bluuebunny/crossref_metadata_2025 huggingface dataset:
T Lymphocytes Are Required for Protection of the Vaginal Mucosae and Sensory Ganglia of Immune Mice Against Reinfection with Herpes Simplex Virus Type 2
Authors: Milligan Gregg N. , Bernstein David I. , Bourne Nigel
Published: January 2023

Intravaginal inoculation of mice with an attenuated strain of herpes simplex virus type 2 (HSV-2) resulted in vigorous HSV-specific immune responses that protected against subsequent challenge with fully virulent HSV-2 strains. Even in the presence of high titers of HSV-specific Ab, T cell-dependent mechanisms were required for protection of the vaginal mucosae of HSV-immune mice and could be detected by 24 h after intravaginal reinoculation. Depletion of specific T cell subsets from HSV-immune mice before HSV-2 reinoculation demonstrated that CD4+ T cells were primarily responsible for this protection. Similarly, optimal protection of the sensory ganglia against reinfection with HSV-2 was dependent on the presence of T cells. Infectious HSV-2 was not detected in the sensory ganglia or spinal cord of HSV-immune mice depleted of only CD4+ or CD8+ T cells, suggesting that the T cell-mediated protection could be provided by either subset. Similarly, neutralization of IFN-γ during challenge of HSV-immune mice resulted in diminished protection of the vaginal mucosa, but not of the sensory ganglia. These results suggest that the ability to induce vigorous HSV-specific T cell responses is an important consideration in the design of vaccines to protect both the vaginal mucosa and sensory ganglia against HSV-2.
Immunization with Purified Natural and Recombinant Allergens Induces Mouse IgG1 Antibodies That Recognize Similar Epitopes as Human IgE and Inhibit the Human IgE-Allergen Interaction and Allergen-Induced Basophil Degranulation
Authors: Vrtala Susanne , Ball Tanja , Spitzauer Susanne , Pandjaitan Budhi , Suphioglu Cenk , Knox Bruce , Sperr Wolfgang R. , Valent Peter , Kraft Dietrich , Valenta Rudolf
Published: January 2023

Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (β-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.
Physiologic Regulation of Postovulatory Neutrophil Migration into Vagina in Mice by a C-X-C Chemokine(s)
Authors: Sonoda Yasuo , Mukaida Naofumi , Wang Jian-bin , Shimada-Hiratsuka Motoko , Naito Makoto , Kasahara Tadashi , Harada Akihisa , Inoue Masaki , Matsushima Kouji
Published: January 2023

Leukocytes, particularly neutrophils, infiltrate into female genital organs after ovulation in both humans and mice. In mice, a female sexual cycle consists of 5 phases: proestrus, estrus, metestrus-1, metestrus-2, and diestrus. Ovulation occurs at estrus; at metestrus-2, a large number of neutrophils infiltrate into the vaginal epithelium accompanied by an increased neutrophil number in vaginal lavage fluid. Concomitantly, concentrations of a functional IL-8 homologue, murine macrophage inflammatory protein (MIP)-2, were increased significantly in vaginal lavage fluid at metestrus-2 as compared with other phases. On the contrary, MIP-2 was not detected in plasma during the whole course of a sexual cycle. Moreover, immunohistochemical analyses demonstrated that MIP-2 protein expression was prominent at the upper layer of the vaginal epithelium at metestrus-2, in contrast to a marginal staining in the vaginal epithelium at proestrus and estrus. These results suggest that a C-X-C chemokine, MIP-2, was produced physiologically in the vaginal epithelium in a sexual cycle-dependent manner. Furthermore, the administration of neutralizing anti-IL-8R homologue Abs at proestrus abrogated leukocyte infiltration into the vagina at metestrus. However, anti-MIP-2 Abs reduced leukocyte influx at metestrus by ∼50%. Thus, a murine IL-8 homologue, MIP-2, and its related molecules physiologically regulate neutrophil migration into the vagina in a sexual cycle-dependent manner.
Antibody-IL-12 Fusion Proteins Are Effective in SCID Mouse Models of Prostate and Colon Carcinoma Metastases
Authors: Gillies Stephen D. , Lan Yan , Wesolowski John S. , Qian Xiuqi , Reisfeld Ralph A. , Holden Sylvia , Super Michael , Lo Kin-Ming
Published: January 2023

IL-12 is a complex cytokine in both its structure and its range of biologic activities. Fusions of this heterodimeric molecule with an intact antitumor Ab were made to test the feasibility and efficacy of targeting IL-12 to tumors to elicit a local immune response. Fusion proteins composed of the human p35 and p40 subunits had IL-12 bioactivities that were nearly as potent on human immune cells as the rIL-12 standard, but were inactive on mouse cells. Hybrid IL-12 fusion proteins composed of mouse p35 and human p40, fused to Ab, were capable of inducing IFN-γ, but were much less active on mouse spleen cells than a mouse IL-12 standard. Despite this relatively low activity, the hybrid fusion protein was as effective in a SCID mouse model as a fully active Ab-IL-2 fusion protein in eliminating established pulmonary metastases of CT26 colon carcinoma. Specific targeting of a human IL-12 fusion protein to metastatic prostate carcinoma xenografts was also shown to be effective in SCID mice transplanted with human lymphocyte-activated killer cells. These results demonstrate the importance of directing this potent cytokine to the tumor microenvironment and suggest an important alternative to systemic IL-12 administration or gene therapy for increasing its therapeutic index.
Reconstitution of EBV-Specific T Cell Immunity in Solid Organ Transplant Recipients
Authors: Haque Tanzina , Amlot Peter L. , Helling Nabeela , Thomas J. Alero , Sweny Paul , Rolles Keith , Burroughs Andrew K. , Prentice H. Grant , Crawford Dorothy H.
Published: January 2023

