bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of experimental allergic encephalomyelitis in mice.
Authors: Baker D , Rosenwasser O A , O'Neill J K , Turk J L
Published: December 2022

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that exhibits many pathologic similarities with multiple sclerosis. While products of the MHC are known to control the development of EAE, it is clear that non-MHC products also influence susceptibility. The chromosomal locations of these were investigated in selective crosses between MHC class II-compatible, EAE-susceptible Biozzi ABH, and low responder nonobese diabetic (NOD) mice. The disease was dominant and highly influenced by gender in the backcross one (BC1) generation. Female mice were significantly more susceptible than male mice. Segregation of disease frequency of female animals in this cross suggested that EAE was controlled by a major locus. Although microsatellite-based exclusion mapping indicated that a number of regions on chromosomes 5, 6, 7, 8, 9, 10, 11, 12, 13, and 18 showed evidence of linkage (p < 0.05) compared with expected random distributions of alleles, disease susceptibility was most strongly linked (p < 0.001) to chromosome 7. However, by selectively analyzing animals that were either severely affected or almost normal, additional susceptibility loci were mapped on chromosomes 18 and 11 that were linked (p < 0.001) to resistance and the development of severe disease, respectively. The data indicate a major locus on chromosome 7, affecting initiation and severity of EAE that is probably modified by several other unlinked loci. These localizations may provide candidate loci for the analysis of human autoimmune-demyelinating disease.
Cytokine patterns during progression to AIDS in children with perinatal HIV infection.
Authors: Hyjek E , Lischner H W , Hyslop T , Bartkowiak J , Kubin M , Trinchieri G , Kozbor D
Published: December 2022

Patterns of cytokine expression were analyzed in polyclonal and antigenic responses in children with perinatal HIV infection. Responses of PBL to PMA and A23187 calcium ionophore studied in patients in different stages of HIV infection revealed reduced levels of IL-2 in HIV-infected children beginning before 6 mo of age, and age-dependent increases in expression of IL-4, IL-10, and IFN-gamma. The levels of IL-4, IL-10, and IFN-gamma expression did not differ significantly between HIV-infected and age-matched uninfected children of HIV-seropositive mothers, except for a small reduction in HIV-infected children in late stages of infection. Responses to PHA, HLA alloantigens, HIV envelope peptides T1 and P18, and tetanus toxoid were studied in PBMC derived from asymptomatic and mildly symptomatic HIV-infected children. IL-2, IFN-gamma, IL-4, and IL-5 expression was detected in PHA-stimulated PBMC from all analyzed patients. HIV-infected children who failed to respond to HLA alloantigens, tetanus toxoid, or the envelope peptides had lower numbers of CD4+ cells and expressed, on PHA stimulation, higher levels of IL-4 and IL-5 and lower levels of IL-2 and IFN-gamma than patients who responded to the antigenic stimulation. Results of these analyses suggest that cytokine expression in HIV-infected children depends on the character of the stimuli as well as the phenotype of PBMC, and indicate possible prevalence of Th2 Ag-specific responses during the progression of HIV-induced immunodeficiency.
Both a precursor and a mature population of dendritic cells can bind HIV. However, only the mature population that expresses CD80 can pass infection to unstimulated CD4+ T cells.
Authors: Weissman D , Li Y , Orenstein J M , Fauci A S
Published: December 2022

Dendritic cells (DC) are the principle APC involved in primary immune responses; their major function is to obtain Ag in tissues, migrate to lymphoid organs, and activate T cells. DC are also the first immune cells to arrive at sites of inflammation on mucous membranes, the major site of sexual transmission of HIV. We have demonstrated previously that three populations of cells that can develop a dendritic morphology are present in peripheral blood. Two of these populations can express CD83, a marker of DC, and appear to be at different stages of maturation: 1) a precursor population and 2) a mature immunostimulatory DC. Precursor-derived DC express high levels of CD86 (B7-2) and HLA-DR but no CD80 (B7-1), whereas mature DC have high levels of expression of all three markers. Mature DC in peripheral blood bind HIV to their surface and induce infection when added to autologous CD4+ T cells in the absence of added stimuli, such as mitogens. These mature DC, when isolated directly from peripheral blood, appear to be conjugated to T cells, and these conjugates are infected easily and productively with HIV. These findings suggest a role for DC in early HIV infection in which they bind virus and interact with T cells locally or after migrating to a lymphoid organ, thus establishing a productive infection. Furthermore, they likely play a role in the propagation of HIV infection by activating T cells in the presence of HIV, which leads to viral replication and immune cell destruction.
The Ig-related killer cell inhibitory receptor binds zinc and requires zinc for recognition of HLA-C on target cells.
Authors: Rajagopalan S , Winter C C , Wagtmann N , Long E O
Published: December 2022

