differentiation of encephalitogenic T cells confers resistance to an inhibitory anti-CD4 monoclonal antibody.

The anti-CD4 mAb W3/25 inhibits experimental autoimmune encephalomyelitis (EAE) in Lewis rats by blocking Th cell responses to encephalitogenic determinants of myelin basic protein (MBP). However, it has yet to be resolved how W3/25 modulates CD4 to inhibit EAE-associated T cell responses. This study revealed that W3/25 profoundly inhibited MBP-stimulated proliferation by sensitized lymph node cells but only partially inhibited the respective response of uncloned and cloned lines of MBP-specific T cells. That is, low concentrations of W3/25 blocked 30 to 60% of MBP-stimulated proliferation, but 100-fold higher concentrations did not result in additional inhibition. W3/25 also inhibited MBP-induced acquisition of EAE transfer activity, but only in cultures of freshly isolated lymph node cells and not in cultures of continuously propagated T cells. Studies focusing on the GP2.E5 T cell line revealed that the lack of sensitivity to W3/25 in encephalitogenic and proliferative assays was nevertheless associated with an effective blockage of MBP-stimulated IL-2 production. Importantly, W3/25 specifically inhibited antigenic but not mitogenic stimulation of IL-2 production. Reverse transcriptase/polymerase chain reaction analyses revealed that MBP-activated GP2.E5 T cells produced mRNA for both IL-2 and IL-4, and that W3/25 selectively inhibited accumulation of IL-2 as compared to IL-4 mRNA. Thus, GP2.E5 T cells apparently express a IL-4-dependent pathway that confers resistance to the inhibitory activity of W3/25. Studies focusing on two CD4+ T cell hybridomas revealed that W3/25 profoundly inhibited MBP-stimulated IL-2 production but did not affect the alternative response of MBP-induced growth inhibition. Several other hybrids also mediated MBP-stimulated IL-2 production but did not express CD4 and were not affected by W3/25. These results indicate that: 1) interactions of W3/25 with CD4 do not necessarily block class II MHC-restricted recognition of MBP; and 2) expression of CD4 is not necessary for Ag recognition by several clonotypes of MBP-reactive T cells. Rather, the results of this study are consistent with the concept that W3/25 inhibits transduction of costimulatory signals that are required specifically for initiation of IL-2 production. These findings may have important implications for understanding the therapeutic potential of anti-CD4 mAb in autoimmune disease.

Animals↗

PubMed1993

A unique costimulatory pathway defined with T cell hybridomas specific for myelin basic protein: third party costimulators restrict antigenic responses in time and space.

Costimulatory signals regulate T cell responses to myelin basic protein (MBP) during induction of EAE in Lewis rats. However, the physiology of these costimulatory pathways has yet to be resolved. In this study, costimulatory pathways were defined by comparing MBP-stimulated IL-2 production by two types of T cell hybridoma (designated THYB-1 and THYB-2). THYB-1 hybrids required presentation of MBP/Ia complexes to stimulate IL-2 production, whereas THYB-2 hybrids also required the additional presence of costimulatory signals from radiosensitive (RS), nonadherent (NAdh) splenocytes (SPL). This study shows that allogeneic SPL, syngeneic B cells, or syngeneic T cells provided costimulatory signals to THYB-2 hybrids, although only syngeneic B cells were able to provide APC activity. Transduction of costimulatory signals did not occur across a microporous membrane but rather required close proximity of T cell hybrids with RS accessory cells. Simultaneous presence of costimulatory signals together with MHC-restricted antigen were required to initiate and maintain IL-2 production by THYB-2 hybrids. These findings support a model in which costimulatory molecules expressed by effector cells serve to restrict lymphokine production by T-helper cells. That is, THYB-2-like T-helper cells may mediate a paracrine pathway of IL-2 production that provides "help" to antigen-activated effector cell types that coexpress IL-2 receptors with appropriate costimulatory molecules.

