bluuebunny/crossref_metadata_2025 huggingface dataset:
Markers of Lymphocyte Homing Distinguish CD4 T Cell Subsets That Turn Over in Response to HIV-1 Infection in Humans
Authors: Hengel Richard L. , Jones Bonnie M. , Kennedy M. Susan , Hubbard Marjorie R. , McDougal J. Steven
Published: December 2022

In HIV-1 infection, the abrupt rise in CD4 T cells after effective antiretroviral therapy has been viewed as a measure of HIV-1-related CD4 T cell turnover in the steady state. The early (2–4 wk) response is reportedly dominated by CD4 T cells with a memory (CD45RO) phenotype. It is controversial whether the measurement of steady-state kinetics identifies cells that otherwise would have been recruited into a short-lived, virus-producing pool or reflects lymphoid redistribution/sequestration. We performed detailed phenotypic and kinetic analysis of CD4 T cell subsets in 14 patients. Turnover occurs in memory (CD45RO) as well as naive (CD45RA) cells, if the latter are present at baseline. Most of the turnover occurs in those memory (CD45RO) and naive (CD45RA) cells that are programmed for recirculation through lymphoid organs (CD62L+ and CD44low), whereas very little turnover occurs in memory cells (CD45RO) destined for recirculation from blood to tissue (CD62L− and CD44high). Turnover occurs in both activated (CD25+ and HLA-DR+) and nonactivated populations, although it is restricted to CD38-positive cells, indicating that turnover does not measure cells that are already infected. More likely, turnover occurs in cells that replace infected cells or are on their way to becoming infected. Taken together, markers of lymphocyte trafficking better describe cell turnover related to virus replication than do naive and memory markers per se, and lymph organs, not tissue-destined cells or peripheral blood cells, appear to be the important site of virus replication and CD4 T cell turnover, destruction, and redistribution.
Cutting Edge: Paradigm Revisited: Antibody Provides Resistance toListeriaInfection
Authors: Edelson Brian T. , Cossart Pascale , Unanue Emil R.
Published: December 2022

Listeriolysin O (LLO) is a secreted pore-forming toxin of the facultative intracellular bacterium Listeria monocytogenes. We assessed the ability of a murine anti-LLO mAb to affect the course of infection in mice challenged with Listeria. This mAb was previously shown to be capable of neutralizing LLO-mediated pore formation in vitro, and here we show that the passive administration of this Ab to mice before infection provides increased resistance. Mice treated with the mAb were protected from a lethal challenge with virulent Listeria and showed a significant reduction in Listeria burden during the first hours to days postinfection. These effects of the Ab were independent of host B or T cells, since treatment with the mAb provided enhanced resistance to SCID mice. The titer of anti-LLO Abs during the regular infection of mice with Listeria was found to be low to negative.
Cytokine Requirements for Production of a Novel Anti-CD8-Resistant CTL Population
Authors: Lamousé-Smith Esi S. N. , Dougall David S. , McCarthy Susan A.
Published: December 2022

A population of CD8+ CTL can be generated in vitro in the presence of anti-CD8 mAb. Due to their apparent high avidity characteristic, these anti-CD8-resistant CD8+ CTL may have important functional in vivo roles in graft rejection, and may be important in antiviral and antitumor responses. We have previously reported that this anti-CD8-resistant subset of CD8+ CTL demonstrates functional differences from anti-CD8-sensitive CD8+ CTL. One important difference between the subsets is the markedly greater dependence of anti-CD8-resistant CTL upon exogenous cytokines for their generation in vitro. In this study, we have investigated in detail the cytokine requirements for the generation of allospecific CD8+ CTL in vitro and have found that IL-4 can augment the generation of anti-CD8-sensitive but not anti-CD8-resistant CTL, whereas IL-2 or IL-12 can augment the generation of both anti-CD8-sensitive and anti-CD8-resistant CTL. However, anti-CD8-resistant CTL require at least 10-fold higher concentrations of IL-2 than do anti-CD8-sensitive CTL. This more stringent IL-2 requirement precludes the efficient generation of anti-CD8-resistant CTL in vitro in the absence of exogenous IL-2 because they cannot produce sufficient IL-2 to meet their needs, in contrast to anti-CD8-sensitive CTL. By providing exogenous cytokines to allospecific CTL generation cultures, we further demonstrate that anti-CD8-resistant CTL can be functionally skewed to the Tc1 subset, but differ from anti-CD8-sensitive conventional CTL in that they cannot be skewed to the Tc2 subset.
Estrogen Receptor α Is Necessary in Thymic Development and Estradiol-Induced Thymic Alterations
Authors: Staples J. Erin , Gasiewicz Thomas A. , Fiore Nancy C. , Lubahn Dennis B. , Korach Kenneth S. , Silverstone Allen E.
Published: December 2022