EBV-specific autologous CTL were grown in vitro from three adults (two liver transplant recipients and one patient on hemodialysis awaiting kidney retransplant). All CTL lines were TCR αβ, CD8 positive cells, EBV specific, and MHC class I restricted. The CTL lines were expanded in vitro and infused in three escalating doses (5 × 107, 1 × 108, and 2 × 108) at monthly intervals. Weekly blood samples were collected following each infusion. EBV-specific CTL precursor cells in peripheral blood were quantitated by limiting dilution analysis, and their effect on EBV load in vivo was assessed by semiquantitative PCR. In all three patients, the numbers of CTL precursor cells increased during the weeks following the infusions and were highest after the third infusion. This level gradually declined but remained above the preinfusion levels for up to 3 mo. EBV genome copy number, on the other hand, dropped following the first infusion and became undetectable thereafter. The EBV DNA level remained lower than the pretransplant level in all patients for up to 3 mo after the last infusion. Our study shows that it is feasible to generate and expand EBV-specific CTL from pretransplant blood samples of solid organ transplant recipients, that these CTL can be stored and infused posttransplant, and that they remain cytotoxic and EBV specific in vivo. The aim of this study is to use these CTL for prevention and treatment of lymphoproliferative disease in solid organ transplant recipients.
Thymosin α1 Stimulates Endothelial Cell Migration, Angiogenesis, and Wound Healing
Authors: Malinda Katherine M. , Sidhu Gurmel S. , Banaudha Krishna K. , Gaddipati Jaya P. , Maheshwari Radha K. , Goldstein Allan L. , Kleinman Hynda K.
Published: January 2023

In wound healing, lymphoid cells release soluble factors that attract fibroblasts and macrophages, initiating repair, endothelial cell migration, angiogenesis, and matrix production. We analyzed the effect of thymosin α1 (Tα1) on endothelial cell migration, angiogenesis, and wound healing. Tα1, a 28 amino acid peptide initially isolated from the thymus, enhanced the morphologic differentiation of endothelial cells and was a potent chemoattractant for endothelial cells and monocytes in vitro. In vivo, Tα1 stimulated angiogenesis in a subcutaneous model. When given either topically or i.p., it accelerated wound healing in a punch model, demonstrating that Tα1 promotes angiogenesis and wound healing.
Rejection of Allogeneic and Syngeneic But Not MHC Class I-Deficient Tumor Grafts by MHC Class I-Deficient Mice
Authors: Freland Sofia , Chambers Benedict J. , Andersson Malena , Van Kaer Luc , Ljunggren Hans-Gustaf
Published: January 2023

The ability of TAP1−/−, β2m−/−, and TAP1/β2m−/− mice to mount rejection responses against allogeneic, syngeneic, and MHC class I-deficient tumor grafts was examined. The results demonstrate a potent ability of TAP1−/− and β2m−/− as well as TAP1/β2m−/− mice to reject allogeneic tumors. In contrast to published data, rejection of syngeneic MHC class I-expressing tumors was also observed. This response was specific for the MHC class I-deficient mice, since wild-type mice did not reject syngeneic MHC class I-positive tumors under identical experimental conditions. The rejection response of syngeneic tumors required preimmunization of the mice and was MHC class I specific at the level of priming as well as at the level of the tumor target. Finally, MHC class I-deficient tumor grafts were accepted in MHC class I-deficient mice while similar grafts were rejected in wild-type mice. In summary, while MHC class I-deficient mice have retained a capacity to reject allogeneic tumors, they have gained an ability to reject syngeneic MHC class I-positive tumors and lost the ability to reject MHC class I-negative tumors. The present results are discussed in relation to the role of MHC class I molecules in selecting functional CD8+ T and NK cell repertoires, and the development of cell-mediated immunity.
MHC Class II Expression in Double Mutant Mice Lacking Invariant Chain and DM Functions
Authors: Kenty George , Martin W. David , Van Kaer Luc , Bikoff Elizabeth K.
Published: January 2023