Animals↗

PubMed1993

Emergence of a radioresistant population of co-stimulatory splenocytes during remission of experimental autoimmune encephalomyelitis in Lewis rats.

T-cell hybridomas specific for myelin basic protein (MBP) were used to assess regulation of co-stimulatory signals during remission of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Both THYB-1 and THYB-2 subsets of T-cell hybridomas recognize class II major histocompatibility complex-restricted determinants in the 72-86 encephalitogenic region of MBP. However, THYB-2 hybrids uniquely express additional requirements for co-stimulatory signals from radiosensitive splenocytes (SPL) to support the response of MBP-stimulated IL-2 production. Hence, this subset provides a means to study regulation of THYB-2 specific co-stimulatory signals during the course of EAE. This study revealed that sensitization of Lewis rats with MBP in complete Freund's adjuvant induced a radioresistant subpopulation of co-stimulatory SPL that emerged during the remission phase of EAE. These radioresistant SPL provided specific accessory cell activities that fulfilled the co-stimulatory requirements of THYB-2 hybrids. These findings support the hypothesis that in vivo activation events elicit radioresistance in an emergent clonally expanding population of antigen-specific lymphocytes. A central prediction of this hypothesis is that cellular activation should confer radioresistance to co-stimulatory lymphocytes. This prediction was verified by the observation that in vitro activation of naive SPL with different B- and T-cell mitogens conferred radioresistance to co-stimulatory SPL. Mitogenic activation not only induced radioresistance but also dramatically augmented co-stimulatory activity of purified B cells. In summary, the results of this study support the hypothesis that in vivo activation of co-stimulatory lymphocytes may regulate activities of encephalitogenic T-helper cells during progression and remission of EAE.

Animals↗

PubMed1993

Immune discrimination of self and nonself: a unified theory for the induction of self tolerance among thymocytes and mature peripheral T cells.

A comprehensive model of immunological self tolerance is described which is based on the unique tenet that interactions between T cell antigen receptors (TcR) and specific MHC ligands may vary in efficacy (the ability of an MHC ligand to catalyze TcR-mediated activation). Based on this postulate, two interrelated mechanisms are described to explain how self tolerance is induced among immature thymocytes and mature peripheral T cells, respectively. In the thymus, APC apparently present a diverse array of self MHC ligands (complexes of self peptides and MHC glycoproteins) to clonotypic T cells. According to the first mechanism, immature thymocytes that efficaciously bind specific MHC ligands undergo TcR-mediated activation and programmed cell death whereas those that nonefficaciously bind MHC ligands are not activated and thereby escape negative selection. The latter T cells undergo positive selection and eventually constitute the mature T cell repertoire. This model of thymic selection ensures that interactions of mature T cells with self in peripheral tissues are predominantly nonefficacious. According to the second mechanism, clonotypically diverse T cells and individual APC comprise an integrative unit that measures antigenic complexity of the local environment as a basis to enable or disable immunogenic responses by mature T cells. T cells recognize efficacious MHC ligands (E) via the TcR/CD3 complex but are also able to detect nonefficacious MHC ligands (N) by conserved signal transduction pathways that are initiated upon cell-cell contact with APC. Clonotypic T cells relay E or N signals by conserved feedback pathways back to APC. APC integrate and compare large numbers of E or N signals to derive an E/N ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

An alternative pathway of B cell activation: stilbene disulfonates interact with a Cl- binding motif on AEn-related proteins to stimulate mitogenesis.

