Neonatal death in mice lacking cardiotrophin-like cytokine is associated with multifocal neuronal hypoplasia.

Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Ralpha), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Ralpha ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Ralpha or Clf bolsters the hypothesis that Clc participates in Cntf-II.

In vivo activation of antigen-specific CD4 T cells.

Physical detection of antigen-specific CD4 T cells has revealed features of the in vivo immune response that were not appreciated from in vitro studies. In vivo, antigen is initially presented to naïve CD4 T cells exclusively by dendritic cells within the T cell areas of secondary lymphoid tissues. Anatomic constraints make it likely that these dendritic cells acquire the antigen at the site where it enters the body. Inflammation enhances in vivo T cell activation by stimulating dendritic cells to migrate to the T cell areas and display stable peptide-MHC complexes and costimulatory ligands. Once stimulated by a dendritic cell, antigen-specific CD4 T cells produce IL-2 but proliferate in an IL-2--independent fashion. Inflammatory signals induce chemokine receptors on activated T cells that direct their migration into the B cell areas to interact with antigen-specific B cells. Most of the activated T cells then die within the lymphoid tissues. However, in the presence of inflammation, a population of memory T cells survives. This population is composed of two functional classes. One recirculates through nonlymphoid tissues and is capable of immediate effector lymphokine production. The other recirculates through lymph nodes and quickly acquires the capacity to produce effector lymphokines if stimulated. Therefore, antigenic stimulation in the presence of inflammation produces an increased number of specific T cells capable of producing effector lymphokines throughout the body.

Highly efficient selection of CD4 and CD8 lineage thymocytes supports an instructive model of lineage commitment.

We undertook a kinetic analysis of the generation of mature T cells in TCR and coreceptor transgenic mice using BrdU labeling. We observed that the selection efficiency of mature CD4-CD8+ and CD4+CD8- thymocytes could be as high as 40% and 90% of CD4+CD8+ precursors, respectively. The surprisingly high efficiency of selection favors an instructional model of lineage commitment and is incompatible with a stochastic model in which the efficiency of selection would be no greater than 100% in both lineages combined.

The cytoplasmic domain of CD8 beta regulates Lck kinase activation and CD8 T cell development.

Previous studies have shown that CD8 beta plays a role in both enhancing CD8 alpha-associated Lck kinase activity and promoting the development of CD8-lineage T cells. To examine the role of this enhancement in the maturation of CD8-lineage cells, we assessed CD8 alpha-associated Lck kinase activity in both T cell hybridomas and thymocytes of mice expressing CD8 beta mutations known to impair CD8 T cell development. Lack of CD8 beta expression or expression of a cytoplasmic domain-deleted CD8 beta resulted in a severalfold reduction in CD8 alpha-associated Lck kinase activity compared with that observed with cells expressing wild-type CD8 beta chain. This analysis indicated a critical role for the cytoplasmic domain of CD8 beta in the regulation of CD8 alpha-associated Lck activity. Decreased CD8 alpha-associated Lck activity observed with the various CD8 beta mutations also correlated with diminished in vivo cellular tyrosine phosphorylation. In addition, analysis of CD8 beta mutant mice (CD8 beta-/- or cytoplasmic domain-deleted CD8 beta transgenic) indicated that the degree of reduction in CD8 alpha-associated Lck activity associated with each mutation correlated with the severity of developmental impairment. These results support the importance of CD8 beta-mediated enhancement of CD8 alpha-associated Lck kinase activity in the differentiation of CD8 single-positive thymocytes.

An activated form of Notch influences the choice between CD4 and CD8 T cell lineages.

Notch is a transmembrane receptor that controls cell fate decisions in Drosophila and whose role in mammalian cell fate decisions is beginning to be explored. We are investigating the role of Notch in a well-studied mammalian cell fate decision: the choice between the CD8 and CD4 T cell lineages. Here we report that expression of an activated form of Notch1 in developing T cells of the mouse leads to both an increase in CD8 lineage T cells and a decrease in CD4 lineage T cells. Expression of activated Notch permits the development of mature CD8 lineage thymocytes even in the absence of class I major histocompatability complex (MHC) proteins, ligands that are normally required for the development of these cells. However, activated Notch is not sufficient to promote CD8 cell development when both class I and class II MHC are absent. These results implicate Notch as a participant in the CD4 versus CD8 lineage decision.

The cytoplasmic domain of CD4 promotes the development of CD4 lineage T cells.

