Prenatal alcohol exposure decreases the number of nitric oxide synthase positive neurons in rat superior colliculus and periaqueductal gray.

Because nitric oxide (NO) is involved in the development and refinement of axonal projections and synapses, it is of interest to know if developmental alcohol exposures affect NO producing neurons. Pregnant rats were fed artificial liquid diet throughout gestation as the only fluid or caloric source. The diet for experimental dams contained ethanol (6.7% v/v) while the pair-fed diet for control dams contained isocaloric maltose-dextrin instead of ethanol. This ethanol diet regime is known to produce peak blood alcohol concentrations of approximately 140 mg%. Cells stained histochemically for nitric oxide synthase (NOS) were counted at postnatal day 15 (P15) and 35 (P35) in cross-sections of the stratum griseum superficiale (SGS) of the superior colliculus (SC) and in the dorsolateral column of the periaqueductal gray (dlPAG). Compared to control tissues, alcohol caused the following effects: In the SC, the areal density of NOS+ neurons was decreased 24% at P15 but a similar decrease in means at P35 was not statistically significant (P=0.10); soma size was unaffected at either P15 or P35. In the dlPAG, both the areal density and the total number of NOS+ neurons per section were unaffected at P15 but were decreased at P35 (33% and 37% decreases); soma size was unaffected at either P15 or P35. The decrease in NOS+ neurons in the SC at P15 could be expected to have a negative impact on the refinement of neuronal connections while the decreases in NOS+ neurons in the dlPAG at P35 likely represent more permanent effects that could alter the function of that nucleus.

Interleukin-12 enhances clinical experimental autoimmune myasthenia gravis in susceptible but not resistant mice.

Experimental autoimmune myasthenia gravis (EAMG) is induced by antibodies against the nicotinic acetylcholine receptor (AChR). Studies indicate a role for interferon-gamma (IFN-gamma) in EAMG. We examined the effect of IL-12, a major inducer of IFN-gamma production, on EAMG in C57BL/6 mice. Five doses of IL-12 accelerated and enhanced clinical disease in AChR-immunized mice. Control B6 mice, IFN-gamma gene-knockout mice, and EAMG-resistant bm12 mice showed no enhancement of disease. Shifting to a Th1-type antibody isotype distribution was insufficient to cause disease. Other factors, such as direct effects of Th1 cytokines on muscle tissue, may be involved in EAMG susceptibility.

Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells.

The development of the blood-brain barrier in alcohol-exposed rats.

Circulating horseradish peroxidase (HRP) was used as a tracer to determine if the blood-brain barrier to protein was altered by dietary prenatal alcohol exposure. Animals were prepared for light microscopic visualization of HRP after HRP infusion on gestational days 16, 18, 20, 22 and postnatal day 4. There was no consistent evidence of HRP leakage through the BBB in the alcohol-exposed animals compared to control animals. Capillary endothelial cells and perivascular astrocytic endfeet were morphologically characterized by electron microscopy in rat optic nerve and cerebellum following dietary prenatal and postnatal ethanol exposure. Photomontages of optic nerve capillaries from G20 and P5 animals and cerebellar capillaries from P15 animals were examined for evidences of effects of alcohol on the development of the capillaries and adjacent astroglial endfeet. There was no consistent evidence of any alcohol-induced effect that could indicate a disruption of the vessel, the endothelial tight junctions, the perivascular glial limiting membranes, or the extent of vascular ensheathment by astrocytic endfeet.

Restricted T cell receptor repertoire for acetylcholine receptor in murine myasthenia gravis.

Immunization of C57BL/6 mice with AChR provokes symptoms similar to those seen in the disease myasthenia gravis. To elucidate the structural requirements for T cell recognition of AChR and to identify TcR features which might provide targets for immunotherapy, a panel of T cell hybridomas was generated after immunization of mice with the immunodominant peptide of the AChR alpha chain. The TcR genes expressed by these hybridomas were sequenced. TcR-V beta 6 was preferentially employed, but other V beta genes were also observed. A conserved acidic residue was present in all CDR3 regions, regardless of the V beta. The TcR-V alpha repertoire was somewhat skewed with three V alpha families accounting for 82% of the sequences. The utilization of multiple T cell receptor V beta genes may contribute to the inability to inhibit EAMG by elimination of V beta 6+ T cells.

Combined pre- and postnatal ethanol exposure alters the development of Bergmann glia in rat cerebellum.

