bluuebunny/crossref_metadata_2025 huggingface dataset:
Cutting Edge: Apoptosis Induced by a Chimeric Fas/FLICE Receptor: Lack of Requirement for Fas- or FADD-Binding Proteins
Authors: Memon Sarfraz A. , Hou Jing-zhou , Moreno M. Belen , Zacharchuk Charles M.
Published: January 2023

Current models for Fas (CD95)-mediated apoptosis suggest that FLICE/caspase-8 is recruited and activated, which results in cell death. However, the role of additional molecules in Fas signaling and FLICE activation is not clear. A chimeric Fas/FLICE (F/F) receptor, containing the extracellular/transmembrane portion of Fas and the caspase region of FLICE, mediated anti-Fas apoptosis. FLICE protease subunits were generated from the F/F precursor. Killing induced by Fas, but not F/F, was blocked by a dominant negative FADD. Apoptosis triggered through Fas and F/F was inhibited by coexpression of CrmA and p35, but not Bcl-xL. F/F bypassed Fas resistance in COS-7 cells and blocking by the death effector domain (DED)-containing viral protein MC159. These results show that: 1) F/F induces cell death, indicating that FLICE activation is sufficient for apoptosis and does not require additional Fas- or FADD-binding proteins; and 2) F/F bypasses proximal defects in Fas signaling that prevent FLICE recruitment or activation.
Antigen-Specific IgG Responses from Naive Human Splenocytes: In Vitro Priming Followed by Antigen Boost in the SCID Mouse
Authors: Brams Peter , Nguyen Mai-Lan , Chamat Soulaima , Royston Ivor , Morrow Phillip R.
Published: January 2023

High titers of Ag-specific human IgG were consistently achieved in SCID mice reconstituted with human splenocytes that had been primed with Ag in vitro and then boosted with Ag after engraftment into SCID mice. Specific human IgG titers in the hu-SPL-SCID mice reached approximately 1:4 × 105 when the mice were immunized with a neo-antigen, whereas titers reached 1:2 × 106 when recall responses were induced. Booster immunizations with Ag 21 days after the initial in vivo boost further enhanced this response, and specific human IgG titers of 1:17 × 106 were achieved. This represented an essentially monospecific IgG population. These responses were CD4+ T cell dependent. In addition, affinity maturation of the human Ab responses was observed. Spleens of hu-SPL-SCID mice with Ag-specific titers ≤1:1 × 106 were often significantly enlarged and often displayed visible tumors. Fourteen of sixteen B cell tumors removed from spleens of five such hu-SPL-SCID mice, produced Abs that were specific for the immunizing Ags. From such tumor, cloned cell lines were established. One such mAb, MLN-7 (γ1,κ), was raised to tetanus toxoid and had no identified cross-reactivity.
IFN-γ Is Critical for Long-Term Allograft Survival Induced by Blocking the CD28 and CD40 Ligand T Cell Costimulation Pathways
Authors: Konieczny Bogumila T. , Dai Zhenhua , Elwood Eric T. , Saleem Sohail , Linsley Peter S. , Baddoura Fady K. , Larsen Christian P. , Pearson Thomas C. , Lakkis Fadi G.
Published: January 2023

It is postulated that IFN-γ production hinders long-term acceptance of transplanted organs. To test this hypothesis, we compared survival of skin and heart allografts in wild-type (IFN-γ+/+) mice to that in IFN-γ gene knockout (IFN-γ−/−) mice. We found that perioperative blockade of the CD28 and/or CD40 ligand T cell costimulation pathways induces long-term skin and heart allograft survival in IFN-γ+/+ recipients but fails to do so in IFN-γ−/− mice or in wild-type mice treated with IFN-γ-neutralizing Ab at the time of transplantation. In vitro studies showed that endogenously produced IFN-γ down-regulates T cell proliferation and CTL generation in MLCs. These actions of IFN-γ were not mediated by TNF-α production or Fas-Fas ligand interactions. In vivo studies revealed exaggerated expansion and, subsequently, impaired deletion of superantigen-reactive T lymphocytes in IFN-γ−/− mice injected with staphylococcal enterotoxin B. Taken together, our findings indicate that IFN-γ does not hinder but instead facilitates induction of long-term allograft survival possibly by limiting expansion of activated T cells.
Requirement for Jak3 in Mature T Cells: Its Role in Regulation of T Cell Homeostasis
Authors: Sohn Sue J. , Forbush Katherine A. , Nguyen Nhan , Witthuhn Bruce , Nosaka Tetsuya , Ihle James N. , Perlmutter Roger M.
Published: January 2023

The tyrosine kinase Jak3 plays a key role in transducing signals from the IL-2, -4, -7, -9, and -15 receptors. Mice lacking Jak3 exhibit a profound, early block in both B and T cell development. To examine the mechanisms whereby Jak3 influences T cell function, we have reconstituted thymic development in Jak3−/− animals by introducing a Jak3 transgene in which expression was driven by the lck proximal promoter. Thymic reconstitution required Jak3 kinase activity, as catalytically inactive Jak3 did not restore early thymic development. Furthermore, the thymus-restricted expression pattern of the transgene allowed us to assess the requirement for Jak3 in peripheral T cells. In these mice, loss of Jak3 expression was associated with a failure to proliferate in response to antigen receptor crosslinking, the accumulation of T cells manifesting an activated cell surface phenotype, and an increased CD4/CD8 ratio among peripheral T cells, all of which are characteristics that were observed in Jak3−/− animals. Finally, we present data which suggest that peripheral T cells proliferate more rapidly in vivo and also undergo apoptosis more rapidly, upon loss of Jak3. Hence Jak3 exerts effects on mature peripheral T lymphocytes, as well as on thymocytes, resulting in the proper maintenance of circulating, quiescent cells.
A Mucosal Intranet: Intestinal Epithelial Cells Down-Regulate Intraepithelial, But Not Peripheral, T Lymphocytes
Authors: Yamamoto Masafumi , Fujihashi Kohtaro , Kawabata Keiko , McGhee Jerry R. , Kiyono Hiroshi
Published: January 2023