Invariant (Ii) chain and DM functions are required at distinct stages during class II maturation to promote occupancy by diverse peptide ligands. The class II molecules expressed by mutant mouse strains lacking Ii chain or DM activities display discrete structural and functional abnormalities. The present report describes the cellular and biochemical characteristics of Ii−DM− doubly deficient mice. As for Ii chain mutants, their mature AαbAβb dimers similarly exhibit reduced mobilities in SDS-PAGE, and in functional assays these molecules behave as if empty or occupied by an easily displaced peptide. Additionally, the present experiments demonstrate that the production of floppy AαbAβb dimers is TAP independent. In comparison with Ii chain mutants, Ii−DM− doubly deficient cell populations exhibit increased peptide binding activities and consistently greater presentation abilities in T cell stimulation assays. These functional differences appear to reflect higher class II surface expression associated with their increased representation of B lymphocytes. We also observe defective B cell maturation in mice lacking Ii chain or DM expression, and interestingly, B cell development appears more severely compromised in Ii−DM− double mutants. These mutant mice lacking both Ii chain and DM activities should prove useful for analyzing nonconventional class II Ag presentation under normal physiological conditions in the intact animal.
Activation of Low Avidity CTL Specific for a Self Epitope Results in Tumor Rejection But Not Autoimmunity
Authors: Morgan David J. , Kreuwel Huub T. C. , Fleck Shonna , Levitsky Hyam I. , Pardoll Drew M. , Sherman Linda A.
Published: January 2023

To determine how self-tolerance can alter the ability of the immune system to respond against tumor-associated Ags that are also expressed by normal tissue, we designed experiments in which the same protein was expressed both as a tumor Ag and as a transgene product. Unlike conventional BALB/c mice that rejected renal carcinoma cells transfected with the influenza virus hemagglutinin (Renca-HA), transgenic mice that are tolerant of HA due to its expression as a self-Ag on pancreatic islet β cells, (Ins-HA mice) supported progressive growth of these tumor cells. However, when Ins-HA mice were immunized with a recombinant strain of vaccinia virus expressing the dominant H-2Kd peptide epitope of HA before receiving Renca-HA cells, they too were able to reject the tumor cells. Rejection of Renca-HA cells by immunized Ins-HA mice was found to be associated with the generation of CTL having much lower avidity for target cells presenting the KdHA epitope than CTL from immunized conventional BALB/c mice. Significantly, we show that self-tolerance to the HA Ag is quantitative rather then absolute, and that vaccination of Ins-HA mice can activate low avidity KdHA-specific CD8+ T cells that are able to reject tumor cells expressing high levels of HA, yet these mice remain tolerant of pancreatic islet β cells expressing HA.
Lack of Intraclonal Diversification in Ig Heavy and Light Chain V Region Genes Expressed by CD5+IgM+ Chronic Lymphocytic Leukemia B Cells: A Multiple Time Point Analysis
Authors: Schettino Edward W. , Cerutti Andrea , Chiorazzi Nicholas , Casali Paolo
Published: January 2023

To analyze the modalities of clonal expansion of chronic lymphocytic leukemia (CLL) cells, we sequenced at multiple time points the V(D)J genes expressed by CD5+IgM+CLL B cells in three patients. All three V(D)J gene sequences were found to be point mutated. The mutation frequency in the Ig VH (3.96 × 10−2 and 2.41 × 10−2 change/bp) and Vκ and Vλ (6.67 × 10−2 and 1.74 × 10−2 change/bp) genes of two CLLs (1.19 and 1.32, respectively) was similar, and higher than that in the corresponding gene segments of the third CLL (1.69; 3.4 × 10−3 and 6.67 × 10−3 change/bp). In all three CLLs, there was no preferential representation of nucleotide changes yielding amino acid replacement (R mutations), nor was there any preferential segregation of R mutations within the Ig V gene complementarity-determining regions. In all three CLLs, the somatic mutations were all identical in multiple Ig VHDJH transcripts at any given time point, and were all conserved at multiple time points throughout a 2-yr period. The lack of concentration of R mutations in the complementarity-determining regions and the lack of intraclonal heterogeneity suggest that Ag may no longer be able to play a significant role in the clonal expansion of these cells. This conclusion would be strengthened further by the germline configuration of the bcl-1 and bcl-2 proto-oncogenes that are translocated in neoplastic B cells that display significant traces of intraclonal diversification and Ag-dependent selection, such as B-prolymphocytic leukemia and low grade follicular non-Hodgkin lymphoma.
Distinct Patterns of Folding and Interactions with Calnexin and Calreticulin in Human Class I MHC Proteins with Altered N-Glycosylation
Authors: Zhang Qing , Salter Russell D.
Published: January 2023

Calnexin is a lectin-like chaperone that binds to class I MHC molecules soon after their synthesis, retaining unassembled heavy chains and also assisting their folding. Following association with β2-microglobulin (β2m) in the endoplasmic reticulum, a large proportion of human class I molecules release from calnexin, whereas mouse class I molecules do not. We asked whether addition of a second N-glycan to the human class I molecule A*0201 at position 176, a site present in mouse, would affect its binding to calnexin. The 176dg mutant with N-glycans at positions 86 and 176, when transfected into CIR cells, demonstrated increased binding to calnexin, detectable both before and after association with β2m, and reduced interaction with calreticulin and TAP relative to wild-type protein bearing a single N-glycan at position 86. Cell surface levels of the mutant were decreased only slightly relative to the wild type, suggesting that the protein is not misfolded or grossly altered structurally. A subpopulation of mutant molecules was retained in the endoplasmic reticulum, and surprisingly, these molecules reacted with w6/32, which recognizes an epitope present on transport-competent class I HLA complexes. Transfection into Daudi cells demonstrated that 176dg reacts with w6/32 in the absence of β2m, suggesting that the Ab epitope can be induced by binding of calnexin. These data may explain previously noted differences between mouse and human class I MHC proteins and demonstrate that the location of N-oligosaccharides within proteins can influence their folding and interactions with chaperones such as calnexin and calreticulin.
Control of Leishmania major by a Monoclonal αβ T Cell Repertoire
Authors: Reiner Steven L. , Fowell Deborah J. , Moskowitz Naomi H. , Swier Kevin , Brown Daniel R. , Brown Charles R. , Turck Christoph W. , Scott2 Phillip A. , Killeen Nigel , Locksley3 Richard M.
Published: January 2023