Estrogens affect the development, maturation, and function of multiple organ systems, including the immune system. One of the main targets of estrogens in the immune system is the thymus, which undergoes atrophy and phenotypic alterations when exposed to elevated levels of estrogen. To determine how estrogens influence the thymus and affect T cell development, estrogen receptor α (ERα) knockout (ERKO) mice were examined. ERKO mice have significantly smaller thymi than their wild-type (WT) littermates. Construction of ER radiation bone marrow chimeras indicated that the smaller thymi were due to a lack of ERα in radiation-resistant tissues rather than hemopoietic elements. ERKO mice were also susceptible to estradiol-induced thymic atrophy, but the extent of their atrophy was less than what was seen in WT mice. The estradiol-treated ERKO mice failed, however, to manifest alterations in their thymic CD4/CD8 phenotypes compared with WT mice. Therefore, ERα is essential in nonhemopoietic cells to obtain a full-sized thymus, and ERα also mediates some of the response of the thymus to elevated estrogen levels. Finally, these results suggest that in addition to ERα, another receptor pathway is involved in estradiol-induced thymic atrophy.
The Self-Directed T Cell Repertoire Against Mouse Lysozyme Reflects the Influence of the Hierarchy of Its Own Determinants and Can Be Engaged by a Foreign Lysozyme
Authors: Moudgil Kamal D. , Southwood Scott , Ametani Akio , Kim Kasey , Sette Alessandro , Sercarz Eli E.
Published: December 2022

The T cell repertoire is shaped by the processes of positive and negative selection. We have previously shown that mice are tolerant to a native self-Ag, mouse lysozyme (ML), but they respond vigorously when challenged with different ML peptides (“cryptic” self-determinants). In this study, we have addressed the issue of the physiological significance of both the hierarchy (dominance/crypticity) of self-determinants within ML and the anti-cryptic, self (ML)-directed T cell repertoire. Our results demonstrate that there are several ML peptides that bind well to MHC but are totally nonimmunogenic when tested for proliferative T cell response and cytokine secretion: a subset of these peptides presumably represent the originally dominant self-determinants of ML, which have rendered the T cells tolerant during thymic selection. Other ML peptides, which bind well to MHC and are immunogenic, correspond to the cryptic determinants of ML: T cells against cryptic ML determinants escape tolerance induction. Thus, the mature T cell repertoire against ML bears the direct imprint of the hierarchy of self (ML)-determinants. Interestingly, hen egg white lysozyme could prime T cells in vivo that were cross-reactive with certain cryptic ML determinants, and vice versa, without requiring any coimmunization with the foreign lysozyme and ML peptide(s). Moreover, repeated, deliberate priming and expansion of T cells by hen egg white lysozyme immunization concomitantly enhanced T cell response to such cross-reactive ML determinants. This reciprocal self-foreign determinant cross-reactivity may play a previously unrecognized, but crucial, role in the expansion and diversification of self-reactive clones in the autoimmune response.
Ligation of CD28 In Vivo Induces CD40 Ligand Expression and Promotes B Cell Survival
Authors: Yin Deling , Zhang Liying , Wang Ruoxiang , Radvanyi Laszlo , Haudenschild Christian , Fang Qiding , Kehry Marilyn R. , Shi Yufang
Published: December 2022

