bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies to horse cytochrome c expressing four distinct idiotypes distribute among two sites on the native protein.
Authors: Carbone F R , Paterson Y
Published: December 2022

Antibody-secreting hybridoma cell lines produced from BALB/c mice that had been immunized with horse cytochrome c (cyt c) conjugated to hemocyanin yielded six hybridoma subclones that produced four monoclonal antibodies (mAb) with different patterns of cross-reactivity with a panel of evolutionarily variant cyt c. The recognition sites for three of these mAb lay in the same region of the intact molecule, because two of the mAb were sensitive to the amino acid residue present at sequence position 44, with one requiring threonine at position 47. The fourth mAb bound in another region of the molecule at a site that involves either residue 60 or residue 89. Synthetic peptides that included these residues did not react with these mAb, indicating that these sites may require interactions from noncontiguous regions of the molecule to bind antibody. The association constants for the interaction of the mAb with horse cyt c were very similar and of the order of 10(10) M-1. Specificity studies with anti-idiotypic sera and competition assays between mAb for binding to horse cyt c confirmed that the six positive hybridoma subclones produced from this fusion produced mAb that had one of these four distinct specificities. The idiotypes of these four mAb were serologically distinct, and were derived from Vh genes of the J558 family.
Lipid-induced recognition of a conformational determinant (residues 65 to 83) in myelin basic protein.
Authors: Boggs J M , Hashim G A , Day E D , Moscarello M A
Published: December 2022

The precipitation by antibodies to intact myelin basic protein (BP) and to synthetic peptides containing a sequence based on the region 65 to 83 of bovine BP, S82, S81, S79, and S24, of intact BP in solution or bound to lipid vesicles was compared, using 125I-BP or 14C-DPPC-labeled lipid-BP vesicles. The antipeptide antibodies were shown earlier to recognize conformational determinants which are not expressed in the intact protein in solution. Several anti-BP antibodies precipitated more of the BP free in solution than when bound to lipid vesicles, suggesting that some of the determinants recognized by these antibodies were either sequestered in the bilayer or were altered in conformation. In contrast, one anti-peptide antisera, which had a high titer for the conformational determinant in two of these peptides, S82 and S81, precipitated the protein to a significant degree when it was bound to PG vesicles, even though it did not react with the intact protein in solution. These results indicated that PG was able to confer on the protein the unique peptide conformation recognized by this antibody. PS was less effective, and other lipids were ineffective at conferring this conformation on the protein, supporting earlier results which showed that the conformation of the protein is influenced by the lipid composition of its environment. None of the other anti-peptide antibodies studied bound to the protein either in solution or in lipid vesicles. These results indicate that the lipid environment can sequester or alter the conformation of some antigenic determinants, preventing recognition by some anti-BP antibodies, and can expose or generate other conformational determinants, allowing recognition by an anti-peptide antiserum.
Ia molecular localization of the DRw52 allodeterminant and the DRw52-like determinants defined by monoclonal antibodies.
Authors: Karr R W , Lipman L K , Alber C , Koning F , Goeken N E
Published: December 2022

DRw52 (formerly MT2) is a human Ia alloantigen that is expressed in linkage disequilibrium with DR3, 5, w6, and w8. Although there is general agreement that the DRw52 determinant resides on biochemically defined DR molecules, conflicting evidence exists regarding whether DRw52 resides on one or both DR molecules, DQ and DR molecules, or DR and BR molecules. Six anti-DRw52 allosera and three DRw52-like monoclonal antibodies were used to identify the Ia molecules that bear the DRw52 and DRw52-like determinants from DR5 and DRw6 homozygous cells. Based on these two-dimensional gel studies, the DRw52 allodeterminant appears to reside on a subset of DR molecules from DR5 and DRw6 cells. In contrast, the determinants defined by the three anti-DRw52-like monoclonal antibodies were found to reside on one DR molecule, on the second DR molecule, or on both DR molecules, respectively. Therefore, there is considerable complexity of Ia antigenic determinants that are associated with DR3, 5, w6, and w8 at the population level.
Complement receptor type three-dependent degradation of opsonized erythrocytes by mouse macrophages.
Authors: Rothlein R , Springer T A
Published: December 2022

