bluuebunny/crossref_metadata_2025 huggingface dataset:
Conserved and variable residues within the Bw4 motif of HLA-B make separable contributions to recognition by the NKB1 killer cell-inhibitory receptor.
Authors: Gumperz J E , Barber L D , Valiante N M , Percival L , Phillips J H , Lanier L L , Parham P
Published: December 2022

Allotypes from four divergent HLA-B families (B8, B15, B16, and B27) were compared for their inhibition of cytolysis by NK cells expressing the NKB1 receptor. Allotypes differing solely at the Bw4/Bw6 region were examined as were a more divergent subset of B15 allotypes. The capacity to interact with NKB1 correlated precisely with possession of a Bw4 sequence motif at residues 77-83, whereas no correlation was made with the peptide-binding specificities of two Bw4 and four Bw6 allotypes of the B15 family. HLA-B allotypes having four different Bw4 motifs were examined and all interact with NKB1. In contrast, HLA-A allotypes, which have a Bw4 motif identical with one of those present in HLA-B, do not. Mutation at leucine 82 and arginine 83, the residues common to Bw4 motifs, shows they contribute to NKB1 interaction but are not essential. Three types of polymorphism are implicated in formation of the ligand recognized by NKB1: ones shared by Bw4 motifs; ones distinguishing Bw4 motifs; and ones outside the Bw4/Bw6 region that distinguish HLA-B from HLA-A.
Cloning and characterization of the human granulocyte chemotactic protein-2 gene.
Authors: Rovai L E , Herschman H R , Smith J B
Published: December 2022

We recently described a novel murine CXC chemokine, designated lipopolysaccharide-induced CXC chemokine (LIX). In an ongoing search for new human chemokines related to LIX, we cloned the gene for human granulocyte chemotactic protein-2 (GCP-2) as well as previously described CXC chemokine genes, including epithelial cell-derived neutrophil-activating peptide-78 (ENA-78). Both coding and noncoding portions of the GCP-2 gene have very high nucleotide similarity to ENA-78, except for the occurrence of a long interspersed DNA-1 sequence 5' of the GCP-2 gene. The GCP-2 gene encodes a propeptide of 114 amino acid residues. The predicted 77-residue mature peptide is identical with the GCP-2 protein previously isolated from MG-63 osteosarcoma cells, except for two additional residues at the carboxyl terminus. We confirmed expression of the gene by Northern analysis and by cloning a portion of the cDNA from reverse transcribed MG-63 cell RNA. Despite 85% identity of the first 270 nucleotides 5' of the transcription start sites, GCP-2 and ENA-78 show cell-specific differences in regulation. GCP-2 is induced in MG-63, but not A549 cells by TNF-alpha, IL-1beta, and LPS, while ENA-78 is expressed in both cell types. Analysis of nucleotide sequence relationships does not support the proposal, by others, that LIX is murine GCP-2. LIX is no more closely related to human GCP-2 than to human ENA-78 and is more distant from both human genes than is porcine alveolar macrophage chemotactic factor-II.
Role of T lymphocyte subsets in immunity to spotted fever group Rickettsiae.
Authors: Feng H , Popov V L , Yuoh G , Walker D H
Published: December 2022

To evaluate the roles of CD4 and CD8 T lymphocytes in immunity to disseminated endothelial infection with Rickettsia conorii (Malish 7 strain), these T cell subsets were depleted or adoptively transferred into subsequently infected C3H/HeN mice. CD4 T lymphocyte-depleted and sham-depleted mice underwent a similar course of illness with a sublethal rickettsial dose, cleared the infection by day 10, and recovered on days 10 to 11. In contrast, mice depleted of CD8 lymphocytes or CD4 and CD8 lymphocytes died or remained persistently infected through day 15 with the ordinarily sublethal dose. Endothelium was the major site of rickettsial persistence, including sites in the vital organs, brain, and lungs of CD8 lymphocyte-depleted mice. In nondepleted animals, CD8 T lymphocytes were observed in apposition to endothelial cells on day 10 at the time of rickettsial clearance. Adoptive transfer of immune CD4 or CD8 T lymphocytes protected mice against a lethal dose of R. conorii in the disseminated endothelial target model. Nonimmune CD4 or CD8 lymphocytes and immune lymphocytes that had passed through columns that depleted both CD4 and CD8 lymphocytes failed to protect mice against R. conorii. These studies represent the first analysis of the role of T lymphocyte subsets in immunity to spotted fever group rickettsiae and the first demonstration that clearance of spotted fever group rickettsiae from endothelial cells requires immune CD8 T lymphocytes.
Oral immunization with simian immunodeficiency virus p55gag and cholera toxin elicits both mucosal IgA and systemic IgG immune responses in nonhuman primates.
Authors: Kubota M , Miller C J , Imaoka K , Kawabata S , Fujihashi K , McGhee J R , Kiyono H
Published: December 2022