Stilbene disulfonates are known to competitively inhibit Cl-/HCO3- flux through Band 3-related anion exchange (AE) proteins. To study the role of AE in lymphocyte activation, stilbene disulfonates were added to cultures of rat splenocytes (SPL). Four different stilbene derivatives were tested and each directly stimulated mitogenic proliferative responses of SPL. The mitogenic activity of these analogs paralleled their known patterns of interaction with Band 3-related AE proteins, as measured by; (a) their effective mitogenic concentrations, (b) their rank order of mitogenic potency [DIDS greater than SITS greater than DNDS congruent to DAzDS], (c) their patterns of nonreversible binding to the mitogenic receptor [DIDS much greater than SITS, DNDS], and (d) the specific, noncompetitive inhibition of their activity by the antagonist niflumic acid. Stilbene disulfonates directly activated purified B cell populations but not isolated T cells and furthermore, acted in synergy with anti-IgM to stimulate proliferation of SPL. These findings show that stilbene disulfonates represent a novel class of mitogens that interact with AEn-related proteins to stimulate an alternative activation pathway in B cells. These studies also indicate that immunomodulating activities of nonsteroidal anti-inflammatory drugs such as niflumic acid may be mediated, in part, by their interactions with AEn-related proteins.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Subset-specific co-stimulatory signals are required for IL-2 production but not growth inhibition responses by T cell hybrids specific for myelin basic protein.

Two distinct types of T cell hybridomas (designated THYB-1 and T-HYB-2) were derived by fusing BW5147 thymoma cells with encephalitogenic T helper cells from Lewis rats. Both subsets required MHC-restricted presentation of determinants within the 72-86 peptide sequence of myelin basic protein (MBP) as a requisite signal for IL-2 production. Unlike THYB-1 hybrids, however, THYB-2 hybrids required additional accessory cell activities that were mediated by radiosensitive nonadherent (RS-NAdh) splenocytes (SPL). In this study, we describe two observations indicating that RS-NAdh SPL enable MBP-specific responses of THYB-2 hybrids by providing subset-specific co-stimulatory signals that act independently of antigen recognition pathways. First, RS-NAdh SPL were required by THYB-2 hybrids for MBP-stimulated IL-2 production but were not needed when MBP-specific inhibition of hybrid growth was used as an alternative measure of cellular activation. Second, PMA and ionomycin induced optimal IL-2 production by both THYB-1 hybrids and BW5147 thymoma cells but only stimulated low or marginal levels of IL-2 production by THYB-2 hybrids. Together, these observations indicate that RS-NAdh SPL were required for the specific response of IL-2 production regardless of whether the response was stimulated by antigen or by mitogens that bypass initial antigen recognition events. This study thereby provides additional evidence that distinct stimulus-response relationships define two T-helper cell lineages in experimental autoimmune encephalomyelitis.

Animals↗

PubMed1991

A unified model for T cell antigen recognition and thymic selection of the T cell repertoire.

Positive selection of T cells during thymic differentiation predisposes mature T cells to recognize glycoproteins encoded by "self" alleles of the major histocompatibility complex (MHC) as "restricting elements" for antigen presentation. Yet, negative selection also occurs during thymic differentiation resulting in the clonal deletion of T cells reactive with "self" MHC glycoproteins. Thymic processes of positive and negative selection represent a paradox because the "altered self" view of T cell receptor (TcR) recognition indicates that the same type of TcR-MHC glycoprotein binding interaction mediates both positive and negative selection of the T cell repertoire. Most contemporary models of thymic selection attempt to explain these paradoxical observations by quantitative differences of affinity. That is, TcR interactions with MHC ligands that are of medium affinity lead to positive selection whereas those that are of high affinity lead to negative selection. The purpose of this manuscript is to provide an alternative model of thymic selection based on the efficacy (the ability of a ligand to catalyze receptor mediated biological activity) of TcR-MHC ligand interactions. The "efficacy" model predicts that among those thymocytes exhibiting affinity for self MHC ligands, some clones bind self MHC ligands without efficacy whereas others bind these ligands with efficacy. Immature T cells that bind MHC ligands without efficacy do not undergo TcR mediated activation and thereby escape clonal deletion. Instead, these T cells compete for growth--promoting sites on thymic antigen presenting cells (APC) based upon their clonotypic TcR affinity for self MHC ligands. These T cells experience positive thymic selection and eventually dominate a repertoire of mature T cells predisposed to exhibit non-efficacious binding to "self" MHC ligands. In contrast, immature T cells that exhibit efficacious binding to self MHC ligands are deleted from the T cell repertoire during thymic maturation. By this mechanism, the mature T cell repertoire is selected so that clonotypic T cells are predisposed to bind the very sites on MHC glycoproteins responsible for antigen presentation without risk of autoimmunity. Given the clonotypic diversity of the mature repertoire, complexes of foreign peptides and self MHC glycoproteins would be recognized by clones of the appropriate specificity as highly efficacious ligands. In summary, the "efficacy" model is entirely consistent with the "altered self" concept of T cell antigen recognition and readily accounts for both positive and negative selection of the T cell repertoire.

