bluuebunny/crossref_metadata_2025 huggingface dataset:
Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.
Authors: Lai M Z , Jang Y J , Chen L K , Gefter M L
Published: December 2022

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.
Identification of a human protein homologous to the mouse Lyb-2 B cell differentiation antigen and sequence of the corresponding cDNA.
Authors: Von Hoegen I , Nakayama E , Parnes J R
Published: December 2022

We have isolated and sequenced cDNA clones encoding the human homolog of the mouse Lyb-2 B cell differentiation Ag. Previous data suggest that Lyb-2 might represent a growth factor or lymphokine receptor. Human Lyb-2 mRNA is expressed in normal human tonsils and bone marrow cells, in the pre-B cell line REH, in three Burkitt lymphoma cell lines, and in some EBV-transformed B cell lines, but not in antibody-secreting myeloma cell lines, T cell lines, or a promyelocytic leukemia cell line. These data indicate that expression of human Lyb-2 is restricted to B lineage cells and turned off in antibody-secreting plasma cells. A polyclonal mouse antiserum was raised against human Lyb-2 and immunoprecipitates a Mr 42,000 protein from REH, Raji, and Daudi cells and from mouse L(tk) cells transfected with the human Lyb-2 cDNA in an expression vector. The human Lyb-2 protein is related to both the asialoglycoprotein receptor and CD23, the B cell-specific FcR for IgE. These data demonstrate that human B cells express a previously undescribed cell surface protein that is homologous to mouse Lyb-2 and has a similar pattern of expression during B cell development.
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.
Authors: Mannie M D , Paterson P Y , Thomas D W , Nairn R
Published: December 2022

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.
Development of the autoimmune B cell repertoire in MRL-lpr/lpr mice.
Authors: Klinman D M , Eisenberg R A , Steinberg A D
Published: December 2022

The processes responsible for the production of autoantibodies have been shown to include both Ag-specific and generalized (polyclonal) forms of B cell activation. The relative contribution and temporal association of these processes to the genesis of systemic autoimmunity are incompletely understood. To study this relationship, the B cell repertoires of MRL-lpr/lpr mice were analyzed by ELISA spot assay over an 8-mo period. Between 6 and 12 wk of age, the number of splenic lymphocytes producing antibodies reactive with both autoantigens and conventional Ag increased proportionately. The repertoires of MRL-lpr/lpr mice under 12 wk were dominated by IgM-secreting B cells that showed no bias toward the production of specific autoantibodies. From 12 to 38 wk of age, an increasing proportion of animals developed repertoires dominated by IgG-secreting B cells that were skewed toward reactivity against one or very few (auto)antigens. Although there was no single Ag against which all mice developed skewed reactivity, 55% of MRL-lpr/lpr adults had increased numbers of B cells producing antibodies to the Sm Ag and 13 to 16% developed increased reactivity toward DNA, myosin, histone, thyroglobulin, or T cells. These data indicate that generalized (polyclonal) B cell activation dominates early repertoire development whereas (auto)-antigen-specific responses become increasingly important during the latter stages of disease in these autoimmune-prone mice.
HIV-gp120 can block CD4-class II MHC-mediated adhesion.
Authors: Rosenstein Y , Burakoff S J , Herrmann S H
Published: December 2022

A possible component of the immune dysfunction associated with infection by HIV is the inhibition of CD4 function resulting from the avid binding of soluble HIV envelope glycoprotein (gp120) to cell surface CD4. We assessed CD4 function by measuring the ability of CD4+ T cells to form conjugates with cell size lipid vesicles, artificial target cells (ATC), bearing the natural ligand for CD4, MHC class II proteins. Conjugate formation was a transient process with the greatest number of specific cell to ATC conjugates found after approximately 30 min of incubation at 37 degrees C. Addition of gp120 specifically blocked conjugates between CD4+ cells and class II ATC in a concentration-dependent manner. These data indicate that T lymphocyte adhesion mediated by CD4 is a dynamic event and that binding of gp120 to CD4 is able to disrupt the normal progression of the interaction between CD4+ T lymphocytes and class II+ APC.
IL-2 dependence of the promotion of human B cell differentiation by IL-6 (BSF-2).
Authors: Splawski J B , McAnally L M , Lipsky P E
Published: December 2022