Little is known regarding the diversity of the host T cell response that is required to maintain immunologic control of microbial pathogens. Leishmania major persist as obligate intracellular parasites within macrophages of the mammalian host. Immunity is dependent upon activation of MHC class II-restricted T cells to an effector state capable of restricting growth and dissemination of the organisms. We generated α-β Leishmania-specific (ABLE) TCR transgenic mice with MHC class II-restricted T cells that recognized an immunodominant Leishmania Ag designated LACK. Naive T cells from ABLE mice proliferated in vitro after incubation with recombinant LACK or with Leishmania-parasitized macrophages and in vivo after injection into infected mice. Infected ABLE mice controlled Leishmania infection almost as well as wild-type mice despite a drastic reduction in the T cell repertoire. ABLE mice were crossed to mice with disruption of the TCR constant region α gene to create animals with a single αβ T cell repertoire. Although mice deficient in all αβ T cells (TCR-Cαo mice) failed to control L. major, mice with a monoclonal αβ T cell repertoire (ABLE TCR-Cαo mice) displayed substantial control. The immune system is capable of remarkable efficiency even when constrained to recognition of a single epitope from a complex organism.
TNF Receptor-Deficient Mice Reveal Divergent Roles for p55 and p75 in Several Models of Inflammation
Authors: Peschon Jacques J. , Torrance Dauphine S. , Stocking Kim L. , Glaccum Moira B. , Otten Carol , Willis Cynthia R. , Charrier Keith , Morrissey Philip J. , Ware Carol B. , Mohler Kendall M.
Published: January 2023

The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.
Dynamic Regulation of α- and β-Chemokine Expression in the Central Nervous System During Mouse Hepatitis Virus-Induced Demyelinating Disease
Authors: Lane Thomas E. , Asensio Valérie C. , Yu Naichen , Paoletti Alyssa D. , Campbell Iain L. , Buchmeier Michael J.
Published: January 2023

Infection of C57BL/6 mice with the V5A13.1 strain of mouse hepatitis virus (MHV-V5A13.1) results in an acute encephalomyelitis and chronic demyelinating disease with features similar to the human demyelinating disease multiple sclerosis. Chemokines are a family of proinflammatory cytokines associated with inflammatory pathology in various diseases. The kinetics and histologic localization of chemokine production in the central nervous system of MHV-infected mice were examined to identify chemokines that contribute to inflammation and demyelination. Transcripts for the chemokines cytokine-response gene-2 (CRG-2), regulated on activation, normal T cell expressed and secreted (RANTES), macrophage-chemoattractant protein-1 and protein-3 (MCP-1, MCP-3), macrophage-inflammatory protein-1β (MIP-1β), and MIP-2 were detected in the brains of MHV-infected mice at 3 days postinfection (p.i.), and these transcripts were increased markedly in brains and spinal cords at day 7 p.i., which coincides with the occurrence of acute viral encephalomyelitis. By day 35 p.i., RANTES, CRG-2, and MIP-1β were detected in brains and spinal cords of mice with chronic demyelination. CRG-2 mRNA expression colocalized with viral RNA and was associated with demyelinating lesions. Astrocytes were the predominant cell type expressing CRG-2 mRNA. These observations suggest a role for chemokines, notably CRG-2, in the initiation and maintenance of an inflammatory response following infection with MHV, which is important in contributing to demyelination.
Gene Organization and Promoter Function for CC Chemokine Receptor 5 (CCR5)
Authors: Guignard Florence , Combadiere Christophe , Tiffany H. Lee , Murphy Philip M.
Published: January 2023

CC chemokine receptor 5 (CCR5) functions physiologically as a receptor for the leukocyte chemoattractants macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, and RANTES, and functions pathologically as a key cell entry coreceptor for HIV-1. The factors that regulate CCR5 expression may be useful therapeutic targets for HIV-1 infection. To identify nuclear regulatory factors, we have located and functionally characterized the CCR5 gene promoter. The gene consists of two exons separated by a 1.9-kb intron. Exon 1 contains 43 bp of the 5′-untranslated region; exon 2 contains 11 bp of the 5′-untranslated region and the complete open reading frame. Primer extension analysis identified two adjacent transcriptional start points (tsp) that map to the first 2 bp found in the longest known CCR5 cDNA sequence. A TATA box is present 31 bp upstream from the first tsp. CCR5 mRNA was detected constitutively in both primary human myeloid and lymphoid cells by Northern blot hybridization. Consistent with this, transcription of a chloramphenicol acetyltransferase reporter gene was constitutively activated in both transiently transfected myeloid and lymphoid cell lines by the 80-bp gene fragment located immediately upstream of the tsp. Deletion analysis located a strong silencer element between nucleotides −244 and −80, and a strong enhancer element between −486 and −244. These results suggest that the gene region between −486 and −1 may regulate the expression of CCR5 in monocyte/macrophages and T lymphocytes.
Functional Roles of Fas and Bcl-2-Regulated Apoptosis of T Lymphocytes
Authors: Van Parijs Luk , Biuckians Andre , Abbas Abul K.
Published: January 2023