Members of the Ig superfamily are predominantly receptors that mediate interactions between cells or provide signals to cells when binding specific ligands. Here we describe an Ig-related receptor that requires zinc for its function. Killer cell inhibitory receptors (KIR) belonging to the Ig superfamily mediate inhibition of NK cells upon recognition of HLA-C molecules on target cells. An abundance of histidine residues in the first extracellular domain of KIR, including the signature zinc binding motif HEXXH, suggested that this receptor may bind zinc. Two distinct KIR molecules that mediate recognition of HLA-Cw4 and -Cw8, respectively, bound specifically to zinc affinity columns. Furthermore, addition of the zinc chelator 1,10-phenanthroline during chromium release assays reversed the inhibition of killing by NK clones specific for HLA-Cw4 or HLA-Cw8, demonstrating that zinc is necessary for the inhibitory function of KIR. Such functionally relevant zinc binding has not been described for other members of the Ig superfamily and may represent a novel regulatory mechanism for Ag receptor-ligand interactions.
Divergent induction of apoptosis and IL-8 secretion in HT-29 cells in response to TNF-alpha and ligation of Fas antigen.
Authors: Abreu-Martin M T , Vidrich A , Lynch D H , Targan S R
Published: December 2022

Colonic epithelial cell injury is the common manifestation of inflammatory diseases of the bowel. One form of epithelial injury is apoptosis. In our study, we investigated the mechanism leading to apoptosis in HT-29 cells in response to TNF-alpha and ligation of Fas Ag. HT-29 displayed a dual response to TNF-alpha and Fas Ag ligation: in combination with IFN-gamma, HT-29 cells underwent apoptosis, whereas independently, these factors stimulated secretion of IL-8. We used this model of immune-mediated epithelial cell injury to elucidate the signals leading to apoptosis in response to TNF-alpha and Fas Ag ligation compared with the signals leading to induction of IL-8 secretion. The model was further used to distinguish signaling differences between TNF-alpha receptors and the Fas Ag in this cell line. The experiments presented here demonstrate that Fas Ag ligation alone led to production of IL-8 by colonic epithelial cells and represented another function mediated by Fas Ag in addition to apoptosis. This study shows that the pathways leading to cell death and IL-8 production in response to Fas Ag ligation and TNF-alpha were similar with regard to their requirements for new gene expression, protein synthesis, and protein kinase activity. Specifically, new gene expression and protein synthesis were not necessary for TNF-alpha- and Fas Ag-mediated apoptosis, but were necessary for TNF-alpha- and Fas Ag-mediated IL-8 secretion. Tyrosine protein kinase phosphorylation was necessary to signal secretion of IL-8 in response to both agonists but it was not necessary for apoptosis. In spite of the similarities between these two agonists, the kinetics of apoptosis via Fas Ag were significantly more rapid than through the TNF-alpha receptor and serve to distinguish these two signals.
A nonlethal conditioning approach to achieve durable multilineage mixed chimerism and tolerance across major, minor, and hematopoietic histocompatibility barriers.
Authors: Colson Y L , Wren S M , Schuchert M J , Patrene K D , Johnson P C , Boggs S S , Ildstad S T
Published: December 2022

Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic (A+B-->A) bone marrow results in multilineage mixed allogeneic chimerism, donor-specific transplantation tolerance, superior immunocompetence and resistance to graft-vs-host disease. However, the morbidity and mortality associated with lethal irradiation would be a major limitation to the clinical application of chimerism to induce tolerance for solid organ grafts or treat other nonmalignant hematologic diseases. We report here that durable multilineage mixed allogeneic chimerism and donor-specific transplantation tolerance for skin and primarily vascularized allografts can be achieved across multiple histocompatibility barriers using a nonmyeloablative radiation-based approach. The percentage of B10 mouse recipients that engrafted directly correlated with the degree of disparity between donor and recipient and the dose of total body irradiation administered. Although the occurrence of engraftment following conditioning with doses of total body irradiation of > or = 600 cGy was similar for animals receiving bone marrow disparate at MHC or MHC, minor and hematopoietic (Hh-1) loci (67% vs 78%), the level of donor chimerism was significantly less when multiple histocompatibility barriers were present (94.6 +/- 3.8% vs 37.5 +/- 12.5%). Treatment of the recipient with cyclophosphamide 2 days following allogeneic bone marrow transplantation reduced the dose of radiation sufficient for reliable engraftment to only 500 cGy of total body irradiation, regardless of MHC and Hh-1 disparity. Donor chimerism was stable and present in all lineages, with production of lymphoid (T and B cell), NK, and myeloid (erythrocyte, platelet, granulocyte, and macrophage) cells. Mixed chimeras exhibited donor-specific tolerance in vitro, as assessed by mixed lymphocyte culture (MLR) and cytotoxicity (CML) assays, and in vivo to skin and primarily vascularized cardiac allografts. The observation that engraftment and tolerance can be achieved across multiple histocompatibility barriers using nonmyeloablative recipient conditioning may allow allogeneic bone marrow transplantation to be applied to nonmalignant disease states in which lethal conditioning cannot be justified, including the induction of donor-specific tolerance for solid organ transplantation and the treatment of hemoglobinopathies and enzyme deficiency states.
Peptide-induced anergy in allergen-specific human Th2 cells results in lack of cytokine production and B cell help for IgE synthesis. Reversal by IL-2, not by IL-4 or IL-13.
Authors: Fasler S , Aversa G , Terr A , Thestrup-Pedersen K , de Vries J E , Yssel H
Published: December 2022