The effect of rIL-6 on the growth and differentiation of highly purified human peripheral blood B cells was examined. IL-6 alone induced minimal incorporation of [3H]thymidine by unstimulated or Staphylococcus aureus (SA)-stimulated B cells and did not augment proliferation induced by SA and IL-2. Similarly, IL-6 alone did not support the generation of Ig-secreting cells (ISC) or induce the secretion of Ig by unstimulated or SA-stimulated B cells. However, IL-6 did augment the generation of ISC and the secretion of all isotypes of Ig induced by SA and IL-2. Maximal enhancement of B cell responsiveness by IL-6 required its presence from the initiation of culture. Delaying the addition of IL-6 to B cells stimulated with SA and IL-2 beyond 24 h diminished its effect on ISC generation. However, increased Ig production but not ISC generation was observed when IL-6 was added to B cells that had been preactivated for 48 h with SA and IL-2. This effect was most marked when the activated B cells were also stimulated with IL-2. IL-6 in combination with other cytokines such as IL-1 and IL-4 did not induce the secretion of Ig or generation of ISC in the absence of IL-2. Moreover, antibody to IL-6 did not inhibit the effect of IL-2 on the growth and differentiation of B cells stimulated with SA, but did inhibit the IL-6-induced augmentation of Ig secretion by B cells stimulated with SA and IL-2. IL-6 alone enhanced T cell dependent induction of B cell differentiation stimulated by PWM. Part of this enhancement was related to its capacity to increase the production of IL-2 in these cultures. These results indicate that IL-6 has several direct enhancing effects on the differentiation of B cells, all of which are at least in part dependent on the presence of IL-2. In addition, IL-6 can indirectly increase B cell differentiation by increasing IL-2 production by T cells.
Generation and use of site-specific antibodies to serum amyloid A for probing amyloid A development.
Authors: Miura K , Ju S T , Cohen A S , Shirahama T
Published: December 2022

Serum amyloid A (SAA) is an acute phase reactant and the putative precursor of an amyloid fibril protein (AA). During amyloidogenesis in mice, the C-terminal portion of SAA that consists of 28 amino acids is cleaved off to produce the 75 amino acid AA. Of three known isotypes of SAA, SAA2 has been identified as amyloidogenic. For differential immunologic recognition of SAA and AA, antisera were generated in rabbits against AA protein and a synthetic peptide corresponding to the C-terminal (amino acids 84 to 103) of murine SAA2. The anti-AA antiserum reacted with AA and SAA, but the anti-peptide antiserum reacted with SAA only. Immunohistochemically anti-peptide antiserum predominantly stained the rims of murine amyloid deposits. In contrast, anti-AA antiserum generated an overall homogeneous staining of amyloid deposits. The data lend support to a view that SAA are concentrated in the vicinity of the surrounding cells (mostly macrophages) where they are processed to AA, or that SAA are deposited initially as components of the amyloid fibrils onto the outside margin of amyloid deposits and the C-terminus is cleaved off as maturation of the amyloid fibril progresses.
Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity.
Authors: Marra M N , Wilde C G , Griffith J E , Snable J L , Scott R W
Published: December 2022

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.
Short term exposure to cis unsaturated free fatty acids inhibits degranulation of cytotoxic T lymphocytes.
Authors: Richieri G V , Mescher M F , Kleinfeld A M
Published: December 2022

Degranulation of CTL stimulated by alloantigen-bearing target cells is shown to be inhibited by short term exposure to low concentrations of long chain cis unsaturated free fatty acids (FFA), whereas saturated FFA have no effect. The Ag-specific (TCR mediated) stimulation of cloned murine CTL was monitored by changes in intracellular calcium concentrations [( Ca2+]i) using the fluorescence indicator acetoxymethylester of fura-2 and by degranulation as measured by the release of BLT-esterase. Treatment of the CTL cells with any of the physiologically important FFA; oleic (18:1), linoleic (18:2), linolenic (18:3), or arachidonic (20:4) acid, at concentrations between 1 and 10 microM inhibits the target cell-mediated rise in [Ca2+]i which occurs within seconds of stimulation and the release of BLT-esterase, which occurs over a period of 1 to 3 h. These inhibitory effects are observed within seconds to minutes of FFA addition. Inhibition can be reversed by treating cells with fatty acid free BSA and, in agreement with our previous studies, indicates that the effects of FFA are due to physical perturbations of cellular components. To determine the locus of this perturbation, the effect of FFA on the lipid order of CTL plasma membrane was determined using fluorescence polarization of the membrane impermeable probe trimethylammoniumdiphenylhexatriene. Cis unsaturated FFA were found to disorder the lipid acyl chains and the degree of disorder was found to increase with the degree of cis unsaturation. These results, together with the previous studies, suggest that inhibition results from a physical perturbation of plasma membrane lipid order. Moreover, because degranulation requires elevated levels of [Ca2+]i, it is likely that inhibition of degranulation results from a FFA-induced decrease in Ca2+ permeability through the membrane.
Natural antibodies to IFN-gamma in man and their increase during viral infection.
Authors: Caruso A , Bonfanti C , Colombrita D , De Francesco M , De Rango C , Foresti I , Gargiulo F , Gonzales R , Gribaudo G , Landolfo S
Published: December 2022