Apoptotic cell death is an important mechanism for maintaining homeostasis in the immune system and for regulating the fates of lymphocytes following encounters with self and foreign Ags. To study the physiologic roles of the proapoptotic Fas pathway and the antiapoptotic protein, Bcl-2, in T cell maturation and homeostasis, a TCR transgene has been bred into mice lacking functional Fas and mice that express Bcl-2 constitutively. In vitro, Fas-deficient T cells are resistant to activation-induced cell death, whereas Bcl-2-overexpressing T cells are resistant to death induced by withdrawal of growth factors. In vivo, Bcl-2-overexpressing mice accumulate T cells in the thymus and peripheral lymphoid tissues in the absence of Ag, but these cells are deleted normally after Ag administration. In contrast, Fas-deficient mature T cells are present in normal numbers in the absence of Ag, but are resistant to Ag-induced deletion. Both Fas-deficient and Bcl-2 overexpressing thymocytes are deleted when exposed to transgene-encoded circulating self Ag, indicating that the pathways of apoptosis controlled by these proteins are not critical for negative selection of developing thymocytes. Moreover, deficiency of Fas, but not Bcl-2 overexpression, results in the accumulation of autoreactive T cells in peripheral lymphoid tissues. These results demonstrate that Fas and Bcl-2 regulate different pathways of apoptosis that may serve distinct functions in lymphocyte homeostasis and in the maintenance of T cell tolerance.
Synergistic Activation of CD4+ T Cells by IL-16 and IL-2
Authors: Parada Nereida A. , Center David M. , Kornfeld Hardy , Rodriguez Wilma L. , Cook Jennifer , Vallen Margaret , Cruikshank William W.
Published: January 2023

IL-16, in a CD4-dependent manner, induces high affinity IL-2R (CD25) selectively on CD4+ T cells. Based on this observation, we determined the relative effects of IL-16 on IL-2Rα, β, and γ expression on CD4+ T cells and of IL-16/IL-2 cotreatment of resting human PBMC obtained from normal individuals on CD4+ T cell proliferation and cytokine production, in vitro. IL-16 increased CD4+ T cell IL-2Rα and β expression, but had no effect on expression of IL-2Rγ. There was marked synergy of thymidine uptake and expansion of CD4+ T cell numbers in the presence of IL-16 and IL-2 or IL-16 and IL-15 compared with the responses to any of the cytokines alone. By 4 wk, IL-16/IL-2-cotreated PBMC cultures were predominantly CD4+, CD25+ CD45RO T cells. Of the cytokines measured, IL-16 treatment alone was sufficient to induce synthesis of granulocyte-macrophage CSF by 2 wk. IL-16/IL-2 cotreatment did not appear to induce selective proliferation of any Th subset, as cytokines of both Th1 (e.g., IFN-γ) and Th2 (e.g., IL-5) types were synthesized by the expanded cell populations at 2 and 4 wk. These results suggest that IL-16 can prime CD4+ T cells for IL-2 responsiveness, and therefore may be a useful adjunct to IL-2 therapy for immune reconstitution in disease or therapeutic conditions resulting in CD4+ T cell depletion.
CD40 Ligand and Appropriate Cytokines Induce Switching to IgG, IgA, and IgE and Coordinated Germinal Center and Plasmacytoid Phenotypic Differentiation in a Human Monoclonal IgM+IgD+ B Cell Line
Authors: Cerutti Andrea , Zan Hong , Schaffer Andras , Bergsagel Leif , Harindranath Nagaradona , Max Edward E. , Casali Paolo
Published: January 2023

B lymphocytes are induced to undergo Ig class switching and a complex phenotypic differentiation by the milieu of the germinal center. Partly as a result of the lack of a suitable in vitro B cell model, the relationship between these processes in the humans has never been formally established in vitro. We have identified a human monoclonal B cell line, CL-01, that expresses surface IgM and IgD and, upon induction with CD40 ligand, IL-4, and IL-10, switches to all seven downstream isotypes, showing typical DNA switch recombination preceded by germline transcription of targeted CH regions. In CL-01 cells, switch-inducing stimuli trigger concomitant changes in expression of surface IgD, CD23, CD38, and CD77 that parallel those reported in ex vivo isolated tonsillar centroblasts, centrocytes, and memory B cells. Eventually, in the presence of IL-6, CL-01 cells express CD56 and accumulate cytoplasmic IgG and IgA, both traits of plasmacytoid differentiation. Analysis of transcription and recombination of the Ig H locus in sorted CL-01 cells suggest that Ig class switching begins in centroblasts, it extends to all isotypes in centrocytes, and it is extinct in memory B cells. Thus, we have induced coordinated Ig class switching, progression through germinal center phenotypic stages, and differentiation to memory B cells and plasma cells at the level of a single B clonotype. Our data suggest that these processes are likely regulated by a common maturation program, the activation of which may require CD40 ligand, IL-4, IL-10, and IL-6 only.
Fetal Mouse NK Cell Clones Are Deficient in Ly49 Expression, Share a Common Broad Lytic Specificity, and Undergo Continuous and Extensive Diversification In Vitro
Authors: Manoussaka Maria S. , Smith Rachel J. , Conlin Victoria , Toomey Jennifer A. , Brooks Colin G.
Published: January 2023