The induction of anergy in T cells is believed to be the result of triggering of the TCR in the absence of adequate costimulation mediated through the interaction of CD28 and its ligands, CD80 and CD86. Here, we demonstrate that stimulation of human group I allergen in Dermatophagoides pteronyssinus extract (Der p 1)-specific CD4+ Th2-like T cell clones with Der p 1-derived peptides in the absence of professional APC results in a state of nonresponsiveness. The induction of anergy occurred despite the expression of high levels of CD28, CD80, and CD86 on the surface of the T cell clones and was not prevented by the addition of anti-CD28 mAb. The anergic, Der p 1-specific, Th2 cells failed to mobilize calcium from intracellular stores, to proliferate, and to produce IL-2, IL-4, IL-13, GM-CSF, and TNF-alpha following optimal stimulation with Der p 1-derived peptide and autologous APC. However, they mobilized intracellular calcium following stimulation with Ca(2+)-ionophore and produced all of the above cytokines, including IFN-gamma, when stimulated with phorbol ester and Ca2+ ionophore. These results indicate that the anergic T cell clones are capable of responding to signals circumventing the TCR/CD3 complex activation pathway. In contrast to T cell clones optimally activated with peptide and APC, anergic T cells failed to induce IgG4 and IgE synthesis when cocultured with B cells, even in the presence of exogenous IL-4 or IL-13. Anergic T cells expressed normal levels of CD40L, suggesting that their inability to help in Ig production by B cells is due to conditions other than a lack of expression of this molecule. Finally, exogenous IL-2 restored the helper function of anergic Th2 T cells for IgE production by B cells, which was greatly enhanced by the addition of IL-4 or IL-13. These data suggest that induction of anergy in allergen-specific Th2 T cells by allergen-derived peptides may play an important role in the successful desensitization of allergic patients.
Removal of the majority of epidermal Langerhans cells by topical or systemic steroid application enhances the effector phase of murine contact hypersensitivity.
Authors: Grabbe S , Steinbrink K , Steinert M , Luger T A , Schwarz T
Published: December 2022

Whereas epidermal Langerhans cells (LC) are thought to be the principal APCs for initiation of contact hypersensitivity (CHS) responses, their role as APC in the effector phase of CHS is still unclear. It is currently thought that LC elicit the CHS response by presenting Ag to trafficking Ag-specific T cells within the skin. To test this hypothesis, we removed the majority of resident LC at the site of CHS challenge by topical application of various steroid creams to one ear in BALB/c mice (> 85% LC depletion). Either 2 days before or 4 days after steroid treatment of the ear, mice were sensitized on the abdomen with the hapten trinitrophenyl and challenged 10 days later at the steroid-pretreated ear. At that time point, direct anti-inflammatory effects of the steroid were no longer present. Surprisingly, CHS responses were markedly stronger at the sites of prior steroid application when compared with vehicle-treated controls, indicating that depletion of most of the resident LC not only fails to impair, but enhances, the expression of CHS significantly. UV irradiation or application of croton oil at the challenge site, as well as systemic steroid application, all of which are alternative methods of diminishing the number of epidermal LC, also significantly up-regulated CHS. In contrast, irritant dermatitis and sensitization or elicitation of CHS in steroid-treated mice at distant sites, as well as delayed-type hypersensitivity responses against the same hapten, were unaffected by topical steroid pretreatment. In conclusion, our data suggest that resident LC are not the relevant APC in the effector phase of CHS and that they may even provide down-regulatory, rather than stimulatory, signals during elicitation of CHS.
Administration of recombinant human IL-1 by Staphylococcus enterotoxin B prevents tolerance induction in vivo.
Authors: Nakata Y , Matsuda K , Uzawa A , Nomura M , Akashi M , Suzuki G
Published: December 2022

Peripheral tolerance is important to prevent autoimmunity of T cells against tissue-specific autoantigens in peripheral organs. In certain pathologic situations, peripheral tolerance breaks for unknown reasons and autoimmune diseases occur. Anergy is one of the mechanisms of peripheral tolerance that down-modulates IL-2 synthesis and IL-4 responsiveness by helper T cell clones. In this report, we utilized a model system in which V beta 8+ CD4 T cells were anergized by administration of high doses of Staphylococcus enterotoxin B (SEB) in vivo and investigated an effect of recombinant human (rh) IL-1 on the tolerance induction. RhIL-1 was used because of its ability to induce IL-4 responsiveness in T cells. When rhIL-1 was administered within 24 h after SEB inoculation, the cytokine interfered with tolerance induction; V beta 8+CD4 T cells from mice that had been treated with both SEB and IL-1 proliferated in response to SEB and produced IL-2, IL-4, and IFN-gamma upon TCR/CD28 cross-linking. Delayed administration of rhIL-1 by 48 h failed to do this; T cells did not proliferate in response to SEB, but they retained an ability to produce IL-4 upon TCR/CD28 cross-linking. Administration of rhIL-1 induced better proliferation of V beta 8+CD4 T cells in response to SEB in vivo but did not prevent cell death after proliferation. These results suggest a potential role of inflammatory cytokine IL-1 in the course of autoimmunity via interference with tolerance.
IL-4 induces germ-line IgE heavy chain gene transcription in human fetal pre-B cells. Evidence for differential expression of functional IL-4 and IL-13 receptors during B cell ontogeny.
Authors: Punnonen J , Cocks B G , de Vries J E
Published: December 2022