The role of the complement receptor type 3 (CR3) on thioglycollate-elicited peritoneal macrophages (TG-PM) in the destruction of opsonized particles was studied. We found that sheep red blood cells (E) that were opsonized with an IgM monoclonal anti-Forssman antibody and complement (E-IgM-C) were lysed by TG-PM, whereas there was little lysis of E pretreated with either the antibody or the complement source alone. Furthermore, this lysis could be inhibited by anti-CR3 monoclonal antibodies that had previously been shown to inhibit binding of E-IgM-C to the CR3. Kinetic studies of phagocytosis and lysis indicated that lysis of E-IgM-C occurs after phagocytosis, suggesting that lysis is an intracellular event. Further findings suggested that intra-cellular lysis was promoted by CR3 bound to the phagocytosed target, because a monoclonal anti-CR3 antibody decreased the rate of phagocytosis of E-IgM-C but not its magnitude, whereas the rate and extent of lysis were strikingly inhibited. Furthermore, TG-PM that had already internalized unopsonized E selectively lysed E-IgM-C that were added later. These data confirm that the interaction of the CR3 with its ligand on E-IgM-C promotes rapid phagocytosis, and further suggest that the CR3 facilitates degradation of the target particle once internalization has occurred.
Endogenous somatostatin-like peptides of rat basophilic leukemia cells.
Authors: Goetzl E J , Chernov-Rogan T , Cooke M P , Renold F , Payan D G
Published: December 2022

The tetradecapeptide somatostatin (SOM 14) and a 28-amino acid biosynthetic precursor (SOM 28) are constituents of diverse neuroendocrine tissues that are released by noxious stimuli from a subset of sensory nerve endings, and substantially modify the functions of basophils and mast cells. SOM-like factors were detected initially in the fluid phase of suspensions of immunologically challenged rat basophilic leukemia cells (RBL), and were purified from ethanol/0.2 M acetic acid (3/1, v/v) extracts of replicate portions of 3 X 10(9) RBL. Sephadex G-25 columns resolved factors of over 10,000, 2000 to 4000, and 300 to 1200 daltons that are antigenically related to SOM 14, as assessed by a radioimmunoassay specific for SOM 14. Only the two larger factors were detected by a radioimmunoassay for SOM 28(1-14), which binds to prepro-SOM and SOM 28 but not SOM 14. Reverse-phase high performance liquid chromatography distinguished the two smaller SOM peptides of RBL from SOM 28 and SOM 14, respectively. Amino acid analyses showed major differences in composition between the 2000 to 4000 dalton SOM of RBL and SOM 28. Picomolar to nanomolar concentrations of both of the smaller SOM peptides of RBL inhibited the IgE-dependent release of histamine from basophils to the same extent as SOM 14. The finding of 3 to 5 ng of structurally unique SOM-like peptides per 10(8) RBL suggests that endogenous mechanisms analogous to those of specialized sensory neurons may regulate the expression of hypersensitivity.
The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation.
Authors: Hamilos D L , Wedner H J
Published: December 2022

The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.
In vitro parasite antigen-induced antibody responses in human helminth infections.
Authors: Nutman T B , Withers A S , Ottesen E A
Published: December 2022

Helminth parasites characteristically induce vigorous antibody responses in human infections, but the immunoregulatory mechanisms determining the level of these responses are not at all understood. To investigate these mechanisms, peripheral blood mononuclear cells were obtained from 10 patients with parasitic helminth infections (three with schistosomiasis, three with onchocerciasis, and four with loiasis), along with six normal controls. These cells were then cultured in vitro and the capacity of the cells to respond to a specific parasite antigenic stimulus was examined by measuring the amount of parasite-specific antibody produced. Parasite antigen alone, without exogenous mitogen, induced an IgG anti-parasite antibody response in vitro. Optimal responses were obtained at extremely low antigen concentrations--concentrations at which little if any polyclonal immunoglobulin production occurred. Additionally, the in vitro induction of parasite-specific antibody was antigen dose-dependent, requiring much lower antigen concentrations than those necessary to induce lymphocyte blastogenesis. Antibody production was shown to require the cooperative interaction of B and T cells. These studies demonstrate that in vitro responses to antigens from naturally acquired parasitic infections, like those in individuals postimmunization, can be utilized to dissect the cellular and humoral factors that regulate antibody production to naturally acquired human pathogens.
Stimulation of a cell-mediated cytotoxic response to FeLV-induced T cell lymphomas in the cat.
Authors: Tompkins M B , Tompkins W A
Published: December 2022

