bluuebunny/crossref_metadata_2025 huggingface dataset:
Patterns of mutations and selection in antibodies to the phosphocholine-specific determinant in Proteus morganii.
Authors: Claflin J L , George J , Dell C , Berry J
Published: December 2022

The contribution of somatic mutation to the generation of an antibody response was investigated by using the phosphocholine (PC) determinant in the bacterium Proteus morganii as the model Ag. The response to this determinant is restricted to a single VH/VL pair and apparently is derived from only one or two precursors per mouse. In this study we examined hybridoma antibodies from nine individual mice which produced representatives of 12 different clones. We found that all antibodies reactive with the PC Ag of P. morganii contained somatic mutations; the number ranged from 2 to 20. Two clusters of mutations were observed, one in complementarity-determining residue (CDR) 2 and the other in CDR 3 of VH. Examination of a three-dimensional model of M603, an antibody with the same V region composition as the anti-PC antibodies under study, showed that these clusters occupied an area of the binding site which presumably interacts with carrier elements of the PC epitope in P. morganii. A high incidence of recurring mutations were found in both clusters, and one of these was invariant, leading to an Asn for Asp substitution at 95. Ag binding studies with these antibodies and an additional one, which was unmutated except for the invariant substitution at 95, showed that: 1) antibodies having only the 95Asn mutation failed to bind the PC Ag of P. morganii, 2) addition of a second recurring mutation, at 52a (CDR 2), was sufficient to create strong binding to the P. morganii Ag, and 3) accumulation of mutations was directly correlated with increased binding activity for Ag. These results show that somatic mutations play a critical, if not essential, role in generating specificity for this PC Ag, and that Ag, and most likely a carrier element of the epitope, is a primary force in the continued selection and expansion of Ag-reactive B cells.
Protection against murine neonatal herpes simplex virus infection by lymphokine-treated human leukocytes.
Authors: Kohl S
Published: December 2022

One-week-old mice were protected against a uniformly lethal herpes simplex virus (HSV) infection by IL-2 alone, but especially by the addition of human mononuclear cells (MC) plus IL-2. The dose response of IL-2 was biphasic. The addition of MC from cord blood did not enhance IL-2-mediated survival. Because the effect of IL-2 alone, or IL-2 plus MC, was ablated by anti-IFN-gamma and human neonates have an IFN-gamma production defect, the protective effect of MC plus human IFN-gamma (HuIFN-gamma) was tested. MC from adults cultured for 5 days in HuIFN-gamma afforded protection. At least 1 x 10(6) HuIFN-gamma-treated MC were required with increasing survival to 1 x 10(7) MC. The effector cell activity was ablated by adherence, silica, L-leucine methyl ester treatment or treatment with Leu-M3 plus C (all macrophage markers), and OKT4 plus C treatment (CD4 marker). Use of Leu-11, Leu-7, OKT3, or OKT8 plus C did not inhibit protection and excluded NK or T cell participation. In addition to survival, the ability to produce anti-HSV antibody was reconstituted. For the first time protection was afforded by human cord blood MC after treatment with HuIFN in vitro. We have identified an IFN-gamma-driven protection system against murine neonatal HSV infection mediated by human adult- or cord blood-derived CD4-positive macrophages. Protection is associated with enhanced effector cell function and reconstitution of the neonatal antibody production defect.
Phenotypic and functional characteristics of human newborns' B lymphocytes.
Authors: Durandy A , Thuillier L , Forveille M , Fischer A
Published: December 2022

It has been demonstrated two major facts concerning human newborns' B lymphocytes: 1) they differentiate poorly into Ig-producing cells and 2) they express CD5 and CD1c membrane proteins. We have further analyzed human newborns' B cell characteristics and found that approximately half of them express activation Ag, i.e., 4F2 and IL-2R, both associated in significant proportions with CD23 and Bac-1. These membrane Ag were found both on CD5(+) and CD5(-) B cells. Newborns' B cells do not exhibit other activation markers because they express surface IgD, and because their size, RNA, and DNA contents do not differ from those of adults' B cells, indicating that they are in the G0/G1 cell cycle phase. Newborns' B cell proliferation can be induced by rIL-2, rIL-4, low m.w. B cell growth factor, and by Staphylococcus aureus protein A. It is presently difficult to build a hypothesis accounting for all the specific findings made on newborns' B cells. It is not known for instance whether CD5(+) and (-) B cells belong to distinct subsets as suggested by the fluorescence intensity curve obtained with an anti-CD5 antibody or to distinct stages in a unique pattern of B cell maturation during fetal and newborn life. This may indicate that partially activated B cells actually produce natural polyspecific autoantibodies of the IgM isotype found in newborns' human serum.
Immunosuppression and lymphoid hypoplasia associated with chronic graft versus host disease is dependent upon IFN-gamma production.
Authors: Klimpel G R , Annable C R , Cleveland M G , Jerrells T R , Patterson J C
Published: December 2022