Rhesus macaques were orally immunized with a mucosal vaccine consisting of two different concentrations (1 mg vs 250 microg) of recombinant SIV p55gag (p55) with or without cholera toxin (CT, 50 microg) as a mucosal adjuvant. The plasma from macaques receiving the higher dose of p55 (1 mg) and CT had higher p55-specific IgG and IgA Ab titers compared with macaques that received the lower dose of p55 (250 microg) and CT. Further, high levels of p55-specific IgG and IgA Abs were present in external secretions from both groups. The level of p55-induced T cell responses was elevated in PBMCs isolated from the high dose group compared with the low dose group. When culture supernatants from these p55-stimulated PBMCs were examined for Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, both IFN-gamma and IL-10 were present, but IL-4 was absent. CD4+ T cells isolated from these p55-stimulated PBMCs contained IFN-gamma spot-forming cells (SFCs) but not IL-4 SFCs. These results were further confirmed by cytokine-specific reverse transcriptase PCR analysis, where p55-specific CD4+ T cells expressed mRNA for IFN-gamma, IL-6, and IL-10, but not IL-4. These findings suggest that oral immunization of nonhuman primates induced both IFN-gamma-secreting Th1 and select Th2 cytokine (e.g., IL-6 and IL-10)-producing CD4+ Th cells, which accounted for the generation of p55-specific systemic and mucosal Ab responses.
Protection of mice against Trypanosoma cruzi by immunization with paraflagellar rod proteins requires T cell, but not B cell, function.
Authors: Miller M J , Wrightsman R A , Stryker G A , Manning J E
Published: December 2022

Previous studies have shown that immunization of mice with the paraflagellar rod proteins (PAR) of Trypanosoma cruzi induces an immune response capable of protecting mice against an otherwise lethal challenge with this parasite. Herein, we define immunologic responses that do or do not play a critical role in PAR-mediated protection. Firstly, PAR-immunized Ab-deficient (muMT) strain mice survived an otherwise lethal T. cruzi challenge, indicating that a B cell response is not required for PAR-induced immunity. However, beta2m -/- mice, which are severely deficient in MHC class I and TCR alphabeta+ CD8+ CD4- T cells, did not survive challenge infection following PAR immunization, indicating that MHC class I/CD8+ T cell function is necessary for protection induced by PAR immunization. Surprisingly, PAR-immunized mice depleted of CD4+ T cells survived a T. cruzi challenge for >84 days postinfection while maintaining a parasitemia that is generally thought to be lethal (i.e., >10(6) trypomastigotes/ml), thus associating CD4+ T cell function with the process of parasite clearance. Consistent with this association, CD4+ T cells from PAR-immunized mice released INF-gamma and stimulated T. cruzi-infected macrophages to release nitric oxide. The importance of IFN-gamma in PAR-induced protective immunity is further indicated by the observation that PAR-immunized INF-gamma knockout mice developed an extremely high parasitemia and did not survive a challenge infection. Thus, while Ab-mediated immune mechanisms are not required for protection induced by PAR immunization, T cell responses are necessary for both elimination of bloodstream parasites and survival.
Thrombin induces endothelial type II activation in vitro: IL-1 and TNF-alpha-independent IL-8 secretion and E-selectin expression.
Authors: Kaplanski G , Fabrigoule M , Boulay V , Dinarello C A , Bongrand P , Kaplanski S , Farnarier C
Published: December 2022

In addition to its role in coagulation, thrombin is involved in the inflammatory process by inducing vessel neutrophilic infiltration. Thrombin induces endothelial P-selectin expression and platelet activating factor release, which participate to induce early neutrophil adhesion and activation. We employed HUVEC and now show that thrombin induces the production of the chemokine IL-8 in a time- and dose-dependent fashion. Similarly, thrombin induced E-selectin expression on HUVEC. Both IL-8 secretion and E-selectin expression were preceded by an increase in steady state levels of the respective mRNAs. Thrombin action on HUVEC was inhibited by the specific thrombin inhibitor, hirudin. In addition, these effects of thrombin on HUVEC were mimicked by the 14-amino acid thrombin receptor agonist peptide, which triggers the native thrombin receptor in a similar fashion to thrombin itself. Although IL-1 and TNF-alpha also induce IL-8 and E-selectin, the thrombin effects in these experiments were not mediated by those cytokines, since neither IL-1 receptor antagonist nor anti-TNF-alpha Ab inhibited the effects of thrombin. Furthermore, IL-1alpha, IL-1beta, and TNF-alpha were not detected in the supernatants of thrombin-activated HUVEC. Although intracellular IL-1alpha was found in thrombin-activated HUVEC, antisense IL-1alpha had no inhibitory effect on IL-8 secretion. These results demonstrate that in addition to short term endothelial activation, thrombin also functions as a long acting proinflammatory agent by inducing endothelial synthesis of the mediators required for neutrophils activation and extravazation during inflammation.
Eosinophil adhesion regulates RANTES production in nasal epithelial cells.
Authors: Terada N , Maesako K , Hamano N , Houki G , Ikeda T , Sai M , Yamashita T , Fukuda S , Wakita A , Yoshimura K , Konno A
Published: December 2022

Among the many known chemotactic factors for eosinophils, the proinflammatory chemokine RANTES is particularly important, because it is potently and selectively chemotactic for eosinophils. Throughout the process of the migration of eosinophils from the blood vessels into the nasal cavity, eosinophil functions are assumed to be regulated by surface adhesion molecules. Conversely, the messages conferred by the eosinophils to the endothelial and epithelial cells are also of great interest. In the present study, we showed that eosinophil adhesion to human nasal epithelial cells (HNECs) inhibits RANTES production in HNECs. Eosinophils were isolated from peripheral blood obtained from patients with allergic rhinitis. Human mucosal microvascular endothelial cells and HNECs were isolated from human nasal mucosa specimens. After stimulation of the HNECs in the presence of eosinophils, the secretion of RANTES, induced by a combination of TNF-alpha and IFN-gamma, appeared to have decreased. The amount of the decrease was a function of the number of involved eosinophils. On the other hand, the presence of eosinophils did not affect RANTES production by the endothelial cells. After pretreatment of the eosinophils with anti-CD18 mAb or coculture with HNECs in Transwell culture inserts, these cells did not inhibit the TNF-alpha- and IFN-gamma-induced RANTES production. These results were virtually identical with those observed on RANTES mRNA expression. The adhesion of eosinophils to HNECs plays a key role in the inhibition of RANTES production. Our data indicate that a certain established system causes the signal transfer from eosinophils to HNECs to inhibit RANTES production, thus decreasing the eosinophil infiltration.
Control of multiple autoantibodies linked with a lupus nephritis susceptibility locus in New Zealand black mice.
Authors: Vyse T J , Rozzo S J , Drake C G , Izui S , Kotzin B L
Published: December 2022