Nonspecific cell-mediated cytotoxicity was examined in the peripheral blood and spleens of normal and vaccinia virus-infected adult domestic cats. Natural cytotoxic (NC)-like cells, as measured by lysis of vaccinia- or HSV-infected, adherent cat tongue cells, were found in both the spleen and peripheral blood of normal, nonimmune cats. Cytotoxicity was expressed in a 16-hr assay but not in a 4-hr assay. Natural killer (NK)-like cells, as measured by lysis of an FeLV-induced lymphoid tumor cell line (FL-74) growing in suspension, were found in the spleen but not PBL, and required a 16-hr assay for expression. Infection with vaccinia virus did not increase the activity of feline NC-like cells in either the peripheral blood or the spleen. NK-like function, however, was increased. Cytotoxicity peaked 6 days post-infection and required a 16-hr assay for maximal expression of cell lysis. Furthermore, a cell with cytotoxic characteristics of the spleen NK-like cell appeared at low levels in the circulation at 6 days post-vaccinia infection. NK-like cells from vaccinia-infected cats showed some cytotoxicity for FL74 targets in a 4-hr assay. The cat thus possesses at least two functionally different populations of naturally cytotoxic cells. NC-like cells are found in the spleen and peripheral blood, lyse virus-infected monolayer targets, and are not activated by infection. NK-like cells are found in the spleen, lyse-lymphoid tumor targets, and can be activated by infection, with their peak activity occurring 6 days after infection.
Orally induced tolerance generates an efferently acting suppressor T cell and an acceptor T cell that together down-regulate contact sensitivity.
Authors: Gautam S C , Battisto J R
Published: December 2022

Intragastric administration of the hapten trinitrochlorobenzene (TNCB) suppresses development of contact sensitivity (CS) to attempted epicutaneous sensitization with TNCB. Suppression induced by feeding TNCB is hapten specific and can be transferred to normal animals with lymphoid cells from fed mice. The lymphoid cells in hapten-fed mice that cause suppression of CS have been identified as Thy-1.2-positive cells in spleen and mesenteric nodes. The suppression with Peyer's patch cells from hapten-fed mice appears to be attributable to cells bearing Thy-1.2 antigen (T cell) and to cells with surface Ig (B cell). Feeding TNCB induces an efferent-acting suppressor T cell (Ts eff), as well as an intermediary acceptor T cell (T acc) with which it interacts to block adoptive transfer of CS with immune cells. Ts eff emanating from hapten-fed mice was identified by its specificity for the hapten, insensitivity to pretreatment with cyclophosphamide (CY), ability to produce soluble suppressor factor (SSF), and requirement for T acc to be functional. The presence of T acc in hapten-fed mice, on the other hand, was confirmed by its sensitivity to treatment with CY, interaction with Ts eff or SSF, and the ability to produce nonspecific inhibitor of TDTH cells. Thus, the suppressor T cells that are induced by administering the hapten intragastrically appear to function much like the cells of the suppressor T cell cascade that are induced by giving hapten via parenteral routes.
Two subpopulations of stem cells for T cell lineage.
Authors: Katsura Y , Amagai T , Kina T , Sado T , Nishikawa S
Published: December 2022

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.
Interactions and quantitative analysis of immunoregulatory cells in the chicken thymus.
Authors: Boyd R L , Hàla K , Wick G
Published: December 2022

Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions. The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells. The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM. When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high. When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter. Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus. With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses. Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed. The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL. If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement. The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells. An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.
Differences in antibody repertoires for (4-hydroxy-3-nitrophenyl)acetyl (NP) in splenic vs immature bone marrow precursor cells.
Authors: Riley R L , Klinman N R
Published: December 2022

To evaluate the contribution of environmental regulatory mechanisms in fashioning the primary B cell repertoire, we have compared the repertoire of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific primary splenic B cells with that of precursor cells present as surface immunoglobulin-negative (sIg-) cells in adult bone marrow of C.B20 (Ighb) mice. Previous analyses using a variety of antigens have led to the conclusion that the antibody repertoire expressed in the spleen is similar to that expressed in newly generated B cell precursors with respect to both repertoire diversity and the representation of various predominant clonotypes. However, in the response to NP of C.B20 precursor cells, two marked disparities have been identified between the repertoire of sIg- bone marrow cells vs splenic precursor cells. The first concerns precursor cells that give rise to lambda-bearing NP-specific antibodies with heteroclitic fine specificity. Such antibodies normally dominate the primary response of Ighb mice; however, the representation of precursor cells giving rise to lambda-bearing antibodies is disproportionately low in the sIg- bone marrow cell population of C.B20 mice. Thus, during the maturation of these cells, post-sIg receptor expression, there is an apparent increase in the proportionate representation of lambda-bearing NP-specific cells. The second disparity concerns precursor cells whose antibody products bear kappa-light chains and exhibit high affinity and homoclitic binding for the NP haptenic determinant. Such precursor cells are poorly represented in the spleen, but represent a sizeable proportion of the sIg- NP-specific precursor cell population. Thus, there seems to be a selective elimination of high affinity, kappa-homoclitic anti-NP antibody-bearing cells as they acquire their sIg receptors. The elimination of this cell population could partially account for the dominance of lambda-heteroclitic antibodies in the serum responses to NP of C.B20 mice.
Characterization of the requirements for human T cell mitogenesis by using suboptimal concentrations of phytohemagglutinin.
Authors: Mills G B , Lee J W , Cheung R K , Gelfand E W
Published: December 2022

