bluuebunny/crossref_metadata_2025 huggingface dataset:
The Absence of IL-6 Does Not Affect Th2 Cell Development In Vivo, But Does Lead to Impaired Proliferation, IL-2 Receptor Expression, and B Cell Responses
Authors: La Flamme Anne Camille , Pearce Edward J.
Published: January 2023

The role of IL-6 in Th2 cell differentiation and response development after the injection of eggs from Schistosoma mansoni was investigated using wild-type (WT) and IL-6−/− mice. IL-6 was induced in the lymph nodes (LN) of WT mice within 24 h of egg injection, and IL-4 production by WT LN cells and CD4 T cells isolated 24 h after egg injection and stimulated in vitro was observed. In the absence of IL-6, this early production of IL-4 by LN cells and purified CD4 T cells was not abolished; although the level of IL-4 produced by IL-6−/− LN cells was similar to WT, IL-4 production by purified IL-6−/− CD4 T cells was reduced compared with WT. Despite the difference in CD4 T cell production of IL-4, the development of egg-specific Th2 cells 7 days after egg injection was not affected by the absence of IL-6. Nevertheless, Ab production was impaired and the in vitro proliferative response of whole LN cell populations, CD4 and CD8 T cells, and B cells from IL-6−/− mice was poor compared with WT. The proliferative defect in the IL-6−/− cells correlated with decreased IL-2R expression, and addition of exogenous IL-6 enhanced IL-2R expression as well as proliferation of LN cells from IL-6−/− mice. These studies demonstrate that Th2 differentiation and response development in vivo is not dependent on IL-6, but that Th-dependent and independent B cell responses are. Our results also emphasize the importance of IL-6 for lymphoproliferation, possibly through induction of IL-2R expression.
Regulation of Apoptosis in Mature αβ+CD4−CD8− Antigen-Specific Suppressor T Cell Clones
Authors: Khan Qasim , Penninger Josef M. , Yang Liming , Marra Luciano E. , Kozieradzki Ivona , Zhang Li
Published: January 2023

The regulation of apoptosis in mature CD4+ or CD8+ αβ+ T cells has been well studied. How the survival and death is regulated in peripheral CD4−CD8− (double negative, DN) αβ+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of αβ+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-α/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.
The TCR ζ-Chain Immunoreceptor Tyrosine-Based Activation Motifs Are Sufficient for the Activation and Differentiation of Primary T Lymphocytes
Authors: Geiger Terrence L. , Leitenberg David , Flavell Richard A.
Published: January 2023

The TCR complex signals through a set of 10 intracytoplasmic motifs, termed immunoreceptor tyrosine-based activation motifs (ITAMs), contained within the γ-, δ-, ε-, and ζ-chains. The need for this number of ITAMs is uncertain. Limited and contradictory studies have examined the ability of subsets of the TCR’s ITAMs to signal into postthymic primary T lymphocytes. To study signaling by a restricted set of ITAMs, we expressed in transgenic mice a chimeric construct containing the IAs class II MHC extracellular and transmembrane domains linked to the cytoplasmic domain of the TCR ζ-chain. Tyrosine phosphorylation and receptor cocapping studies indicate that this chimeric receptor signals T cells independently of the remainder of the TCR. We show that CD4+ and CD8+ primary T cells, as well as naive and memory T cells, are fully responsive to stimulation through the IAs-ζ receptor. Further, IAs-ζ stimulation can induce primary T cell differentiation into CTL, Th1, and Th2 type cells. These results show that the ζ-chain ITAMs, in the absence of the γ, δ, and ε ITAMs, are sufficient for the activation and functional maturation of primary T lymphocytes. It also supports the isolated use of the ζ-chain ITAMs in the development of surrogate TCRs for therapeutic purposes.
Fibroblast-Like Synoviocytes from Rheumatoid Arthritis Patients Have Intrinsic Properties of Follicular Dendritic Cells
Authors: Lindhout Ernst , van Eijk Marco , van Pel Melissa , Lindeman Jan , Dinant Huibert J. , de Groot Cornelis
Published: January 2023

The production of IgG rheumatoid factors in the inflamed synovium of many patients with rheumatoid arthritis (RA) implies that local sites exist where plasma cell precursors undergo isotype switching and affinity maturation by somatic mutation and selection. Lymphonodular infiltrates of the synovium-containing germinal centers (GCs), are candidates to fulfill such function in the rheumatoid patient. It has been suggested that these GCs are organized around, obviously ectopic, follicular dendritic cells (FDCs). The present study attempts to find out whether these putative FDCs 1) are specific for RA, 2) have the same phenotype and functional capacity as FDCs in lymphoid organs, and 3) may locally differentiate from fibroblast-like synoviocytes (FLS). Synovial biopsies from patients with RA versus non-RA, yet arthritic backgrounds, were compared. Cells with the FDC phenotype were found in both RA and non-RA tissues as well as in single cell suspensions thereof. When FLS were cultured in vitro, part of these cell lines could be induced with IL-1β and TNF-α to express the FDC phenotype, irrespective of their RA or non-RA background. By contrast, the FDC function, i.e., stable binding of GC B cells and switching off the apoptotic machinery in B cells, appeared to be the prerogative of RA-derived FLS only. The present data indicate that FDC function of FLS in RA patients is intrinsic and support the idea that synovial fibroblast-like cells have undergone some differentiation process that is unique for this disease.
Differential Induction of Adhesion Molecule and Chemokine Expression by LTα3 and LTαβ in Inflammation Elucidates Potential Mechanisms of Mesenteric and Peripheral Lymph Node Development
Authors: Cuff Carolyn A. , Sacca Rosalba , Ruddle Nancy H.
Published: January 2023