The present study demonstrates that IL-4 induces germ-line IgE heavy chain (epsilon) gene transcription in human fetal splenic mononuclear cells; fetal bone marrow cells; highly purified sorted surface (s) mu+, CD10+, CD19+ immature B cells; and s mu-, cytoplasmic mu+, CD10+, CD19+ pre-B cells derived from human fetal bone marrow. Similar to observations in normal adult B cells, TGF-beta and IFN-gamma inhibited IL-4-induced germ-line epsilon RNA synthesis in fetal pre-B cells, whereas anti-CD40 mAbs and TNF-alpha had enhancing effects, suggesting that the general mechanisms regulating germ-line epsilon transcription in adult B cells and pre-B cells are similar. IL-13 also induced germ-line epsilon RNA synthesis in s mu+, CD10+, CD19+ immature B cells, but the level of transcription induced by IL-13 was significantly less than that induced by IL-4. Anti-CD40 mAbs strongly synergized with both IL-4 and IL-13 in inducing germ-line epsilon RNA synthesis by fetal immature B cells. Interestingly, IL-13 failed to induce germ-line epsilon RNA synthesis in s mu- pre-B cells even in the presence of anti-CD40 mAbs. These distinct effects of IL-4 and IL-13 suggest that functional IL-13R are expressed at a later stage of B cell ontogeny than IL-4R, and that IL-13, in contrast to IL-4, does not regulate pre-B cell differentiation. Given the fact that IL-4 production appears to be enhanced in atopic individuals, the capacity of IL-4 to induce germ-line epsilon transcription in human fetal immature B cells and pre-B cells suggests that commitment of B cell precursors to IgE-producing cells may occur during intrauterine life and may explain the increased IgE production in neonates with a family history of atopy.
Entry of B cell antigen receptor and antigen into class II peptide-loading compartment is independent of receptor cross-linking.
Authors: Song W , Cho H , Cheng P , Pierce S K
Published: December 2022

The processing and presentation of Ag by B lymphocytes are initiated by Ag binding to the B cell Ag receptor (BCR). Using subcellular fractionation, we recently identified a compartment in B cells in which functional, processed Ag-class II complexes are formed following BCR-mediated Ag internalization, referred to as the peptide-loading compartment. These studies, however, did not address the transport of Ag or BCR from the cell surface to the peptide-loading compartment. In this work, we describe the intracellular trafficking of Ag and surface Ig (sIg) in B cells and evaluate the effect of cross-linking sIg on this intracellular movement. We show that sIg constitutively transports Ag from the plasma membrane, through endosomes, to the MHC class II peptide-loading compartment. The cross-linking of the BCR increases the rate of internalization of sIg and bound Ag, but does not alter the trafficking pathway. Thus, the delivery of Ag to the class II peptide-loading compartment by the sIg is independent of BCR cross-linking, but can be influenced by BCR cross-linking.
Cloning and characterization of a mouse IL-12 receptor-beta component.
Authors: Chua A O , Wilkinson V L , Presky D H , Gubler U
Published: December 2022

Using DNA cross-hybridization, we have isolated and characterized cDNA clones encoding a mouse (mo) IL-12R beta component. Two forms of cDNA were found. The first form encodes a receptor protein that has an overall structure very similar to that of the known human (hu) IL-12R beta with 54% amino acid identity, whereas in the second type of mouse cDNA, the equivalent of the transmembrane region has been deleted. This presumed alternative splicing event also gives rise to a frame shift that results in a receptor with an identical extracellular domain but a different C-terminal sequence. Whether this alternative C terminus is capable of signaling is not yet known. Both types of receptors when expressed in COS-7 cells are membrane associated and bind moIL-12 with an affinity of 1 nM, similar to the affinity of huIL-12R beta for huIL-12. The monomeric size of both moIL-12R beta proteins is about 100 kDa. Similar to huIL-12R beta, moIL-12R beta is expressed at the surface of COS-7 and Ba/F3 cells as a dimer/oligomer whose formation is independent of IL-12 binding. When expressed in Ba/F3 cells, moIL-12R beta binds moIL-12 with two affinities of 50 and 470 pM, corresponding to the medium and high affinity IL-12 binding sites previously identified on mouse Con A lymphoblasts. Despite the higher affinity displayed by the moIL-12R beta in Ba/F3 cells, this receptor alone is not sufficient to transduce a signal, suggesting that another subunit is probably required to generate a functional moIL-12R complex.
Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.
Authors: Moreno C S , Emery P , West J E , Durand B , Reith W , Mach B , Boss J M
Published: December 2022

The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.
Influence of coding-end sequence on coding-end processing in V(D)J recombination.
Authors: Nadel B , Feeney A J
Published: December 2022

The large diversity of the Ig and TCR repertoires is accounted for by combinatorial assembly of the germ-line-encoded V, D, and J gene segments, as well as extensive modification at the junctions during the recombination process. Those modifications, termed coding-end processing, consist of removal and addition of an apparently random number of nucleotides. To obtain further insights into the mechanism of the coding-end processing, we constructed a large data base of several Ig and TCR coding ends obtained in vivo, using conditions that avoid potential bias by cellular selection events. We show that the processing patterns are not random, but rather specific for each coding end, suggesting that specific motifs in the coding-end sequence influence the processing. We found a good correlation between the presence of internal stretches of at least three A.T nucleotides, absence of stretches of G.C nucleotides, and high average nucleotide deletion. Based on a detailed analysis of the processing patterns, we propose that nicks of the hairpin intermediate take place preferentially in potential open structures formed by weaker pairings of A.T stretches. Together, these findings indicate that the sequence of the coding end plays an important role in nonrandom aspects of the recombination mechanism. This suggests that coding-end sequences might have been selected throughout evolution to participate in an early control of the development of the primary repertoire.
Multiple proteins physically interact with PU.1. Transcriptional synergy with NF-IL6 beta (C/EBP delta, CRP3).
Authors: Nagulapalli S , Pongubala J M , Atchison M L
Published: December 2022