To characterize the requirements for T cell proliferation, we have studied the response of purified populations of human T cells to varying concentrations of the mitogen phytohemagglutinin (PHA). Concentrations of PHA which induce optimal proliferative responses induce increases in cytosolic free calcium ([Ca2+]i), expression of interleukin 2 (IL 2) receptors, and production of IL 2. As the concentration of PHA is decreased, each of these processes decreases in parallel. At suboptimal concentrations of PHA, the addition of exogenous IL 2 reconstitutes both the proliferative response and the expression of the IL 2 receptor, as measured by immunofluorescence with antibodies directed against the TAC/IL 2 receptor molecule, but without reconstituting the increase in [Ca2+]i. Therefore, the concentration dependence of responses to PHA appears to be secondary to an absence of IL 2 production due to a failure to induce an increase in [Ca2+]i. The addition of the calcium ionophores A23187 and ionomycin or of accessory cells to low concentrations of PHA induces increases in [Ca2+]i and subsequent proliferative responses, suggesting that the two events are linked. The proliferative response can be inhibited by antibodies directed towards IL 2 or the IL 2 receptor, indicating that the proliferative response was at least partially dependent on the production and action of IL 2. This suggests that, although increases in [Ca2+]i are an integral event in the induction of proliferation by PHA, the increase in [Ca2+]i is required for the production but not the action of IL 2. In addition, low concentrations of PHA deliver an additional signal to cells, independent of an increase in [Ca2+]i, which induces IL 2 receptor expression and allows a proliferative response in the presence of exogenous IL 2.
Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus.
Authors: Uytdehaag F G , Loggen H G , Logtenberg T , Lichtveld R A , van Steenis B , van Asten J A , Osterhaus A D
Published: December 2022

An in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization assay and ELISA-SPOT test, respectively. After booster immunization with polio vaccine, anti-poliovirus-neutralizing ASC were present in circulation. This response was measurable between 5 and 12 days after booster vaccination. At between 12 and 90 days, another subset of B cells was found in peripheral blood that only produced poliovirus-specific neutralizing antibody after in vitro antigenic stimulation. The in vitro virus-induced response required B cells, monocytes, and T4+ (T helper) cells, and was shown to result from de novo protein synthesis. The anti-poliovirus-neutralizing response in vitro could be dissected in a type-specific and intertypic cross-reactive response by using various antigen concentrations for in vitro stimulation. Evidence was obtained by absorption studies for the existence of intertypic cross-reactive neutralization-inducing epitopes.
Autoaggressive T lymphocyte lines recognizing the encephalitogenic region of myelin basic protein: in vitro selection from unprimed rat T lymphocyte populations.
Authors: Schluesener H J , Wekerle H
Published: December 2022

Autoimmune T lymphocyte lines have been established from unprimed normal rat lymph node cell populations. In a first, negative-selection round, spontaneously proliferating (SMLR) T cells were eliminated by a pulse of BUdR followed by short wave light irradiation. In a second, positive-selection round, the SMLR-depleted populations were confronted with MBP presented by syngeneic spleen adherent cells. Reactive T cells were propagated until stable, permanent T lines were established. All lines were exclusively specific for the selecting antigen, MBP, and were restricted in recognition by determinants of the own MHC. All lines expressed the differentiation marker W3/25, but not OX8. Line vLe, which was derived from Lewis (LEW) rat lymphocytes, and which recognized the encephalitogenic sequence 48-88 of MBP, was extremely efficient in mediating EAE to normal untreated LEW rats. Doses of 1 X 10(6) and greater transferred lethal EAE, whereas transient although definite disease was caused by a minimum of 1 X 10(4) cells. Rats recovering from disease were resistant against subsequent active induction of EAE. In contrast, BN rat-derived line vBN was completely incapable of transferring EAE to syngeneic rats. This lack of encephalitogenicity was a property of the T line, because vLe cells transferred severe EAE to (LEW X BN)F1 hybrid rats, whereas none of hybrid rats injected with vBN cells showed any sign of disease. The data provide strong evidence in favor of the presence of potentially autoaggressive T clones in the normal immune system, and they might suggest that the actual proportion of these clones within the natural T cell repertoire is genetically determined.
Purification and partial characterization of a monoclonal "second order" suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10.
Authors: Turck C W , Kapp J A , Webb D R
Published: December 2022