Lymphotoxin (LT) is a member of the proinflammatory TNF family of cytokines that plays a critical role in the development of lymphoid tissue. It has previously been reported that the presence of the LTα transgene under the control of the rat insulin promoter results in inflammation at the sites of transgene expression. LTα transgene expression results in expression of the adhesion molecules VCAM, ICAM, peripheral node addressin (a marker of peripheral lymph nodes), and mucosal addressin cellular adhesion molecule (a marker of mucosal lymphoid tissue, including mesenteric lymph nodes). In this study to determine the mechanisms by which LT promotes inflammation and lymphoid tissue organization, we analyzed the regulation of expression of adhesion molecules and chemokines in LT transgenic mice. The results demonstrate that LTα3 induces expression of the adhesion molecules VCAM, ICAM, and mucosal addressin cellular adhesion molecule as well as the chemokines RANTES, IFN-inducible protein-10, and monocyte chemotactic protein-1, while LTαβ is required for the induction of peripheral node addressin that may contribute to the recruitment of L-selectinhigh CD44low naive T cells. These data provide candidate mediators of LT-induced inflammation as well as potential mechanisms by which LTα and LTαβ may differentially promote the development of mesenteric and peripheral lymph nodes.
USF/c-Myc Enhances, While Yin-Yang 1 Suppresses, the Promoter Activity of CXCR4, a Coreceptor for HIV-1 Entry
Authors: Moriuchi Masako , Moriuchi Hiroyuki , Margolis David M. , Fauci Anthony S.
Published: January 2023

Transcription factors USF1 and USF2 up-regulate gene expression (i.e., HIV-1 long terminal repeats) via interaction with an E box on their target promoters, which is also a binding site for c-Myc. The c-Myc oncoprotein is important in control of cellular proliferation and differentiation, while Yin-Yang 1 (YY1) has been shown to control the expression of a number of cellular and viral genes. These two proteins physically interact with each other and mutually inhibit their respective biological functions. In this study, we show that USF/c-Myc up-regulates, while YY1 down-regulates the promoter activity of CXCR4, a coreceptor for T cell-tropic HIV-1 entry. We have identified an E box around −260 and a YY1 binding site around −300 relative to the transcription start site. Mutation of the E box abolished USF/c-Myc-mediated up-regulation of CXCR4 promoter activity, and mutation of the YY1 binding site was associated with unresponsiveness to YY1-mediated inhibition. These data suggest that USF/c-Myc and YY1 may play an important role in the HIV-1-replicative cycle, by modulating both the viral fusion/entry process and viral expression.
IgM Heavy Chain Complementarity-Determining Region 3 Diversity Is Constrained by Genetic and Somatic Mechanisms Until Two Months After Birth
Authors: Shiokawa Satoshi , Mortari Frank , Lima Jose O. , Nuñez César , Bertrand Fred E. , Kirkham Perry M. , Zhu Shigui , Dasanayake Ananda P. , Schroeder Harry W.
Published: January 2023

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an “inappropriate” length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.
Macrophage-Inflammatory Protein-1α Receptor Expression on Normal and Chronic Myeloid Leukemia CD34+ Cells
Authors: Nicholls Sian E. , Lucas Guy , Graham Gerard J. , Russell Nigel H. , Mottram Rachel , Whetton Anthony D. , Buckle Anne-Marie
Published: January 2023

We have assessed expression of MIP-1α binding sites on the surface of CD34+ cells from normal bone marrow (NBM) and chronic myeloid leukemia (CML) peripheral blood. This study has highlighted a small subpopulation of CD34+ (15.7 ± 6.2% in NBM and 9 ± 4% in CML), which has specific macrophage-inflammatory protein-1α (MIP-1α) cell surface binding sites. Further phenotypic characterization of the receptor-bearing cells has shown that they do not express the Thy-1 Ag, suggesting that they are committed progenitor cells rather than CD34+ Thy+ stem cells. However, more than 80% of methanol-fixed CD34+ cells do bind MIP-1α, suggesting that these cells may possess a pool of internal receptors, although we were unable to induce cell surface expression by cytokine stimulation. The percentage of these CD34+, MIP-1α-R+ cells present in the CD34 compartment of NBM is significantly higher than in CML, implicating lack of binding sites as part of the mechanism for the loss of response to this chemokine seen in CML. Specific Ab to the MIP-1α receptor implicated in HIV infection, CCR5, revealed that very few CD34+ cells expressed these receptors and that expression was confined to the CD34+ Thy− progenitor population. Data presented in this work suggest that active binding sites for the stem cell growth inhibitor MIP-1α are not constitutively expressed on the surface of most resting primitive multipotent cells, and that these cells are not potential targets for HIV-1 infection through CCR5.
Bleomycin Stimulates Lung Fibroblasts to Release Neutrophil and Monocyte Chemotactic Activity
Authors: Takamizawa Akemi , Koyama Sekiya , Sato Etsuro , Masubuchi Takeshi , Kubo Keishi , Sekiguchi Morie , Nagai Sonoko , Izumi Takateru
Published: January 2023