We have previously shown that both IFN-gamma and IFN-beta are produced in vivo and in vitro by spleen cells obtained from mice experiencing a chronic form of graft vs host disease (GVHD). Further, we have shown that in vitro production of IFN-beta by spleen cells from GVHD mice may play a role in the suppressed in vitro mitogen responsiveness of these cells. This study was undertaken to investigate if treatment of such mice with mAb to IFN-gamma or IFN-beta could alter the immunosuppression or lymphoid hypoplasia associated with chronic GVHD. GVHD was induced across minor histocompatibilities by the i.v. injection of B10.D2 spleen cells into sublethally irradiated BALB/c mice. These mice were given daily injections for 20 days of one of the following: 1) mAb to IFN-gamma, 2) mAb to IFN-beta, or 3) control IgG. Histologic examination of these mice at 21 to 22 days post transplantation revealed that mice treated with mAb to IFN-beta or control IgG had dramatic hypoplasia of the thymus, spleen, and lymph nodes which was similar to untreated GVHD mice. Mice given mAb to IFN-gamma, however, had no lymphoid hypoplasia and had a near normal gross and histologic appearance of their thymus, spleen, and lymph node tissue when compared with syngeneic controls. In vitro mitogen-induced proliferative responses of spleen and lymph node cells obtained from GVHD mice or GVHD mice treated with mAb to IFN-beta were severely suppressed or absent. In contrast, spleen and lymph node cells from GVHD mice given mAb to IFN-gamma were capable of giving a significant in vitro proliferative response to Con A, PHA, and LPS. Further, natural suppressor cell activity and spontaneous production of IFN-beta, a characteristic of this form of GVHD, was absent in spleen cells obtained from GVHD mice treated with mAb to IFN-gamma. These results further identify the IFN as playing critical roles in the pathogenesis of GVHD.
Cytopathic effect determined by the amount of CD4 molecules in human cell lines expressing envelope glycoprotein of HIV.
Authors: Koga Y , Sasaki M , Yoshida H , Wigzell H , Kimura G , Nomoto K
Published: December 2022

A plasmid which has an envelope gene from the human T lymphotropic virus IIIB isolate of HIV under the transcriptional control of the human metallothionein IIA promoter was constructed and transfected into human CD4+ and CD4- cell lines. When the expression of the env gene was induced with metal ions in cloned cell lines from such transfected cells, the viral envelope protein precursor, gp160, was produced in both the CD4+ and CD4- cell clones in the same amounts. An extensive cytopathic effect developed resulting in cell killing; however, this occurred only in CD4+ and not in CD4- cell clones. The formation of syncytium was not found in those cells showing cytopathic effect. Immunofluorescence microscopy showed the envelope proteins localized in the periphery, forming a mass in the CD4+ cells but not in the CD4- cells after induction. Moreover, immunoprecipitation and immunoblot analyses demonstrated the formation of a complex between gp160 and CD4 molecules in the cytoplasm of those cells from whose surface CD4 Ag had already disappeared. Therefore, it is proposed that the formation of such a complex is directly related to the cell killing of CD4+ cells by envelope proteins in our cell clones as well as in infections with HIV.
Adoptively transferred antigen-specific T cells can be grown and maintained in large numbers in vivo for extended periods of time by intermittent restimulation with specific antigen plus IL-2.
Authors: Chen W , Reese V A , Cheever M A
Published: December 2022

The aim of the current study was to determine whether cultured tumor Ag-specific T cells could be induced to grow and maintained functional in large numbers in vivo by intermittent restimulation in vivo with specific Ag plus IL-2. T cells derived from spleens of B6 mice (Thy-1.2) immune to FBL-3, a Friend virus-induced leukemia, were activated by in vitro stimulation with irradiated FBL-3 and expanded by culture for 14 days with low concentrations of IL-2. The resultant FBL-3-specific T cell lines were adoptively transferred into cyclophosphamide pretreated congenic hosts (B6/Thy-1.1), and restimulated every 14 days by an injection of irradiated FBL-3 plus a 7-day course of IL-2. Donor T cells residing in the host were identified and quantified by use of antibody to the Thy-1.2 allele. The results confirmed that stimulation with FBL-3 on the day of transfer (day 0) plus IL-2 on days 0 to 6 induced rapid growth of donor T cells to approximately an 11-fold increase in total donor T cell number recoverable from host ascites and spleen by day 7. However, prolonging the course of IL-2 administration to 35 days did not maintain the number or the specific cytolytic function of donor T cells. By contrast, intermittent restimulation with specific Ag plus IL-2 induced intermittent regrowth of donor T cells in vivo, maintained the number of donor T cells in vivo at greater than the number input for longer than 1 mo, and allowed detection of substantially augmented donor T cell-mediated specific antitumor function over that period of time.
Generation of cytolytic activity with anti-CD3 monoclonal antibodies involves both IL-2-independent and -dependent components.
Authors: Stohl W , Tovar Z , Talal N
Published: December 2022

We have generated potent Ag-nonspecific cytolytic activity in PBMC cultures without exogenous IL-2 by stimulating the cells with anti-CD3 mAb. Three anti-CD3 mAb (147, 446, 454) each induced cytolytic activity against both K562 and Daudi targets, albeit to different degrees. The differences among the anti-CD3 mAb could not be explained by differences in their isotypes or avidity constants, by the number of anti-CD3 mAb molecules initially bound per cell, or by preferential or differential binding to TCR alpha/beta+ vs TCR gamma/delta+ cells. Each anti-CD3 mAb appeared to induce cytolytic activity via, in part, an IL-2-independent component, as evidenced by: 1) the generation of potent cytolytic activity in mAb 446-stimulated cultures despite undetectable IL-2 levels; 2) the frequent inability of exogenous IL-2 (10 U/ml) to generate as much cytolytic activity as that induced by mAb 147 or 454, despite the low levels of IL-2 (less than U/ml) in the latter cultures; and 3) the different kinetics in generation of cytolytic activity between IL-2 and anti-CD3 mAb. Moreover, exogenous IL-2 enhanced anti-CD3-induced cytolytic activity for each anti-CD3 mAb. However, IL-2-dependent processes also contributed to the generation of cytolytic activity, because anti-p55 IL-2R mAb in combination with anti-p75 IL-2R mAb partially inhibited generation of anti-CD3-induced cytolytic activity, albeit to a lesser degree than the inhibition by these anti-IL-2R mAb of generation of IL-2-induced cytolytic activity. By demonstrating the generation of potent cytolytic activity in the absence of exogenous IL-2, these studies may assist in the development of more effective and less toxic clinical adoptive immunotherapy protocols.
Abnormalities of lymphokine gene expression in patients with common variable immunodeficiency.
Authors: Sneller M C , Strober W
Published: December 2022