A GAT-specific "second order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 has been purified and biochemically characterized. The protein has a m.w. of approximately 66,000, an isoelectric point of 6.8 to 6.9, and elutes from a reversed phase HPLC column in two peaks, one in 55% acetonitrile, the other in 70% propanol. Amino acid analysis of both forms gave similar molar ratios, suggesting that the two forms are closely related and may differ mainly in the degree of posttranslational modification. SDS-PAGE electrophoresis under reducing conditions gave two chains of the apparent m.w. of 42,000 and 35,000.
Purification and analysis of isoforms of soluble immune response suppressor (SIRS).
Authors: Webb D R , Mason K , Semenuk G , Aune T M , Pierce C W
Published: December 2022

Soluble immune response suppressor (SIRS) isolated from the T cell hybrid 393D2.6 was originally reported to exist as at least two m.w. forms and to migrate on reverse-phase high-performance liquid chromatography columns as three separate species. In experiments presented here, a further analysis of the different chromatographic forms of SIRS has been carried out. SIRS-alpha elutes from C-18 reverse-phase columns in 20% propanol. When SIRS-alpha is subjected to isoelectric focusing, three biologically active species are isolated at approximately pH7, approximately pH6, and approximately pH5 (SIRS-alpha 7, SIRS-alpha 6, and SIRS-alpha 5, respectively). SIRS-beta elutes in 30% propanol, and on isoelectric focusing the biologic activity is found only at approximately pH7 (SIRS-beta 7). Both the alpha and beta forms of SIRS have nearly identical m.w. when subjected to molecular sieve chromatography and migrate with a m.w. of 11,000. The molecular basis for these isoforms is not yet clear but is consistent with earlier studies showing two separate messenger RNA species coding for SIRS.
Delineation of functional sites in HLA-B27 antigens. Molecular analysis of HLA-B27 variant Wewak I defined by cytolytic T lymphocytes.
Authors: Vega M A , Wallace L , Rojo S , Bragado R , Aparicio P , López de Castro J A
Published: December 2022

An HLA-B27 positive, Epstein Barr virus-transformed cell line Wewak I was not lysed by Epstein Barr virus-specific cytolytic T lymphocytes restricted by HLA-B27. This line is weakly reactive with a B27-specific monoclonal antibody, M2, which recognizes a majority, but not all, of the HLA-B27-positive cells. To establish the molecular basis for this lack of recognition, the structure of the variant HLA-B27 antigen was compared with the known structure of HLA-B27 from the LG-2 cell line, which is representative of a major subtype of this antigen. Both molecules were almost indistinguishable by isoelectric focusing. However, comparative peptide mapping and sequencing of the difference peptides revealed two amino acid changes: At position 77, Asp in donor LG-2 had changed to Ser in the variant, and at position 152, Val in LG-2 had changed to Glu in the variant. The nature of these substitutions was consistent with the extreme similarity of the isoelectric focusing pattern. An evaluation of these findings in the context of studies on other HLA variants and H-2Kb mutants suggests that both positions 77 and 152 contribute to the determinants recognized by B27-specific cytolytic T lymphocytes. The change at position 152 adds to previous evidence suggesting that the segment 149-156 is critical for cellular recognition. In addition, it is proposed on the basis of structural comparisons that residue 77 may also participate in the epitope recognized by the B27M2 antibody.
Induction of suppressor cells from peripheral blood T cells by 15-hydroperoxyeicosatetraenoic acid (15-HPETE).
Authors: Gualde N , Rigaud M , Goodwin J S
Published: December 2022