We determined whether human lung fibroblasts might release chemotactic activity for neutrophils (NCA) and monocytes (MCA) in response to bleomycin. The human lung fibroblasts supernatant fluids were evaluated for chemotactic activity by a blind well chamber technique. Human lung fibroblasts released NCA and MCA in a dose- and time-dependent manner in response to bleomycin. Checkerboard analysis of supernatant fluids revealed that both NCA and MCA were chemotactic. Partial characterization revealed that NCA was partly heat labile, trypsin sensitive, and predominantly ethyl acetate extractable. In contrast, MCA was partly trypsin sensitive and ethyl acetate extractable. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by leukotriene B4 receptor antagonist and anti-IL-8 and G-CSF Abs. MCA was attenuated by leukotriene B4 receptor antagonist, and monocyte chemoattractant protein-1, GM-CSF, and TGF-β Abs. Leukotriene B4 receptor antagonist and these Abs inhibited the corresponding m.w. chemotactic activity separated by column chromatography. The concentrations of IL-8, G-CSF, monocyte chemoattractant protein-1, GM-CSF, and TGF-β in the supernatant fluids significantly increased in response to bleomycin. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to bleomycin.
IL-4 Induces Mucin Gene Expression and Goblet Cell Metaplasia In Vitro and In Vivo
Authors: Dabbagh Karim , Takeyama Kiyoshi , Lee Heung-Man , Ueki Iris F. , Lausier James A. , Nadel Jay A.
Published: January 2023

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-α and IL-1β levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.
Tissue-Specific Segregation of CD1d-Dependent and CD1d-Independent NK T Cells
Authors: Eberl Gérard , Lees Rosemary , Smiley Stephen T. , Taniguchi Masaru , Grusby Michael J. , MacDonald H. Robson
Published: January 2023

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.
Glucocorticoids Affect Human Dendritic Cell Differentiation and Maturation
Authors: Piemonti Lorenzo , Monti Paolo , Allavena Paola , Sironi Marina , Soldini Laura , Leone Biagio Eugenio , Socci Carlo , Di Carlo Valerio
Published: January 2023

Because dendritic cells (DC) play a major role in the initiation of T cell-mediated immunity, we studied the effects of glucocorticoids, well-known inhibitors of the immune and inflammatory response, on the differentiation and maturation of human DC. DC were differentiated from human monocytes by culture with GM-CSF and IL-4 for 7 days with and without dexamethasone (Dex). Cells treated with Dex (10−8 M) (Dex-DC) developed a characteristic dendritic morphology; however, membrane phenotype analysis demonstrated that they were not fully differentiated. Dex-DC expressed low levels of CD1a and, unlike untreated cells, high levels of CD14 and CD16. Molecules involved in Ag presentation (CD40, CD86, CD54) were also impaired. In contrast, molecules involved in Ag uptake (mannose receptor, CD32) and cell adhesion (CD11/CD18, CD54) were up-regulated. After exposure to TNF-α or CD40 ligand, Dex-DC expressed lower levels of CD83 and CD86 than untreated cells. Dex-DC showed a higher endocytic activity, a lower APC function, and a lower capacity to secrete cytokines than untreated cells. Overall, these results indicate that DC differentiated in the presence of Dex are at a more immature stage. Moreover, Dex also partially blocked terminal maturation of already differentiated DC. In conclusion, our data suggest that glucocorticoids may act at the very first step of the immune response by modulating DC differentiation, maturation, and function.
Altered Expression Level of a Systemic Nuclear Autoantigen Determines the Fate of Immune Response to Self
Authors: Kawahata Kimito , Misaki Yoshikata , Komagata Yoshinori , Setoguchi Keigo , Tsunekawa Shinji , Yoshikawa Yasuji , Miyazaki Jun-ichi , Yamamoto Kazuhiko
Published: January 2023