Natural antibodies to IFN-gamma were found in healthy individuals ranging from newborn babies to adults and, at higher levels, in patients suffering from different viral infections. During a viral infection, the titer of anti-IFN-gamma antibodies was observed to be correlated with the stage of the disease. Antibodies specific to IFN-gamma were affinity purified both from sera taken from healthy individuals and sera from viral-infected patients, by using a rIFN-gamma-coupled CNBr-activated Sepharose 4B column. The antibodies were found to be of the IgG class, and maintained their ability to bind rIFN-gamma. They were then tested for neutralizing activity and none of the IgG preparations we analyzed impaired the antiviral activity of rIFN-gamma. This finding suggests that the antigenic determinants recognized by these antibodies on the IFN-gamma molecule are located outside the site, on the IFN-gamma molecule, responsible for its antiviral activity.
Depletion of CD8+ T cells increases susceptibility and reverses vaccine-induced immunity in mice infected with Trypanosoma cruzi.
Authors: Tarleton R L
Published: December 2022

The role of CD8+ T cells in immune control of Trypanosoma cruzi infection in mice was examined by using in vivo depletion of CD8+ T cells with antibodies. Both the resistant C57BL/6J and the highly susceptible C3H/HeSnJ developed higher parasitemias and increased or earlier mortality when depleted of CD8+ T cells before infection with the Brazil strain of T. cruzi. CD8 depletion also affected the induction and expression of immunity to T. cruzi after vaccination with the avirulent Corpus Christi strain of T. cruzi. C57BL/6J mice depleted of CD8+ T cells either before or after vaccination showed a near total loss of vaccine induced protection. C3H mice were only partially protected by the vaccination procedure but the protection was totally reversed by anti-CD8 treatment. Chronically infected mice that had survived the acute infection were unaffected by CD8 depletion and showed neither a recrudescence of parasitemia nor an increased susceptibility to reinfection. These results suggest that CD8+ T cells play a role in immunity to T. cruzi in the acute phase but possibly not during the chronic phase of infection. Also, immunization with Corpus Christi strain T. cruzi induces an immunity that is distinct from that induced by survival of the acute phase of infection. The mechanism by which CD8+ T cells contribute to control of T. cruzi infection is not known. However, CD8 depletion had no effect on suppressed immune responses, suggesting their function in T. cruzi-infected mice is related more to the cytotoxic activity or cytokine-producing capacity of this subpopulation of T cells.
A membrane-associated form of C-reactive protein is the galactose-specific particle receptor on rat liver macrophages.
Authors: Kempka G , Roos P H , Kolb-Bachofen V
Published: December 2022

Rat liver macrophages express a galactose-specific receptor which mediates endocytosis of particles or neuraminidase-treated blood cells. From rat serum we now have isolated and purified a galactose-specific lectin by affinity chromatography. Comparative analysis of this serum galactose-binding protein with the galactose-particle receptor protein purified from rat liver macrophages and with C-reactive protein (CRP) reveals close relation or identity of these proteins. An apparent m.w. of 30,000 was determined for all three proteins by SDS-PAGE under reducing conditions and m.w. of about 130,000 by native PAGE. All three proteins exhibit the same pentameric, ring-shaped structure in electron microscopy after negative staining. Antibodies raised against the serum galactose-binding protein or against the macrophage receptor cross-react. A mAb specific for rat neo-CRP labels liver macrophages but not hepatocytes and reacts with the isolated protein in a Western blot assay. Furthermore, the galactose-particle receptor can be functionally replaced by purified CRP: the binding capacity for neuraminidase-treated E of receptor-depleted liver macrophages can be restored by preincubation with purified rat CRP. We therefore conclude that CRP occurs as a membrane-associated protein constitutively expressed on liver macrophages functioning as a receptor mediating galactose-specific binding of particulate ligands.
Protection against Leishmania major in BALB/c mice by adoptive transfer of a T cell clone recognizing a low molecular weight antigen released by promastigotes.
Authors: Scott P , Caspar P , Sher A
Published: December 2022