Functional activation of T cells requires ligation of Ag receptors with specific peptides presented by MHC molecules on APCs concurrent with appropriate contacts of cell surface accessory molecules. Among these accessory molecules, interactions between CD28/CTLA-4 with B7 family members (CD80 and CD86) and CD40 with CD40 ligand (CD40L) play a decisive role in regulating the progression of balanced immune responses. However, most information regarding the role of accessory molecules in immune responses has been derived in the context of signals from the TCRs. Little understanding has been achieved regarding the consequence of ligation of costimulation molecules in absence of signals from the TCR. By employing an in vivo murine system, we show, herein, that ligation of CD28 alone with anti-CD28 Abs leads to a dramatic enlargement of the peripheral lymphoid organs characterized primarily by the expansion of B cells. B cells from anti-CD28-treated mice are resistant to spontaneous and anti-IgM-induced apoptosis. These cells are also unsusceptible to FasL-mediated apoptosis. Interestingly, this in vivo effect of CD28 on B cells is largely mediated by inducing the expression of CD40L, since coadministration of a blocking Ab against CD40L inhibited CD28-mediated B cell survival and expansion. Therefore, CD28-mediated expression of CD40L may play an important role in the regulation of lymphocyte homeostasis.
Role of IL-12 in Intrathymic Negative Selection
Authors: Lúdvíksson Björn R. , Ehrhardt Rolf O. , Strober Warren
Published: December 2022

Cytokines are central regulatory elements in peripheral lymphocyte differentiation, but their role in T cell ontogeny is poorly defined. In the present study, we evaluated the role of IL-12 in thymocyte selection more directly by determining its role in two models of in vivo negative selection. In initial studies we demonstrated that abundant intrathymic IL-12 synthesis occurs during OVA peptide-induced negative selection of thymocytes in neonatal OVA-TCR transgenic mice, and such synthesis is associated with increased IL-12R β2-chain expression as well as STAT4 intracellular signaling. In further studies, we showed that this form of negative selection was occurring at the αβTCRlowCD4lowCD8low stage and was prevented by the coadministration of anti-IL-12. In addition, the IL-12-dependent thymocyte depletion was occurring through an intrathymic apoptosis mechanism, also prevented by administration of anti-IL-12. Finally, we showed that IL-12 p40−/− mice displayed aberrant negative selection of double positive CD4+CD8+ thymocytes when injected with anti-CD3 mAb. These studies suggest that intact intrathymic IL-12 production is necessary for the negative selection of thymocytes occurring in relation to a high “self” Ag load, possible through its ability to induce the thymocyte maturation and cytokine production necessary for such selection.
Functional Synergism of STAT6 with Either NF-κB or PU.1 to Mediate IL-4-Induced Activation of IgE Germline Gene Transcription
Authors: Stütz Adrian M. , Woisetschläger Maximilian
Published: December 2022

Ig heavy chain class switching to IgE is directed by IL-4 and IL-13 by inducing transcription from the IgE germline promoter. A crucial transcription factor in this process is STAT6, which binds to a specific DNA element upon cytokine activation. In this paper it is shown that the B cell- and monocyte-specific factor PU.1 interacts with a closely spaced sequence in the human IgE germline promoter that overlaps with a previously described binding site for NFκB/rel. The authenticity of PU.1 was demonstrated by specific competition and supershifts in EMSA experiments. In addition, in vitro translated PU.1 could interact with an oligonucleotide derived from the IgE germline promoter containing the PU.1 binding site and migrated with the same mobility compared with the complex formed with nuclear extracts. Transient transfection experiments using IgE germline promoter reporter gene constructs demonstrated that mutations affecting DNA binding of PU.1 or NFκB/rel had no or little effect on IL-4 inducibility of these plasmids. However, point mutations that abolished binding of both factors abrogated cytokine inducibility. No strict spacing of the STAT6 and the composite PU.1/NF-κB elements is required for IL-4 induction. IL-4-induced STAT6 DNA binding was retained in PU.1−/NFκB/rel− double mutants. The data demonstrate that cooperation of STAT6 with at least PU.1 or NFκB/rel is necessary for IL-4-induced activation of IgE germline gene transcription.
The Human Antibody Response to Porcine Xenoantigens Is Encoded by IGHV3-11 and IGHV3-74 IgVH Germline Progenitors
Authors: Kearns-Jonker Mary , Swensson Joyce , Ghiuzeli Cristina , Chu Wilson , Osame Yuka , Starnes Vaughn , Cramer Donald V.
Published: December 2022