Epithelial cells and lymphocytes, including γδ and αβ T cells, in the gastrointestinal tract epithelium represent a major host defense intranet that is incompletely understood. Cell-to-cell interactions between intraepithelial lymphocytes (IELs) and intestinal epithelial cells (IECs) comprise this intranet, and we have assessed the role of IECs in the regulation of γδ and αβ T cell responses. When highly purified CD3+ IEL T cells were stimulated via the TCR-CD3 complex, high proliferative responses and cytokine synthesis were induced. However, the addition of viable IECs or purified IEC membranes (mIEC) down-regulated T cell proliferative and cytokine responses. Further, the inhibitory effect of mIEC was not restored by antibodies to TGF-β, CD1d, E-cadherin, or MHC class I or II. This inhibitory effect was noted for both γδ and αβ T cell subsets from IELs, and mRNA levels were reduced for both Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-5) cytokines in γδ and αβ IELs. In contrast, a purified membrane fraction obtained from thymocytes did not inhibit IEL proliferative responses. Further, mIEC did not inhibit splenic αβ T cell proliferative responses. These findings show that cell-to-cell interactions between intraepithelial γδ and αβ T cells and IECs occur via cell surface molecules, suggesting an intranet to prevent potential inflammatory responses at the intestinal mucosal surface.
Different Protein Kinase C Isoenzymes Regulate IL-2 Receptor Expression or IL-2 Synthesis in Human Lymphocytes Stimulated via the TCR
Authors: Szamel Marta , Appel Annette , Schwinzer Reinhard , Resch Klaus
Published: January 2023

Stimulation of purified human PBL with mAbs raised against the T cell receptor resulted in an immediate and transient activation of protein kinase C-α (PKC-α) and PKC-θ, peaking at 10 min, whereas PKC-β, -δ, and -ε were translocated with a delay of >90 min and remained activated for up to 2 h. To characterize specific functions of distinct PKC isoenzymes, Abs against different PKC isoenzymes were introduced by means of electropermeabilization. Neutralization of PKC-α and -θ resulted in the complete inhibition of IL-2R expression, whereas anti-PKC-β, -δ, and -ε Abs inhibited IL-2 synthesis. Extensive control experiments have shown that neither electropermeabilization nor control Ig influenced PKC activity and cellular functions. Our data thus clearly show that specific PKC isoenzymes regulate different cellular functions in stimulated human lymphocytes.
Inhibition of Cell Cycle Progression by a Synthetic Peptide Corresponding to Residues 65–79 of an HLA Class II Sequence: Functional Similarities but Mechanistic Differences with the Immunosuppressive Drug Rapamycin
Authors: Boytim Michelle L. , Lyu Shu-Chen , Jung Ron , Krensky Alan M. , Clayberger Carol
Published: January 2023

A synthetic peptide corresponding to a region of the α1 α-helix of DQA03011 (DQ 65–79) inhibits the proliferation of human PBL and T cells in an allele-nonspecific manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71–73, and 75–79 are critical for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65–79 peptide does not affect expression of IL-2 or IL-2R; however, IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin-dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism.
CD38 Functions Are Regulated Through an Internalization Step
Authors: Funaro Ada , Reiniš Milan , Trubiani Oriana , Santi Spartaco , Di Primio Roberto , Malavasi Fabio
Published: January 2023

The endocytosis of the human CD38 molecule has been investigated in normal lymphocytes and in a number of leukemia- and lymphoma-derived cell lines. CD38 internalization was followed using radioiodinated Abs in an acidic elution endocytosis assay to monitor the effects of cross-linking on internalization processes and to quantify the ratio of the internalized molecule. Second, conventional, confocal, and electron microscopies were used to evaluate the morphologic effects induced by ligation of the molecule with Abs mimicking the natural ligand(s). The results demonstrated that internalization is a reproducible phenomenon following CD38 ligation with both agonistic and nonagonistic specific Abs and involving only a fraction of the entire amount of the surface molecule. It is independent from signal transduction as can be inferred by the observation that 1) both agonistic and non agonistic Abs are effective and 2) the dynamic of internalization is much slower than that of cellular signaling. Morphologic studies demonstrated that endocytosis induced as a result of CD38 ligation presents a very specific pathway consisting of subcellular organelles fundamental to the processing of the complex. Our data indicate that down-regulation by endocytosis may be, in parallel with shedding, a regulatory element in activation and adhesion processes mediated by CD38. However, internalization seems not to be a key step in triggering intracellular signaling; more likely, it is a negative feedback control mechanism which interrupts signal transduction or cell-cell cross-talks mediated by membrane CD38.
PU.1/Spi-1 Is Essential for the B Cell-Specific Activity of the Mouse CD72 Promoter
Authors: Ying Han , Chang Ju-Fay , Parnes Jane R.
Published: January 2023