The development and maturation of Bergmann glial cells in the rat cerebellum was evaluated on postnatal day 15 by glial fibrillary acidic protein (GFAP) immunocytochemistry, following combined gestational and 10-day postnatal ethanol exposure (a full three trimester human equivalency). GFAP-positive Bergmann glial fibers of lobules I, III, VIb, VII and X of the cerebellar vermis were examined and counted in the molecular layer (ML), the external granular layer (EGL) and the external limiting membrane (ELM). Ethanol exposure reduced: (1) the number of GFAP-positive fibers (per unit length of folia surface) at all three levels; (2) the percentage of mature fibers; and (3) the cross-sectional area in all lobules examined. When data from the five lobules were pooled, there were 7% fewer GFAP-positive fibers in the ML, 15% fewer in the EGL and 20% fewer in the ELM; the percentage of mature fibers was reduced by 16%; and the cross-sectional areas of lobules were reduced by 16%. The altered development of Bergmann glia could be one of the factors causing delayed migration of granular neurons and reductions in the number of granule cells reported in other studies following developmental ethanol exposures and could help to explain some of the motor dysfunctions reported in FAS victims.

A disease-related epitope of Torpedo acetylcholine receptor. Residues involved in I-Ab binding, self-nonself discrimination, and TCR antagonism.

Residues 146-162 of the Torpedo californica acetylcholine receptor alpha-subunit contain the immunodominant T cell epitope for experimental autoimmune myasthenia gravis-susceptible C57BL/6 mice. To develop potential therapeutic peptides, a detailed analysis of the epitope was undertaken. Truncated and substituted synthetic peptides were tested as stimulators of T cell clones and immune lymph node cells. Critical residues spanned positions 151-159. Y151 and V156 were critical for MHC binding. The results indicated a general motif for binding to I-Ab to be an aromatic or hydrophobic residue (preferentially Y or F) at position i followed by an uncharged residue at position (i + 5). Lysine was not tolerated at position (i + 8). Residues D152, K155, S157, and I158 were important T cell contact residues. A peptide corresponding to the murine 146-162 sequence, which differs from the Torpedo sequence at 5 residues, bound to I-Ab but was nonimmunogenic, consistent with the assigned TCR and I-Ab contact residues. These results suggest that tolerance is responsible for the lack of T cell cross-reactivity with the murine acetylcholine receptor. Substituted peptides were tested for the inhibition of T cell clone responses and for TCR antagonism. Although peptides substituted at residues 157 and 158 inhibited most clones, no single peptide could completely inhibit all clones or primed lymph node cells. These findings indicate that antagonist peptides may be useful in inhibiting T cell responses to complex Ag displaying a single immunodominant epitope. Multiple antagonists used in combination may be required for maximum inhibition of the response.

Preferential use of a T cell receptor V beta gene by acetylcholine receptor reactive T cells from myasthenia gravis-susceptible mice.

Experimental autoimmune myasthenia gravis (EAMG) is an important model for testing current concepts in autoimmunity and novel immunotherapies for autoimmune diseases. The EAMG autoantigen, acethylcholine receptor (AChR), is structurally and immunologically complex, a potential obstacle to the application of therapeutic strategies aimed at oligoclonal T cell populations. Inasmuch as we had previously shown that the clonal heterogeneity of T cell epitope recognition in EAMG was unexpectedly limited, we examined TCR V beta expression. AChR primed lymph node T cells and established AChR reactive T cell clones from EAMG-susceptible C57BL/6 (B6; H-2b, Mls-1b) mice showed preferential utilization of the TCR V beta 6 segment of the TCR. After in vivo priming and in vitro restimulation for 7 days with AChR or a synthetic peptide bearing an immunodominant epitope, V beta 6 expressing lymph node cells (LNC) were expanded several-fold, accounting for up to 75% of recovered viable CD4+ cells. The LNC of B6.C-H-2bm12 (bm12; H-2bm12, Mls-1b) mice, which proliferated in response to AChR but not to the B6 immunodominant peptide, failed to expand V beta 6+ cells. Inasmuch as nonimmune bm12 and B6 animals had similar numbers of V beta 6+ LNC (4-5%), this suggested that structural requirements for TCR recognition of Ag/MHC complexes dictated V beta usage. Results concerning peptide reactivity and V beta 6 expression among T cells from (B6 x bm12)F1 animals also suggested that structure-function relationships, rather than negative selection or tolerance, accounted for the strain differences between B6 and bm12. To examine the potential effects of thymic negative selection of V beta 6+ cells on the T cell response to AChR, CB6F1 (H-2bxd, Mls-1b; V beta 6-expressing) and B6D2F1 (H-2bxd, Mls-1axb; V beta 6-deleting) strains were analyzed for AChR and peptide reactivity and V beta 6 expression. Both F1 strains responded well to AChR but the response of B6D2F1 mice to peptide was significantly reduced compared to CB6F1. Short and long term cultures of peptide-reactive B6D2F1 LNC showed no expansion of residual V beta 6+ cells, although similar cultures of CB6F1 LNC were composed of more than 60% V beta 6+ cells. The results from the F1 strains further indicated that the T cell repertoire for peptide was highly constrained and that non-V beta 6 expressing cells could only partially overcome Mls-mediated negative selection of V beta 6+ TCR capable of recognizing peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

Application of an N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine-substituted peptide as a heterobifunctional cross-linking agent in a study of protein O-glycosylation in yeast.