One of the hallmarks of systemic autoimmune diseases is immune responses to systemic nuclear autoantigens. We have examined the fate of the immune response against a nuclear autoantigen using human U1 small nuclear ribonucleoprotein-A protein (HuA) transgenic (Tg) mice by adoptive transfer of autoreactive lymphocytes. We obtained two Tg lines that have different expression levels of the transgene. After spleen cells from HuA-immunized wild-type mice were transferred to Tg mice and their non-Tg littermates, these recipients were injected with HuA/IFA to induce a recall memory response. HAB69, which expressed a lower amount of HuA, exhibited a vigorous increase in the autoantibody level and glomerulonephritis. Moreover, the autoreactivity spread to 70K autoantigen. Alternatively, in HAB64, which expressed a higher amount of HuA, the production of autoantibody was markedly suppressed. The immune response to HuA autoantigen was impaired as demonstrated in a both delayed-type hypersensitivity response and proliferation assay. This inhibition was Ag-specific and was mediated by T cells. These data suggest that the expression level of systemic autoantigens influences the outcome of the immune response to self.
Plasma Membrane Potential in Thymocyte Apoptosis
Authors: Dallaporta Bruno , Marchetti Philippe , de Pablo Manuel A. , Maisse Carine , Duc Huynh-Thien , Métivier Didier , Zamzami Naoufal , Geuskens Maurice , Kroemer Guido
Published: January 2023

Apoptosis is accompanied by major changes in ion compartmentalization and transmembrane potentials. Thymocyte apoptosis is characterized by an early dissipation of the mitochondrial transmembrane potential, with transient mitochondrial swelling and a subsequent loss of plasma membrane potential (ΔΨp) related to the loss of cytosolic K+, cellular shrinkage, and DNA fragmentation. Thus, a gross perturbation of ΔΨp occurs at the postmitochondrial stage of apoptosis. Unexpectedly, we found that blockade of plasma membrane K+ channels by tetrapentylammonium (TPA), which leads to a ΔΨp collapse, can prevent the thymocyte apoptosis induced by exposure to the glucocorticoid receptor agonist dexamethasone, the topoisomerase inhibitor etoposide, γ-irradiation, or ceramide. The TPA-mediated protective effect extends to all features of apoptosis, including dissipation of the mitochondrial transmembrane potential, loss of cytosolic K+, phosphatidylserine exposure on the cell surface, chromatin condensation, as well as caspase and endonuclease activation. In strict contrast, TPA is an ineffective inhibitor when cell death is induced by the potassium ionophore valinomycin, the specific mitochondrial benzodiazepine ligand PK11195, or by primary caspase activation by Fas/CD95 cross-linking. These results underline the importance of K+ channels for the regulation of some but not all pathways leading to thymocyte apoptosis.
Molecular Cloning and Characterization of a Novel CD1 Gene from the Pig
Authors: Chun Taehoon , Wang Kai , Zuckermann Federico A. , Gaskins H. Rex
Published: January 2023

Much effort is underway to define the immunological functions of the CD1 multigene family, which encodes a separate lineage of Ag presentation molecules capable of presenting lipid and glycolipid Ags. To identify porcine CD1 homologues, a cosmid library was constructed and screened with a degenerate CD1 α3 domain probe. One porcine CD1 gene (pCD1.1) was isolated and fully characterized. The pCD1.1 gene is organized similarly to MHC class I and other CD1 genes and contains an open reading frame of 1020 bp encoding 339 amino acids. Expression of pCD1.1 mRNA was observed in CD3− thymocytes, B lymphocytes, and tissue macrophages and dendritic cells. The pCD1.1 cDNA was transfected into Chinese hamster ovary cells, and subsequent FACS analysis demonstrated that mAb 76-7-4, previously suggested to be a pig CD1 mAb, recognizes cell surface pCD1.1. Structurally, the pCD1.1 α1 and α2 domains are relatively dissimilar to those of other CD1 molecules, whereas the α3 domain is conserved. Overall, pCD1.1 bears the highest similarity with human CD1a, and the ectodomain sequences characteristically encode a hydrophobic Ag-binding pocket. Distinct from other CD1 molecules, pCD1.1 contains a putative serine phosphorylation motif similar to that found in human, pig, and mouse MHC class Ia molecules and to that found in rodent, but not human, MHC class-I related (MR1) cytoplasmic tail sequences. Thus, pCD1.1 encodes a molecule with a conventional CD1 ectodomain and an MHC class I-like cytoplasmic tail. The unique features of pCD1.1 provoke intriguing questions about the immunologic functions of CD1 and the evolution of Ag presentation gene families.
Characteristics of Sequences Around Individual Nucleotide Substitutions in IgVH Genes Suggest Different GC and AT Mutators
Authors: Spencer Jo , Dunn Mark , Dunn-Walters Deborah K.
Published: January 2023