We have shown previously that BALB/c mice can be protected against a fatal infection with Leishmania major by adoptive transfer of a T cell line recognizing a protective soluble fraction (fraction 9) of promastigotes. We now describe the isolation and characterization of a T cell clone (9.1-2) that also transfers protective immunity against Leishmania. After Ag or mitogen stimulation, this clone secrets IL-2 and IFN-gamma, but not IL-4 or IL-5. The clone preferentially recognizes L. major fraction 9, and in addition, soluble Ag from Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis, but not from the related flagellates, Trypanosoma cruzi or Crithidia fasciculata. Besides being contained in fraction 9, the stimulatory Ag is also released from the parasite, because concentrated promastigote culture supernatants induced IFN-gamma production by 9.1-2. By means of T cell immunoblotting, we determined that 9.1-2 recognized a protein with a relative m.w. between 8,000 and 12,000, and within this region is a predominant protein contained in fraction 9 of approximately 10,000 m.w. These data identify a new candidate Ag for immunization against protozoa belonging to the genus Leishmania.
Antigen recognition by T cells. Quantitative effects of augmentation by antibodies providing accessory interactions.
Authors: Kane K P , Mescher M F
Published: December 2022

Although engagement of the TCR via antibody can be sufficient to trigger T cells, responses to Ag-bearing cells require additional "accessory" interactions in many cases. A method has been developed which allows preparation of surfaces bearing both purified class I alloantigen and coimmobilized antibodies. With this approach, it is possible to mimic such "accessory" interactions and to examine their quantitative effects on triggering via TCR-Ag interaction. Experiments are described which use this approach to examine triggering of degranulation by cloned, allogeneic CTL lines. Coimmobilization of antibodies specific for any of a variety of CTL surface proteins, including CD8, class I MHC proteins, CD45 (T200) and Thy-1, had the effect of decreasing the critical threshold density of Ag necessary to trigger responses, and decreasing by an order of magnitude the density required to stimulate a half-maximal response. Furthermore, in comparison with the 30-min lag seen with Ag alone, response was initiated immediately when an antibody specific for a CTL surface component was present. These results are consistent with the hypothesis that any CTL surface molecule having sufficient affinity for a component of the target surface can contribute to activation via the Ag-specific TCR; and at low Ag density could determine whether any response occurs.
Role of phosphatidylinositol-anchored proteins in T cell activation.
Authors: Presky D H , Low M G , Shevach E M
Published: December 2022

A novel class of cell surface proteins are attached to the plasma membrane via a phosphatidylinositol (PI)-glycan anchoring structure, and these proteins can be selectively removed from the cell surface by the enzyme PI-specific phospholipase C (PI-PLC). Enzyme treatment led to a prolonged reduction in cell surface expression of several PI-anchored proteins. Activation of T cells led to a marked decrease in the ability of PI-PLC to remove PI-anchored surface proteins from the activated T cells. This decrease in PI-PLC sensitivity may reflect an alteration in the PI-glycan anchoring structures, or in a general membrane property, which renders the PI-anchored proteins inaccessible to the enzyme. When murine T lymphocytes were treated with PI-PLC and then stimulated with either Con A, the calcium ionophore A23187 and PMA, or an anti-CD3 mAb, the response to Con A stimulation was inhibited by 90%, whereas the responses to ionophore and PMA or anti-CD3 were not affected. Removal of PI-anchored proteins inhibited an early event in the activation process in response to Con A because both IL-2 production and IL-2R expression were inhibited by the PI-PLC treatment. Inhibition of the Con A response was secondary to removal of a PI-linked protein from the responder T cell population because PI-PLC treatment of T-depleted spleen cells did not alter their ability to act as a source of accessory cells. It is unlikely that removal of the known PI-linked proteins on murine T cells, Thy-1 and Ly-6, can fully account for the inhibition of Con A response because the cell line M2B3, that lacks these surface proteins, responded normally to Con A stimulation. These studies demonstrate that one or more PI-anchored T cell proteins play an important role in an early step of Con A activation, perhaps involving T cell-accessory cell interactions. In contrast, the ability to stimulate T cells by direct cross-linking of TCR/CD3 complex is not dependent on the presence of these PI-anchored proteins.
Antigen-specific sIalo B cells do not secrete IgG2a in response to TH1 cells and antigen: responsiveness can be restored by IL-4.
Authors: Stevens T L , Vitetta E S
Published: December 2022