In order to investigate the O-mannosyltransferase involved in the initial O-mannosylation of glycoproteins in Saccharomyces cerevisiae, a photoactive hexapeptide, [125I]-N-(4-azido-2,3,5,6-tetrafluorobenzoyl)-3-iodo-Tyr-Asn-Pro-T hr-Ser-Val ([125I]azidoTyr-peptide), was synthesized by solid-phase techniques using a new photoactive cross-linking reagent, N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosine, and resin-bound Asn-Pro-Thr(tBu)-Ser(tBu)-Val. When this modified hexapeptide substrate was incubated with O-mannosyltransferase preparations, the hexapeptide was an acceptor of [14C]-mannose from dolichol phosphate-[14C]mannose. After partially purifying the O-mannosyltransferase and photolabeling these enzyme preparations with [125I]azidoTyr-peptide, a ca. 82-kDa protein was shown to be the only apparent photolabeled protein that was protected by unmodified hexapeptide. This ca. 82-kDa protein may be the catalytic subunit of the O-mannosyltransferase. The susceptibility of the N-(4-azido-2,3,5,6-tetrafluorobenzoyl) moiety to reducing agents in aqueous buffers was also examined.

Determinant selection in murine experimental autoimmune myasthenia gravis. Effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes.

C57BL/6 (B6) mice respond to immunization with acetylcholine receptor (AChR) from Torpedo californica as measured by T cell proliferation, antibody production, and the development of muscle weakness resembling human myasthenia gravis. The congenic strain B6.C-H-2bm12 (bm12), which differs from B6 by three amino acid substitutions in the beta-chain of the MHC class II molecule I-A, develops a T cell proliferative response but does not produce antibody or develop muscle weakness. By examining the fine specificity of the B6 and bm12 T cell responses to AChR by using T cell clones and synthetic AChR peptides, we found key differences between the two strains in T cell epitope recognition. B6 T cells responded predominantly to the peptide representing alpha-subunit residues 146-162; this response was cross-reactive at the clonal level to peptide 111-126. Based on the sequence homology between these peptides and the T cell response to a set of truncated peptides, the major B6 T cell epitope was determined to be residues 148-152. The cross-reactivity of peptides 146-162 and 111-126 could also be demonstrated in vivo. Immunization of B6 mice with either peptide primed for T cell responses to both peptides. In contrast, immunization of bm12 mice with peptide 111-126 primed for an anti-peptide response, which did not cross-react with 146-162. Peptide-reactive T cells were not elicited after immunization of bm12 mice with 146-162. These results define a major T cell fine specificity in experimental autoimmune myasthenia gravis-susceptible B6 mice to be directed at alpha-subunit residues 148-152. T cells from disease-resistant bm12 mice fail to recognize this epitope but do recognize other portions of AChR. We postulate that alpha-148-152 is a disease-related epitope in murine experimental autoimmune myasthenia gravis. In this informative strain combination, MHC class II-associated determinant selection, rather than Ag responsiveness per se, may play a major role in determining disease susceptibility.

Purification and properties of arylmannosidases from mung bean seedlings and soybean cells.