Somatic hypermutation affects Ig genes during T-dependent B cell responses and is characterized by a high frequency of single base substitutions. Hypermutation is not a completely random process; a study of mutations in different systems has revealed the presence of sequence motifs that target mutation. In a recent analysis of the sequences surrounding individual mutated bases in out-of-frame human IgVH genes, we found that the target motifs around mutated G’s and C’s are reverse complements of each other. This finding suggests that hypermutation acts on both strands of DNA, which contradicts evidence of a strand-dependent mechanism as suggested by an observed bias in A and T mutations and the involvement of transcriptional machinery. We have now extended our database of out-of-frame genes and determined the sequence motifs flanking mutated A and T nucleotides. In addition, we have analyzed the flanking sequences for different types of nucleotide substitutions separately. Our results confirm the relationship between the motifs for G and C mutations and show that the motifs surrounding mutated A’s and T’s are weaker and do not have the same relationship. Taken together with our observation of A/T strand bias in out-of-frame genes, this observation suggests that there is a semitargeted G/C mutator that is strand-independent and a separate A/T mutator that is strand-dependent and is less reliant on the local target sequence.
Antigen-Induced Somatic Diversification of Rabbit IgH Genes: Gene Conversion and Point Mutation
Authors: Winstead Candace R. , Zhai Shi-Kang , Sethupathi Periannan , Knight Katherine L.
Published: January 2023

During T cell-dependent immune responses in mouse and human, Ig genes diversify by somatic hypermutation within germinal centers. Rabbits, in addition to using somatic hypermutation to diversify their IgH genes, use a somatic gene conversion-like mechanism, which involves homologous recombination between upstream VH gene segments and the rearranged VDJ genes. Somatic gene conversion and somatic hypermutation occur in young rabbit gut-associated lymphoid tissue and are thought to diversify a primary Ab repertoire that is otherwise limited by preferential VH gene segment utilization. Because somatic gene conversion is rarely found within Ig genes during immune responses in mouse and human, we investigated whether gene conversion in rabbit also occurs during specific immune responses, in a location other than gut-associated lymphoid tissue. We analyzed clonally related VDJ genes from popliteal lymph node B cells responding to primary, secondary, and tertiary immunization with the hapten FITC coupled to a protein carrier. Clonally related VDJ gene sequences were derived from FITC-specific hybridomas, as well as from Ag-induced germinal centers of the popliteal lymph node. By analyzing the nature of mutations within these clonally related VDJ gene sequences, we found evidence not only of ongoing somatic hypermutation, but also of ongoing somatic gene conversion. Thus in rabbit, both somatic gene conversion and somatic hypermutation occur during the course of an immune response.
CD28 Costimulation Augments IL-2 Secretion of Activated Lamina Propria T Cells by Increasing mRNA Stability Without Enhancing IL-2 Gene Transactivation
Authors: Gonsky Rivkah , Deem Richard L. , Lee Doo Han , Chen Alice , Targan Stephan R.
Published: January 2023

The pathways leading to activation in lamina propria (LP) T cells are different from peripheral T cells. LP T cells exhibit enhanced IL-2 secretion when activated through the CD2 pathway. Coligation of CD28 leads to synergistic enhancement of IL-2 secretion. Previous studies have characterized the CD28 augmentation of TCR-mediated signaling in peripheral blood T cells through transcriptional activation of an IL-2 promoter CD28 response element (CD28RE), along with enhanced mRNA stability. This study characterized molecular events involved in CD28 costimulation of IL-2 production in LP mononuclear cells (LPMC). LPMC exhibited increased IL-2 production in response to CD28 costimulation, compared with cells activated through CD2 alone. IL-2 secretion was paralleled by increased expression of IL-2 mRNA, resulting from enhanced IL-2 mRNA stability. In contrast to transcriptional activation in PBMC, EMSA revealed that CD28 coligation of CD2-activated LPMC does not result in increased binding of trans-factors to the CD28RE, nor did Western blots detect changes in I-κBα or I-κBβ levels following CD28 coligation. Furthermore, CD28 coligation fails to enhance IL-2 promoter-reporter or RE/AP construct expression in CD2-activated LPMC. The results reported herein indicate that the molecular mechanisms involved in CD28 cosignaling and regulation of IL-2 secretion in LP T cells are unique to that compartment and differ from those seen in peripheral blood T cells. These observations suggest a biological significance for different mechanisms of IL-2 activation in initiation and maintenance of the cytokine repertoire found in the mucosa.
A Peptide Binding Motif for I-Eg7, the MHC Class II Molecule That Protects Eα-Transgenic Nonobese Diabetic Mice from Autoimmune Diabetes
Authors: Gregori Silvia , Trembleau Sylvie , Penna Giuseppe , Gallazzi Fabio , Hammer Juergen , Papadopoulos George K. , Adorini Luciano
Published: January 2023

The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due a deletion in the Eα gene promoter. Eα-transgenic NOD mice express the EαEβg7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7-restricted T cell epitope corresponding to the sequence 4–13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and Eα-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in Eα-transgenic NOD mice.
Mucosal T Lymphocyte Numbers Are Selectively Reduced in Integrin αE (CD103)-Deficient Mice
Authors: Schön Michael P. , Arya Anu , Murphy Elizabeth A. , Adams Cassandra M. , Strauch Ulrike G. , Agace William W. , Marsal Jan , Donohue John P. , Her Helen , Beier David R. , Olson Sara , Lefrancois Leo , Brenner Michael B. , Grusby Michael J. , Parker Christina M.
Published: January 2023