Common variable immunodeficiency (CVI) is a syndrome characterized by hypogammaglobulinemia, recurrent bacterial infections, and increased occurrence of both autoimmune disease and malignancy. In our study we examine the expression of lymphokine genes in mitogen-activated T cells from four patients with CVI. T cells from patients with CVI did not differ significantly from normals in total T cell number, CD4/CD8 ratio, CD45R expression, or proliferation in response to PHA. However, T cells from this group of patients did exhibit significant abnormalities of mitogen-induced lymphokine gene expression. T cells from patients exhibited significantly decreased expression of IL-2, IL-4, IL-5, and IFN-gamma when compared to normal controls. In contrast to these abnormal findings, mitogen-activated T cells from patients with CVI expressed normal amounts of IL-2R alpha and c-myc suggesting that these patients have a selective abnormality of T cell activation. Furthermore, it is likely that the deficient production of IFN-gamma by patient T cells is partially due to the abnormality of IL-2 production as the levels of IFN-gamma mRNA detected during the initial IL-2-independent phase of T cell activation were normal and the addition of exogenous rIL-2 was able to normalize IFN-gamma production by PHA-stimulated patient cells. Finally, supernatants from PHA-activated cultures of patients PBMC were deficient in their ability to support Ig secretion by Staphylococcus A Cowan's-activated normal B cells suggesting that these T cell abnormalities may contribute to the pathogenesis of this syndrome.
Granulocyte-macrophage colony-stimulating factor stimulates human monocyte accessory cell function.
Authors: Smith P D , Lamerson C L , Wong H L , Wahl L M , Wahl S M
Published: December 2022

We investigated the effect of recombinant human granulocyte-macrophage CSF (rhGM-CSF) on the accessory cell function of purified human monocytes. Compared with untreated monocytes, rhGM-CSF-treated monocytes promoted enhanced mitogen- and Ag-stimulated lymphocyte proliferation. This enhancement was significantly inhibited by mAb to rhGM-CSF. In experiments designed to define the mechanism of rhGM-CSF augmentation of accessory cell function, rhGM-CSF was shown to cause a dose-dependent increase in monocyte expression of surface HLA-DR molecules and stimulated secretion of IL-1, both important in monocyte T cell interactions. Further studies demonstrated that levels of HLA-DR and IL-1 mRNA were increased by rhGM-CSF, indicating transcriptional regulation of gene expression for HLA-DR and IL-1. Thus, rhGM-CSF augments accessory cell function by human monocytes, and this augmentation correlates with rhGM-CSF-induced increases in transcription of the HLA-DR and IL-1 genes leading to increased expression of surface HLA-DR and secretion of IL-1.
Temporal expression of different pathways of 1-arginine metabolism in healing wounds.
Authors: Albina J E , Mills C D , Henry W L , Caldwell M D
Published: December 2022

Arginine can be metabolized by inflammatory cells through at least two pathways. One is an oxidative l-arginine deiminase (OAD) that results in the formation of citrulline and reactive nitrogen intermediates. The other is arginase, which determines the production of ornithine and urea. The temporal expression of these pathways in an experimental wound model (s.c. implanted polyvinyl alcohol sponges in the rat) was investigated by examining the concentrations of amino acids and of nitrite in fluids obtained from the sponges 6 h to 15 day after implantation. These analyses revealed two distinct periods during which the arginine concentration in the fluids was markedly below plasma levels. During the early period (less than 3 days after sponge implantation) wound fluid contained more citrulline and nitrite than at any other time, suggesting OAD activity. In contrast, ornithine accumulated in the fluids during the late decrease in arginine concentration that extended beyond day 3, during which time the wound fluid also contained a high arginase activity. This time-dependent expression of different pathways of arginine metabolism in wounds was confirmed in sponge cultures containing [guanido-14C]-l-arginine. Cells contained in sponges harvested less than 48 h after implantation metabolized labeled arginine mainly to labeled citrulline, whereas labeled urea was produced during culture of sponges harvested after this time. The low arginine content of wound fluid did not appear to be rate limiting for the expression of OAD in late sponges because no OAD activity was evidenced when 4 mM arginine was added to the cultures. These results indicate that the OAD pathway is expressed in this model predominantly during the early, polymorphonuclear leukocyte-predominant, phase of repair. At this time, the reactive nitrogen intermediates resulting from the metabolism of arginine may mediate some of the events characteristic of early inflammation, including microbiostasis, vasodilation, and inhibition/reversal of platelet aggregation. In turn, the late suppression of this pathway and the catabolism of arginine through arginase may promote macrophage function within wounds.
The regulation of resistance to Schistosoma mansoni by auto-anti-idiotypic immunity. II. Global qualitative and quantitative regulation.
Authors: Phillips S M , Lin J J , Walker D J , Linette G P , Fathelbab N G , Perrin P J
Published: December 2022