15-hydroperoxyeicosetetraenoic acid (15-HPETE), a lipoxygenase metabolite of arachidonic acid, inhibited polyclonal IgG and IgM production in pokeweed mitogen (PWM)-stimulated cultures of human peripheral blood mononuclear cells, whereas 15-hydroxyeicosetetraenoic acid (15-HETE) had little effect in this system. T cells preincubated for 18 hr with 15-HPETE caused substantial inhibition of IgG and IgM production of fresh, autologous B and T cells stimulated by PWM. The suppressive effect of the 15-HPETE-treated cells was lost if the cells were irradiated before the PWM culture, but not by treatment with mitomycin C. The suppressive effect was also lost if OKT8+ T cells were removed after, but not before, preincubation of the T cells with 15-HPETE. OKT8- T cells incubated with 15-HPETE for 18 hr showed a large increase in the percentage of cells staining with directly fluoresceinated Leu-2, another marker for suppressor cells. Thus, 15-HPETE induces functional and phenotypic suppressor cells from resting human peripheral blood T cells.
Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro.
Authors: Levi-Schaffer F , Austen K F , Caulfield J P , Hein A , Bloes W F , Stevens R L
Published: December 2022

Rat serosal heparin-containing mast cells (HP-MC) were maintained in vitro for as long as 30 days when co-cultured with mouse skin-derived 3T3 fibroblasts. In contrast, when the mast cells were cultured alone, on fibronectin-, gelatin-, or dermal-collagen-coated dishes, on acid and heat-killed fibroblasts in the presence or absence of 24 hr fibroblast-conditioned medium, or on a monolayer of mouse serosal macrophages, they failed to adhere to the dishes, released significant amounts of their histamine and lactate dehydrogenase, and stained with trypan blue, indicating a loss of viability. The rat serosal HP-MC cultured with the 3T3 fibroblasts became so adherent to the fibroblasts that the two cell types could be separated from one another only by trypsinization. The cultured HP-MC stained with both alcian blue and safranin and continued to synthesize proteoglycan at a rate comparable to that of freshly isolated cells. The 35S-labeled proteoglycan synthesized by these cultured cells, like that produced by freshly isolated rat serosal HP-MC, was a 750,000 to 1,000,000 m.w. proteoglycan containing only heparin glycosaminoglycans of 50,000 to 100,000 m.w. When HP-MC were cultured for 1 wk with the fibroblasts and were then incubated for 5 min with a 1/20 dilution of rabbit anti-rat IgE, they generated and released an average of 22 +/- 10 ng (mean +/- SD, n = 5) of prostaglandin D2 per 10(6) cells and exocytosed a higher net percentage of their total histamine content (44 +/- 11% [mean +/- SD, n = 8]) than did cells just isolated from the animal (6 +/- 4% [mean +/- SD, n = 4]). As assessed by electron microscopy, many of the cultured HP-MC resembled freshly isolated cells except that some secretory granules had fused with one another in some cells. Morphologically, after activation the cultured HP-MC underwent compound exocytosis like freshly isolated cells. These results demonstrate that the in vivo differentiated rat HP-MC maintain their histology, morphology, immunologic responsiveness, histamine content, and ability to synthesize heparin proteoglycan when co-cultured with living fibroblasts.
Implications of the invariant gamma-chain on the intracellular transport of class II histocompatibility antigens.
Authors: Claesson-Welsh L , Peterson P A
Published: December 2022

We have examined the role of the invariant gamma-chain on the intracellular transport of human class II histocompatibility antigens. mRNA was selected by hybridization to cDNA corresponding to class II alpha-, beta-, and gamma-chains, and the obtained mRNA fractions were injected individually and in various combinations into X. laevis oocytes. Translation products were isolated after various periods of chase, and their carbohydrate moieties were analyzed to monitor the subcellular localization of polypeptide chains. A mixture of alpha-, beta-, and gamma-chains, or gamma-chains alone, were transported and glycosylated to the same extent as in a B lymphoblastoid cell line. However, although alpha- and beta-chains formed a complex in the absence of the gamma-chain, the transport of this complex was slowed down. Furthermore, the glycosylation of alpha- and beta-chains appeared incomplete. Thus, the invariant gamma-chain seems to play a crucial role for the rate of transport and glycosylation of class II alpha- and beta-chains.
A human-mouse chimeric immunoglobulin gene with a human variable region is expressed in mouse myeloma cells.
Authors: Tan L K , Oi V T , Morrison S L
Published: December 2022