CD72 is a 45-kDa glycoprotein that is predominantly expressed on cells of the B lineage, except for plasma cells. Its expression pattern is representative of many B cell-specific proteins, which are essential for B cell development and activation but are down-regulated after B cells become terminally differentiated plasma cells. We have examined the promoter region of the mouse CD72 gene to identify sequences responsible for this regulatory pattern. The CD72 gene does not have an obvious TATAA box. Primer extension assays identified multiple transcription initiation sites. Deletion analyses have identified the 255-bp minimal promoter required for tissue-specific and developmental stage-specific expression. DNase I footprinting analysis of the CD72 minimal promoter revealed three protected elements: FP I, FP II, and FP III. Sequences corresponding to FP I or III gave increased reporter gene activity specifically in B cells, but not in T cells or NIH-3T3 cells. Sequences corresponding to FP II gave increased reporter gene activity in mature B cells, but not in plasma cells or non-B cells. Electrophoretic mobility shift assays and DNase I protection analyses revealed that FP I was bound by the transcription factor PU.1/Spi-1. Transient reporter analyses with plasmid bearing the mutated PU.1 binding site showed that binding of PU.1 is necessary for the increase in CD72 promoter activity in B cells. These results suggest that the 255-bp CD72 promoter confers both tissue specificity and developmental stage specificity, and that the B cell and macrophage-specific transcription factor PU.1 is essential for regulating the tissue specificity of the mouse CD72 promoter.
Analysis of BR96 Binding Sites for Antigen and Anti-Idiotype by Codon-Based Scanning Mutagenesis
Authors: Rosok Mae Joanne , Eghtedarzadeh-Kondri Mohammad , Young Kelly , Bajorath Jürgen , Glaser Scott , Yelton Dale
Published: January 2023

We performed a scanning mutagenesis study of heavy chain complementarity-determining region (CDR) residues to identify how mutations affected binding of the anti-carcinoma mAb BR96 to Ag, Lewis Y, and to an anti-Id Ab (anti-Id). By ELISA, we demonstrated that the anti-Id bound close to the Ag binding site of BR96, but the anti-Id and Ag sites were not identical. Immunoblot analysis and screening of light and heavy chain CDR libraries with multiple mutations in each CDR suggested that the heavy chain had greater involvement in anti-Id binding. We then analyzed contributions of individual residues in the heavy chain CDRs to binding of Ag and anti-Id. In a filamentous phage vector containing BR96 V region sequences, mutations were introduced by codon-based mutagenesis at single positions within the three heavy chain CDRs. The resulting libraries of Fab fragments had all amino acids represented at a CDR position. We evaluated the expressed Fabs for binding to Ag and anti-Id by plaque lift assay. We identified the positions with mutations that had the greatest negative effect on binding to the anti-Id and to Ag and analyzed them on the basis of the BR96 x-ray structure. The residues most important for binding to the anti-Id were located in heavy chain CDR1 and CDR2 and were peripheral to the residues within the Lewis Y binding pocket.
Base-Specific Sequences That Bias Somatic Hypermutation Deduced by Analysis of Out-of-Frame Human IgVH Genes
Authors: Dunn-Walters Deborah K. , Dogan Ahmet , Boursier Laurent , MacDonald Connie M. , Spencer Jo
Published: January 2023

Somatic hypermutation introduces mutations into IgV genes during affinity maturation of the B cell response. Mutations are introduced nonrandomly, and are generally targeted to the complementarity determining regions (CDRs). Subsequent selection against mutations that result in lower affinity or nonfunctional Ig increases the relative number of mutations in the CDRs. Investigation of somatic hypermutation is hampered by the effects of selection. We have avoided this by studying out-of-frame human IgVH4.21 and 251 genes, which, being unused alleles, are unselected. By comparison of the frequency of A, C, G, and T nucleotides at positions −3 to +3 around mutated or unmutated A, C, and G nucleotides, we have identified flanking sequences that most commonly surround mutated bases. Distinct trends in flanking sequences that were unique for each base were observed. Statistically significant trends that were common to both IgVH4.21 and 251 were used to deduce motifs that bias somatic hypermutation. The motifs deduced from this data, with targeted bases in regular type, are AANB, WDCH, and DGHD (where W = A/T, B = C/G/T, D = A/G/T, H = A/C/T, and N = any base). Mutations from C and G in two further groups of out-of-frame human IgVH genes, not used in the deduction of the motifs, occurred significantly within the motifs for C and G. The proposed target sequence for G is within the reverse complement of the target sequence for C, suggesting that the hypermutation mechanism may target only G or C. The mutation in the complementary base would appear on the other strand following replication.
A Peptide Binding Motif for HLA-DQA1*0102/DQB1*0602, the Class II MHC Molecule Associated with Dominant Protection in Insulin-Dependent Diabetes Mellitus
Authors: Ettinger Ruth A. , Kwok William W.
Published: January 2023

HLA-DQA1*0102/DQB1*0602 (DQ0602) is observed at a decreased frequency in insulin-dependent diabetes mellitus in different ethnic groups, suggesting a protective role for DQ0602. Analysis of overlapping peptides from human insulin found that insulin B(1–15) bound well to DQ0602 and exhibited a high degree of allelic specificity. Truncation analysis of insulin B(1–15) identified insulin B(5–15) as the minimal peptide for DQ0602 binding. Insulin B(5–15) bound to DQ0602 with an apparent KD of 0.7 to 1.0 μM and peptide binding reached equilibrium at 96 h. Single arginine substitutions at each position of the insulin B(5–15) peptide identified amino acids 6, 8, 9, 11, and 14 (relative positions P1, P3, P4, P6, and P9) as important for binding. Extensive substitutions for each of these amino acids revealed that amino acids 11 and 14 (P6 and P9) exhibited the highest specificity. Amino acid 11 (P6) preferred large aliphatic amino acids, while amino acid 14 (P9) preferred smaller aliphatic and hydroxyl amino acids. Binding of an overlapping series of peptides from a randomly chosen protein, the herpes simplex virus-2 tegument protein UL49, correlated completely with the presence or absence of the DQ0602 peptide binding motif. Peptides 11 amino acids long were selected from GAD65, IA-2, and proinsulin, that contained the DQ0602 peptide binding motif. Of these, 79% (19 of 24) were able to bind DQ0602. This study identifies a peptide binding motif for DQ0602 and peptides from insulin-dependent diabetes mellitus autoantigens that bind DQ0602 in vitro.
Type 1- and Type 2-Like Lesional Skin-DerivedMycobacterium leprae-Responsive T Cell Clones Are Characterized by Coexpression of IFN-γ/TNF-α and IL-4/IL-5/IL-13, Respectively
Authors: Verhagen Claudia E. , van der Pouw Kraan Tineke C. T. M. , Buffing Anita A. M. , Chand Mohamed A. , Faber William R. , Aarden Lucien A. , Das Pranab K.
Published: January 2023