An NZB locus on distal chromosome 1 has been linked to murine lupus nephritis in backcross analyses of New Zealand mice. This locus, designated Nba2 for New Zealand Black autoimmunity 2, was found to colocalize in both (NZB x SM/J)F1 x NZW and (B6.H2z x NZB)F1 x NZB backcrosses, and was most likely situated between 92 and 97 cM from the centromere. This region of mouse chromosome 1 encodes several candidate genes, including the low affinity Fc gamma receptor genes. Both backcrosses were examined by interval mapping for quantitative trait loci linked with autoantibody and total Ig production. Nba2 was linked with elevated serum levels of multiple autoantibodies, including a variety of antinuclear Abs (anti-dsDNA, anti-chromatin and anti-histone) and autoantibodies to gp70, in both backcrosses. Nba2 was also linked (or showed a trend for linkage) with hypergammaglobulinemia and IgG1, IgG2a, and/or IgG3 levels in each backcross. In the (B6.H2z x NZB)F1 x NZB backcross, MHC was an additional genetic contribution that interacted with Nba2 in the production of autoantibodies and the development of nephritis. Together, these data provide new insight into the nature of one important genetic contribution to murine lupus and suggest that Nba2 may act as an immune response gene that influences Ag-driven B cell responses to self and possibly to exogenous Ags.
Autocrine and paracrine regulation of human T cell IL-10 production.
Authors: Cohen S B , Parry S L , Feldmann M , Foxwell B
Published: December 2022

We confirm that IL-10 is produced comparatively late after the activation of human T cells via CD3 stimulation, after IL-2 and IFN-gamma. Furthermore we show that IL-10 production by human T cell lines, such as IFN-gamma and IL-2, is inhibited by the immunosuppressive drugs cyclosporin A and FK506. However, a third immunosuppressive drug, rapamycin, normally associated with inhibiting the effects, but not the production, of cytokines, inhibited IL-10, but not IFN-gamma, production. This implies that IL-10 induction may be a secondary event in T cell activation. Since IL-2 is the major growth factor for T cells and is detected before IL-10, we focused on this factor as a potential activator of IL-10 production. We showed that IL-10 production by human T cell lines stimulated by immobilized anti-CD3 in the presence of neutralizing Abs to IL-2 and IL-2R (anti-CD25) was inhibited, whereas addition of exogenous IL-2 enhanced IL-10 production, indicating that IL-10 production by human T cells can be driven by stimulation via IL-2. As the IL-2R shares the common gamma-chain component with IL-4, IL-7, and IL-15, we investigated the possibility that they may all enhance anti-CD3-induced IL-10 production and established that this was the case. Since IL-10 has been previously described as a direct inhibitor of Ag presentation and of IL-2 production, our finding that IL-2 is capable of inducing its own inhibitor demonstrates a feedback mechanism within the immune system that could limit ongoing T cell activation and possibly inflammation.
Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation.
Authors: Lu L , Qian S , Hershberger P A , Rudert W A , Lynch D H , Thomson A W
Published: December 2022

Activation of T cells is induced efficiently by dendritic cells (DC), but little is known about the role of DC in the regulation of T cell death. In this study, highly purified DC (DEC-205+, MHC class II(high), B7-1+ [CD80+], B7-2high [CD86high], CD40+, CD11c+) grown from normal mouse bone marrow in granulocyte-macrophage CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells, but not DC propagated from FasL-deficient (B6.gld) mice, induced dose-dependent increases in DNA fragmentation in Fas+ Jurkat T cells over 18 h coculture. Addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations, however, B7-2high DC induced only low levels of DNA fragmentation in Con A or alloactivated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick-end labeling. However, in the presence of CTLA4Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4Ig treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional potential (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways may play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions.
The CD94/NKG2-A inhibitory receptor complex is involved in natural killer cell-mediated recognition of cells expressing HLA-G1.
Authors: Pérez-Villar J J , Melero I , Navarro F , Carretero M , Bellón T , Llano M , Colonna M , Geraghty D E , López-Botet M
Published: December 2022