We have constructed and obtained expression of a chimeric human-mouse immunoglobulin gene after transfection into mouse myeloma cells. A human VDJH gene segment was joined to a mouse C kappa gene in the plasmid vector pSV2-gpt, and the construct was transfected into J558L cells by protoplast fusion. Analyses of six transformants by RIA and SDS-PAGE indicated that the chimeric protein was synthesized in large amounts in five. A kappa-specific transcript was observed by Northern blot analysis. Four out of five clones were stable producers of this chimeric chain over a period of 10 mo.
Antigen presentation by Hodgkin's disease cells.
Authors: Fisher R I , Cossman J , Diehl V , Volkman D J
Published: December 2022

The L428 tumor cell line is a long-term tissue culture of Reed-Sternberg cells which was derived from the pleural effusion of a patient with Hodgkin's disease. The L428 cells express all known cell surface antigens, cytochemical staining, and cytologic features of freshly explanted Reed-Sternberg cells. In addition to the previously described HLA-DR cell surface antigens, the L428 cells are now demonstrated to express both DS and SB alloantigens. Thus, the L428 cells express all of the known subclasses of the human immune response genes that are located in the major histocompatibility complex. Furthermore, the L428 cells are capable of presenting soluble antigen to T cells in a genetically restricted fashion. T cell lines were established from normal donors previously immunized with tetanus toxoid. The T cells utilized were incapable of tetanus toxoid-induced proliferation unless antigen-presenting cells were added to the cultures. However, T cells from the two normal donors, which like the L428 cells expressed HLA-DR 5, demonstrated significant proliferative responses when cultured with tetanus toxoid and L428 cells. No proliferative response was observed when the L428 cells were used as antigen-presenting cells for a DR (4,-), DR (2,-) or DR (1,7) T cell line. The tetanus toxoid dose-response curve was similar regardless of whether autologous mononuclear leukocytes or L428 cells were used as antigen-presenting cells. The T cell proliferation induced by soluble antigen was also blocked by anti-HLA-DR antibody. Thus, functionally, Hodgkin's disease may be classified as a tumor of antigen-presenting cells.
Characterization of the molecules on SJL/J lymphomas which stimulate syngeneic T cells.
Authors: Brown P H , Thorbecke G J
Published: December 2022

I-A antigens isolated from SJL reticulum cell sarcoma (RCS) cells show greater heterogeneity with respect to charge and size of the A alpha chains than do I-A antigens isolated from normal SJL spleen cells. The relevance of these structural changes in RCS I-A to the previously shown syngeneic T cell stimulatory properties of RCS was investigated. It was shown that RCS cells expressed acidic forms of the A alpha chain on their cell surface which were not present on SJL spleen cells, peritoneal cells, or dendritic cells. The only normal cells which showed A alpha chain heterogeneity approaching that of RCS cells were LPS-induced B cell blasts. Treatment with tunicamycin completely abolished the RCS A alpha chain heterogeneity, whereas exposure to neuraminidase removed the charge heterogeneity, but not the size heterogeneity. Parallel studies of the syngeneic T cell proliferative response to RCS showed that tunicamycin abolished, while neuraminidase enhanced, the ability of RCS cells to stimulate syngeneic T cells. It is suggested that polysaccharide chains on RCS I-A molecules are particularly important for the biologic properties of these lymphoma cells. The possibility that highly glycosylated I-A antigens on normal B cell blasts are also involved in the stimulation of syngeneic T cells is discussed.
Monoclonal antibodies for carcinoembryonic antigen (CEA) as a model system: identification of two novel CEA-related antigens in meconium and colorectal carcinoma tissue by Western blots and differential immunoaffinity chromatography.
Authors: Neumaier M , Fenger U , Wagener C
Published: December 2022

In a previous study, five monoclonal antibodies against the carcinoembryonic antigen (CEA) with different epitope specificities were delineated. One of these antibodies which exhibits a high affinity for CEA binds to different carcinoma tissues, to liver tissue, and to granulocytes. This antibody was selected for the immunoaffinity purification of CEA and related antigens from colorectal carcinoma tissue, from spleen tissues, from bile, and from meconium. After elution from the immunosorbent, the antigens were separated by SDS-PAGE, were transferred to nitrocellulose, and were incubated with the five different antibodies. Antibody T84.1 bound to the following antigens: 177 kD and 128 kD from colonic carcinoma, 81 kD from bile, 49 kD from spleen, as well as 165 kD and 100 kD from meconium. Two additional antibodies showed a similar binding pattern. The fourth antibody (CEA.11) bound to the 165 kD meconium antigen and to the two colorectal carcinoma antigens. The fifth antibody (T84.66) showed a strong reaction with the 177 kD colorectal carcinoma antigen and a faint reaction with a 183 kD antigen in meconium. As judged from m.w. and immunochemical properties, the 128 kD colorectal carcinoma antigen and the 100 kD meconium antigen are two novel CEA-related antigens. Because antibody CEA.11 did not bind to the 100 kD meconium antigen in Western blots, the 165 kD antigen could be eluted from a CEA.11 immunosorbent without contamination by the 100 kD antigen. Similarly, as predicted from the binding pattern in the Western blots, the two colorectal carcinoma antigens were separated from each other by a T84.66 immunosorbent.
In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene.
Authors: Ishikawa H , Suzuki H , Hino T , Kubota E , Saito K
Published: December 2022