The mucosal lymphocyte integrin αE(CD103)β7 is thought to be important for intraepithelial lymphocyte (IEL) localization or function. We cloned the murine integrin gene encoding αE, localized it to chromosome 11, and generated integrin αE-deficient mice. In αE−/− mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of αEβ7 to E-cadherin expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E-cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer’s patch, and splenic T lymphocyte numbers were not reduced in αE-deficient mice. Thus, αEβ7 was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated organized lymphoid tissues. Finally, the impact of αE deficiency upon intestinal IEL numbers was greater at 3–4 wk of life than in younger animals, and affected the TCR αβ+ CD8+ T cells more than the γδ T cells or the TCR αβ+ CD4+CD8− population. These findings suggest that αEβ7 is involved in the expansion/recruitment of TCR αβ+ CD8+ IEL following microbial colonization. Integrin αE-deficient mice will provide an important tool for studying the role of αEβ7 and of αEβ7-expressing mucosal T lymphocytes in vivo.
CD40-CD40 Ligand Interaction Is Central to Cell-Mediated Immunity Against Toxoplasma gondii: Patients with Hyper IgM Syndrome Have a Defective Type 1 Immune Response That Can Be Restored by Soluble CD40 Ligand Trimer
Authors: Subauste Carlos S. , Wessendarp Matthew , Sorensen Ricardo U. , Leiva Lily E.
Published: January 2023

Cell-mediated immunity that results in IL-12/IFN-γ production is essential to control infections by intracellular organisms. Studies in animal models revealed contrasting results in regard to the importance of CD40-CD40 ligand (CD40L) signaling for induction of a type 1 cytokine response against these pathogens. We demonstrate that CD40-CD40L interaction in humans is critical for generation of the IL-12/IFN-γ immune response against Toxoplasma gondii. Infection of monocytes with T. gondii resulted in up-regulation of CD40. CD40-CD40L signaling was required for optimal T cell production of IFN-γ in response to T. gondii. Moreover, patients with hyper IgM (HIGM) syndrome exhibited a defect in IFN-γ secretion in response to the parasite and evidence compatible with impaired in vivo T cell priming after T. gondii infection. Not only was IL-12 production in response to T. gondii dependent on CD40-CD40L signaling, but also, patients with HIGM syndrome exhibited deficient in vitro secretion of this cytokine in response to the parasite. Finally, in vitro incubation with agonistic soluble CD40L trimer enhanced T. gondii-triggered production of IFN-γ and, through induction of IL-12 secretion, corrected the defect in IFN-γ production observed in HIGM patients. Our results are likely to explain the susceptibility of patients with HIGM syndrome to infections by opportunistic pathogens.
Improved Clearance of Mycobacterium avium Upon Disruption of the Inducible Nitric Oxide Synthase Gene
Authors: Gomes M. Salomé , Flórido Manuela , Pais Teresa F. , Appelberg Rui
Published: January 2023

Mice genetically deficient in the inducible NO synthase gene (iNOS−/−) were used to study the role played by NO during infection by Mycobacterium avium. iNOS−/− macrophages were equally able to restrict M. avium growth in vitro following stimulation by IFN-γ and TNF-α as macrophages from wild-type mice. In vivo, the infection progressed at similar rates in wild-type and NO-deficient mice during the first 2 mo of infection, but the latter mice were subsequently more efficient in clearing the mycobacteria than the former. The increased resistance of iNOS−/− mice was associated with higher IFN-γ levels in the serum and following in vitro restimulation of spleen cells with specific Ag, increased formation of granulomas and increased survival of CD4+ T cells. We show that NO is not involved in the antimycobacterial mechanisms of M. avium-infected macrophages and, furthermore, that it exacerbates the infection by causing the suppression of the immune response to the pathogen.
Mice Lacking Two or All Three Selectins Demonstrate Overlapping and Distinct Functions for Each Selectin
Authors: Jung Unsu , Ley Klaus
Published: January 2023

Selectins support the capture and rolling of leukocytes in venules at sites of inflammation and in lymphocyte homing. Gene-targeted mice with null mutations at the L-, E-, or P-selectin locus develop normally and show mild (E−/−) to moderate (P−/−, L−/−) defects in inflammatory cell recruitment. Mice lacking both P- and E-selectin (E/P−/−) have severe neutrophilia and spontaneous skin infections that limit their life span. Other combinations of selectin deficiency have not been investigated. We have generated novel mice lacking L- and P-selectin (L/P−/−), L- and E-selectin (L/E−/−), or all three selectins (E/L/P−/−) by bone marrow transplantation. L/P−/− mice (only E-selectin present) show an absence of leukocyte rolling after trauma and severely reduced rolling (by ∼90%) in inflammation induced by TNF-α. Residual rolling in L/P−/− mice was very slow (3.6 ± 0.2 μm/s after TNF-α). L/E−/− mice (only P-selectin present) showed rolling similar to that of L−/− at increased velocities (15.1 ± 0.3 μm/s). The number of adherent leukocytes after 2 or 6 h of TNF-α treatment was not significantly reduced in L/E−/− or L/P−/− mice. E/L/P−/− mice showed very little rolling after TNF-α, all of which was blocked by mAb to α4 integrin. Adherent and emigrated neutrophils were significantly reduced at 6 h after TNF-α. We conclude that any one of the selectins can support some neutrophil recruitment but eliminating all three selectins significantly impairs neutrophil recruitment.
Promotion of Neutrophil Chemotaxis Through Differential Regulation of β1 and β2 Integrins
Authors: Harler Mary Beth , Wakshull Eric , Filardo Edward J. , Albina Jorge E. , Reichner Jonathan S.
Published: January 2023