Thymocytes must bind major histocompatibility complex (MHC) proteins on thymic epithelial cells in order to mature into either CD8+ cytotoxic T cells or CD4+ helper T cells. Thymic precursors express both CD8 and CD4, and it has been suggested that the intracellular signals generated by CD8 or CD4 binding to class I or II MHC, respectively, might influence the fate of uncommitted cells. Here we test the notion that intracellular signaling by CD4 directs the development of thymocytes to a CD4 lineage. A hybrid protein consisting of the CD8 extracellular and transmembrane domains and the cytoplasmic domain of CD4 (CD884) should bind class I MHC but deliver a CD4 intracellular signal. We find that expression of a hybrid CD884 protein in thymocytes of transgenic mice leads to the development of large numbers of class I MHC-specific, CD4 lineage T cells. We discuss these results in terms of current models for CD4 and CD8 lineage commitment.

Functional commitment to helper T cell lineage precedes positive selection and is independent of T cell receptor MHC specificity.

Thymocyte differentiation proceeds from double positive CD4+CD8+ to single positive T cells. It has been proposed that this process occurs by an instructive or a stochastic mechanism. In this report, we show that in recombination-deficient mice (RAG-1-I-) constitutive expression of a CD8 transgene allows maturation of CD4+(CD8tg+) cells, which express mature levels of a transgenic class I-restricted T cell receptor, F5. Rescued F5+CD4+(CD8tg+) cells have equivalent levels of T cell receptor expression as CD8end+ cells, respond to cognate antigen and, upon stimulation, they exhibit a phenotype characteristic of CD4+ helper T cells. These data are consistent with a model of differentiation that predicts that thymocytes become functionally committed to a helper or cytotoxic lineage before the final step of positive selection and independently of MHC specificity of their T cell receptor.

A role for the cytoplasmic tail of the beta chain of CD8 in thymic selection.

The CD8 coreceptor plays a critical role in the recognition of foreign antigens by mature T cells and in the development of class I-restricted T cells. CD8 can be expressed on the surface of T cells as either a heterodimer composed of an alpha and beta chain, or as a homodimer composed of two alpha chains. In this report, we show that a CD8 beta transgene that lacks a cytoplasmic domain can suppress expression of wild-type endogenous CD8 beta and act as a dominant negative mutation. We show that this dominant negative CD8 beta transgene interferes with the development of mature CD8 T cells to different extents depending on the individual class I-restricted TCR. These data suggest CD8 beta plays a role in thymic development, and that different class I-restricted TCRs differ in their dependence on the cytoplasmic tail of CD8 beta.

Constitutive CD8 expression allows inefficient maturation of CD4+ helper T cells in class II major histocompatibility complex mutant mice.

Although mature CD4+ T cells bear T cell receptors (TCRs) that recognize class II major histocompatibility complex (MHC) and mature CD8+ T cells bear TCRs that recognize class I MHC, it is possible that the initial commitment of an immature thymocyte to a CD4 or CD8 lineage is made without regard to the specificity of the TCR. According to this model, CD4+ cells with class I TCR do not mature because the CD8 coreceptor is required for class I MHC recognition and positive selection. If this model is correct, constitutive expression of CD8 should allow CD4+ T cells with class I-specific TCRs to develop. In this report, we show that mature peripheral CD4+ cells are present in class II MHC-deficient mice that express a constitutive CD8.1 transgene. These cells share a number of properties with the major class II MHC-selected CD4 population, including the ability to express CD40 ligand upon activation. Although mature CD4 cells are also detectable in the thymus of class II MHC mutant/CD8.1 transgenic mice, they represent a small fraction of the mature CD4 cells found in mice that express class II MHC. These results indicate that some T cells choose the CD4 helper lineage independent of their antigen receptor specificity; however, the inefficiency of generating class I-specific CD4 cells leaves open the possibility that an instructive signal generated upon MHC recognition may bias lineage commitment.

Stochastic component to development of class I major histocompatibility complex-specific T cells.

The mechanism by which an initially uncommitted cell chooses between alternative fates is a central issue in developmental biology. In the mammalian thymus, CD4 helper T cells and CD8 cytotoxic T cells arise from a common precursor that expresses both CD4 and CD8. The choice between the CD4 and CD8 lineage is linked to the specificity of the T-cell antigen receptor expressed by a thymocyte, but whether lineage commitment is stochastic or instructed has not been definitively resolved. We present evidence that expression of a constitutive CD8 transgene during thymic selection permits development of mature CD4 cells bearing the class I-restricted F5 T-cell antigen receptor. These results suggest that there is a stochastic component to the development of class I major histocompatibility complex-restricted T cells.

Chronic murine experimental myasthenia gravis: strength testing and serology.