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.
A murine cytotoxic T lymphocyte clone from the intestinal mucosa that is antigen specific for proliferation and displays broadly reactive inducible cytotoxic activity.
Authors: Klein J R , Lefrancois L , Kagnoff M F
Published: December 2022

Thy-1+, Lyt-1-,2+, asialo GM1+ cytotoxic T lymphocyte (CTL) clones have been isolated from the intestinal mucosa of mice primed with alloantigens. Two different types of cytotoxic clones have been obtained. The first type is functionally similar to most splenic and lymph node-derived CTL clones in that they are strictly antigen specific with respect to proliferation and cytolytic activity. The second type of CTL clone has several unique characteristics. Although these clones are also antigen specific with regard to proliferation, they are not cytolytic under standard growth conditions in medium containing 4% rat concanavalin A-induced spleen cell supernatant. After culture for 4 days in the presence of high concentrations of interleukin 2, cells become activated and exhibit broad lytic potential. Moreover, during the activation process, these CTL begin to express a murine T cell surface antigen, CT-1, which is associated with activated cytotoxic cells. The findings reported in this report should now allow us to precisely define, both phenotypically and functionally, specific lymphocyte populations that make up the gut-associated lymphoid tissues. These data also describe a new type of effector CTL that differs from other cytotoxic cells reported to date, because it is antigen dependent for proliferation, but requires signals mediated by lymphokines for lytic activation.
Immune complex effects on murine macrophages. I. Immune complexes suppress interferon-gamma induction of Ia expression.
Authors: Virgin H W , Wittenberg G F , Unanue E R
Published: December 2022

We have studied the effects of immune complexes on the expression of macrophage surface proteins in vitro. Increased expression of the H-2 molecules I-A, I-E, and K on the macrophage membrane was induced by in vitro culture with crude lymphokine or interferon-gamma. Expression of all three of the molecules was additionally increased by stimulating the cultures with heat-killed Listeria monocytogenes. Addition of soluble immune complexes to the cultures did not have any effect on macrophage expression of these proteins. However, significant inhibition of lymphokine or interferon-gamma induction of I-A, I-E, and H-2K was observed when macrophages were cultured on plates to which immune complexes had been bound. This inhibition was dose dependent, required an immunoglobulin (Ig) molecule with an intact Fc portion, did not require the presence of T cells, and occurred in the presence of indomethacin. Complexes containing IgG1, IgG2a, IgG2b, and IgE, but not IgM or IgA, antibodies mediated the inhibitory effect.
The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle.
Authors: Golay J T , Clark E A , Beverley P C
Published: December 2022

Two monoclonal antibodies, 1F5 and B1, directed against the CD20 (Bp35) antigen were found to have both stimulatory and inhibitory effects on B cells. 1F5, but not B1, induces small resting tonsillar B cells and prolymphocytic leukemia cells to enlarge, to rapidly increase their RNA synthesis, and to become responsive to growth factors present in mixed lymphocyte reaction supernatants. In addition, 1F5 induces a moderate increase in thymidine uptake, which is accompanied by enhanced viability of the cells, but not by any increase in total cell number or by any detectable entry into S phase or mitosis. Taken together, these observations suggest that 1F5 can initiate transition from the G0 to the G1 phase of the cell cycle. The fact that all the changes observed can be inhibited by low concentrations (I50 = 50 ng/ml) of cyclosporin A is further evidence that 1F5 is involved at an early stage of B cell activation. Because both 1F5 and B1 belong to the IgG2a subclass, differences in their activities are likely to reflect their different epitope specificities. Although only 1F5 had stimulatory activity, both 1F5 and B1 strongly inhibited B cell differentiation to immunoglobulin secretion. Possible explanations for the dual activities of 1F5 and implications for the role of the CD20 antigen in B cell differentiation are discussed.