NK cells obtained by exposing mouse fetal thymocytes to appropriate combinations of IL-4, IL-2, and PMA are phenotypically indistinguishable from cultured adult splenic NK cells with the exception that they generally lack measurable expression of all of the inhibitory Ly49 molecules that can currently be detected with Abs (Ly49A, -C, -G, and -I) and of the activating molecule Ly49D. Despite this deficiency, fetal NK cells have a similar specificity to Ly49-expressing adult splenic NK cells. Individual fetal NK cell clones display an essentially invariant and broad specificity similar to that of polyclonal populations of fetal or adult NK cells, although significant differences in the fine specificity of clones can occasionally be detected. Most remarkably, cloned fetal NK cell lines display heterogeneous expression of a restricted set of surface molecules that includes 10A7, Ly6C, 3C2, CD8, certain isoforms of CD45, and also, occasionally, Ly49 molecules. This heterogeneity is not related to the cell cycle or activation status of the cells, and micromanipulation recloning demonstrates unambiguously that it is not due to a lack of a single cell origin. Diversity is generated rapidly and the capacity for diversification appears to persist indefinitely in vitro. The expression of individual variable Ags is independent and stochastic, resulting in fetal NK “clones” being potentially composed of hundreds of phenotypically distinct cells. We hypothesize that fetal NK cells behave as progenitor cells that are undergoing a process of rapid, extensive, and continuous diversification and that are individually capable of generating and regenerating a complex NK cell repertoire.
CD28 Receptor Endocytosis Is Targeted by Mutations That Disrupt Phosphatidylinositol 3-Kinase Binding and Costimulation
Authors: Céfaï Daniel , Schneider Helga , Matangkasombut Oranart , Kang Hyun , Brody Joshua , Rudd Christopher E.
Published: January 2023

Although the lipid kinase phosphatidylinositol 3-kinase (PI-3K) binds at high levels to the cytoplasmic tail of CD28, controversy exists regarding its role in CD28 costimulation. Potentially, the kinase could be linked to a signaling cascade or be needed indirectly in events such as receptor endocytosis. Indeed, little is known regarding both the fate of CD28 following receptor ligation and the events that control the process. In this study, we help to resolve this issue by providing evidence that PI-3K plays a role in regulating CD28 endocytosis. We show that ∼25 to 35% of wild-type CD28 becomes endocytosed following Ab binding (t1/2 = 10 min), followed by segregation into two pools; one pool is destined for degradation in lysosomal compartments and is blocked by chloroquine, and another pool that is recycled to the cell surface (t1/2 = 2.5 h). Recycling of CD28 could have an important impact on CD80/86-mediated costimulation by replenishing functionally active receptors on the cell surface. Several findings implicate PI-3K in the control of endocytosis. Modulation experiments indicate that CD28-PI-3K complexes are preferentially endocytosed, and mutations that alter PI-3K binding concordantly affect the efficacy of endocytosis. Importantly, mutations that inhibit receptor internalization also block cosignaling. Therefore, previous results documenting a requirement for PI-3K may be explained by a blockage of receptor internalization.
Identification of an IL-7-Associated Pre-Pro-B Cell Growth-Stimulating Factor (PPBSF). I. Production of the Non-IL-7 Component by Bone Marrow Stromal Cells from IL-7 Gene-Deleted Mice
Authors: McKenna Sean D. , Chen Fangqi , Lai Laijun , Goldschneider Irving
Published: January 2023