Preformed and induced Ab responses present a major immunological barrier to the use of pig organs for human xenotransplantation. We generated IgM and IgG gene libraries established from lymphocytes of patients treated with a bioartificial liver (BAL) containing pig hepatocytes and used these libraries to identify IgVH genes that encode human Ab responses to pig xenoantigens. Genes encoded by the VH3 family are increased in expression in patients following BAL treatment. cDNA libraries representing the VH3 gene family were generated, and the relative frequency of expression of genes used to encode the Ab response was determined at days 0, 10, and 21. Ig genes derived from the IGHV3-11 and IGHV3-74 germline progenitors increase in frequency post-BAL. The IGHV3-11 gene encodes 12% of VH3 cDNA clones expressed as IgM Abs at day 0 and 32.4–39.0% of cDNA clones encoding IgM Abs in two patients at day 10. IGHV3-11 and IGHV3-74 genes encoding IgM Abs in these patients are expressed without evidence of somatic mutation. By day 21, an isotype switch occurs and IGHV3-11 IgVH progenitors encode IgG Abs that demonstrate somatic mutation. We cloned these genes into a phagemid vector, expressed these clones as single-chain Abs, and demonstrated that the IGHV3-11 gene encodes Abs with the ability to bind to the gal α (1,3) gal epitope. Our results demonstrate that the xenoantibody response in humans is encoded by IgVH genes restricted to IGHV3-11 and IGHV3-74 germline progenitors. IgM Abs are expressed in germline configuration and IgG Abs demonstrate somatic mutations by day 21.
An Antibody-Avidin Fusion Protein Specific for the Transferrin Receptor Serves as a Delivery Vehicle for Effective Brain Targeting: Initial Applications in Anti-HIV Antisense Drug Delivery to the Brain
Authors: Penichet Manuel L. , Kang Young-Sook , Pardridge William M. , Morrison Sherie L. , Shin Seung-Uon
Published: December 2022

In the present study a novel Ab-avidin fusion protein has been constructed to deliver biotinylated compounds across the blood brain barrier. This fusion molecule consists of an Ab specific for the transferrin receptor genetically fused to avidin. The Ab-avidin fusion protein (anti-TfR IgG3-CH3-Av) expressed in murine myeloma cells was correctly assembled and secreted and showed both Ab- and avidin-related activities. In animal models, it showed much longer serum half-life than the chemical conjugate between OX-26 and avidin. Most importantly, this fusion protein demonstrated superior [3H]biotin uptake into brain parenchyma in comparison with the chemical conjugate. We also delivered a biotinylated 18-mer antisense peptide-nucleic acid specific for the rev gene of HIV-1 to the brain. Brain uptake of the HIV antisense drug was increased at least 15-fold when it was bound to the anti-TfR IgG3-CH3-Av, suggesting its potential use in neurologic AIDS. This novel Ab fusion protein should have general utility as a universal vehicle to effectively deliver biotinylated compounds across the blood-brain barrier for diagnosis and/or therapy of a broad range of CNS disorders such as infectious diseases, brain tumors as well as Parkinson’s and Huntington’s diseases.
Binding of Longer Peptides to the H-2Kb Heterodimer Is Restricted to Peptides Extended at Their C Terminus: Refinement of the Inherent MHC Class I Peptide Binding Criteria
Authors: Hörig Heidi , Young Aideen C. M. , Papadopoulos Nicholas J. , DiLorenzo Teresa P. , Nathenson Stanley G.
Published: December 2022