Human NK cells bear surface receptors that inhibit their cytolytic activity upon specific recognition of MHC class Ia Ags; little is known about the capacity of class Ib molecules to regulate NK cell function. We have studied the roles of different NK inhibitory receptors in recognition of the class Ib HLA-G. To this end, we analyzed the ability of an HLA-defective tumor cell line (721.221) transfected with the membrane form of HLA-G1, which contains the three external domains, to inhibit the cytolytic activity mediated by a panel of NK clones from several donors. A substantial proportion of peripheral blood NK clones appeared to be significantly inhibited by the HLA-G1-transfected cell line (referred to as .221-G1); nevertheless, no relation was observed between the expression and the function of serologically identifiable Ig-SF receptors (p58/p70) and specific recognition of .221-G1 cells. Moreover, p58 killer cell inhibitory receptor-IgG soluble fusion proteins specifically bound to 721.221 transfectants bearing their corresponding HLA-C ligands, but only a weak reactivity with .221-G1 cells was detectable. By contrast, most NK clones blocked by HLA-G1 expressed the CD94/NKG2-A inhibitory receptor, and moreover, CD94-specific mAbs reconstituted their cytolytic activity comparably to anti-HLA class I mAbs. These data support the idea that the CD94/NKG2 receptor complex is involved in the recognition of cells expressing HLA-G1.
Intermediate TCR cells in mouse lung: their effector function to induce pneumonitis in mice with autoimmune-like graft-versus-host disease.
Authors: Watanabe T , Kawamura T , Kawamura H , Haga M , Shirai K , Watanabe H , Eguchi S , Abo T
Published: December 2022

The lung comprises lymphocytes even under noninflammatory conditions. We investigated the presence of NK cells, extrathymic T cells, and thymus-derived T cells in this organ. As shown previously in mouse liver, two-color staining for CD3 and IL-2R beta-chain (IL-2Rbeta) identifies CD3- IL-2Rbeta+ NK cells, CD3int IL-2Rbeta+ cells (int indicates intermediate; of extrathymic origin), and CD3high IL-2Rbeta- cells (high indicates high; of thymic origin). These populations were present in the lungs of normal mice in a unique manner (i.e., NK cells > CD3high cells > CD3int cells). The proportion of CD3int cells increased in the lung with age. In athymic mice, only CD3int cells were detected in the lung. In contrast to CD3int cells in the liver, the majority of these cells in the lung did not express NK1.1 Ags. Other properties of CD3int cells in the lung were the same as those in the liver, e.g., double-negative CD4- 8- cells and gammadelta T cells. CD3int cells in the lung contained forbidden clones similar to those in other organs. When (B6 x bm12) F1 mice (Ly5.2) were injected with CD3high cells of B6-Ly5.1 origin, F1 mice fell victim to chronic graft-vs-host disease and pneumonitis, showing the expansion of CD3int cells. Almost all of them were of recipient origin (Ly5.2+). More importantly, such CD3int cells induced autoimmune-like pneumonitis when injected into irradiated F1 mice. CD3int cells isolated from the lung in mice with graft-vs-host disease exerted autologous cytotoxicity against thymocytes in vitro. These results suggest that extrathymic T cells exist in the lung and that their expansion may be responsible for inflammatory lung diseases.
A tandem GC box motif is necessary for lipopolysaccharide-induced transcription of the type II TNF receptor gene.
Authors: Bethea J R , Ohmori Y , Hamilton T A
Published: December 2022

LPS induces the expression of the gene encoding the type II TNF-alpha receptor in mononuclear phagocytes. To elucidate the nuclear signaling mechanisms involved in this response, a 772-bp fragment of the TNFRII gene promoter was analyzed by deletion and site-specific mutagenesis following transient transfection in the macrophage-like cell line RAW264.7. A region located between -100 and -75 relative to the transcription start site was found to be essential for LPS sensitivity. This contained a GC-rich region composed of two tandemly arrayed Sp-1 sites. While mutation of this region confirmed that it was essential for LPS sensitivity, the sequence was not able to confer LPS sensitivity upon a heterologous promoter. Internal deletion and site-specific mutagenesis of the 100-bp fragment identified regions immediately flanking an initiator region (Inr) site that were also necessary for sensitivity to LPS. Electrophoretic mobility shift assays demonstrated that the GC box bound Sp-1 and Sp-3, although the level of binding activity did not vary with LPS stimulation. An oligonucleotide probe containing nucleotide positions -45 to -18 also bound several protein complexes, but these were not enhanced by LPS. These findings indicate that the tandem GC box is necessary, but not sufficient, for LPS-mediated transcription of the TNFRII gene. A second apparently novel motif, located within 15 nucleotides of the Inr, is also necessary.
The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells.
Authors: Kraj P , Rao S P , Glas A M , Hardy R R , Milner E C , Silberstein L E
Published: December 2022

The expressed human Ig repertoire is not an equal representation of all V(H) segments present in genomic DNA. Studies have shown that a restricted set of V(H) gene segments are over-represented in Ab repertoires of fetal/neonatal and adult B cells. Additionally, this restricted set of V(H) genes is frequently expressed by autoimmune and tumor B cells. To investigate at which developmental stage a bias in the repertoire begins, we compared the V(H)3 and V(H)4 family repertoires of pre-B and immature B cells from bone marrow and mature B cells from peripheral blood of two adults. We found that the V4-34 and V4-59 gene segments of the V(H)4 family and the V3-23 gene segment of the V(H)3 family dominate the repertoires of the surface Ig-negative early pre-B as well as immature and mature B cells. Furthermore, the pattern of utilization of other V(H)3 family members suggests that certain genes that are frequently rearranged during early stages of B cell development are subsequently disfavored during later stages of B cell maturation. We conclude that the over-representation of certain V genes could arise from sequential mechanisms operating at both early and later stages of B cell development. These V(H)-mediated mechanisms might include preferential rearrangement and/or efficiency of pairing with the surrogate light chain at the surface Ig-negative, early pre-B cell stage and ligand selection at more mature, surface Ig-positive, B cell stages.
Regulatory factor X, a bare lymphocyte syndrome transcription factor, is a multimeric phosphoprotein complex.
Authors: Moreno C S , Rogers E M , Brown J A , Boss J M
Published: December 2022