These studies explore the suppression of resistance to schistosomiasis mansoni through interactions of autologous immune functions derived from an induced anti-idiotypic response. This anti-clonotypic response is induced by immunization with syngeneic L3T4+ receptor-bearing lymphoblasts and for the sake of description is termed "auto-anti-idiotypic". It is antigenically restricted and cannot be induced by allogeneic cells. Anti-idiotypic immunization profoundly suppressed the development of protective immunity after exposure to irradiated cercariae and altered a wide variety of functional humoral and cellular immune responses to the parasite. In addition to quantitative suppressive effects, the anti-idiotypic network also regulated qualitative aspects of the immune response by increasing the heterogeneity and reducing the functional binding avidity of antibody for Ag. These effects also were reflected in analogous alterations in cellular reactivity, using the criteria of the Ag mediated blast transformation and delayed type hypersensitivity. Thus idiotypic regulation can mold the specificity and sensitivity of the immune response to Schistosoma mansoni by affecting quantitative and qualitative responses. Manipulation of idiotypic recognition provides an approach to optimize the expression of protective resistance to schistosomiasis.
Regulation of B cell responses to the variant surface glycoprotein molecule in trypanosomiasis. II. Down-regulation of idiotype expression is associated with the appearance of lymphocytes expressing antiidiotypic receptors.
Authors: Theodos C M , Mansfield J M
Published: December 2022

The current study examines the idiotypic expression and regulation of variant surface glycoprotein (VSG)-specific B cell responses during African trypanosomiasis. Utilizing competitive inhibition RIA analysis, we detected antibodies in the serum of BALB/cByJ mice infected with Trypanosoma brucei rhodesiense clone LouTat 1.5 that recognized the same VSG epitopes as three VSG 1.5-specific mAb. These epitope-specific antibody responses were detectable by day 5 of infection, peaked by day 10, and then declined slowly through day 15 of infection. VSG-specific antibodies detectable in the serum of infected BALB/cByJ mice included those that were idiotypically cross-reactive with the VSG 1.5-specific mAb. These idiotypically defined, VSG-specific antibody responses appeared to peak around day 7 of infection, but then declined to near preimmune levels by day 15 of infection, demonstrating that the aggregate epitope-specific response was composed only in part by the idiotypically cross-reactive responses. Although corresponding antiidiotypic antibodies could not be detected in infected sera during periods of up- or down-regulation of idiotypically defined antibodies, flow cytometry analysis of lymphocytes isolated from the spleens of LouTat 1.5-infected BALB/cByJ mice revealed the presence of antiidiotypic receptor-bearing cells. These cells were detectable primarily during days 10 to 12 of infection and subsequently down-regulated their receptors, or declined in numbers, to near preimmune levels by day 15 of infection. The appearance of these antiidiotypic receptor-bearing cells coincides with the decline of idiotypic antibody present in the serum of the LouTat 1.5-infected mice and may represent nascent evidence for idiotypic regulation of trypanosome-specific immune responses in infected animals.
Rejection of mouse sarcoma cells after transfection of MHC class II genes.
Authors: Ostrand-Rosenberg S , Thakur A , Clements V
Published: December 2022

Th cells are stimulated by peptide Ag presented in the context of MHC class II molecules. We have reasoned that immune responses against tumors may be more efficient if tumor cells were class II Ag positive, and thereby able to directly function as APC to stimulate tumor-specific Th cell proliferation. We have tested this hypothesis by using DNA-mediated gene transfer to generate syngeneic MHC class II Ag-expressing mouse Sal sarcoma cells (Sal/Ak transfectants). Autologous A/J mice challenged i.p. or s.c. with Sal/Ak transfectants do not develop tumors, whereas A/J mice challenged with the class II negative parental Sal tumor have a high tumor incidence. Furthermore, immunization of the autologous host with Sal/Ak transfectants completely protects against subsequent challenge with wild-type Sal cells. MHC class II-expressing tumor cells, therefore, stimulate an improved tumor-specific immune response, and the immunity is cross-reactive with the class II negative tumor. Inasmuch as the transfected MHC class II gene product is not functioning as a target molecule for autologous tumor rejection, the improved immunogenicity of the Sal/Ak cells is probably due to stimulation of a tumor-specific Th cell population. The increased immunogenicity of Sal/Ak cells is, therefore, probably the result of direct presentation of Sal tumor-associated Ag in the context of tumor cell MHC class II molecules to Th lymphocytes. These studies demonstrate that induction of tumor cell MHC class II Ag expression is a potential strategy for tumor-specific immunotherapy, and suggest that tumor immunity may be enhanced by improved Th cell generation.
Role of ICAM-1 in antigen presentation demonstrated by ICAM-1 defective mutants.
Authors: Dang L H , Michalek M T , Takei F , Benaceraff B , Rock K L
Published: December 2022