The ability of Th cells, type 1 (TH1), to activate and induce differentiation of B cells into antibody-secreting cells is controversial because 1) some clones of TH1 cells provide help while others do not, and 2) by using the same TH1 clone, different laboratories disagree on whether they provide help to B cells. One possible explanation for the latter is the variability in the activation status of the B cells used in different laboratories. In the present studies, we have used Ag-specific B cells from athymic (nu/nu) mice, or sterilely housed nu/+ mice to study the TH1-mediated activation of B cells that had received little or no prior help from T cells and/or antigen in vivo. These B cells express low levels of surface Ia (sIa) Ag, and fail to secrete IgG2a in response to TH1 cells plus Ag; in contrast, responses to TH2 cells plus Ag are normal. To explore this observation further, we prepared "surface(s) Ia1o" B cells from conventionally housed BALB/c mice by sorting spleen cells on the fluorescence-activated cell sorter. This sIalo population also failed to produce IgG2a in response to TH1 cells plus Ag. In contrast, the sIahi, (presumably more mature) B cells, responded to both the TH1 and TH2 cells. The addition of LPS, TH2 cells or the lymphokine, IL-4, to cultures of sIalo B cells from normal or nu/nu mice (plus Ag and TH1 cells), restored IgG2a responses to control levels. Low sIa levels were not the sole cause of nonresponsiveness of the nu/nu B cells because a 24-h pulse with IL-4 restored sIa to control levels without restoring IgG2a production after activation with TH1 cells plus Ag. These data support the conclusion that sIalo B cells are immature and require an activation/maturation signal from IL-4 in vivo in order to respond to TH1 cells and Ag in vitro.
Murine Thy-1+ dendritic epidermal cells induce immunologic tolerance in vivo.
Authors: Welsh E A , Kripke M L
Published: December 2022

To study the potential immunologic functions of murine Thy-1+ dendritic epidermal cells (Tdec), we used long term, IL-2-dependent, Tdec lines derived from the skin of C3H mice. Cells of the Tdec lines AU16 or AU4 were conjugated in vitro with the hapten FITC and injected s.c. into the footpad (ifp.) or i.v. into syngeneic recipient mice to assess their ability to induce or inhibit contact hypersensitivity (CHS). FITC-conjugated Tdec were unable to induce CHS after ifp. or i.v. injection. Furthermore, when mice were sensitized epicutaneously with FITC after the injection of FITC-conjugated Tdec, they were unable to mount a CHS response. These results indicated that hapten-conjugated Tdec are capable of inducing tolerance upon ifp. or i.v. injection. In contrast, ifp. injection of normal lymphocytes conjugated with FITC in vitro produced no impairment of the CHS response to FITC, although i.v. injection of these cells, as with that of FITC-conjugated Tdec, induced down-regulation of CHS. The immunologic tolerance induced by FITC-conjugated Tdec was Ag specific, but not H-2 restricted. Immunization with unconjugated Tdec either ifp. or i.v. did not induce tolerance, indicating that unconjugated Tdec by themselves are not suppressive. In addition, Tdec were unable to mediate a local graft-vs-host reaction in an F1 host. These results demonstrate that Tdec can induce immunologic tolerance and suggest that these cells may play a role in the down-regulation of cutaneous immune responses.
Elucidation of discontinuous linear determinants in peptides.
Authors: Appel J R , Pinilla C , Niman H , Houghten R
Published: December 2022

Synthetic peptides, made by the method of simultaneous multiple peptide synthesis, were coupled to the protein carrier keyhole limpet hemocyanin and used to raise mAb. Omission and substitution analogs of the original peptides were tested by ELISA to characterize their reactivity with the respective mAb. Linear antigenic determinants were located for 18 different peptides by using omission analogs. The length of the antigenic determinants ranged from 2 to 8 residues, with an average of 6 residues. The three aromatic amino acids, phenylalanine, tryptophan, and tyrosine, the charged hydrophilic amino acids, aspartic acid and lysine, and the neutral amino acid alanine were found to occur most often in the determinant region of the peptides tested, whereas asparagine, cysteine, and histidine occurred the least often. Alanine substitution analogs provided more information than omission analogs by enabling the determination of which side chain groups of the antigenic determinant residues were not critical for binding to the mAb. Detailed, "fingerprint" information about the interaction of the peptide, GASPYPNLSNQQT, and its mAb was obtained by synthesizing a complete series of analogs with individual substitutions for each position of the antigenic determinant, PYPNLS, with the 19 other amino acids. These results suggest that, at the amino acid level, all antigenic determinants of synthetic peptides defined by mAb can be considered discontinuous linear determinants.
Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone.
Authors: Liu C C , Joag S V , Kwon B S , Young J D
Published: December 2022