In an earlier study, we generated a large number of Mycobacterium leprae-responsive and M. leprae-nonresponsive T cell clones (TCC) from the lesional skin of immunologic unstable borderline leprosy patients. In that study, we divided TCC into type 1- and type 2-like on the basis of their IFN-γ and IL-4 expression. To explore whether other cytokines are coproduced along with IFN-γ and IL-4, we investigated the secretion of a panel of other cytokines (TNF-α, IL-5, IL-6, IL-10, and IL-13) by a large number of these TCC. Upon analysis of 139 M. leprae-responsive TCC, we observed a positive correlation in the coproduction of IFN-γ/TNF-α (r = 0.81), and in that of IL-4/IL-5 (r = 0.83), IL-4/IL-13 (r = 0.80), and IL-5/IL-13 (r = 0.82). Polarized type 1-like TCC produced dominantly IFN-γ/TNF-α, and polarized type 2-like TCC predominantly IL-4/IL-5/IL-13. Most type 0-like TCC produced both sets of cytokines. In contrast, type 1- and type 2-like subsets of M. leprae-nonresponsive TCC (n = 58) did not show the same coexpression of these cytokines. Furthermore, when the differential expression of a broad panel of cytokines by individual M. leprae-responsive TCC is considered, it appeared that additional phenotypes could be recognized. These results suggested that distinct isotypes of type 1- and type 2-like T cells, based on the secretion of a panel of cytokines, may reflect M. leprae-specific characteristics.
IL-5 Is Required for Eosinophil Recruitment, Crystal Deposition, and Mononuclear Cell Recruitment During a Pulmonary Cryptococcus neoformans Infection in Genetically Susceptible Mice (C57BL/6)
Authors: Huffnagle Gary B. , Boyd Michael B. , Street Nancy E. , Lipscomb Mary F.
Published: January 2023

CBA/J (highly resistant), BALB/c (moderately resistant), and C57BL/6 (susceptible) mice displayed three resistance patterns following intratracheal inoculation of Cryptococcus neoformans 52. The inability to clear the infection correlated with the duration of the eosinophil infiltrate in the lungs. The role of IL-5 in promoting the pulmonary eosinophilia and subsequent inflammatory damage in susceptible C57BL/6 mice was investigated. C57BL/6 mice developed a chronic alveolar, peribronchiolar, and perivascular eosinophilia following C. neoformans infection. This resulted in the accumulation of intracellular Charcot-Leyden-like crystals in alveolar macrophages by wk 4 and the extracellular deposition of these crystals in the bronchioles with associated destruction of airway epithelium by wk 6. IL-5 mRNA was expressed in the lungs, and injections of anti-IL-5 mAb prevented eosinophil recruitment and crystal deposition but did not alter cryptococcal clearance. Depletion of CD4+ T cells (but not CD8+) ablated IL-5 production by lung leukocytes in vitro and eosinophil recruitment in vivo. Neutralization of IL-5 also inhibited the recruitment of macrophages, CD8+ T lymphocytes, and B lymphocytes by 47 to 57%. Anti-IL-5 mAb inhibited CD4+ T lymphocyte recruitment by 30% but did not affect neutrophil recruitment. Thus, the development of a chronic eosinophil infiltrate in the lungs of C. neoformans-infected C57BL/6 mice is a nonprotective immune response that causes significant lung pathology. Furthermore, IL-5 promotes the recruitment and activation of eosinophils, resulting in the recruitment of additional macrophages and lymphocytes into the lungs.
Lymphocyte-Dependent Inhibition of Growth of VirulentMycobacterium tuberculosisH37Rv Within Human Monocytes: Requirement for CD4+ T Cells in Purified Protein Derivative-Positive, But Not in Purified Protein Derivative-Negative Subjects
Authors: Silver Richard F. , Li Qing , Boom W. Henry , Ellner Jerrold J.
Published: January 2023

Protective human immunity to Mycobacterium tuberculosis(M. tb) has proven difficult to characterize, in part because of technical obstacles to in vitro infection of human cells with virulent M. tb. We established a reproducible method of infecting human monocytes (MN) with the virulent M. tb strain H37Rv that did not reduce MN viability. TNF-α had no effect on replication of H37Rv within MN, and IFN-γ mediated only a 1.9-fold reduction in bacterial growth. In contrast, nonadherent cells (NAC) from purified protein derivative (PPD)-positive and PPD-negative subjects reduced intracellular growth of H37Rv by 6- and 10.6-fold, respectively (p = 0.007 and p = 0.005). CD4+ T cells were essential to growth inhibition mediated by NAC of PPD-positive subjects, whereas containment of M. tb by NAC of PPD-negative subjects did not require CD4+ cells. CD8+ T cells did not contribute to protection mediated by NAC of either group. Supernatants of cocultured H37Rv-infected MN and NAC only partially reduced intracellular growth of M. tb despite containing nanogram concentrations of TNF-α and IFN-γ. Neutralizing antibodies to TNF-α, IFN-γ, and IL-12 failed to affect the NAC-mediated growth limitation. NAC treated with emetine retained approximately 40% of their capacity to contain intracellular H37Rv, however. These studies indicate that protective human recall responses to M. tb are mediated primarily by CD4+ T cells, whereas CD4−CD8− lymphocytes may contribute to innate immunity to M. tb. The ability of NAC to activate M. tb-infected MN is only partly attributable to soluble mediators and may also involve contact-mediated mechanisms.
Development of a Murine Mutant Ras CD8+ CTL Peptide Epitope Variant That Possesses Enhanced MHC Class I Binding and Immunogenic Properties
Authors: Bristol J. Andrew , Schlom Jeffrey , Abrams Scott I.
Published: January 2023