PU.1 is a transcription factor that belongs to the ets family of DNA binding proteins. In this study, we show by Far Western blot analyses that multiple nuclear proteins are capable of physically interacting with PU.1. Using radiolabeled PU.1 protein as a probe, we screened a B cell cDNA expression library and isolated a number of clones encoding PU.1 interacting proteins. Three of these clones encode DNA binding proteins (NF-IL6 beta, HMG I/Y, and SSRP), one clone encodes a chaperone protein, and another clone encodes a multifunctional phosphatase. We have characterized the physical and functional interactions between PU.1 and NF-IL6 beta, a leucine zipper transcription factor implicated in inflammatory responses. We found that deletion of the carboxyl-terminal 28 amino acids of PU.1 disrupted PU.1-NF-IL6 beta physical interaction. This deletion disrupts the PU.1 Ets domain. Deletion of the NF-IL6 beta leucine zipper domain also greatly diminished the interaction between these two proteins. In transient expression assays, we found that PU.1 and NF-IL6 beta can functionally cooperate to synergistically activate transcription. Electrophoretic mobility shift assays showed that PU.1 and NF-IL6 beta can simultaneously bind to adjacent DNA binding sites, but apparently do not influence the kinetics or affinity of each other's DNA binding. These results suggest that transcriptional synergy is due to each protein independently influencing the basal transcription complex.
Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues.
Authors: Shastri N , Serwold T , Gonzalez F
Published: December 2022

Processing of intracellular proteins yields 8-11 residue peptides that are displayed on the APC surface as peptide/MHC class I complexes. The remarkably precise excision of antigenic peptides from their precursor polypeptides raises the question of whether specific flanking residues influence presentation efficiency. Here we addressed this question by analyzing the generation of OVA/Kb or influenza nucleoprotein/Db complexes in APC expressing precursors with varying N- or C-terminal flanking residues. We find that T cell responses were not significantly affected by varying the N-terminal flanking residue in the precursors. In contrast, presentation of peptide/MHC complexes was inhibited with the addition of a single C-terminal flanking residue. The most dramatic inhibition was observed with isoleucine, leucine, cysteine, and proline as the C-terminal flanking residues. These residue-specific variations in presentation activity could not be accounted for by differences in the stimulatory activity of corresponding synthetic peptides but were proportional to the relative amounts of naturally processed peptides recovered in the cell extracts. These findings suggest differences in the susceptibility of N- vs C-terminal flanking residues to proteolytic cleavage during Ag processing. The strong influence of specific C-terminal flanking residue(s) could be an important factor affecting the choice of peptides presented to T cells on the APC surface.
Host cell-derived complement control proteins CD55 and CD59 are incorporated into the virions of two unrelated enveloped viruses. Human T cell leukemia/lymphoma virus type I (HTLV-I) and human cytomegalovirus (HCMV).
Authors: Spear G T , Lurain N S , Parker C J , Ghassemi M , Payne G H , Saifuddin M
Published: December 2022

The current study was undertaken to determine whether the human T cell leukemia/lymphoma oncovirus type I (HTLV-I) and the herpesvirus human cytomegalovirus (HCM) incorporate host cell-derived C regulatory proteins. Our experiments showed that both CD59 and CD55 were associated with the external membrane of HTLV-I derived from MT2 cells, since virus could be captured by mAbs to these proteins, and antisera to CD55 and CD59 induced C-mediated lysis of HTLV-I virions. Additionally, both CD55 and CD59 were detected by immunoblot analysis of purified HTLV-I. Purified HCMV produced in human foreskin fibroblasts (HFF) also contained both CD55 and CD59, as detected by immunoblot analysis. However, treatment with anti-CD55, but not anti-CD59, reduced the HCMV infectious titer in the presence of C. Additional studies determined whether HTLV-I-associated CD55 and CD59 participated in the resistance of the virus to C-mediated lysis. Treatment of virus with phosphatidylinositol-specific phospholipase C (PI-PLC), which removes glycosylphosphatidylinositol-anchored CD55 and CD59, increased the sensitivity of HTLV-I to C-mediated destruction in the presence of anti-HTLV-I Abs. Reconstitution of PI-PLC-treated virus with purified CD55 and CD59 restored resistance to C. These experiments show that HTLV-I and HCMV acquire C control proteins from host cells. Together with our previous experiments showing that both CD55 and CD59 are present on HIV-1, these studies demonstrate a mechanism by which a variety of enveloped viruses may acquire resistance to C-mediated destruction.
Incorporation of a 35-kilodalton purified protein from Loxosceles intermedia spider venom transforms human erythrocytes into activators of autologous complement alternative pathway.
Authors: Tambourgi D V , Magnoli F C , Von Eickstedt V R , Benedetti Z C , Petricevich V L , da Silva W D
Published: December 2022