CB6 (Balb/c x C57Bl/6 F1) and C57Bl/6 (B6) mice were hyperimmunized with Torpedo acetylcholine receptor (AChR) for 7 months. Control groups were hyperimmunized with bovine serum albumin. Antibody titers against Torpedo AChR rose quickly, reaching plateau levels by 3-4 months, while antibody to mouse AChR lagged by a few months, reaching plateau levels in 5 months. After the last immunization the mice maintained a state of stable autoimmunity for 9 months with high levels of antibodies against Torpedo and mouse AChR. Fatigability was measured on a programmable treadmill and remained present through the 9 months after the last immunization. CB6 mice had less weakness than the B6 mice, but the latter strain when immunized with BSA had more "false-positive" weakness. Titers of antibodies did not correlate with the degree of weakness measured on the treadmill. Despite the weakness and the high titers of anti-AChR antibodies, sera from myasthenic mice, in contrast to sera from myasthenic humans, were not able to block bungarotoxin binding to native AChR on the surface of BC3H1 cells.

Olfactory classical conditioning in neonates.

One-day-old, awake infants underwent an olfactory classical conditioning procedure to assess associative learning within the olfactory system of newborns. Experimental infants received ten 30-second pairings of a novel olfactory conditioned stimulus (a citrus odor of neutral value) and tactile stimulation provided by stroking as the reinforcing unconditioned stimulus (a stimulus with positive properties). Control babies received only the odor, only the stroking, or the stroking followed by the odor presentation. The next day, all infants, in either the awake or sleep state, were given five 30-second presentations of the odor. Results were analyzed from video tapes scored by an observer unaware of the infants' training condition. The results indicate that only those infants who received the forward pairings of the odor and stroking exhibited conditioned responding (head turning toward the odor) to the citrus odor. The performance of the conditioned response was not affected by the state of the baby during testing, because both awake and sleeping infants exhibited conditioned responses. Furthermore, the expression of the conditioned response was odor specific; a novel floral odor presented during testing did not elicit conditioned responses in the experimental babies. These results suggest that complex associative olfactory learning is seen in newborns within the first 48 hours of life. These baseline findings may serve as normative data against which observation from neonates at risk for neurological sequelae may be compared.

Anti-ligand antibodies as potential autoantigens: in vitro and in vivo characteristics of anti-bungarotoxin antibodies in the idiotype network.

Some antibodies to ligands of a receptor will have combining sites that structurally resemble the receptor's binding site for that ligand. The network hypothesis predicts that anti-idiotypic antibodies to these anti-ligand antibodies will also bind to the receptor. We pursued these hypotheses in the well-defined ligand-receptor system, alpha-bungarotoxin(BTX)-acetylcholine receptor (AChR). Monoclonal antibodies (mAbs) to BTX were generated; native BTX was used as the immunogen to optimize the probability of obtaining mAbs to the AChR binding site. These mAbs were then characterized for their ability to "mimic" AChR in the following in vitro assays: neutralization of BTX binding to native AChR on the surface of the cell line TE671, formation of a ternary complex with 125BTX-AChR, and ability of cholinergic ligands to interfere with binding to BTX. Three aBTX mAbs which had in vitro attributes of the AChR on the basis of these assays, were injected into C3H mice and serial sera tested for antibodies to Torpedo and murine AChR. Anti-AChR antibodies directed primarily to the gamma and delta subunits of the Torpedo AChR were detected, as well as low amounts of anti-mouse AChR antibody. The generation of anti-AChR antibodies by immunization with aBTX antibodies supports the network hypothesis and provides a theoretical basis for initiation of autoimmunity to cell receptors.

Borrelia burgdorferi infection of the brain: characterization of the organism and response to antibiotics and immune sera in the mouse model.

To learn more about the neurologic involvement in Lyme disease, we inoculated inbred mice with the causative agent of Lyme disease, Borrelia burgdorferi. We cultured brains and other organs, and measured anti-B burgdorferi antibody titers. We further studied a brain isolate for its plasmid DNA content and its response in vitro to immune sera and antibiotics. One strain of B burgdorferi, N40, was consistently infective for mice, and resulted in chronic infection of the bladder and spleen. SJL mice developed fewer culture-positive organs and had lower antibody titers than Balb/c and C57Bl/6 mice. Organism was cultured from the brain early in the course of infection, and this isolate, named N40Br, was further studied in vitro. The plasmid content of N40Br was different from that of the infecting strain, implying either a highly selective process during infection or DNA rearrangement in the organism in vivo. N40Br was very sensitive to antibiotics, but only after prolonged incubation. Immune sera from both mice and humans infected with B burgdorferi were unable to completely kill the organism by complement-mediated cytotoxicity. These data demonstrate that B burgdorferi infects the brain of experimental animals, and is resistant to immune sera in vitro but sensitive to prolonged treatment with antibiotics.