We report a methodology for selecting APC with mutations that have impaired their ability to present Ag to T cells. A20 B lymphoblastoid cells were mutagenized and then repeatedly cocultured with murine T-T hybridomas in the presence of specific Ag. During these cocultures, the T-T hybridomas kill the competent APC, allowing the outgrowth of inactive variants. Two variants, A20.M1 and A20.M2, were isolated and studied in detail. These variants are impaired in their ability to present multiple Ag to T cells. This defect is also observed for the presentation of processing independent peptides by fixed APC indicating that a lesion exists in a post-Ag processing step. The level of expression of MHC molecules is unaffected and the functional defect in the APC is not localized to a particular MHC molecule. In contrast, these mutants were found to have a selective decrease in the expression of the murine homolog of ICAM-1, and the residual ability of these cells to present Ag was not blocked by anti-ICAM-1 mAb. Conversely, Ag presentation by the wild-type A20 is inhibited by anti-ICAM-1 mAb. Similarly, anti-LFA-1 mAb inhibited the response of T cells to Ag presented by the wild-type A20 to a much greater degree than by the mutant cells, indicating that LFA-1 is involved in interaction of T cells with the former, but not latter, APC. In the apparent absence of a contribution of LFA-1 to the T cell-APC interaction, either as a result of mAb blocking or the disruption of the APC membrane, the mutant and wild-type APC have a similar level of Ag-presenting activity. Reconstitution of ICAM-1 expression in these mutants by transfection with murine ICAM-1 cDNA fully restores their ability to present Ag. Together these results demonstrate that a murine ICAM-1 homolog is expressed on A20 B cells, where it functions as a major cell interaction molecule. The degree of functional impairment in these mutant APC gives insight into the contribution of cell interaction molecules to efficient Ag presentation and T cell-B cell interaction. Finally, these results also demonstrate the feasibility of selecting APC with mutations affecting Ag presentation.
Evidence implicating utilization of different T cell receptor-associated signaling pathways by TH1 and TH2 clones.
Authors: Gajewski T F , Schell S R , Fitch F W
Published: December 2022

We have reported recently that high concentrations of anti-CD3 mAb inhibited IL-2-dependent proliferation of TH1 but not TH2 clones. The selective inhibitory effect on TH1 clones suggested that the two helper T lymphocyte subsets might utilize different TCR-associated signal transduction mechanisms. In the present study, we demonstrate that this distinction was not due to a gross difference in the level of TCR expression by TH1 and TH2 clones. Inhibition of TH1 proliferation by anti-CD3 mAb appeared to depend on calcium for maximal effect, suggesting that a substantial elevation of intracellular free calcium concentration ([Ca2+]i) might not occur after ligation of the TCR complex of TH2 clones. Calcium ionophore inhibited IL-2-dependent proliferation of both subsets, suggesting that receptor/ligand systems which stimulate elevated [Ca2+]i would be expected to inhibit proliferation. Although elevated [Ca2+]i and generation of inositol phosphates were readily detected in TH1 clones, these second messengers were not detected following stimulation of TH2 clones via the TCR complex. In addition, lymphokine production by TH1 clones was more sensitive to inhibition by cholera toxin, 8-bromoadenosine 3':5'-cyclic monophosphate, and cyclosporin A than was lymphokine production by TH2 clones. Collectively, these results suggest that TH1 and TH2 clones utilize distinct TCR-associated signal transduction mechanisms for lymphokine gene expression. The difference in signaling mechanisms suggests a potential pharmacologic target for intervention in situations where inappropriate activation of TH1 or TH2 cells occurs in vivo.
Requirement of the thymus for the recovery of anti-alloantigen helper T cells from tolerance induced by intravenous presensitization with allogeneic cells.
Authors: Kitagawa S , Sato S , Azuma T , Hori S , Hamaoka T , Fujiwara H
Published: December 2022

Intravenous presensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) whole spleen cells and class II H-2-disparate B6-C-H-2bm12 (bm12) spleen cells depleted of APC resulted in almost complete elimination of the respective anti-bm1 and anti-bm12 reactivities. However, the reduced alloreactivities as assessed by Th cell capacities (proliferative responses and IL-2 production) were recovered around 8 wk after the i.v. presensitization in euthymic B6 mice. In contrast, background levels of bm1- or bm12-specific reactivities were revealed to last more than 12 wk after the presensitization in B6 mice which had been thymectomized prior to the i.v. presensitization. Such a reduced alloreactivity was also observed in the capacity to reject bm1 or bm12 skin grafts, and prolongation of graft survival was strikingly enhanced in the thymectomized group compared to that induced in the nonthymectomized group. However, there was an important difference in such prolongation in the thymectomized hosts between bm1 and bm12 grafts; a considerable percentage (greater than 80%) of bm12 skin grafts continued to take more than 5 mo, whereas about 90% of bm1 grafts were rejected by around 5 mo along with the emergence of weak, but detectable anti-bm1 Th and cytotoxic T cell activities. These results indicate that 1) i.v. presensitization regimen is capable of eliminating in vitro and in vivo alloreactive capabilities but these alloreactivities can be recovered in euthymic hosts with T cell repopulating potential and 2) there are differential requirements of the thymus for repopulating anti-bm1 (Lyt-2+) and anti-bm12 (L3T4+) T cell activities.
Monoclonal antibodies block murine IL-4 receptor function.
Authors: Beckmann M P , Schooley K A , Gallis B , Vanden Bos T , Friend D , Alpert A R , Raunio R , Prickett K S , Baker P E , Park L S
Published: December 2022