Cutaneous inoculation of Loxosceles spp. spider venoms produces local necrosis, occasionally accompanied by systemic intravascular clotting and hemolysis. In this work, we analyzed the role of the C system on the lysis of human erythrocytes (Eh) induced by Loxosceles venoms in vitro. Eh were treated with whole venom of Loxosceles laeta, Loxosceles gaucho, or Loxosceles intermedia, or with purified venom proteins, and incubated with C-sufficient (Cs-NHS) or C9-depleted autologous (C9d-NHS) serum. Hemolysis was determined spectrophotometrically, and deposition of C components or removal of C regulatory proteins was analyzed by FACS. Eh suspensions exposed to venoms or to a purified 35-kDa protein from L. intermedia were lysed after incubation with Cs-NHS, but not with C9d-NHS. Lysis was blocked by heating the serum at 50 degrees C or Ca2+/Mg2+ chelation by EDTA, but not by Ca2+ chelation with EGTA. Deposition of C1, C2, C3, C4, C5, and factor B on the venom-treated Eh occurred during activation of autologous C. Regulatory proteins decay-accelerating factor (DAF) and CD59 were not altered significantly. Conversion of C-resistant Eh into C-susceptible Eh by the L. intermedia venom was accompanied by incorporation of a 35-kDa venom protein onto the cell surface. Thirty-five-kilodalton-related proteins were detected in the two other Loxosceles venoms by ELISA, using rabbit antiserum against the L. intermedia 35-kDa protein. These data suggest that the C system mediates the lysis of human erythrocytes and, by extension, of other cell types able to incorporate the lytic factor of Loxosceles venoms on their cell surfaces.
Tumor-specific and HLA-A2-restricted cytolysis by tumor-associated lymphocytes in human metastatic breast cancer.
Authors: Linehan D C , Goedegebuure P S , Peoples G E , Rogers S O , Eberlein T J
Published: December 2022

A tumor-specific cytotoxic T lymphocyte (CTL) immune response has been well documented in melanoma, renal cell carcinoma, and ovarian cancer. Conflicting evidence exists regarding the existence of tumor-specific CTL populations in breast cancer. Tumor cells and tumor-associated lymphocytes (TAL) were isolated from the pleural effusions of six consecutive patients with metastatic breast cancer. After solid-phase anti-CD3 stimulation, TAL cultures were expanded with weekly autologous tumor stimulation and low-dose IL-2 for 3 wk. T cell populations were characterized using flow cytometric analysis and ranged from 49 to 91% CD8+, > 98% CD3+, and < 3% CD16+. Functionally, tumor-stimulated TAL showed tumor-specific recognition of autologous tumor cells (241 +/- 142 LU20/10(7)) and no detectable lysis of autologous fibroblasts, Daudi or K562. Cytotoxicity of TAL against HLA-A2+ allogeneic targets was significantly higher when compared with HLA-A2- tumor cell lines (127 +/- 76 vs 6 +/- 18 LU, p = 0.0001). This cytotoxicity against autologous and allogeneic tumor cells was blocked by anti-HLA-A2 mAb and cold HLA-A2+ targets in cold-target inhibition assays. TAL from all HLA-A2+ patients recognized GP2, a known, HER2/neu-derived tumor-associated peptide Ag that is HLA-A2 restricted. We have shown that TAL obtained from metastatic effusions of breast cancer patients contain lymphocytes that can recognize and lyse autologous and allogeneic tumor cells in a tumor-specific, HLA-A2-restricted fashion. In addition, tumor-specific TAL derived from breast cancer patients can selectively lyse HLA-A2+ pancreatic and ovarian tumor cell targets, suggesting a common HLA-A2-restricted tumor-associated Ag between these distinct epithelial cancers. Further elucidation of the cell-mediated immune response to breast cancer and the identification of shared TAA could result in the development of broadly applicable vaccine therapies for many cancers.
Treatment with bacterial LPS renders genetically resistant C57BL/6 mice susceptible to Theiler's virus-induced demyelinating disease.
Authors: Pullen L C , Park S H , Miller S D , Dal Canto M C , Kim B S
Published: December 2022

Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease in susceptible strains, which clinically and histopathologically resembles human multiple sclerosis. Since bacterial LPS produced by Gram-negative bacteria is known to potentiate an immune response and trigger resident central nervous system cells to produce various inflammatory cytokines, we examined the ability of LPS to affect resistance to TMEV-induced demyelinating disease (TMEV-IDD). Intraperitoneal injection of LPS, concomitant with intracerebral of genetically resistant C57BL/6 mice with TMEV, resulted in clinical symptoms in approximately 50% of the group. The increase in susceptibility following LPS treatment correlated with the enhanced levels of TMEV-specific delayed-type hypersensitivity and T cell proliferative responses. Similar treatment with LPS, however, did not accelerate the clinical course of susceptible (SJL/J) or intermediately susceptible (C3H) mice. The LPS-treated C57BL/6 mice displayed an increased viral persistence in the central nervous system when compared with nontreated control mice. Intraperitoneal administration of IL-1 beta could mimic the LPS effect in C57BL/6 mice, suggesting that the increase in susceptibility to TMEV-IDD may function via IL-1 produced following LPS stimulation.
Different patterns of TCR transgene expression in single-positive and double-negative T cells. Evidence for separate pathways of T cell maturation.
Authors: Cheng L , Dejbakhsh-Jones S , Liblau R , Zeng D , Strober S
Published: December 2022