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.
"CD3low" human thymocyte populations can readily be triggered via the CD2 and/or CD28 activation pathways whereas the CD3 pathway remains nonfunctional.
Authors: Pierres A , Cerdan C , Lopez M , Mawas C , Olive D
Published: December 2022

We have investigated the role of the CD2 and the CD28 Ag-independent pathways of activation on CD3low thymocytes. We previously showed that anti-CD28 mAb synergized with anti-CD2 mAb directed against epitopes T11.1 and T11.2, in the activation of purified resting T cells or unseparated thymocytes. Proliferation induced via CD2 plus CD28 was mediated via an IL-2-dependent pathway and was not affected by prior modulation of the CD3-TCR complex. Here, we show that a subset of CD3low thymocytes, although unresponsive to CD3 activation, can be activated to proliferate through the CD2 or the CD28 pathways, in the presence of exogenous IL-2. The mitogenic combination of mAb to CD2 and CD28 induces a proliferation of thymocytes which, in absence of exogenous lymphokines, is restricted to the more mature intrathymic subpopulation, CD1a-. However, CD3low thymocytes can also be triggered through the CD2 plus CD28 activation pathways but require at least addition of exogenous IL-2 to proliferate. This study demonstrates that a fraction of immature CD3low thymocytes possesses functional CD2 and CD28 surface molecules at a time when CD3 is not yet functional.
Anti-CD3 antibody induces unresponsiveness to IL-2 in Th1 clones but not in Th2 clones.
Authors: Williams M E , Lichtman A H , Abbas A K
Published: December 2022

Culture of murine T cells with immobilized (platebound) anti-CD3 antibody results in autocrine growth factor secretion in both Th1 (IL-2 producing) and Th2 (IL-4 producing) cells. Using a panel of murine T cell clones, we demonstrate that the IL-2-induced proliferation of Th1 clones is dramatically inhibited by immobilized anti-CD3 antibody, whereas that of Th2 clones is not. This unresponsiveness of Th1 clones to IL-2 is not due to decreases in IL-2R expression. Supernatants from Th1 or Th2 cell cultures fail to alter the effects of anti-CD3 on the two types of clones, suggesting that unresponsiveness induced in Th1 clones or the lack thereof in Th2 clones is not mediated by a stable cytokine(s). Accessory cells enhance the proliferation of Th1 cells exposed to low concentrations of anti-CD3, but the unresponsiveness induced by high concentrations of anti-CD3 is not prevented by accessory cells. Finally, soluble anti-CD4 antibody prevents the induction of the unresponsive state even at high concentrations of anti-CD3. These experiments demonstrate that two subsets of cloned CD4+ T cells differ in their responses to anti-CD3, and that CD4 molecules may play a critical role in regulating the outcome of receptor-mediated stimulation.
Human polysaccharide-specific B cells are responsive to pokeweed mitogen and IL-6.
Authors: Ambrosino D M , Delaney N R , Shamberger R C
Published: December 2022

The responsiveness of polysaccharide-specific B cells to PWM was examined in vitro. Spleen cells from six patients immunized with Haemophilus influenzae type b-diphtheria toxoid, pneumococcal and meningococcal vaccines were T cell-depleted and separated by Percoll density gradient centrifugation. In each B cell fraction, spontaneous antibody production was demonstrated to capsular polysaccharides as well as diphtheria toxoid. The peak of spontaneous antibody production was demonstrated to be five to seven days after immunization. When T cells and PWM were added, the total Ig secretion increased in all B cell fractions. PWM also enhanced IgG antibody directed to each of three polysaccharide Ag measured. This enhancement was most noticeable for nonresting B cells. The PWM effect was not confined to IgG, as IgM and IgA to Neisseria meningitidis type C were measured and also enhanced. The kinetics of the PWM response demonstrated the most IgG antibody to polysaccharide Ag from spleens immunized five to seven days before splenectomy. When the patients were immunized either 2 days or 4 mo before splenectomy, no spontaneous IgG antibody to polysaccharides was detected although PWM induced small amounts of antibody. Finally, anti-IL-6 antibody blocked PWM-induced total and polysaccharide-specific antibody production. We conclude that human polysaccharide-specific B cells are responsive to PWM and IL-6. We suggest that polysaccharide B cells are not truly "T cell-independent" and may respond to T cell lymphokines and thus are similar to protein-specific B cells.
T cells in inductive and effector compartments of the intestinal mucosal immune system of nonhuman primates differ in lymphokine mRNA expression, lymphokine utilization, and regulatory function.
Authors: James S P , Kwan W C , Sneller M C
Published: December 2022