IL-4 is a cytokine which can induce B-lymphocyte proliferation, increase cell-surface Ia expression, and induce some activated B cells to differentiate and begin to secrete IgE. IL-4 binds specifically to a cell-surface receptor (IL-4R) on cells from a variety of lineages including T and B cells. In general both primary cells and in vitro cell lines express less than 5000 receptors per cell. Utilizing a subclone of the cytotoxic T cell line CTLL-2 expressing a high level of IL-4R, mAb against the murine IL-4R were prepared. Two mAb have been identified which have different properties. These antibodies, designated M1 and M2, recognize sequences specific to the murine IL-4R. Immunoprecipitation studies with M1 and M2 on CTLL-2 cells have identified the receptor as a Mr = 145,000 cell-surface protein. Similar results have been obtained with the recently isolated full length murine IL-4R cDNA expressed in COS-7 cells. In addition the antibodies are capable of inhibiting IL-4 binding. One antibody, M1, is also a potent inhibitor of IL-4-induced proliferation. These antibodies will be useful in dissecting a wide array of activities attributed to IL-4.
Transcriptional modulation of human IL-6 gene expression by verapamil.
Authors: Walz G , Zanker B , Barth C , Wieder K J , Clark S C , Strom T B
Published: December 2022

Calcium channel-blocking agents interfere with the initial increase of cytosolic calcium that follows mitogenic stimulation of T lymphocytes. In cultures of mitogen-stimulated PBMC, verapamil also blocked T cell accumulation of cytoplasmic IL-2-encoding mRNA. In sharp contrast, the addition of verapamil to PHA and PMA-stimulated PBMC augmented the mitogen-stimulated increases in nuclear transcription of IL-6-encoding mRNA, steady state levels of IL-6 encoding mRNA, and release of IL-6 bioactivity. These experiments indicated that an increased IL-6 transcriptional rate rather than stabilization of transcripts accounted for the increased cytoplasmic IL-6 mRNA levels and subsequent expression of IL-6 bioactivity. These effects were not produced by nicardipine, another potent calcium channel blocker, or EGTA. We suggest that a non-calcium-dependent, IL-6 regulatory factor, absent or inactive in verapamil-treated cultures, inhibits IL-6 gene activation in mitogen-stimulated PBMC. Failure to express this inhibitory factor would result in IL-6 gene superinduction at a transcriptional level.
T cell subsets and IFN-gamma production in resistance to systemic candidosis in immunized mice.
Authors: Cenci E , Romani L , Vecchiarelli A , Puccetti P , Bistoni F
Published: December 2022

In addition to previous evidence for the roles of T cell-dependent immunity and delayed-type hypersensitivity in acquired resistance to systemic candidosis in mice, in the present study we have investigated the relative contributions of L3T4+ and Lyt-2+ lymphocytes in the protective immunity induced by vaccination with low virulence Candida albicans cells. We have also addressed the issue of the mode of Candida Ag recognition by specific T cells leading to cytokine release. Spleen cells from immunized mice produced high levels of IFN-gamma in vitro in response to Candida Ag, and this activity was abolished only by the combined treatment of the responder population with anti-L3T4 and anti-Lyt-2.2 mAb plus C. Positively selected L3T4+ and Lyt-2+ cells also produced IFN-gamma in vitro, provided accessory cells (plastic-adherent and Thy-1- Ia- splenocytes, respectively) were added to the lymphocyte-yeast cell cocultures. The production of IFN-gamma by purified L3T4+ and Lyt-2+ cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to the cultures. In vivo, administration of anti-L3T4, anti-Lyt-2.2 mAb or a combination of both significantly impaired the resistance of immunized mice to challenge with virulent C. albicans, as manifested by increased recovery of the yeast from the mouse kidneys. A similar effect was observed upon neutralization of endogenous IFN-gamma by treatment with rat mAb. These results suggest that both L3T4+ and Lyt-2+ T cells play a role in acquired immunity to systemic C. albicans infection, and that their activity may involve IFN-gamma-mediated stimulation of candidacidal mechanisms.
Characterization and utilization of a monoclonal antibody inhibiting porcine natural killer cell activity for isolation of natural killer and killer cells.
Authors: Dato M E , Kim Y B
Published: December 2022

A mAb, porcine NK-inhibitory mAb (PNK-I) that inhibits porcine NK activity without affecting antibody-dependent cellular cytotoxicity (ADCC) has been developed. PNK-I acts at the level of the effector cell and inhibition of NK activity is independent of complement. Inhibitory effects are seen against various human and murine NK-susceptible targets. Addition of PNK-I antibody up to 60 min after assay initiation was effective at inhibiting NK activity. Furthermore PNK-I does not inhibit E:T conjugation and inhibits during the Ca2(+)-dependent phase of NK cytolysis. PNK-I Ag is present on virtually all PBL showing a bimodal distribution with 74% "dim" and 15% "bright" by flow cytometry. Monocytes and granulocytes stain with an intermediate intensity with greater than 90% and 95% staining positively, respectively. F(ab')2 fragments of PNK-I antibody show identical staining and functional activity as the whole molecule indicating that PNK-I acts independently of FcR. PNK-I immunoprecipitates molecules of molecular mass of 166, 155, 95 kDa under reducing and nonreducing conditions. PNK-I appears to be recognizing an epitope on a CD18 molecule. The CD18 molecule (beta-chain of CD11a,b,c) is ubiquitous on the surface of leukocytes and is implicated in a variety of cellular functions. Dim and bright populations were sorted and assessed functionally for NK and ADCC activity. It is demonstrated that PNK-I+ bright lymphocytes contain all detectable NK and ADCC activity in porcine PBL. Furthermore PNK-I+ bright lymphocytes contain the cytokine responsive NK cells capable of stimulation by IL-2, porcine NK-activating factor, and porcine natural killer-enhancing mAb. PNK-I+ dim cells were devoid of all baseline as well as inducible NK and ADCC activity. Giemsa stain of sorted populations show PNK-I+ bright cells containing the large granular lymphocytes whereas dim are devoid of these. Two color analysis show that PT4+ cells are PNK-I+ dim whereas PT8+ lymphocytes are divided between PNK-I+ bright and dim populations. Our results indicate that we are able to isolate all active as well as inducible NK and ADCC effector cells from porcine PBL based on relative Ag expression of CD18. Therefore quantitative as well as qualitative antigen expression is important in NK/ADCC-mediated cytotoxicity.
Specificity and frequency of primary anti-HLA cytotoxic T lymphocytes in normal and HLA-B27.2-, HLA-B27.5-, and HLA-Cw3-transgenic mice. A transgenic model for MHC xenoantigen recognition.
Authors: Kievits F , Boerenkamp W J , Lokhorst W , Ivanyi P
Published: December 2022