Antigens, CD↗

PubMed1990

Distinct accessory cell requirements define two types of rat T cell hybridomas specific for unique determinants in the encephalitogenic 68-86 region of myelin basic protein.

Six clonotypically unique T cell hybridomas from Lewis rats were used to study accessory cell activities required for class II MHC restricted T cell responses to the 68-86 encephalitogenic sequence of myelin basic protein (MBP). T cell hybrids which were cultured with GP68-86 68-86 sequence of guinea pig MBP (GPMBP) and naive splenocytes (SPL) were induced to produce IL-2 as measured by the CTLL indicator cell line. The hybrids were categorized into two subsets (designated THYB-1 and THYB-2), because two distinct subset-specific pathways of communication between accessory cells and T cells were involved in GPMBP-induced IL-2 production. These pathways were distinguished by the following six observations. First, when the duration of a pulse of SPL with GPMBP was lengthened from 1 to 4 h, these SPL lost their ability to induce IL-2 production by THYB-2 hybrids yet nevertheless retained full stimulatory activity for THYB-1 hybrids. Second, paraformaldehyde fixation of GPMBP-pulsed SPL abrogated an activity necessary for Ag-induced IL-2 production by THYB-2 hybrids. These fixed SPL were nevertheless able to stimulate THYB-1 hybrids, albeit to a lesser extent than viable unfixed SPL. Third, the addition of either cycloheximide, cytochalasin B, or 2-deoxyglucose to an Ag pulse of SPL with GPMBP dramatically inhibited the subsequent responses of THYB-2 hybrids yet had little or no effect upon the reactivity of THYB-1 hybrids. Fourth, thymocytes lacked necessary activities for GPMBP evoked IL-2 production by THYB-2 hybrids yet strongly promoted THYB-1 hybrid responses. Fifth, exposure of SPL to as little as 500 rad of gamma-irradiation markedly attenuated THYB-2 hybrid response to GPMBP but did not affect THYB-1 responses. Sixth, anti-GPMBP responses by THYB-2 hybrids were observed only in the presence of both radioresistant adherent SPL and a distinct population of radiosensitive nonadherent SPL. Conversely, THYB-1 hybrids exhibited full reactivity to GPMBP in the presence of adherent radioresistant SPL. Together, these observations reveal that two distinct accessory cell-T cell pathways mediate immune recognition of the 68-86 encephalitogenic region of MBP. Furthermore, these results indicate that subsets of Th cells can be defined by the accessory cell type-specific interactions that are necessary for Ag-mediated responses.

Animals↗

PubMed1990

The N- and C-terminal boundaries of myelin basic protein determinants required for encephalitogenic and proliferative responses of Lewis rat T cells.