MHC class I molecules usually bind short peptides of 8–10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/β2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.
Tumor Regression After Adoptive Transfer of Effector T Cells Is Independent of Perforin or Fas Ligand (APO-1L/CD95L)
Authors: Winter Hauke , Hu Hong-Ming , Urba Walter J. , Fox Bernard A.
Published: December 2022

The adoptive transfer of tumor-specific effector T cells can result in complete regression and cure mice with systemic melanoma, but the mechanisms responsible for regression are not well characterized. Perforin- and Fas ligand (APO-1/CD95 ligand)-mediated cytotoxicity have been proposed as mechanisms for T cell-mediated tumor destruction. To determine the role of perforin and Fas ligand (FasL) in T cell-mediated tumor regression in a murine melanoma model, B16BL6-D5 (D5), we generated D5-specific effector T cells from tumor vaccine-draining lymph nodes of wild type (wt), perforin knock out (PKO), or FasL mutant (gld) mice and treated established D5 metastases in mice with the same genotype. Effector T cells from wt, PKO and gld mice induced complete regression of pulmonary metastases and significantly prolonged survival of the treated animals regardless of their genotype. Complete tumor regression induced by PKO effector T cells was also observed in a sarcoma model (MCA-310). Furthermore, adoptive transfer of PKO and wt effector T cells provided long-term immunity to D5. Therapeutic T cells from wt, PKO, or gld mice exhibit a tumor-specific type 1 cytokine profile; they secrete IFN-γ, but not IL-4. In these models, T cell-mediated tumor regression and long-term antitumor immunity are perforin and FasL independent.
TNF Is Essential for the Cell-Mediated Protective Immunity Induced by the Radiation-Attenuated Schistosome Vaccine
Authors: Street Miyoko , Coulson Patricia S. , Sadler Clare , Warnock Lorna J. , McLaughlin Deborah , Bluethmann Horst , Wilson R. Alan
Published: December 2022

C57BL/6 mice exposed to the radiation-attenuated schistosome vaccine exhibit high levels of protective immunity. The cell-mediated pulmonary effector mechanism involves IFN-γ-producing CD4+ T cells in a focal response around challenge larvae. IFN-γ can promote production of TNF and can synergize with this cytokine in its actions on responder cells. We have examined whether TNF plays a role in lung phase immunity to schistosomes using mice with a disrupted gene for TNFRI (TNFRI−/−). The most dramatic finding was that the schistosome vaccine elicited no protection whatsoever in these mice. However, this could not be attributed to a lack of responder cells, because more lymphocytes were lavaged from the airways of TNFRI−/− than wild-type mice. Furthermore, CD4+ T cells were equally represented in airway populations from the two groups and produced IFN-γ upon Ag stimulation in vitro. In contrast, pulmonary macrophage function was defective in TNFRI−/− mice, as indicated by a failure to up-regulate inducible NO synthase mRNA. Histopathological analysis revealed that focal infiltrates were of similar size and cell composition in the two groups but that more parasites were free of foci in the TNFRI−/− mice. These animals had a greatly impaired IgG response to schistosomes, which may explain their lack of residual protection due to Ab in a situation where cell-mediated immunity is disabled. We suggest that the absence of protective immunity could result from a retarded build-up of leukocytes around migrating lung worms and/or a deficit in accessory cell function within a focus, both of which would permit parasite escape.
Activated Lymphocytes Promote Endothelial Cell Detachment from Matrix: A Role for Modulation of Endothelial Cell β1 Integrin Affinity
Authors: Phan Cecile , McMahon Alan W. , Nelson Randall C. , Elliott John F. , Murray Allan G.
Published: December 2022