Two arylmannosidases (signified as A and B) were purified to homogeneity from soluble and microsomal fractions of mung bean seedlings. Arylmannosidase A from the microsomes appeared the same on native gels and on SDS gels as soluble arylmannosidase A, the same was true for arylmannosidase B. Sedimentation velocity studies indicated that both enzymes were homogeneous, and that arylmannosidase A had a molecular mass of 237 kd while B had a molecular mass of 243 kd. Arylmannosidase A showed two major protein bands on SDS gels with molecular masses of 60 and 55 kd, and minor bands of 79, 39 and 35 kd. All of these bands were N-linked since they were susceptible to digestion by endoglucosaminidase H. In addition, at least the major bands could be detected by Western blots with antibody raised against the xylose moiety of N-linked plant oligosaccharides, and they could also be labeled in soybean suspension cells with [2-3H]mannose. Arylmannosidase B showed three major bands with molecular masses of 72, 55 and 45 kd, and minor bands of 42 and 39 kd. With the possible exception of the 45 and 42 kd bands, all of these bands are glycoproteins. Arylmannosidases A and B showed somewhat different kinetics in terms of mannose release from high-mannose oligosaccharides, but they were equally susceptible to inhibition by swainsonine and mannostatin A. Polyclonal antibody raised against the arylmannosidase B cross-reacted equally well with arylmannosidase A from mung bean seedlings and with arylmannosidase from soybean cells. However, monoclonal antibody against mung bean arylmannosidase A was much less effective against arylmannosidase B. Antibody was used to examine the biosynthesis and structure of the carbohydrate chains of arylmannosidase in soybean cells grown in [2-3H]mannose. Treatment of the purified enzyme with Endo H released approximately 50% of the radioactivity, and these labeled oligosaccharides were of the high-mannose type, i.e. mostly Man9GlcNAc. The precipitated protein isolated from the Endo H treatment still contained 50% of the radioactivity, and this was present in modified structures that probably contain xylose residues.

Inhibitors of glycoprotein processing alter T-cell proliferative responses to antigen and to interleukin 2.

Most of the cell-surface molecules involved in T-cell immune responses are N-linked glycoproteins. We have investigated the effects of inhibitors of glycoprotein processing on specific T-cell functions, with the dual aims of examining the functional role of carbohydrate and of testing the usefulness of such compounds as immunomodulators. Treatment of a cloned murine helper T-cell line with these inhibitors differentially affects the proliferative response of the cell, depending upon the nature of the stimulus. Treatment with the plant alkaloid swainsonine, which inhibits the processing mannosidase II and causes the accumulation of glycoproteins bearing hybrid-type oligosaccharide structures, enhances the proliferative response of the T-cell clone to antigen and to the mitogen concanavalin A. Treatment with another plant alkaloid, castanospermine, which inhibits glucosidase I and causes the accumulation of glucose-containing high-mannose structures, has the opposite effect and inhibits the proliferative response of the T cell to antigen. Cell-surface oligosaccharide alteration does not affect antigen recognition, as judged by the lack of effect of either drug on interleukin 2 production following antigen stimulation. Cells treated with either alkaloid proliferate poorly to exogenous interleukin 2 and may have defective interleukin 2 receptor function. Swainsonine-treated cells apparently have compensatory alterations that can overcome the reduced responsiveness to interleukin 2. Antibody-binding studies indicate that normal quantities of many cell-surface molecules, including the T-cell receptor for antigen, are expressed by the treated cells.

A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin.

Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.

Light activates the reaction of bacteriorhodopsin aspartic acid-115 with dicyclohexylcarbodiimide.

Conditions for a light-induced reaction between the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) and bacteriorhodopsin in Triton X-100 micelles were previously reported [Renthal, R., Dawson, N., & Villarreal, L. (1981) Biochem. Biophys. Res. Commun. 101, 653-657]. We have now located the DCCD site in the bacteriorhodopsin amino acid sequence. [14C]DCCD-bacteriorhodopsin (0.67 mol/mol of bacteriorhodopsin) was cleaved with CNBr. The resulting peptides were purified by gel filtration and reverse-phase high-performance liquid chromatography (HPLC). One major 14C peptide (50%) and two minor fractions were obtained. The modified peptides were completely absent in the absence of DCCD, and 10 times less was obtained when the reaction was run in the dark. Amino acid analysis and sequence analysis showed that the major fraction contained residues 69-118. This region includes six carboxyl side chains. Quantitative sequence analysis ruled out significant amounts of DCCD at Glu-74, Asp-85, Asp-96, Asp-102, and Asp-104. The major 14C peptide was also subjected to pepsin hydrolysis. HPLC analysis of the product gave only a single major radioactive subfragment. Amino acid analysis of the peptic peptide showed that it contained residues 110-118. The only carboxyl side chain in this region is Asp-115. Thus, we conclude that Asp-115 is the major DCCD site. The light sensitivity of this reaction suggests that Asp-115 becomes more exposed or that its environment becomes more acidic during proton pumping. The DCCD reaction blue-shifts the retinal chromophore. Such a result would be expected if Asp-115 is the negative point charge predicted to be near the cyclohexene ring of retinal.

Cell-surface modification with an iodinatible imidoester to enhance radiolabeling.

A procedure to enhance incorporation of radioactive iodine into cell-surface proteins is described. The effects of chemical modification of cells with the iodinatible haptens N-succinimidyl-3-(4-hydroxyphenyl) propionate (Bolton-Hunter reagent) and methyl-p-hydroxybenzimidate HC1 (Wood reagent) are compared. The Wood reagent, that does not alter the charge of the modified amino group, is superior in retention of cell viability and function. Cell-surface modification with the Wood reagent prior to radiolabeling is particularly useful for detecting antigens not readily iodinated using standard techniques.