Migration of neutrophils requires sequential adhesive and deadhesive interactions between β1 and β2 integrins and components of the extracellular matrix. Prompted by reports that describe interaction of soluble β-glucan with the β2 integrin Mac-1, a role for β-glucan in regulation of integrin-mediated migration was investigated. Neutrophil migration in response to fMLP was assessed using an agarose overlay method with slides precoated with fibronectin (Fn) ± β-glucan. On Fn, random migration in excess of directed migration was observed. In contrast, migration on Fn + β-glucan was directional, with marked diminution of random migration. This conversion of random to directed migration was seen neither when Fn was supplemented with alternative polysaccharides nor when β-glucan was applied to other components of the extracellular matrix. This effect of β-glucan was shown to be cation dependent and to be effected by Arg-Gly-Asp-containing peptides consistent with an integrin-mediated event. mAb inhibition studies demonstrate that β-glucan effects this shift toward directed migration through suppression of migration mediated by Mac-1 and very late Ag 5 and enhancement of very late Ag 3-mediated migration. Adhesion assays suggest that the prochemotactic influence of β-glucan is due, in part but not entirely, to modulation of PMN adhesion to Fn. In summary, these data support a novel role for β-glucan in regulation of β1- and β2-mediated neutrophil migration on Fn.
Paclitaxel Enhances Macrophage IL-12 Production in Tumor-Bearing Hosts Through Nitric Oxide
Authors: Mullins David W. , Burger Carol J. , Elgert Klaus D.
Published: January 2023

Tumor-induced macrophages (Mφs) mediate immunosuppression, in part, through increased production of factors that suppress T cell responsiveness and underproduction of positive regulatory cytokines. Pretreatment of tumor-bearing host (TBH) Mφs with the anticancer agent paclitaxel (Taxol) partially reverses tumor-induced Mφ suppressor activity, suggesting that paclitaxel may restore TBH Mφ production of proimmune factors. Because paclitaxel demonstrates LPS-mimetic capabilities and increased production of the LPS-induced immunostimulatory cytokine IL-12 could account for enhanced T cell responsiveness, we investigated whether paclitaxel induces Mφ IL-12 production. Tumor growth significantly down-regulated Mφ IL-12 p70 production through selective dysregulation of IL-12 p40 expression. LPS stimulation failed to overcome tumor-induced dysregulation of p40 expression. In contrast, paclitaxel significantly enhanced both normal host and TBH Mφ IL-12 p70 production in vitro, although TBH Mφ IL-12 production was lower than that of similarly treated normal host Mφs. Paclitaxel enhanced p40 expression in a dose-dependent manner. Through reconstituted Mφ IL-12 expression, paclitaxel pretreatment relieved tumor-induced Mφ suppression of T cell alloreactivity. Blocking Mφ NO suppressed paclitaxel’s ability to induce IL-12 production. This suggests that paclitaxel-induced activities may involve a NO-mediated autocrine induction pathway. Collectively, these data demonstrate that paclitaxel restores IL-12 production in the TBH and ascribe a novel immunotherapeutic component to the pleiotropic activities of NO. Through its capacity to induce IL-12 production, paclitaxel may contribute to the correction of tumor-induced immune dysfunction.
Regulation of Pulmonary T Cell Responses to Inhaled Antigen: Role in Th1- and Th2-Mediated Inflammation
Authors: Lee Shiour-Ching , Jaffar Zeina H. , Wan Kong-Sang , Holgate Stephen T. , Roberts Kevan
Published: January 2023

DO11.10 transgenic mice, expressing an OVA-specific TCR, were used to study pulmonary T cell responses to inhaled Ags. Before OVA inhalation, the activation of lung parenchymal T cells elicited both strong proliferative responses and IL-2 production. However, following Ag inhalation the proliferative responses of the lung T cells, when restimulated in vitro with OVA323–339 peptide or immobilized anti-CD3, were severely attenuated and associated with a decrease in the level of production of IL-2 but not IFN-γ. Such immune regulation was tissue-specific, because T cell responses in the lymph nodes and spleens were normal. This dramatic aerosol-induced attenuation of parenchymal T cell proliferation was also observed in BALB/c mice immunized with OVA and in BALB/c mice following adoptive transfer of DO11.10 T cells bearing either a Th1 or Th2 phenotype. In mice that had received Th2 cells, the reduced proliferative responses were associated with a decrease in IL-2 expression but augmented IL-4 and IL-5 production. Invariably, the inhibition of proliferation was a consequence of the action of F4/80+ interstitial macrophages and did not involve alveolar macrophages or their products. These observations demonstrate that clonal expansion of T cells in the lung compartment is prevented following the onset of either Th1- or Th2-mediated inflammation. This form of immune regulation, which appears as a selective defect in IL-2-driven proliferation, may serve to prevent the development of chronic pulmonary lymphoproliferative responses.
HLA-Class II-Associated Control of Antigen Recognition by T Cells in Leprosy: A Prominent Role for the 30/31-kDa Antigens
Authors: Thole Jelle E. R. , Janson Anneke A. M. , Cornelisse Yolanthe , Schreuder Geziena M. T. , Wieles Brigitte , Naafs Ben , de Vries René R. P. , Ottenhoff Tom H. M.
Published: January 2023