To define further the basis of T cell function in the inductive and effector limbs of the normal intestinal immune system, the capacity of mucosal lymphocytes to produce and use lymphokines and their effects on regulation of Ig production were determined in normal nonhuman primates. Northern blots of RNA from mitogen-activated lamina propria T cells contained more mRNA for IL-2 and IFN-gamma than did mesenteric lymph node T cells. In comparison with lymphocytes from peripheral sites, there was high expression of IL-4 and IL-5 mRNA in both mesenteric lymph node and lamina propria T cells. In studies of lymphokine utilization, T cells from lamina propria had high IL-2-induced but no IL-4-induced proliferative responses. In contrast, mesenteric lymph node T cells had high IL-4-induced and lower IL-2-induced proliferative responses compared with lamina propria T cells. Lamina propria T cells had higher helper activity in PWM-stimulated cultures and exhibited less inhibition by IL-4 than did mesenteric lymph node T cells. These data and previous studies suggest that T cells in an inductive site such as the mesenteric lymph node are a mixed population containing both "naive" cells with low potential for IFN-gamma and IL-2 production and differentiated cells with high potential for IL-4 and IL-5 production. In contrast, the data suggest that T cells in the effector compartment of the lamina propria are comprised primarily of differentiated "memory" cells that produce high levels of IL-2, IFN-gamma, IL-4, and IL-5, have high helper activity, and have a more limited ability to proliferate in response to lymphokines such as IL-4.
Detection by in situ hybridization and phenotypic characterization of cells expressing IL-6 mRNA in human stimulated blood.
Authors: Kato K , Yokoi T , Takano N , Kanegane H , Yachie A , Miyawaki T , Taniguchi N
Published: December 2022

IL-6 has manifold biologic functions in immune and inflammatory responses and is produced by a variety type of cells. In this work, we used the whole blood culture to identify the cells expressing IL-6 gene/protein after various stimulation. When the whole blood was incubated with LPS or Con A, much IL-6 activity, measured by the growth promoting assay using a murine IL-6-dependent hybridoma clone, was detected in the plasma as early as 4 h of culture and continued to increase with time, reaching a plateau around 12 h. Immunocytochemical analysis with anti-rIL-6 antiserum revealed that a proportion of mononuclear cells (MNC) contained intracytoplasmic IL-6 in LPS- or Con A-stimulated blood. Northern blot analysis for MNC from the blood stimulated with these stimuli showed that their transcripts for IL-6 peaked at 4 h, then rapidly declined and was undetectable after 24 h of stimulation. In situ hybridization technique with radiolabeled antisense RNA probe for IL-6 demonstrated that a fraction of MNC from LPS- as well as Con A-stimulated blood expressed IL-6 mRNA. With the combined use of in situ hybridization and immunofluorescence by corresponding mAb, it was confirmed that IL-6 mRNA expressing cells in stimulated blood were exclusively monocytes. In the whole blood culture, it was shown that expression of IL-6 mRNA by monocytes was inhibited by dexamethasone, but not by cyclosporin A. These results suggest that monocytes are the major cells expressing IL-6 gene/protein in the circulation after exposure to external stimuli.
Immunogenic Ia-binding peptides immobilize the Ia molecule and facilitate its aggregation on the B cell membrane. Control by the M1s-1 gene.
Authors: Mecheri S , Edidin M , Dannecker G , Mittler R S , Hoffmann M K
Published: December 2022

Aggregation (e.g., through cross-linkage) of cell surface molecules is in various biologic systems a necessary event in cellular activation. Examining the Ia molecule on B cells we found that aggregation is a function of the surface Ag mobility; the higher the fraction of immobile molecules on the plane of the membrane, the better Ia forms aggregates and patches. We identify two factors that control Ia mobility and aggregability. One factor is the M1s-1a gene product; the other factor is an Ia-reactive immunogenic peptide. Both factors increase Ia aggregability and reduce the MHC Ag mobility.
Loss of a major idiotype (CRIA) after repopulation of irradiated mice.
Authors: Willems F , Vansanten-Urbain G , De Wit D , Slaoui M , Urbain J
Published: December 2022