Previous studies have shown that the lymphocytes of naive mice produce a strong primary CTL responses in vitro to human MHC class I Ag presented by HLA-transgenic mouse (TGM) cells. A limiting dilution (LD) assay was used to analyze this xenoreactive CTL repertoire in mice. Frequencies of HLA class I-specific CTL precursors (CTLp) were estimated in naive normal and HLA-B27.2-, -B27.5- and HLA-Cw3-double TGM (i.e., mice expressing HLA and human beta 2-microglobulin (hu beta 2m]. The xenoreactive CTLp frequencies were compared to frequencies of CTLp to H-2 alloantigens estimated in naive normal mice. The results showed that the frequencies of HLA class I-specific CTLp are comparable with those of alloreactive CTLp. This overlap in CTLp frequencies suggests that HLA class I xenoantigens are recognized by primary mouse CTL as allelic variants of H-2K and H-2D. This was confirmed in split well analysis by the observation that the xenoreactive response was not restricted by self-MHC of the responding mouse. Thus, primary HLA class I-specific mouse CTL clones recognized their target Ag regardless of whether they were expressed on H-2-mismatched mouse cells or on human cells. The frequencies of HLA class I-specific CTLp in HLA-TGM were comparable to those in normal mice. We propose that MHC allo- and xenoreactive CTL responses are not caused by the activation of CTLp specific for self-MHC plus peptide but to the activation of CTLp recognizing MHC allo- and xenoantigens directly or as peptides presented by their native MHC molecules.
Ligand binding to the LFA-3 cell adhesion molecule induces IL-1 production by human thymic epithelial cells.
Authors: Le P T , Vollger L W , Haynes B F , Singer K H
Published: December 2022

We have shown that human thymic epithelial (TE) cells produce IL-1 alpha, IL-1 beta, and TE cells bind to thymocytes by CD2 and LFA-1 molecules on thymocytes and LFA-3, ICAM-1 on TE cells. We investigated whether ligand binding to LFA-3 on human TE cells can modulate TE cell IL-1 production. First, we investigated the ability of human thymocytes to regulate IL-1 release by TE cells. Both autologous and allogenic emetine-treated thymocytes when cultured with TE cells augmented IL-1 release by TE cells. The augmentation of IL-1 release was cell density dependent. Inasmuch as the interaction between thymocytes and TE cells is mediated in part by CD2 molecules on thymocytes and LFA-3 molecules on TE cells we next determined the effect on IL-1 release of ligand binding (anti-LFA-3 mAb TS2/9) to TE cell surface LFA-3. Purified anti-LFA-3 mAb augmented IL-1 release in a concentration-dependent fashion. The anti-LFA-3-mediated augmentation of IL-1 release required both new protein and RNA synthesis as shown by the ability of cycloheximide and actinomycin-D to inhibit augmentation of IL-1 production by TE cells, and by direct quantitation of IL-1 alpha and IL-1 beta mRNA by Northern blot analysis. Both F(ab)'2 and Fab' fragments of anti-LFA-3 mAb augmented IL-1 alpha and IL-1 beta mRNA production, indicating that monovalent binding to cell surface LFA-3 was sufficient to provide the inducing signal. The identification of LFA-3, the cell surface ligand for thymocyte CD2 molecules, as a molecule via which TE cell-derived cytokine production may be regulated suggests a mechanism at the cell surface by which direct TE cell-thymocyte interaction might result in the triggering of local IL-1 release within the human thymic microenvironment.
Evidence for tumor necrosis factor-alpha involvement in the optimal induction of class I allospecific cytotoxic T cells.
Authors: Robinet E , Branellec D , Termijtelen A M , Blay J Y , Gay F , Chouaib S
Published: December 2022