We recently identified a murine mutant Ras p21 CD8+ CTL epitope reflecting residues 4 to 12, containing the mutation of Gly to Val at codon 12, that bound weakly to H-2Kd in vitro and generated a weak primary CTL response in immunized BALB/c mice. Here, we explored the hypothesis that specific modifications to the Ras4–12 peptide sequence can improve MHC binding, leading to enhanced immunogenicity without altering immune specificity. We synthesized Ras4–12 peptides in which Val at residue 12 was replaced with the more dominant H-2Kd C-terminus anchor residue Leu or Ile. In functional H-2Kd binding assays, Ras4–12(L12 or I12) peptide variants competed more effectively than the Ras4–12(V12) peptide. Ras4–12(L12 or I12) peptide variants enhanced both in vitro cytotoxicity and proliferation responses of anti-Ras4–12 CTL compared with the mutant Ras4–12(V12) peptide. Additionally, the Ras4–12(L12) peptide variant induced a quantitatively greater T cell response in vivo compared with that produced by Ras4–12(V12) as determined by IFN-γ production. Mice immunized with Ras4–12(L12) peptide elicited CD8+ CTL activity specific for target cells presenting the Ras4–12(V12) epitope exogenously and endogenously. Moreover, both anti-Ras4–12(V12)-derived and anti-Ras4–12(L12)-derived CTL lines were similar insofar as their TCR usage and amino acid contact residues in the Ras4–12(V12) peptide. These experiments demonstrate that modifications can be introduced in tumor-specific peptide epitopes to enhance both in vitro and in vivo immunogenicity. The design of oncogene-specific peptide epitope variants as immunogens may accelerate the generation of anti-tumor T cell responses for cancer immunotherapy.
A Helminth-Induced Mucosal Th2 Response Alters Nonresponsiveness to Oral Administration of a Soluble Antigen
Authors: Shi Hai Ning , Ingui Christian J. , Dodge Ingrid , Nagler-Anderson Cathryn
Published: January 2023

A fascinating feature of the intestinal mucosal immune system is its ability to guard against invasion by pathogens while avoiding a response to the many potential Ags present in food. The phenomenon of systemic tolerance after oral administration of protein Ags is well documented, but the cellular and molecular basis for the observed nonresponsiveness is not fully understood. To gain insight into the role of the mucosal microenvironment in the induction of orally induced nonresponsiveness, we attempted to induce tolerance to OVA in mice primed for a Th2-biased mucosal immune response by infection with the nematode parasite Heligmosomoides polygyrus. We found that oral tolerance for Th1-type responses to OVA is maintained when OVA is fed during the peak of the mucosal immune response to H. polygyrus. Tolerance for Th2 cytokine responses or a Th2-dependent isotype of IgG is not induced in this Th2-biased mucosal environment. Treatment of infected mice with rIL-12 to reverse the Th2 polarity of the parasite-specific immune response restores tolerance of both Th1 and Th2 responses to OVA. We conclude that the polarized Th2 response induced by this enteric infection plays a central role in determining whether or not systemic tolerance is induced. Our results imply that attempts to use oral administration of Ag to suppress systemic immune responses will be influenced strongly by the presence of mucosal infection.
Lymphoproliferative Disease in Human Peripheral Blood Mononuclear Cell-Injected SCID Mice. IV. Differential Activation of Human Th1 and Th2 Lymphocytes and Influence of the Atopic Status on Lymphoma Development
Authors: Coppola Vincenzo , Veronesi Arianna , Indraccolo Stefano , Calderazzo Francesca , Mion Marta , Minuzzo Sonia , Esposito Giovanni , Mauro Daniele , Silvestri Barbara , Gallo Paolo , Falagiani Paolo , Amadori Alberto , Chieco-Bianchi Luigi
Published: January 2023

Intraperitoneal transfer of PBMC from EBV+ donors into SCID mice leads to high human Ig levels in mouse serum and B cell lymphoproliferative disease. As these events depend on the activation of coinjected human T cells, we addressed the behavior of the Th1 and Th2 subsets in this model. Production of IFN-γ, but not of Th2 cytokines such as IL-4, was detected in culture supernatants of PBMC stimulated in vitro with mouse splenocytes. Moreover, anti-CD3 stimulation of the human cells recovered from mice brought about IFN-γ, but not IL-4, synthesis; on the other hand, PCR and in situ hybridization analysis of ex vivo-recovered cells disclosed the presence of mRNA for both cytokines following in vitro restimulation, thus suggesting post-transcriptional regulation of IL-4 gene expression. When SCID mice were inoculated with PBMC from atopic donors, whose Th1/Th2 profile displays an imbalance toward Th2 cells, tumor development rates were lower, and tumor latency was higher, compared with those in mice injected with PBMC from normal donors. Isotypic analysis of human Ig in mouse serum showed the exclusive presence of IFN-γ-driven IgG subclasses; in addition, human IgE were low or undetectable in most cases. These findings indicate that following transfer into SCID mice, human Th1 lymphocytes undergo preferential activation, whereas Th2 function is down-regulated. Th1 lymphocytes probably are a major component in promoting EBV+ B cell expansion and tumor development; the individual Th1/Th2 profile could in part account for the as yet unexplained donor variability in tumor generation in this experimental model.
Malaria Elicits Type 1 Cytokines in the Human Placenta: IFN-γ and TNF-α Associated with Pregnancy Outcomes
Authors: Fried Michal , Muga Richard O. , Misore Ambrose O. , Duffy Patrick E.
Published: January 2023