The normal immune response of A/J mice against arsonate coupled to hemocyanin is characterized by a major recurrent cross-reactive Id, the CRIA. This Id is encoded by a single gene segment combination: VHidcr11-DFL16.1e-JH2 for the H chain and Vkidcr-Jk1 for the L chain. In this report, we show that lethal irradiation of A/J mice followed by reconstitution with autologous or syngeneic lymphoid cells results in loss of major CRIA Id expression in the response to arsonate. Different protocols were performed to repopulate the irradiated mice. First, lethally irradiated A/J mice were reconstituted by the transfer of syngeneic bone marrow cells. Second, A/J mice were lethally irradiated while their hind limbs were partially shielded. Third, lethally irradiated A/J mice received a transfer of syngeneic spleen cells. The three groups of mice produce high titers of antiarsonate antibodies completely devoid of CRIA DH-JH related idiotopes expression. Moreover, a lack of affinity maturation is observed in the secondary antiarsonate response of all irradiated and reconstituted mice. A transfer of syngeneic peritoneal cells or a transfer of primed T cells in irradiated and reconstituted A/J mice do not restore in a significant manner either the recurrent CRIA expression or the affinity maturation of the antiarsonate response. Our data suggest that the choice of this Id is not solely dictated by the Igh locus.
The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells.
Authors: Dumont F J , Melino M R , Staruch M J , Koprak S L , Fischer P A , Sigal N H
Published: December 2022

The structurally related immunosuppressive macrolides FK-506 and rapamycin (RAP) were previously shown to inhibit T cell stimulation through different mechanisms. FK-506 acts similarly to cyclosporin A (CsA) and prevents IL-2 production and IL-2R expression. RAP has little or no effect on these events but markedly impedes the response to IL-2. The present study was initiated to examine the possibility of a complementation between the immunosuppressive actions of RAP and FK-506 or CsA on various murine T cell responses. RAP potentiated the effect of CsA on proliferation and IL-2R expression in T cells stimulated with ionomycin + PMA. However, in the same system, RAP acted as a potent antagonist of FK-506 suppression. RAP also blocked FK-506- but not CsA-mediated inhibition of IL-2 mRNA induction. By using model systems sensitive to inhibition by RAP but not FK-506 we further demonstrated that FK-506 reciprocally behaves as an antagonist of RAP. In one such model, the stimulation of splenic T cells with IL-2 + PMA, FK-506, but not CsA, reversed the suppressive effect of RAP on proliferation. FK-506 also antagonized RAP-mediated inhibition with respect to the induction of Ly-6E Ag expression by IFN in YAC cells. To explore further the competition between the two macrolides at the cellular level, we performed binding experiments with a radiolabeled derivative of FK-506. Both FK-506 and RAP, but not CsA, inhibited the binding of this probe in YAC cells. Taken together, these data demonstrate that FK-506 and RAP antagonize each other's biologic activity and physically interact with a common receptor site(s) in T cells. Moreover, CsA acts at a site distinct from the cellular target(s) of FK-506 or RAP.
Phosphatidylinositol-linked FcRIII mediates exocytosis of neutrophil granule proteins, but does not mediate initiation of the respiratory burst.
Authors: Huizinga T W , Dolman K M , van der Linden N J , Kleijer M , Nuijens J H , von dem Borne A E , Roos D
Published: December 2022

In this report, we present data on the activation of different neutrophil effector functions by two distinct Fc-gamma receptors, FcRII and FcRIII. We and others have shown previously that IgG-dependent activation of phagocytosis and superoxide generation is mediated via FcRII. IgG-dependent exocytosis of granule proteins was assessed with Staphylococcus aureus Oxford opsonized with human IgG or with IgG-coated latex. Both anti-FcRII mAb and anti-FcRIII-F(ab')2 mAb inhibited this release, whereas the combination of these mAb inhibited this process more strongly than either mAb alone. This indicates that both FcRII and FcRIII are involved in IgG-dependent release of granule proteins. Cross-linking of the receptors by anti-FcR mAb and F(ab')2 fragments of goat-anti-mouse-Ig showed again that both FcRII and FcRIII mediate lysozyme release, whereas cross-linking of a control antigen (CD67) did not. By measuring the release of elastase and lactoferrin, we found that cross-linking of either FcRII or FcRIII induced release of both azurophilic and specific granules. Under these conditions, we did not measure any activation of the respiratory burst. When FcRIII was removed by treatment of neutrophils with glycosylphosphatidylinositol-specific phospholipase C, the lysozyme release induced by cross-linking of FcRIII was lower than the release from control neutrophils, whereas the release induced by cross-linking of FcRII was similar. Therefore, we conclude that IgG-dependent activation of neutrophils follows two distinct pathways: one via transmembrane FcRII, activating both the NADPH oxidase and the release of granule proteins (as was demonstrated previously by us and by others), and the other via phosphatidylinositol-linked FcRIII, activating exocytosis of granule proteins.