Regulatory factor X (RFX) is a transcription factor that binds the conserved X1 box of MHC class II promoters and is essential for transcription of class II genes. The subunit structure of the native RFX complex was examined by coimmunoprecipitation using polyclonal antisera to the 75-kDa subunit of RFX, RFX5. Two polypeptides with apparent masses of 41 and 36 kDa coimmunoprecipitated with RFX5 and appear to be subunits of the native RFX complex. Metabolic labeling of wild-type and mutant B cells with [32P]orthophosphate demonstrated that each of the RFX subunits was phosphorylated in vivo and that the phosphorylation of the RFX subunits was independent of the essential MHC class II regulatory factor, CIITA. The trimeric RFX complex was also present in fibroblast cells with or without IFN-gamma treatment. Both the p41 and p36 subunits were absent in immunoprecipitations of RFX5 from lysates of independently established B cell lines from bare lymphocyte syndrome complementation groups B and D. Together, these results suggest that RFX complex assembly is required for class II expression and that the mutations in bare lymphocyte syndrome complementation groups B and D result in an inability to assemble the RFX complex.
TCR beta-chain variable region-driven selection and massive expansion of HLA-class I-restricted antitumor CTL lines from HLA-A*0201+ melanoma patients.
Authors: Maccalli C , Farina C , Sensi M , Parmiani G , Anichini A
Published: December 2022

Recognition of a given melanoma Ag involves a limited array of T cell clones bearing a structurally defined TCR. The aim of this study was to verify whether this information can be used to isolate and expand such anti-tumor effectors from fresh lymphocyte populations. We found that one to three different TCR beta-chain variable (TCRBV) regions were significantly expanded in 4-wk mixed lymphocyte-tumor cultures (MLTC) from six HLA-A*0201+ melanoma patients, and that the T cells expressing the expanded TCRBV regions were involved in HLA class I-restricted lysis of the tumor. T cell activation by mAbs to MLTC-selected TCRBV region and CD28 resulted in large scale expansion (1-10 x 10(9) cells) of T cell lines, highly enriched for the expression of a single TCRBV region and for CD8+ T cells. The TCRBV-driven selection was equally effective when applied to patients' or healthy donors' lymphocytes, and the T cell lines isolated from melanoma patients exerted HLA class I-restricted lysis of the autologous tumor. MLTC and TCRBV-selected lines recognized allogeneic melanomas sharing HLA-A and -B alleles with the autologous tumor, but only two of the HLA-A2-restricted lines were directed to a known peptide from melanoma-associated Ags. Single-strand conformation polymorphism analysis indicated a polyclonal composition of both MLTC and TCRBV-selected lines, but expansion of clonotypes with identical CDR3 length was observed only in the MLTC lines. Thus, TCRBV-driven selection can be exploited to obtain large scale expansion of antitumor CTL lines from melanoma patients.
Restricted TCR V alpha repertoire in the T cell response to a tolerogenic determinant of type II collagen.
Authors: Kawamura S , Miyahara H , Esaki Y , Myers L K , Rosloniec E F , Stuart J M , Kang A H
Published: December 2022

Tolerization of B10.RIII mice (H-2r) with i.v.-injected type II collagen (CII) renders the animals resistant to induction of collagen-induced arthritis (CIA). The B10.RIII mouse is of particular interest, in that the T cell determinants that induce tolerance are different from those that induce arthritis. To characterize T cells that react with the tolerogenic determinant and play a role in regulation of arthritis, we have developed a panel of T cell hybridomas reactive with the tolerogenic T cell epitope, CII 607-621. None of the hybrids cross-reacted with either the arthritogenic CII 445-453 or murine CII. As determined by PCR and immunofluorescence, the T cell response to the tolerogenic determinant was oligoclonal, with evident preferential usage of V alpha. Through the analysis of a large panel of T cell hybridomas, preferential usage of V alpha2, J alpha44, J beta2.7, and D beta2.1 was observed. Characterization of T cells reactive with the immunodominant determinant, CII 607-621, responsible for the induction of tolerance should prove important in developing novel therapeutic approaches for the treatment of autoimmune diseases.
Reduced diversity of CTLs specific for multiple minor histocompatibility antigens relative to allograft rejection in vivo.
Authors: Nevala W K , Paul C , Wettstein P J
Published: December 2022

Minor histocompatibility (H) antigens stimulate in vivo rejection of allografts compatible for the MHC and are recognized by CTLs in short term in vitro assays. CTLs generated by the in vivo priming and in vitro boosting of mice with spleen cells incompatible for multiple minor H Ags are specific for a limited number of dominant Ags (peptides). We have addressed the issue of the identity of the Ags that stimulate rejection of solid tissue allografts vs the H Ags recognized by CTLs. C57BL/6 recipients reject skin grafts from BALB.B and CXB recombinant inbred strains with no significant differences in survival times. Primary grafts from these same strains prime for accelerated rejection of second-set grafts from all CXB strains regardless of inheritance of dominant Ags detected by CTLs. However, CTLs primed by these allografts and boosted in vitro do not exhibit ranges of reactivity with lymphoblast targets from CXB strains predicted by in vivo rejection, suggesting that CTLs primed by multiple Ag-incompatible skin grafts do not recognize all H Ags that stimulate allograft rejection. The fact that first- and second-set BALB.B skin grafts prime for accelerated rejection of 11/13 congenic strains defining single BALB/c minor H Ags indicates that multiple H Ags stimulate allograft rejection. However, CTLs from C57BL/6 mice primed with BALB.B grafts and boosted with BALB.B spleen cells recognize only the H4 Ag from this panel of congenic strains. Limited diversity of the CTL response is corroborated by the recognition of three minor H peptides (including H4 as the most prominent) eluted from Kb molecules from a BALB.B tumor by these CTLs. These results indicate that CTLs recognize only a limited number of Ags operative in vivo and do not accurately reveal the complexity of the antigenic specificity of the in vivo response.
Secreted chondroitin sulfate proteoglycan of human B cell lines binds to the complement protein C1q and inhibits complex formation of C1.
Authors: Kirschfink M , Blase L , Engelmann S , Schwartz-Albiez R
Published: December 2022