Pregnant women, especially primigravidas, are highly susceptible to malaria infection, resulting in maternal anemia and low birth weight infants. Because circulating parasitemia is rare in the newborn, the cause of poor fetal outcomes has been unclear. We measured cytokine concentrations in placentas collected from women delivering in urban hospitals in malaria-holoendemic or nonendemic areas of Kenya. Normal placentas displayed a bias toward type 2 cytokines; type 1 cytokines IFN-γ and IL-2 were absent in placentas not exposed to malaria but present in a large proportion of placentas from a holoendemic area. TNF-α and TGF-β concentrations were significantly higher, and IL-10 concentrations significantly lower, in placentas from the holoendemic area. Among primigravidas, placental TNF-α concentrations were significantly higher in the presence of severe maternal anemia, and both IFN-γ and TNF-α were significantly elevated when a low birth weight, rather than normal weight, infant was delivered. We conclude that maternal malaria decreases IL-10 concentrations and elicits IFN-γ, IL-2, and TNF-α in the placenta, shifting the balance toward type 1 cytokines. This is the first demonstration that these placental cytokine changes are associated with poor pregnancy outcomes in humans.
Cutting Edge: CpG Oligodeoxynucleotides Trigger Protective and Curative Th1 Responses in Lethal Murine Leishmaniasis
Authors: Zimmermann Stefan , Egeter Oliver , Hausmann Susanne , Lipford Grayson B. , Röcken Martin , Wagner Hermann , Heeg Klaus
Published: January 2023

Synthetic oligodeoxynucleotides containing CpG dinucleotides (CpG-ODN) mimic the immunostimulatory qualities of bacterial DNA. We asked whether immunostimulation by CpG-ODN predisposes for a commitment toward a Th1 vs a Th2 response in Leishmania major infection, a model for a lethal Th2-driven disease, in BALB/c mice. CpG-ODN induced Th1 effector T cells in vitro and conveyed protective immunity to disease-prone BALB/c mice in vivo. Conversion to a Th1-driven resistant phenotype was associated with IL-12 production and maintained the expression of IL-12R β2-chains. Most strikingly, CpG-ODN were even curative when given as late as 20 days after lethal L. major infection, indicating that CpG-ODN revert an established Th2 response. These findings imply an important role of bacterial DNA and CpG-ODN in the instruction of adaptive immune responses. They also point to the therapeutic potential of CpG-ODN in redirecting curative Th1 responses in Th2-driven disorders.
Thymic Lineage Commitment Rather Than Selection Causes Genetic Variations in Size of CD4 and CD8 Compartments
Authors: van Meerwijk Joost P. M. , Bianchi Teresa , Marguerat Samuel , MacDonald H. Robson
Published: January 2023

During their development, immature CD4+CD8+ thymocytes become committed to either the CD4 or CD8 lineage. Subsequent complete maturation of CD4+ and CD8+ cells requires a molecular match of the expressed coreceptor and the MHC specificity of the TCR. The final size of the mature CD4+ and CD8+ thymic compartments is therefore determined by a combination of lineage commitment and TCR-mediated selection. In humans and mice, the relative size of CD4+ and CD8+ peripheral T cell compartments shows marked genetic variability. We show here that genetic variations in thymic lineage commitment, rather than TCR-mediated selection processes, are responsible for the distinct CD4/CD8 ratios observed in common inbred mouse strains. Genetic variations in the regulation of lineage commitment open new ways to analyze this process and to identify the molecules involved.
Modulation of Naive CD4 T Cell Activation with Altered Peptide Ligands: The Nature of the Peptide and Presentation in the Context of Costimulation Are Critical for a Sustained Response
Authors: Rogers Paul R. , Grey Howard M. , Croft Michael
Published: January 2023

Altered peptide ligands containing single amino acid substitutions have the potential to be used for modulating immune function. Using a panel of moth cytochrome c peptides, we demonstrate that different phases of naive CD4 T cell response are alternately modulated depending on altered peptide ligand dose and accessory molecule expression by APC. Weak agonists presented at high concentration, and with costimulation, efficiently induced early phase naive T cell activation as assessed by IL-2R/CD69 expression, but could only promote sufficient IL-2 for a short-lived proliferative response. In contrast, strong agonists and heteroclitic peptides induced early phase T cell activation even at low concentrations with costimulation, and allowed sustained IL-2 secretion and proliferation. In the absence of accessory molecule help, early and late phase activation was impaired with weak agonists, whereas strong agonists partially compensated for a lack of costimulation for early phase activation, and also promoted enhanced IL-2 with sustained proliferation. These studies support the hypothesis that the naive T cell response will be determined by the balance between provision of accessory molecule help and the affinity of peptide/MHC complexes for individual TCRs, and suggest that extended IL-2 production is the main facet of naive CD4 activation that is affected by altering the nature of the peptide.
MHC Class I Is Required for Peripheral Accumulation of CD8+ Thymic Emigrants
Authors: Nešić Dragana , Vukmanović Stanislav
Published: January 2023