Highly purified synthetic peptides representing portions of the 68-86 sequence of guinea pig (GP) myelin basic protein (GPMBP) were used to define the N- and C-termini of encephalitogenic determinants that cause experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Each peptide was tested for: (a) induction of EAE, (b)in vitro potentiation of EAE transfer activity by GPMBP-sensitized lymph node cells (LNC), (c) in vitro proliferation of GPMBP-sensitized LNC, and (d) in vitro proliferation of a GPMBP-reactive line of EAE-inducing T cells. In these bioassays, the general rank order of potency was: GPMBP greater than or equal to GP68-86 greater than or equal to GP72-86 greater than [G84]GP68-86 greater than or equal to GP68-84 much greater than GP75-85 greater than or equal to GP75-84 = virtually no activity. These results demonstrate that the encephalitogenic region is bounded by the 72-74 and 84-86 sequences. Further evidence presented herein indicates that the 75-84 sequence contains the primary antigenic features required for specific T cell recognition of the encephalitogenic region.

Animals↗

PubMed1989

Encephalitogenic and proliferative responses of Lewis rat lymphocytes distinguished by position 75- and 80-substituted peptides of myelin basic protein.

The encephalitogenic and proliferative responses of Lewis rat lymphocytes were defined by use of synthetic peptide GP68-84, representing the 68-84 sequence of guinea pig myelin basic protein (GPMBP), and otherwise identical peptides containing substitutions of either A75 or P80 residues. The comparative activities of these peptides were tested in the following bioassays: 1) active induction of experimental autoimmune encephalomyelitis (EAE), 2) potentiation of EAE transfer activity by MBP- or peptide-sensitized lymph node cells (LNC), 3) in vitro proliferation of MBP- or peptide-sensitized LNC, and 4) in vitro proliferation of an encephalitogenic T cell line. The GP68-84 peptide exhibited potent activity in all four bioassays. In contrast, [A75]GP68-84 and [P80]GP68-84 exhibited a selective loss of certain activities while retaining activity in other bioassays. For example, LNC were activated by culture with [A75]GP68-84 to express potentiated EAE transfer activity. Furthermore, [A75]GP68-84 and GP68-84 were equipotent in stimulating the proliferation of the encephalitogenic T cell line. However, [A75]GP68-84 was virtually inactive in assays measuring the induction of EAE or the proliferation of either GPMBP- or [A75]GP68-84-sensitized LNC. Conversely, the [P80]GP68-84 peptide actively induced EAE and potentiated EAE cellular transfer activity but was incapable of stimulating proliferation of either GPMBP-sensitized LNC or an encephalitogenic T cell line. When [P80]GP68-84 was used for sensitization, in vitro proliferation of LNC was stimulated, but only by MBP sequences containing a P80 substitution. Overall, these results indicate that at least two structurally distinct T cell determinants of GP68-84 regulate functionally diverse encephalitogenic and proliferative activities of EAE-associated T cells.

Animals↗

PubMed1989

Indomethacin augments in vitro proliferative responses of Lewis rat lymphocytes to myelin basic protein. Implications for experimental autoimmune encephalomyelitis.

Indomethacin (IM), a specific inhibitor of prostaglandin (PG) synthesis, and PGE2 were studied in terms of their ability to modulate in vitro immune responses associated with experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Lymphoid cells from either the spleens or the draining lymph nodes of myelin basic protein (MBP)-sensitized rats exhibited in vitro immune responses which were enhanced in the presence of IM. Specifically, IM enhanced (i) guinea pig MBP (GPMBP)- and rat MBP (RMBP)-stimulated lymphocyte proliferation, (ii) background proliferation, and (iii) interleukin 2 (IL-2)-stimulated proliferation. Conversely, PGE2 inhibited both GPMBP- and IL-2-stimulated proliferation of MBP-sensitized lymphocytes. Together, these results indicate that PGs secreted by cultured lymphoid cells can directly mitigate MBP- or IL-2-stimulated lymphocyte proliferation. Furthermore, the observation that IM and PGE2 modulate in vitro responses of MBP-specific lymphocytes may provide insight into how the in vivo administration of IM potentiates the severity of EAE (H. Ovadia and P.Y. Paterson, Clin. Exp. Immunol. 49, 386, 1982) and how PGs may be involved in the spontaneous remission of EAE in rats.

Animals↗