We recently characterized a species of proteochondroitin sulfate (CSPG) secreted by human B cell lines that closely resembles in its structure the serum-derived C1q inhibitor (C1qI). These proteoglycans have in common a molecular mass of approximately 130 to 150 kDa with a core protein of 30 kDa to which up to four chondroitin sulfate chains each of approximately 26 kDa are attached. Since this B cell-derived CSPG is a potential source for serum C1qI, we measured its capacity to interact with C1q in solid-phase binding and complex electrophoresis assays. B cell CSPG purified from culture supernatants of the two human B cell lines JOK-1 and U266 strongly bound to C1q. In contrast to the secreted form, cellular proteoglycan of the myeloma cell line U266 did not interact with C1q. Binding of C1q to CSPG was competitively inhibited by free glycosaminoglycans (GAG) in the order dextran sulfate > heparin > heparan sulfate > chondroitin-6-sulfate (CS-C) > dermatan sulfate (CS-B) > chondroitin-4-sulfate (CS-A). B cell CSPG inhibited the hemolytic activity of C1q and C1. In addition, B cell CSPG blocked C1q receptor binding in a dose-dependent manner. The proteoglycans did not influence the activity of C1 complex already bound to EAC4 target cells. By interaction of CSPG with solid-phase-bound C1q, formation of the C1 complex upon the addition of C1r and C1s was impaired. Strong binding of B cell CSPG to C1q, its inhibition of C1q activity, and its structural similarities to the previously described human serum C1qI indicate that B cells produce a soluble CSPG, which may act as C1qI under physiologic conditions.
Local-clonal expansion of infiltrating T lymphocytes in chronic encephalitis of Rasmussen.
Authors: Li Y , Uccelli A , Laxer K D , Jeong M C , Vinters H V , Tourtellotte W W , Hauser S L , Oksenberg J R
Published: December 2022

Rasmussen's syndrome is a progressive and intractable form of epilepsy characterized pathologically by focal brain inflammation with large numbers of infiltrating T lymphocytes. To better understand the nature of the T cell response in this disease, we analyzed TCR expression in the brain lesions using PCR for quantitative assessment of TCRBV gene transcripts, together with size and sequence analysis of the third complementarity-determining region (CDR3) of the dominant TCR rearrangements. Restricted (oligoclonal) BV family usage was not observed, as all of the 22 BV PCR products were usually detected at levels exceeding the background. However, significant individual biases in the frequencies of different TCR families was evident. The distinct pattern of BV expression by infiltrating lymphocytes detected in the original PCR screening suggested a specific immune response. The primary structure of the rearranged CDR3 sequences for the BV family expressed at highest level in each sample was studied by size and sequence analysis. The data showed that predominant TCR BV families expressed in diseased brain tissue displayed limited size heterogeneity and extensive repetition of in-frame CDR3 nucleotide motifs. These findings demonstrate that the local immune response in Rasmussen's syndrome includes restricted T cell populations that have likely expanded from a few precursor T cells responding to discrete antigenic epitopes.
In patients with rheumatoid arthritis IgG binding to denatured collagen type II is in part mediated by IgG-fibronectin complexes.
Authors: Mannik M , Kapil S , Merrill C E
Published: December 2022

The presence of autoantibodies to native and denatured collagen type II has been reported in some patients with rheumatoid arthritis (RA). This study examined the molecular nature of IgG binding to native and denatured collagen type II. The binding of IgG to native and denatured collagen was detected with an ELISA. Serum proteins were separated by size with gel filtration. Gel-filtered fractions were further characterized by binding to staphylococcal protein A. Among plasmas or serums from 60 patients with RA, 12 patients had IgG binding to native collagen type II and 12 had IgG binding to denatured collagen type II. Decomplementing normal serums by heating to 56 degrees C for 30 min markedly increased IgG binding to denatured collagen type II, but not to native collagen. This binding was shown to result from a complex formed between IgG and fibronectin. Nine unheated RA serums were separated by gel filtration. The IgG binding to native collagen was limited to monomeric IgG, but in these serums 65.6 +/- 22.0% of the IgG binding to denatured collagen type II was in the excluded protein fractions. The binding of IgG to denatured collagen type II in the excluded fractions was inhibited by fibronectin and did not bind to staphylococcal protein A. In patients with RA, the IgG binding to native collagen type II represents true autoantibodies. In contrast, in these patients the majority of IgG binding to denatured collagen type II is caused by macromolecular complexes formed between fibronectin and IgG or between fibronectin and IgG-containing immune complexes.
IFN-gamma-inducing factor (IGIF) is a costimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12.
Authors: Kohno K , Kataoka J , Ohtsuki T , Suemoto Y , Okamoto I , Usui M , Ikeda M , Kurimoto M
Published: December 2022