We have investigated the functional interaction between IL-2 and TNF on the generation of alloreactive CTL. The study was performed by using primary mixed cultures of lymphocytes from a MHC-recombinant sibling identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. Our data show that MHC class I disparity can trigger the induction of TNF receptor without promoting significant TNF production. Addition of exogenous TNF at the sensitizing phase of the primary mixed lymphocyte reaction did not result in CTL activation. However, when simultaneously added with IL-2, TNF could promote an optimal induction of cytotoxic T cell generation. The enhanced lytic ability of MHC class-I primed CTL by TNF was associated with a selective up-regulation of Tac Ag and subsequent amplification of cell proliferation. Furthermore, TNF was also found to induce a considerable increase in IL-2-induced intracellular benzyloxycarbonyl-L-lysine thiobenzylester-esterase activity by MHC class I-primed cells. TNF did not affect the expression of LFA1, CD2, CD4, and CD8, molecules that are associated with CTL-target interactions, on responder cells. These results extend our earlier observations on the role of class I MHC molecules that may function to transduce activation signals and suggest that TNF may be a potent mediator involved in the IL-2-induced acquisition of optimal lytic competence by precursor cytotoxic T cells.
IgA-induced avidity maturation of IgA Fc receptors on murine T lymphocytes.
Authors: Sandor M , Waldschmidt T J , Williams K R , Lynch R G
Published: December 2022

The analysis of 30 well characterized murine T lymphocyte populations using a cytofluorometric IgA binding assay has identified many populations that are constitutive and/or inducible for IgA receptor expression, and has identified two distinct mechanisms by which IgA up-regulates the IgA-binding properties of murine T cells. Studies with lymphomas, hybridomas, Ag-specific clones and activated normal splenic T cells identified many examples of CD4 and CD8 lineage cells that constitutively expressed IgA receptors. T cell populations that constitutively expressed IgA receptors exhibited enhanced IgA binding after incubation with oligomeric IgA for 18 h. The IgA-induced up-regulation of IgA binding resulted from two distinct processes: 1) an increase in the number of surface membrane IgA binding sites and 2) an increase in the avidity of IgA binding without a change in the number of binding sites. The IgA-induced avidity increase was reflected by a 5- to 10-fold decrease in the apparent Kd. Depending on the T cell population examined the enhanced binding of IgA involved one or both of these mechanisms. T cell populations that did not constitutively express IgA receptors failed to bind IgA after prolonged incubation with oligomeric IgA suggesting that if such cells can express IgA receptors they require other signals to induce their expression. Consistent with this possibility is the finding that resting splenic T cells did not bind IgA but their activation with Con A or mAb anti-T3 resulted in high level expression of IgA receptors. These studies have identified multiple distinct mechanisms that alter the IgA-binding properties of murine T cells and are discussed in terms of their possible physiologic significance.
In vivo T lymphocyte origin of macrophage-tropic strains of HIV. Role of monocytes during in vitro isolation and in vivo infection.
Authors: Massari F E , Poli G , Schnittman S M , Psallidopoulos M C , Davey V , Fauci A S
Published: December 2022

Previously published isolation techniques with T cell blasts and monocyte-derived macrophages (MDM) were used to recover HIV from the PBMC of a group of 23 asymptomatic seropositive individuals. Viral isolation was more readily accomplished by MDM coculture resulting in 9 isolates being obtained exclusively by this method (macrophage tropic strains). To determine the in vivo cellular source of these isolates we separated PBMC from 5 of these 9 patients into T lymphocyte and monocyte fractions by flow microfluorometry. These fractions were then analyzed by polymerase chain reaction (PCR) for the presence of HIV-1 proviral DNA. In 4 out of these 5 patients HIV-1 proviral DNA could be detected exclusively in T lymphocytes but not in monocytes, although the virus could be isolated only by MDM coculture. In the remaining patient HIV could be amplified in both T lymphocytes and monocytes. Further phenotypic analysis revealed that, among T lymphocytes, only the CD4+ subset was infected with HIV. We conclude that among PBMC the most common in vivo source of HIV strains which preferentially infect macrophages in vitro is the CD4+ T lymphocyte. These data also suggest that the macrophage tropism characteristic of some HIV strains reflects predominantly an in vitro phenomenon.
Evidence for compartmentalization of functional subsets of CD2+ T lymphocytes in atopic patients.
Authors: Wierenga E A , Snoek M , de Groot C , Chrétien I , Bos J D , Jansen H M , Kapsenberg M L
Published: December 2022

Lymphokine secretion profiles were studied of human allergen-specific CD4+ T lymphocyte clones (TLC). To this aim, panels of house dust mite Dermatophagoides pteronyssinus (Dp)-specific TLC were generated from two atopic Dp-allergic patients, suffering from severe atopic dermatitis (AD1) and allergic asthma (AD2), respectively, and from a non-atopic individual (NAD). From AD1 additional TLC were cloned specific for tetanus toxoid or Candida albicans, both Ag that were not relevant for the atopic state of this patient. Secretion of IL-2, IL-4, and IFN-gamma was determined after specific stimulation of these TLC, using autologous monocytes as APC. With respect to the production of IL-4 and IFN-gamma, clearly distinct profiles were observed. All Dp-specific TLC from both atopic donors produced IL-4 but not IFN-gamma, whereas the Dp-specific TLC from NAD, as well as the tetanus toxoid- and C. albicans-specific TLC from AD1, all produced IFN-gamma but not or small quantities of IL-4. Most TLC from all panels produced IL-2. These lymphokine profiles were consistent for at least 3 days and were neither dependent on the dose of allergen nor on the atopic or nonatopic state of the donor of APC. The functional consequence of these restricted lymphokine profiles was stressed by the observation that, whereas Dp-specific TLC from AD1 and AD2 supported in vitro IgE production, this support could be abrogated by a Dp-specific TLC from NAD. The present results suggest that CD4+ T lymphocytes that produce IL-4, but not IFN-gamma, occur in high frequencies in the allergen-specific T cell repertoires of atopic donors, which may have important implications for the pathomechanism of atopic disease.