Control of cloned alloreactive T lymphocyte proliferative responses. A possible role for cell-surface-bound alloantigen.

Previous studies using flow cytofluorometry demonstrated that passively acquired alloantigens encoded by the I-A and H-2K/D regions of the MHC could be identified on the surface of murine cloned T lymphocytes. The present studies were designed to further characterize the nature of that bound alloantigenic material on cloned lines of alloreactive amplifier T cells (Th). Immune precipitation and polyacrylamide gel electrophoresis were used to substantiate that allogeneic I-A and H-2K/D antigens were present on he surface of these cloned Th. Using proteolytic digestion with papain, bound allogeneic I-A and H-2K/D was removed from the surface of a cloned Th designated L2. Allogeneic I-A or surface of a cloned Th designated L2. Allogeneic I-A or H-2K/D antigens were not reexpressed if papain digested cells were incubated overnight in culture medium without addition of fresh alloantigen. Further, cloned cells did not release detectable amounts of interleukin 2 (IL-2) in response to Con A stimulation immediately following enzymatic digestion. After overnight culture, papain-digested cells released amounts of IL-2 similar to untreated controls. The proliferative response of these cells measured by 3H-thymidine incorporation, however, was significantly less than that observed using undigested controls, unless alloantigen was provided in the form of fresh irradiated stimulating cells. The addition of partially purified preparations of monoclonal anti Ia antibodies to cultures of cloned Th resulted in marked reduction in incorporation of 3HTdR. These findings are consistent with the hypothesis that modulation of the Th proliferative response may occur at least partially through binding of allogeneic material to the cell surface.

Comparison of active mutants and wild-type aspartate transcarbamoylase of Escherichia coli.

Two active mutants of aspartate transcarbamoylase from Escherichia coli have been purified from strains which produce large quantities of enzyme. Each enzyme was isolated from a different spontaneous revertant of a pyrimidine auxotrophic strain produced by mutagenesis with nitrogen mustard. Both enzymes exhibit allosteric properties with one having significantly less and the other more cooperativity than wild-type enzyme. Isolated catalytic subunits had different values of Km and Vmax. Studies on hybrids constructed from mutant catalytic and wild-type regulatory subunits (and vice versa) indicate that catalytic chains encoded by pyrB and not the regulatory chains encoded by pyrI were affected by the mutations. Differential scanning calorimetry experiments support these conclusions. Both mutant enzymes undergo ligand-promoted conformational changes analogous to those exhibited by wild-type enzyme: a 3% decrease in the sedimentation coefficient and a 5-fold increase in the reactivity of the sulfhydryl groups of the regulatory chains. Interactions between catalytic and regulatory chains in the mutants are weaker than those in the wild-type enzyme. The gross conformational changes of the mutants upon adding the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, in the presence of the substrate, carbamoylphosphate, and the activator, ATP, correlate with differences in cooperativity. The mutant with lower cooperativity is more readily converted from the low-affinity, compact, T-state to the high-affinity, swollen, R-state than is wild-type enzyme; this conversion for the more cooperative enzyme is energetically less favorable.

Characterization of the murine T cell surface molecule, designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu-3/T4 molecule.

Monoclonal antibody GK1.5 recognizes a previously undescribed murine T cell surface molecule, designated L3T4, which migrates on SDS-PAGE under reducing conditions as a single band with an apparent m.w. of 52,000. L3T4 is expressed by approximately 80% of thymocytes and by approximately 20% of spleen cells. There appears to be poor correlation between expression of L3T4 by functional T cell clones and expression of Lyt-2, expression of the cytolytic phenotype, and class I MHC antigen reactivity. On the other hand, both a class II MHC antigen-reactive HTL clone and an Lyt-1- Mls-reactive HTL clone express L3T4. Analysis of the effect of mAb GK1.5 on PFC responses in adoptive transfer suggests that L3T4 is expressed by the helper/inducer subset of murine T cells. Expression of L3T4 by murine T cells, however, may correlate primarily with class II MHC antigen reactivity rather than with functional phenotype; mAb GK1.5 profoundly blocks antigen-specific cytolysis by the cloned class II MHC antigen-reactive CTL line A15-1.17. Antigen-specific cytolysis by A15-1.17 is blocked by mAb GK1.5 at a step before the lethal hit. Collectively, the flow cytometric, functional, and biochemical data indicate that L3T4 is similar to the human Leu-3/T4 molecule.