MHC molecules influence the fate of T lymphocytes at two important stages of their differentiation. Recognition of self peptide/MHC complexes in the thymus determines whether immature T cells should live and mature into immunocompetent T cells or whether they should die. In the periphery, recognition of Ags presented by MHC molecules induces T cell activation, proliferation, and differentiation into effector/memory T cells. We describe in this work a third role that MHC molecules play in T cell physiology. CD8+ thymic emigrants require presence of MHC class I molecules in the periphery to seed the peripheral lymphoid organs. Numbers of CD8+ T cells are reduced severely in both the thymus and the periphery of β2-microglobulin-deficient (β2m−/−) mice. When grafted with wild-type (β2m+/+) thymic epithelium, immature β2m−/− T cells that populate the graft develop into functional mature CD8+ cells. However, significant numbers of peripheral CD8+ cells in grafted β2m−/− mice can be observed only after injection of MHC class I-expressing cells in the periphery. Thus, naive T cells in the periphery do not passively await antigenic stimulation, but actively engage in interactions with self MHC molecules that may promote their survival.
TCR Usage by Homocysteine-Specific Human CTL
Authors: Chilvers Myo-Myo , Wordsworth Paul , Stubbs Andy , Gao Xiao-Ming
Published: January 2023

We have recently demonstrated that homocysteine can modify HLA class I Ags and induce homocysteine-specific CTL (Hom-CTL) responses in humans. Here, we have investigated TCR usage by Hom-CTL from five patients with ankylosing spondylitis or reactive arthritis. TCR of HLA-A68-restricted Hom-CTL from two unrelated donors share the same TCR Vα, Vβ, and Jβ gene segments (AV4, BV23, and BJ2S1, respectively) with similar third complementarity determining regions (CDR3) of the β-chains. Interestingly, the Vα and Vβ gene segments employed by an HLA-B27-restricted Hom-CTL clone are also closely related to AV4 and BV23, indicating strong selection pressure for AV4, BV23, and related gene products in the homocysteine-specific TCR. An arginine or lysine residue frequently appeared at position α93 in the CDR3 of the TCR α-chains from Hom-CTL restricted by HLA-A68 or -B8. This may suggest a potential salt bridge between the carboxyl group of homocysteine and specific TCR. TCR usage by HLA-B27-restricted Hom-CTL from unrelated individuals appears to be less conserved, although two T cell clones from one individual rearranged the same V gene segments with identical lengths of CDR3. Implications of these data for the molecular mechanisms for homocysteine modification of HLA Ags are also discussed.
Potent Apoptotic Signaling and Subsequent Unresponsiveness Induced by a Single CD2 mAb (BTI-322) in Activated Human Peripheral T Cells
Authors: Dumont Céline , Déas Olivier , Mollereau Bertrand , Hebib Chafika , Giovino-Barry Valérie , Bernard Alain , Hirsch François , Charpentier Bernard , Senik Anna
Published: January 2023

Manipulation of CD2 molecules with CD2 mAb pairs has been shown to deliver apoptotic signals to activated mature T cells. We show that BTI-322, a CD2 mAb directed at a peculiar epitope of CD2, can trigger on its own the apoptotic death of IL-2-activated peripheral T cells and of OKT3-stimulated T cells, contrasting in this respect with a series of other mouse or rat CD2 mAb. F(ab′)2 fragments were as potent as the whole Ab. BTI-322-induced apoptosis proceeded in a few hours and was independent of the Fas/Fas ligand system. Less than 5 ng/ml of BTI-322, added at the begining of culture, were able to eliminate within 4 days most CD3+ cells from OKT3- and IL-2-stimulated lymphocytes, the only cells remaining being CD16+CD2− NK cells. T cell proliferative responses induced by a mitogenic CD2 mAb pair or by PHA-P (which mainly binds to CD2) were not inhibited by BTI-322. In this case, the apoptotic effect was successfully counteracted by simultaneous enhancement of T cell divisions. Thus, the killing effect of BTI-322 was most effective when T cells were exclusively stimulated through the CD3/TCR complex. Apoptosis of the responding T cells may explain why T cells recovered from a primary MLC performed in the presence of BTI-322 responded to third party cells but not to the primary stimulatory cells. These data constitute the rational basis for the use of BTI-322 for inducing tolerance in human allotransplantation.
Nuclear Factor κB Is Required for Peptide Antigen-Induced Differentiation of a CD4+CD8+ Thymocyte Line
Authors: Ochoa-Garay Jorge , Kaye Jonathan , Coligan John E.
Published: January 2023

NF-κB transcription factors are known to regulate the expression of a number of genes involved in T cell activation and function. Some evidence has suggested that they also play a role in T cell development. However, the role of NF-κB in Ag-induced thymocyte differentiation has not been directly addressed to date. Here we critically examine this role by employing DPK, a CD4+CD8+ thymocyte line that undergoes differentiation upon TCR engagement in a process that closely mimics positive selection. Expression of a degradation-resistant form of IκBα in DPK cells results in constitutive inhibition of NF-κB activity. We find that in the absence of NF-κB activity, MHC-peptide-induced differentiation of DPK is blocked. Furthermore, differentiation induced by a nonphysiologic stimulus, anti-TCR Ab, is greatly reduced. Altogether, our data indicate a requirement for NF-κB in the developmental changes associated with positive selection.
Posttranslational Regulation of TCR Vα Allelic Exclusion During T Cell Differentiation
Authors: Alam S. Munir , Gascoigne Nicholas R. J.
Published: January 2023