Mouse bone marrow (BM) stromal cell conditioned medium (CM) from our long-term lymphoid culture system selectively induces the in vitro proliferation and presumptive differentiation of pre-pro-B cells (B220+, HSA−, TdT− or TdT+, cμ−) from adult rat, mouse, and human BM. However, the responsible growth factor(s) has not yet been identified. Inasmuch as IL-7 is one of the cytokines most closely associated with early B-lineage development, we utilized BM adherent cells and stromal cell lines from IL-7 gene-deleted (−/−) mice in combination with rIL-7 and anti-IL-7 mAb to investigate its possible regulatory role in our culture system. The results show that, although rIL-7 and IL-7 (−/−) CM each can maintain the viability of freshly harvested pre-pro-B cells in vitro, neither induces them to proliferate and/or differentiate, even in the presence of recombinant stem cell factor (rSCF) and/or recombinant insulin-like growth factor (rIGF). The results also show that anti-IL-7 mAb fails to neutralize the pre-pro-B cell growth-stimulating activity in IL-7 (+/+) CM. Yet rIL-7 enables IL-7 (−/−) CM to induce proliferation of pre-pro-B cells, and to “prime” them to respond directly to monomeric IL-7. Furthermore, anti-IL-7 mAb adsorbs the pre-pro-B cell growth-stimulating activity from both IL-7 (+/+) CM and rIL-7-supplemented IL-7 (−/−) CM; but rIL-7 does not restore this activity. Lastly, both pre-pro-B cell growth-stimulatory activity and IL-7 are quantitatively recovered by ultrafiltration in the 50 to 100 kDa, rather than the 10 to 50 kDa, apparent molecular mass fraction. These results suggest that the pre-pro-B cell growth-stimulating activity in our culture system is the property of a self-associating complex of IL-7 and a second BM stromal cell-derived cofactor.
Human Purified Protein Derivative-Specific CD4+ T Cells Use Both CD95-Dependent and CD95-Independent Cytolytic Mechanisms
Authors: Lewinsohn David M. , Bement Teresa T. , Xu Jiangchun , Lynch David H. , Grabstein Kenneth H. , Reed Steven G. , Alderson Mark R.
Published: January 2023

CTL, both CD4+ and CD8+, are essential in the eradication of intracellular pathogens. Data generated using murine T cells have suggested a critical role for CD95 (Fas, Apo-1) in CD4+ T cell-induced apoptosis of target cells. In contrast, CD8+ CTL predominantly use the perforin/granzyme lytic pathway. At present little is known about the mechanism of CD4+ CTL lytic function during intracellular infection in humans. We have used human CD4+ T cells specific for purified protein derivative (PPD) of Mycobacterium tuberculosis to explore whether CD95 is the dominant cytolytic mechanism. PPD-reactive CD4+ clones efficiently lysed Ag-pulsed autologous monocytes, adherent macrophages, and EBV-transformed B cells. Addition of an antagonistic CD95 Ab had a minimal effect on cytolysis, whereas addition of MgEGTA to block perforin/granzyme resulted in complete inhibition of killing. In contrast, lysis of activated peripheral blood B cells could be partially blocked with the antagonistic CD95 Ab. Supporting these observations, monocytes, macrophages, and EBV-transformed B cells were not lysed by an agonistic CD95 Ab. Activated B cells were readily lysed by the agonistic CD95 Ab. T cell clones triggered through the TCR with anti-CD3 were capable of lysing the CD95-sensitive Jurkat T cell line in a CD95-dependent manner, but were also able to release granzymes. We conclude that human CD4+ T cells are capable of lysing PPD-pulsed targets using both perforin/granzyme and CD95 pathways. The contribution of CD95 is strictly dependent on target cell susceptibility to CD95-mediated killing.
Post-Translational Processing of Murine IL-1: Evidence that ATP-Induced Release of IL-1α and IL-1β Occurs via a Similar Mechanism
Authors: Perregaux David G. , Gabel Christopher A.
Published: January 2023

In response to LPS, peritoneal macrophages produce IL-1, but, for the most part, newly synthesized cytokine molecules remain cell associated. Externalization and proteolytic processing of pro-IL-1β can be initiated by extracellular ATP. In this study, kinetics and inhibitor sensitivity of the stimulus-coupled mechanism were investigated with [35S]methionine-labeled macrophages. Optimal ATP concentrations required to promote cytokine post-translational processing suggest the involvement of a P2Z type of receptor. Proteolysis of pro-IL-1β initiates within 7.5 min of ATP addition; 17-kDa mature IL-1β is observed first intracellularly and subsequently extracellularly. In contrast, ATP-treated cells do not contain 17-kDa IL-1α. Macrophages exposed to ATP continuously or only for a 15-min pulse release IL-1α, IL-1β, and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1β exceeds that of IL-1α in both formats, but pulsed cells process the externalized cytokines more efficiently. Ethacrynic acid and DIDS (4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid) block ATP-induced proteolysis of pro-IL-1β and prevent release of pro-IL-1α/β and LDH; they do not inhibit ATP-induced K+ (86Rb+) efflux. Ethacrynic acid inhibits release of both forms of IL-1 with a similar concentration dependence; within the arrested cells, procytokines accumulate in a Triton-insoluble fraction. An IL-1β-converting enzyme inhibitor blocks proteolysis of IL-1β, but it does not prevent release of pro-IL-1α, pro-IL-1β, or LDH. These results indicate that ATP stimulates externalization of both IL-1α and IL-1β. The ATP-induced cytokine release mechanism is accompanied by cell death and requires activity of an anion transport inhibitor-sensitive component, but this pathway operates independently of cytokine proteolytic processing.
CD137 (ILA/4-1BB), a Member of the TNF Receptor Family, Induces Monocyte Activation via Bidirectional Signaling
Authors: Langstein Joachim , Michel Jan , Fritsche Jana , Kreutz Marina , Andreesen Reinhard , Schwarz Herbert
Published: January 2023