We have previously reported the cloning of a novel cytokine, IFN-gamma-inducing factor (IGIF), which shared some biologic activities with IL-12. In this study, we analyzed the effects of murine IGIF on the activation of T cells, and compared the effects with those of IL-12. IGIF alone had no effect on the activation of T cell lines or Th1 clones, while IGIF increased the IFN-gamma production by antigen-stimulated T cell lines, but had no effect on IL-4 or IL-10 production. As reported with IL-12, IGIF served as a costimulatory factor for Th1 clones stimulated with Ag on B cell APC, immobilized anti-CD3, Con A, or IL-2 to augment IFN-gamma production and to induce IL-2R alpha-chain expression and proliferation of the Th1 clones, whereas IGIF had little or no effect on the IL-4 production and proliferation of Th2 clones stimulated with anti-CD3 or Ag. However, IGIF synergized with IL-12 to further augment the IFN-gamma production of the Th1 clones. Even in the presence of saturated amounts of IL-12, IGIF still augmented the IFN-gamma production and proliferation and enhanced the IL-2R alpha-chain expression of the Th1 clones. In contrast with IL-12, IGIF induced IL-2 production by Ag- or anti-CD3-stimulated Th1 clones. These two findings indicate that IGIF and IL-12 are utilizing different signal transduction pathways. We also found that IGIF as well as IL-12 was endogenously released through interaction between Th1 cells and spleen cell APC in the presence of specific Ag, and that it regulated IFN-gamma production. These results further suggest that IGIF may act as an immunoregulatory factor in the immune response.
Development of B cells in aged mice: decline in the ability of pro-B cells to respond to IL-7 but not to other growth factors.
Authors: Stephan R P , Lill-Elghanian D A , Witte P L
Published: December 2022

The process of B lymphopoiesis changes with increasing age, resulting in a severe drop in the number of pre-B cells in old mice. We have shown that the ability of freshly isolated pro-B cells to proliferate on stromal cells declines with age. In this study, we wanted to determine the reason for the diminished response of the aged pro-B cells. The functional alterations could arise from changes intrinsic to the pro-B cells or to composition of the precursor population. Changes in the composition of pro-B cells in marrow or long-term bone marrow culture system for B lymphocytes (LTBMC-B) could not account for the functional losses. We then examined the hypothesis that the diminished amount of proliferation on stromal cells was due to changes in the responsiveness to stroma-derived cytokines. The proliferation to IL-7 but not to stem cell factor (SCF) or insulin-like growth factor 1 (IGF-1) was severely impaired by 24 mo of age, independently of the concentration of IL-7 and length of culture time. The reduced IL-7 response could not be explained by an increase in the percentage of cells undergoing apoptosis; rather, a greater frequency of aged cells remained in G0/G1 after IL-7 stimulation. Furthermore, the impaired responsiveness of the aged pro-B cells is not due to diminished expression of the IL-7R alpha-chain or the common gamma-chain. Since only IL-7 responsiveness is impaired, we believe that the underlying molecular mechanism for the cellular alterations is specific to signaling through the IL-7 receptor complex.
A shift from negative to positive selection of autoreactive T cells by the reduced level of TCR signal in TCR-transgenic CD3 zeta-deficient mice.
Authors: Yamazaki T , Arase H , Ono S , Ohno H , Watanabe H , Saito T
Published: December 2022

T cell selection is thought to be determined through the interaction between TCR and Ag/MHC. However, the contribution of the level of TCR signal to thymic selection remains unclear. To address this issue, we analyzed T cell selection of male Ag (HY)-specific TCR transgenic (HYTg) mice crossed with CD3 zeta-deficient (zeta KO) mice (HYTg/zeta KO), which have impaired signaling through TCR. In male HYTg/zeta KO mice, the number of thymocytes was comparable to that in normal mice, and almost all the peripheral T cells were HY specific, although these positively selected cells were anergic to male Ag. From these observations, the decrease in TCR signaling by CD3 zeta deficiency resulted in both the avoidance of negative selection and the acquisition of positive selection of autoreactive T cells in male HYTg/zeta KO mice. There was a shift of T cell selection from positive to no selection of HY-specific T cells in female HYTg/zeta KO mice also. Collectively, these findings suggest that the level of TCR signal directly regulates T cell selection; furthermore, the findings have integrated the models of T cell selection into a concept based on the quantity of TCR signal.
A cyclic hexapeptide is a potent antagonist of alpha 4 integrins.
Authors: Vanderslice P , Ren K , Revelle J K , Kim D C , Scott D , Bjercke R J , Yeh E T , Beck P J , Kogan T P
Published: December 2022