Two lines of transgenic mice were established using the TCR alpha (V alpha 4.4-J alpha 24)- and beta (V beta 9-D beta 1.1-J beta 2.1)-chain genes from a cloned CD4-CD8-alpha beta + (double-negative; DN) T cell line from BALB/c mice. The TCR genes were expressed in CD4+CD8- and CD4-CD8+ (single-positive; SP) and double-positive (DP) T cells in the thymus, and in SP T cells in the peripheral lymphoid tissues, and marrow in one transgenic mouse line, and predominantly in DN T cells in the other. Bone marrow precursor cells from only the DN mouse line generated T cells expressing the V beta 9 transgene during tissue culture. V beta 9+ T cells were found in DN but not SP transgenic mice backcrossed to BALB/c nu/nu mice. The results suggest two separate pathways of T cell maturation, one which generates SP T cells in the thymus, and another which generates DN T cells in both the thymus and bone marrow.
Peptide-dependent expression of HLA-B7 on antigen processing-deficient T2 cells.
Authors: Smith K D , Lutz C T
Published: December 2022

Class I MHC Ag presentation and cell surface expression largely depend on peptide transport into the ER/cis-Golgi by TAP, the transporter associated with Ag processing. Despite this dependency, class I MHC molecules are expressed at low levels on the surface of TAP-deficient T2 cells. We studied the peptide dependency of HLA-B7 expression in transfected T2 cells. HLA-B7 expression was affected by mutations at 19 out of 23 peptide-binding groove residues, but not by nine mutations outside of the peptide-binding groove. T2 cell surface HLA-A2, -B7, and -B51 had similar stabilities, and approximately half of these class I molecules had a long t1/2 consistent with tight peptide binding. Using metabolically labeled T2 cells, HLA-A2-bound peptide eluted as five prominent peaks, but HLA-B7-bound peptide was not detected. In contrast, HLA-B7-eluted peptides were detected spectrophotometrically. These data suggest that HLA-A2 and HLA-B7 molecules utilize distinct TAP-independent peptide supply mechanisms to different degrees. Equivalent amounts of HLA-B7 from TAP- and TAP+ cells yielded similar amounts of peptide, which had the characteristic HLA-B7 peptide-binding motif. The dependency of HLA-B7 cell surface expression on peptide-binding groove residues, the stability of cell surface class I molecules, and the ability to detect HLA-B7-bound peptide indicate that the low level expression on T2 cells is largely peptide dependent. TAP-independent peptide Ag presentation may allow immune recognition of intracellular pathogens that interfere with TAP-dependent peptide transport.
Role of host antigen receptor-bearing and antigen receptor-negative cells in immune response to rat adenocarcinoma 13762.
Authors: Frey A B
Published: December 2022

Involvement of individual immune cell populations in the immune response to rat breast adenocarcinoma 13762 was studied by selective depletion treatments in vivo. Depletion of Ag nonspecific host defense cells from naive animals before tumor challenge resulted in statistically significant acceleration of tumor growth (p less than 0.01 or less), whereas depletion of either CD4+ T cells or CD8+ T cells had no effect on the incidence or kinetics of tumor development. In contrast to naive animals treated before tumor challenge, animals actively immunized by injection of irradiated tumor before depleting treatments were shown to require CD4+ T cells to reject tumorigenic challenge. Depletion of either macrophages or neutrophils from immune animals also increased tumor development, whereas NK cells were not involved. Depletion of CD8+ T cells from immune animals permitted transient growth of tumors that were subsequently rejected, implying a role in tumor rejection. Transfer of immune antiserum to naive animals at the time of tumor challenge was without effect on tumor development. Depletion of CD4+ T cells, neutrophils, or macrophages in the priming phase of antitumor immune response abrogated tumor immunity, but depletion of CD8+ T cells or NK cells was without effect on the ability to prime animals by immunization with irradiated cells. Collectively, these data suggest that host natural defense cells that do not express Ag receptor are primarily responsible for resistance of adenocarcinoma 13762 growth in naive animals. In contrast, tumor immunity induced by active immunization requires Ag receptor-bearing CD4+ T cells and involves participation of CD8+ T cells, neutrophils, or macrophages in elimination of tumor.
Mechanisms of Trypanosoma cruzi-induced down-regulation of lymphocyte function. Inhibition of transcription and expression of IL-2 receptor gamma (p64IL-2R) and beta (p70IL-2R) chain molecules in activated normal human lymphocytes.
Authors: Majumder S , Kierszenbaum F
Published: December 2022

Acute infection with Trypanosoma cruzi, the agent of Chagas' disease, is accompanied by multiple manifestations of immunosuppression, and this parasite has been shown to inhibit T and B lymphocyte functions in vitro through a mechanism involving impaired membrane expression of at least the p55IL-2R (IL-2R alpha) and p70IL-2R (IL-2R beta) components of the multimeric high affinity IL-2R complex. We document in this paper that addition of T. cruzi or a parasite-conditioned medium (trypanosomal immunosuppressive factor (TIF) to cultures of PHA-stimulated normal human blood lymphocytes markedly decreases the cytoplasmic levels of p64IL-2R and p70IL-2R molecules, i.e., the two IL-2R chains that bind IL-2 and participate in signal transduction. These effects highlighted the ability of T. cruzi to curtail biosynthesis of these IL-2R chains and weakened the possibility that decreased membrane IL-2R expression results exclusively from altered chain transportation to the lymphocyte surface. Additional support for impaired lymphocyte production of IL-2R components was derived from northern blot analyses demonstrating TIF-induced decreases in p64IL-2R and p70IL-2R mRNA levels. These reductions are unlikely to be due to conditions affecting mRNA stability, since the rates of decay of both mRNA species were nearly identical in the presence and in the absence of TIF. In addition, nuclear run-on studies revealed that transcription of the p64IL-2R, p70IL-2R, and p55IL-2R genes, but not that of the CD69 gene, was below normal levels in PHA-stimulated lymphocytes incubated with TIF. These results suggest that altered gene transcription is involved in the mechanism by which T. cruzi down-regulates the expression of all the known components of the high affinity IL-2R.
Adoptive immunotherapy with tumor-specific T lymphocytes generated from cytokine gene-modified tumor-primed lymph node cells.
Authors: Ohno K , Yoshizawa H , Tsukada H , Takeda T , Yamaguchi Y , Ichikawa K , Maruyama Y , Suzuki Y , Suzuki E , Arakawa M
Published: December 2022