The recognition of 16 mycobacterial Ags by a panel of T cell lines from leprosy patients and healthy exposed individuals from an endemic population was examined within the context of expressed HLA-DR molecules. Although overall no significant differences were found between the frequencies of Ag recognition in the different subject groups, when Ag-specific T cell responses were examined within the context of HLA-DR, a highly significant difference was found in the recognition of the 30/31-kDa Ag. HLA-DR3 appeared to be associated with high T cell responsiveness to the 30/31-kDa Ag in healthy contacts (p = 0.01), but, conversely, with low T cell responsiveness to this Ag in tuberculoid patients (p = 0.005). Within the group of HLA-DR3-positive individuals, differences in 30/31-kDa directed T cell responsiveness were highly significant not only between healthy individuals and tuberculoid patients (p < 0.0001), but also between healthy individuals and lepromatous patients (p = 0.009), and consequently between healthy individuals compared with leprosy patients as a group (p < 0.0001). A dominant HLA-DR3-restricted epitope was recognized by healthy contacts in this population. It has been proposed that secreted Ags may dominate acquired immunity early in infection. The low T cell response to the secreted, immunodominant 30/31-kDa Ag in HLA-DR3-positive leprosy patients in this population may result in retarded macrophage activation and delayed bacillary clearance, which in turn may lead to enhanced Ag load followed by T cell-mediated immunopathology.
Cellular Immunity to β2-Glycoprotein-1 in Patients with the Antiphospholipid Syndrome
Authors: Visvanathan Sudha , McNeil H. Patrick
Published: January 2023

Patients with antiphospholipid syndrome (APS) suffer recurrent thromboses, thrombocytopenia, and/or fetal loss in association with Abs that can be detected in phospholipid-dependent assays. Despite the name, the Igs associated with APS are predominantly directed against epitopes on phospholipid-binding plasma proteins, such as β2-glycoprotein-1 (β2GP1) and prothrombin. The aim of this study was to examine the cellular immune response to β2GP1 in patients with APS. Using a serum-free stimulation assay, PBMCs from 8 of 18 patients with APS proliferated to purified β2GP1 or to the β2GP1 present in serum, whereas no stimulation was observed by PBMCs from healthy individuals, patients with other autoimmune diseases, or anticardiolipin Ab-positive patients without histories of thromboses or fetal loss. The immune response was Ag-specific, requiring class II molecules, CD4+ T cells, and APCs, and was associated with a selective expansion of CD4+ but not CD8+ T cells. The proliferating T cells produced IFN-γ but not IL-4, indicating a bias toward a type 1 immune response. Chronic low grade stimulation of autoreactive β2GP1-specific, IFN-γ-producing Th1 CD4+ T cells may contribute to the high risk of thromboses and pregnancy failure in patients with APS.
New Class I and II HLA Alleles Strongly Associated with Opposite Patterns of Progression to AIDS
Authors: Hendel Houria , Caillat-Zucman Sophie , Lebuanec Hélène , Carrington Mary , O’Brien Steve , Andrieu Jean-Marie , Schächter François , Zagury Daniel , Rappaport Jay , Winkler Cheryl , Nelson George W. , Zagury Jean-François
Published: January 2023

The genetics of resistance to infection by HIV-1 cohort consists of 200 slow and 75 rapid progressors to AIDS corresponding to the extremes of HIV disease outcome of 20,000 Caucasians of European descent. A comprehensive analysis of HLA class I and class II genes in this highly informative cohort has identified HLA alleles associated with fast or slow progression, including several not described previously. A quantitative analysis shows an overall HLA influence independent of and equal in magnitude (for the protective effect) to the effect of the CCR5-Δ32 mutation. Among HLA class I genes, A29 (p = 0.001) and B22 (p < 0.0001) are significantly associated with rapid progression, whereas B14 (p = 0.001) and C8 (p = 0.004) are significantly associated with nonprogression. The class I alleles B27, B57, C14 (protective), and C16, as well as B35 (susceptible), are also influential, but their effects are less robust. Influence of class II alleles was only observed for DR11. These results confirm the influence of the immune system on disease progression and may have implications on peptide-based vaccine development.