We have previously shown that phenotypic allelic exclusion of TCR α-chain is functional only in mature thymocytes. A significant proportion of immature thymocytes (TCRlow) express more than one cell surface α-chain, but mature thymocytes (TCRhigh) show phenotypic allelic exclusion and express only a single α-chain. We have analyzed thymocytes for both surface and intracellular α-chain expression and find that the majority of mature thymocytes express a second α-chain intracellularly. This result is predicted by a model in which the developmentally regulated allelic exclusion of the TCR α-chain is caused by competition between α-chains for the β-chain rather than by models in which one α-chain is down-regulated or in which selection favors cells with only a single α-chain species. Changes in the relative amounts of α- and β-chains available for pairing may therefore allow competition between the two α-chains for the β-chain. Peripheral T cells also frequently express second α-chains in the cytoplasm (18–27%), despite a rather low frequency of dual α-chain expression on the cell surface (2–4%). The frequency of nonsurface expressed α-chains is reduced somewhat compared with thymocytes, indicating that an additional level of control of allelic exclusion operates during the maturation of peripheral T cells.
Impaired Macrophage Function and Enhanced T Cell-Dependent Immune Response in Mice Lacking CCR5, the Mouse Homologue of the Major HIV-1 Coreceptor
Authors: Zhou Yuhong , Kurihara Takao , Ryseck Rolf-Peter , Yang Yi , Ryan Carol , Loy James , Warr Glenn , Bravo Rodrigo
Published: January 2023

The CC-chemokine receptor CCR5 has been shown to be the major coreceptor for HIV-1 entry into cells, and humans with homozygous mutation in the ccr5 gene are highly resistant to HIV-1 infection, despite the existence of many other HIV-1 coreceptors. To investigate the physiologic function of CCR5 and to understand the cellular mechanisms of these clinical observations, we generated a CCR5-deficient mouse model (ccr5−/−) by targeted deletion of the ccr5 gene. We found that although developed normally in a pathogen-free environment, CCR5-deficient mice showed reduced efficiency in clearance of Listeria infection and exsert a protective effect aganist LPS-induced endotoxemia, reflecting a partial defect in macrophage function. In addition, CCR5-deficient mice had an enhanced delayed-type hypersensitivity reaction and increased humoral responses to T cell-dependent antigenic challenge, indicating a novel role of CCR5 in down-modulating T cell-dependent immune response.
Induction and Enhancement of FcεRI-Dependent Mast Cell Degranulation Following Coculture with Activated T Cells: Dependency on ICAM-1- and Leukocyte Function-Associated Antigen (LFA)-1-Mediated Heterotypic Aggregation
Authors: Inamura Noriaki , Mekori Yoseph A. , Bhattacharyya Siba P. , Bianchine Peter J. , Metcalfe Dean D.
Published: January 2023

Activated mast cells are known to reside in close apposition to T cells in various inflammatory processes. In this regard, we have reported that activated mast cells form heterotypic aggregates with activated lymphocytes. To determine whether this interaction would result in mast cell degranulation, we examined the effect of EL-4, 2B4, or freshly isolated T cells, activated by PMA or immobilized anti-CD3 mAb, on histamine release from murine bone marrow-derived cultured mast cells (BMCMC). Coculturing BMCMC with activated but not with resting T cells resulted in significant histamine release. Also, FcεRI cross-linking-induced degranulation was augmented when BMCMC were cocultured with activated T cells. Supernatants of activated T cells failed to exert the stimulatory effect. Separation of the two cell populations with a porous membrane prevented degranulation, indicating that BMCMC activation was adhesion dependent. Indeed, the kinetics of histamine release paralleled the kinetics of the formation of heterotypic aggregates, which peaked after 12 h of coculture. Introduction of anti-LFA-1 and anti-intercellular adhesion molecule-1 mAb inhibited the adhesion-induced mast cell degranulation. These data suggest a heretofore unrecognized mast cell activation pathway induced by LFA-1/intercellular adhesion molecule-1-mediated heterotypic aggregation with activated T cells.
Posttranslational Modifications Affect the Activity of the Human Monocyte Chemotactic Proteins MCP-1 and MCP-2: Identification of MCP-2(6–76) as a Natural Chemokine Inhibitor
Authors: Proost Paul , Struyf Sofie , Couvreur Mikaël , Lenaerts Jean-Pierre , Conings René , Menten Patricia , Verhaert Peter , Wuyts Anja , Damme Jo Van
Published: January 2023

Chemokines are important mediators in infection and inflammation. The monocyte chemotactic proteins (MCPs) form a subclass of structurally related C-C chemokines. MCPs select specific target cells due to binding to a distinct set of chemokine receptors. Recombinant and synthetic MCP-1 variants have been shown to function as chemokine antagonists. In this study, posttranslationally modified immunoreactive MCP-1 and MCP-2 were isolated from mononuclear cells. Natural forms of MCP-1 and MCP-2 were biochemically identified by Edman degradation and mass spectrometry and functionally characterized in chemotaxis and Ca2+-mobilization assays. Glycosylated MCP-1 (12 and 13.5 kDa) was found to be two- to threefold less chemotactic for monocytes and THP-1 cells than nonglycosylated MCP-1 (10 kDa). Natural, NH2-terminally truncated MCP-1(5–76) and MCP-1(6–76) were practically devoid of bioactivity, whereas COOH-terminally processed MCP-1(1–69) fully retained its chemotactic and Ca2+-inducing capacity. The capability of naturally modified MCP-1 forms to desensitize the Ca2+ response induced by intact MCP-1 in THP-1 cells correlated with their agonistic potency. In contrast, naturally modified MCP-2(6–76) was devoid of activity, but could completely block the chemotactic effect of intact MCP-2 as well as that of MCP-1, MCP-3, and RANTES. Carboxyl-terminally processed MCP-2(1–74) did retain its chemotactic potency. Although comparable as a chemoattractant, natural intact MCP-2 was found to be 10-fold less potent than MCP-1 in inducing an intracellular Ca2+ increase. It can be concluded that under physiologic or pathologic conditions, posttranslational modification affects chemokine potency and that natural MCP-2(6–76) is a functional C-C chemokine inhibitor that might be useful as an inhibitor of inflammation.