CD137 (ILA/4-1BB), a member of the TNF receptor family, was shown previously to inhibit proliferation and to induce apoptosis in T lymphocytes. In this study, we identify CD137 as a novel and potent monocyte activation factor. CD137 protein induces expression of IL-6, IL-8, and TNF-α, and inhibits expression of IL-10. Furthermore, CD137 differentially regulates expression of cell surface receptors. It induces expression of ICAM and reduces expression of FcγRIII, while levels of HLA-DR remain unchanged. CD137 also promotes adherence of monocytes. These effects of CD137 require immobilization of CD137 protein, indicating that they are mediated by cross-linking of a corresponding ligand/coreceptor expressed on monocytes.
A Double Recombinant Adenovirus Expressing the Costimulatory Molecule B7-1 (Murine) and Human IL-2 Induces Complete Tumor Regression in a Murine Breast Adenocarcinoma Model
Authors: Emtage Peter C. R. , Wan Yonghong , Bramson Jonathan L. , Graham Frank L. , Gauldie Jack
Published: January 2023

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL 2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.
Cutting Edge: Predictable TCR Antigen Recognition Based on Peptide Scans Leads to the Identification of Agonist Ligands with No Sequence Homology
Authors: Hemmer Bernhard , Vergelli Marco , Gran Bruno , Ling Nick , Conlon Paul , Pinilla Clemencia , Houghten Richard , McFarland Henry F. , Martin Roland
Published: January 2023

The potential of CD4+ T cells for cross-recognition of self and foreign Ags has important implications for the understanding of thymic selection, lymphocyte survival, and the occurrence of autoimmune diseases. Here, we define the extensive flexibility of Ag recognition for three human CD4+ autoreactive T cell clones (TCC) by using ligands with single and multiple amino acid (aa) substitutions. Our results demonstrate that the spectrum of tolerated ligands and the resulting stimulatory potency of peptides for a TCC can be predicted by the relative influence of each aa. Using this approach, we have identified stimulatory ligands not sharing a single aa in corresponding positions with the Ag used to establish the TCC. These results argue for an independent contribution of each aa in the peptide sequence to the affinity of the MHC/peptide complex to the TCR.
Cross-Reactivity in Murine Fluoroquinolone Photoallergy: Exclusive Usage of TCR Vβ13 by Immune T Cells That Recognize Fluoroquinolone-Photomodified Cells
Authors: Tokura Yoshiki , Seo Naohiro , Yagi Hiroaki , Furukawa Fukumi , Takigawa Masahiro
Published: January 2023

Fluoroquinolone antibacterial agents are well known to elicit photosensitivity as an adverse effect, and their cross-reactivity has been clinically documented. The photoallergenicity of fluoroquinolones is mainly derived from their photohaptenic moiety, and photomodification of skin epidermal cells with fluoroquinolones is thought to be an initial step for this photoallergy. Here we have explored, both in vivo and in vitro, T cell responses to fluoroquinolone-photomodified cells, focusing on their photoantigenic cross-reactivity. Cells were derivatized with fluoroquinolones under exposure to UV-A, and fluoroquinolone photoadducts were detected in photomodified cells by immunostaining, flow cytometry, and cell ELISA using fluoroquinolone-specific mAb. T cell-mediated hypersensitivity induced and elicited by s.c. injection of fluoroquinolone-photomodified epidermal cells was cross-reactive among six fluoroquinolones. In addition, lymph node cells from mice sensitized with fluoroquinolone-photomodified cells proliferated well in vitro not only to Langerhans cell-enriched epidermal cells photoderivatized with corresponding fluoroquinolone, but also to those photomodified with any of five other fluoroquinolones, supporting their cross-reactivity. In three fluoroquinolones tested, Th1 populations that expanded after in vitro photoantigenic stimulation of immune lymph node cells expressed the same Vβ13 of TCR. The sensitivity could be transferred by the i.v. administration of this Vβ13+ T cell line into naive recipients, in which a high percentage of Vβ13+ cells infiltrated at the challenge site. These findings suggest that these fluoroquinolones carry the same photoantigenic epitope, which is recognized by Vβ13+ T cells, leading to fluoroquinolone photosensitivity and cross-reactivity.
Differential Capacities of CD4+, CD8+, and CD4−CD8− T Cell Subsets to Express IL-18 Receptor and Produce IFN-γ in Response to IL-18
Authors: Tomura Michio , Maruo Seiji , Mu Jie , Zhou Xu-Yu , Ahn Hyun-Jong , Hamaoka Toshiyuki , Okamura Haruki , Nakanishi Kenji , Clark Steven , Kurimoto Masashi , Fujiwara Hiromi
Published: January 2023

IL-12 and IL-18 have the capacity to stimulate IFN-γ production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8+, CD4+, and CD4−CD8− T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8+, CD4+, and CD4−CD8− T cells expressed IL-12R, but not IL-18R, and produced IFN-γ in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN-γ production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN-γ production among T cell subsets. CD4+ cells expressed marginal levels of IL-18R and produced small amounts of IFN-γ, whereas CD8+ cells expressed higher levels of IL-18R and produced more IFN-γ than CD4+ cells. Moreover, CD4−CD8− cells expressed levels of IL-18R comparable to those for CD8+ cells but produced IFN-γ one order higher than did CD8+ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN-γ production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.