The alpha 4 integrins mediate leukocyte adhesion to specific counter-receptors, including vascular cell adhesion molecule-1 (VCAM-1), the fibronectin splice variant containing connecting segment 1 (CS1), and mucosal addressin cell adhesion molecule-1. A series of cyclized peptides based on the LDV sequence of CS1 were synthesized and assayed for inhibition of alpha 4 integrin binding. The most potent peptide, C*WLDVC* (where * indicates disulfide-linked residues), inhibited alpha 4 beta 1-dependent binding of lymphocytes to VCAM-1 and CS1 with half-maximal inhibition achieved at 1 to 3 microM of peptide. The peptide proved more potent when the lymphocytes were activated with 1 mM MnCl2; half-maximal inhibition was reached at 0.4 and 0.05 microM for VCAM-1 and CS1, respectively. This represents a 100- to 800-fold increase in potency over a linear CS1 peptide in these same assays. C*WLDVC* also inhibited alpha 4 beta 7-dependent lymphocyte binding to the ligands mucosal addressin cell adhesion molecule-1, VCAM-1 and CS1. Immunoprecipitation of radiolabeled integrin indicated that the peptide could bind alpha 4 beta 1 and alpha 4 beta 7 directly and elute alpha 4 beta 1 from a CS1-conjugated agarose resin. The peptide showed selectivity for alpha 4 integrins in that it effectively inhibited alpha 4 beta 1-dependent, but not alpha 5 beta 1-dependent, binding of cells to intact fibronectin. Due to its small size and potency, C*WLDVC* may serve as a useful tool for the study of alpha 4 integrin biology and the development of small molecule therapeutics.
Non-killer cell-specific transcription factors silence the perforin promoter.
Authors: Zhang Y , Lichtenheld M G
Published: December 2022

Perforin is a pore-forming killer protein exclusively expressed by cells functionally defined as cytolytic lymphocytes. Previous reporter gene investigations have indicated that the cell-type-specific expression of the perforin gene is determined by the promoter and upstream region but the respective cis- and trans-acting molecules remain to be identified. In this investigation, we differentially display DNA-protein interactions of perforin-positive vs perforin-negative cell types and functionally test their biological relevance. Our results provide molecular evidence for the transcriptional repression of the perforin gene in non-killer cells by two novel regulatory elements. One of the respective transcription factors that are exclusively expressed by non-killer cells appears to be an Ets family member. Thus, the development of cells expressing perforin, and perhaps cytolytic lymphocytes in general, may involve a shutdown of the genes for these proteins.
Activation of human macrophage fungistatic activity against Histoplasma capsulatum upon adherence to type 1 collagen matrices.
Authors: Newman S L , Gootee L , Kidd C , Ciraolo G M , Morris R
Published: December 2022

Human monocyte/macrophages (Mphi) were adhered to extracellular matrix proteins, and the intracellular growth of Histoplasma capsulatum (Hc) yeasts were quantified and compared with their growth in Mphi adhered to plastic. Freshly isolated monocytes and cultured monocyte/derived Mphi adhered to type 1 collagen gels, but not to nongelled collagen-, fibronectin-, laminin-, or vitronectin-coated surfaces, demonstrated significant fungistatic activity against Hc yeasts. Activation of Mphi developed immediately upon adherence to the collagen matrices (1 h) and did not require additional time in culture. In addition, many of the yeasts were digested by 24 h postinfection. Mphi adhered to collagen maintained their fungistatic activity for up to 4 days, during which time monolayers cultured on plastic were destroyed. Culture of Mphi in the presence of IFN-gamma or TNF-alpha for 24 h before infection did not augment the fungistatic activity of collagen-adherent Mphi. Likewise, culture of monocytes on collagen gels with IL-3, granulocyte-Mphi CSF (GM-CSF) or Mphi CSF (M-CSF) for 7 days did not enhance Mphi fungistatic activity above that obtained by monocytes cultured on collagen alone. The mechanism(s) of Mphi-mediated fungistasis was not associated with production of toxic oxygen radicals, nitric oxide, or the restriction of intracellular iron. However, experiments with horseradish peroxidase-labeled gold colloids and immunoelectron microscopy demonstrated that phagolysosomal fusion, which is minimal in Hc-infected Mphi adhered to plastic, is enhanced significantly at both 1 h and 24 h postinfection in Mphi adhered to collagen matrices. These data suggest that in vivo, matrix-bound Mphi may express a previously unrecognized antifungal activity that proceeds in the absence of exogenous cytokines and is mediated, in part, by overcoming the capacity of Hc yeasts to inhibit Mphi phagolysosomal fusion.
Fibrin induction of ICAM-1 expression in human vascular endothelial cells.
Authors: Qi J , Kreutzer D L , Piela-Smith T H
Published: December 2022

In acute and chronic inflammatory processes, fibrin deposition, and leukocyte accumulation are classic histopathologic hallmarks. Previous studies have shown that fibrin and fibrin degradation products can have biologic effects on vascular endothelial cells and can induce the expression of several endothelial cell-derived factors that may be important in regulating inflammation and tissue repair. We now demonstrate that coculture of human vascular endothelial cells (EC) with fibrin results in the up-regulation of intercellular adhesion molecule-1 (ICAM-1), thus providing a first link between fibrin deposition and adhesion molecule expression, which may lead subsequently to leukocyte accumulation and extravasation. Increased ICAM-1 expression was demonstrated by ELISA, flow cytometry, and functional adhesion assays. EC ICAM-1 expression increased in a time and dose response fashion. Cell surface levels of ICAM-1 induced by fibrin were comparable to, or exceeded, levels induced by IL-1beta. ICAM-1 expression increased beginning at 4 h post-fibrin formation with sustained elevated expression at 48 h. Fibrin-stimulated EC also bound increased numbers of polymorphonuclear neutrophils in cellular adhesion assays. This increase in adhesion could be blocked by Ab to ICAM-1. Inhibition of fibrin polymerization also inhibited the up-regulation of ICAM-1. Culture medium from fibrin-stimulated EC contained elevated levels of soluble ICAM-1. These data suggest that fibrin deposition on vascular EC, in addition to other reported effects on EC metabolism, may also lead to leukocyte accumulation and extravasation through the induction of leukocyte adhesion molecules such as ICAM-1.