Adoptive immunotherapy with immune T cells mediates regression of established tumors in animal models. We previously demonstrated that precursor lymphocytes of sensitized T cells can develop into mature effector cells after in vitro activation with anti-CD3 mAb and IL-2. We demonstrate here that tumor cells genetically modified to secrete IL-2 can enhance the precursor response in the tumor-bearing host and subsequently augment the antitumor efficacy of adoptive immunotherapy. MCA205 and MCA203, weakly immunogenic fibrosarcomas, were transfected in vitro with cDNA encoding for IL-2, IL-4, or IL-6. Lymph nodes (LN) draining these cytokine-producing tumors for 7 days were harvested, activated in vitro with anti-CD3/IL-2, and adoptively transferred into mice bearing established parental MCA205 pulmonary metastases. The effector cells generated from LN draining the IL-2 producing tumor exhibited enhanced antitumor activity compared with cells from LN draining parental, IL-4-producing, or IL-6-producing tumor. Phenotype analysis of cells from LN draining the IL-2-producing tumor revealed selective expansion of V beta 8+ cells. Depletion of V beta 8+ effector cells abrogated the antitumor efficacy indicating that V beta 8+ cells constituted the majority of antitumor reactivity and that secretion of IL-2 from tumor cells promoted the priming of V beta 8+ precursor cells, which can develop into mature effector cells. These results have important clinical implications that the method presented here could be applicable to the treatment of human cancer as more effective immunotherapy.
Protective immunity in BALB/c mice against the simian virus 40-induced mKSA tumor resulting from injection of recombinant large T antigen. Requirement of CD8+ T lymphocytes.
Authors: Zerrahn J , Utermöhlen O , Warnecke G , Deppert W , Lehmann-Grube F
Published: December 2022

BALB/c mice are often considered "low responders" or even "nonresponders" with regard to cytolytic CD8+ T lymphocytes and SV40 large T Ag (TAg). Large TAg and fragments thereof were produced by recombinant technology and injected into BALB/c mice that were subsequently challenged by i.p. injection of syngeneic TAg-expressing mKSA tumor cells. Two portions of the TAg were found to induce protective immunity, one stretching from amino acid residues 1-272 and the other from amino acid residues 683-708. In mice thus protected, the spleens were virtually free of cytotoxic T cells but CD8+ T lymphocytes obtained from the peritoneal cavity during rejection of the mKSA cells were directly lytic for TAg-expressing target cells. Depleting immune mice of CD4+ or CD8+ T lymphocytes by treatment with mAb abolished their ability to resist tumor development. We conclude that immunity against SV40 TAg-expressing tumor cells in BALB/c mice is dependent on both CD4+ and CD8+ T lymphocytes.
Endometrial T, B, and NK cells in patients with recurrent spontaneous abortion. Altered profile and pregnancy outcome.
Authors: Lachapelle M H , Miron P , Hemmings R , Roy D C
Published: December 2022

Several lines of evidence indicate that immunologic effectors, particularly suppressor T cells and NK cells, may play a role in the pathogenesis of idiopathic repetitive abortions. To investigate the involvement of these immune cell populations, we determined the immunophenotypic characteristics of endometrial leukocytes from nonpregnant recurrent aborters. Habitual aborters with a negative investigation underwent an endometrial biopsy during their secretory phase and were followed prospectively to assess clinical outcome. Endometrial leukocytes were evaluated by two-color flow cytometric analysis. The percentage of endometrial CD8+ T lymphocytes was significantly decreased in recurrent aborters, and their CD4:CD8 ratio was increased. In contrast, the proportion of B lymphocytes (CD20+) was strikingly increased in these patients' endometria. The proportion of NK cells was identical in recurrent aborters and normal controls, but the CD16-CD56 bright NK cell subset, which is predominant in normal decidua and endometrium, was significantly decreased in favor of an important contingent of CD16+CD56 dim NK cells in all habitual aborters. Repetitive aborters who had normal CD8+ and CD20+ cell numbers and a normal CD4:CD8 ratio subsequently underwent successful pregnancies, while patients with continuing abortions presented lymphoid populations observed in the habitual aborters group. In conclusion, endometrial lymphocytes of recurrent spontaneous aborters harbor a distinct immunophenotypic profile that antedates implantation and suggests that endometrial immunologic conditions are intrinsically altered in recurrent aborters. Also, the prognostic impact of CD8 and CD20 expression supports their predominant role in the development of fetal tolerance. Finally, a role for NK cells in the abortion process is suggested by their altered subsets in all repetitive aborters.