In vivo, MHC class I-restricted injury of allogeneic tissue or cells infected by intracellular pathogens occurs in the absence of classical cytolytic effector mechanisms and Ab. Modulation of the target cell adhesion to matrix may be an additional mechanism used to injure vascular or epithelial cells in inflammation. We studied the mechanisms of human umbilical vein endothelial cell (EC) detachment from matrix-coated plastic following contact by concanamycin A-treated lymphocytes as an in vitro model of perforin-independent modulation of EC basement membrane adhesion. Human PBL were depleted of monocytes, stimulated, then added to an EC monolayer plated on either fibronectin or type I collagen matrices. Activated, but not resting, PBL induced progressive EC detachment from the underlying matrix. Injury of the EC monolayer required direct cell contact with the activated lymphocytes because no detachment was seen when the PBL were placed above a Transwell membrane. Moreover plasma membranes prepared from activated but not resting PBL induced EC detachment. Adherent EC stimulated with activated PBL did not show evidence of apoptosis using TUNEL and annexin V staining at time points before EC detachment was observed. Finally, neither the matrix metalloproteinase inhibitors o-phenanthroline and BB-94 nor aprotinin blocked EC detachment. However, activation of EC β1 integrin using mAb TS2/16 or Mg2+ decreased EC detachment. These data indicate that cell-cell contact between activated PBL and EC reduces adhesion of EC to the underlying matrix, at least in part by inducing changes in the affinity of the endothelial β1 integrin.
Cutting Edge: Ig Heavy Chain 3′ HS1–4 Directs Correct Spatial Position-Independent Expression of a Linked Transgene to B Lineage Cells
Authors: Chauveau Christine , Jansson Emmelie Å. , Müller Susanne , Cogné Michel , Pettersson Sven
Published: December 2022

The Ig H chain locus is regulated by a set of cis-acting elements. Hypersensitive sites (HS) located 3′ of the IgH, HS1–4, has been suggested to act as a locus control region (LCR) in cell lines. To assess the proposed role of HS1–4 acting as an LCR, we generated transgenic mice harboring a VH promoter-β-globin reporter gene linked to the Ig H chain HS1–4 3′regulatory sequences. Transgene expression is strictly confined to B lymphocytes, with no detectable expression outside the B cell lineage in all transgenic founder lines. Furthermore, reporter gene activity is integration independent but not copy number dependent. Thus, additional sequences are required to allow the HS1–4 regulatory region to act as a classical LCR in mice. Our data are discussed in the context of tissue-specific gene expression in B lineage cells.
Cutting Edge: Cross-Talk Between Cells of the Innate Immune System: NKT Cells Rapidly Activate NK Cells
Authors: Carnaud Claude , Lee Daniel , Donnars Olivier , Park Se-Ho , Beavis Andrew , Koezuka Yasuhiko , Bendelac Albert
Published: December 2022

α-Galactosylceramide (α-GalCer) is a glycolipid with potent antitumor properties that binds to CD1d molecules and activates mouse Vα14 and human Vα24 NKT cells. Surprisingly, we found that, as early as 90 min after α-GalCer injection in vivo, NK cells also displayed considerable signs of activation, including IFN-γ production and CD69 induction. NK activation was not observed in RAG- or CD1-deficient mice, and it was decreased by pretreatment with anti-IFN-γ Abs, suggesting that, despite its rapid induction, it was a secondary event that depended on IFN-γ release by NKT cells. At later time points, B cells and CD8 T cells also began to express CD69. These findings identify a high-speed communication network between the innate and adaptive immune systems in vivo that is initiated upon NKT cell activation. They also suggest that the antitumor effects of α-GalCer result from the sequential recruitment of distinct innate and adaptive effector lymphocytes.
Cutting Edge: KAP10, a Novel Transmembrane Adapter Protein Genetically Linked to DAP12 but with Unique Signaling Properties
Authors: Chang Chiwen , Dietrich Jes , Harpur Ailsa G. , Lindquist Jonathan A. , Haude Anja , Loke Y. W. , King Ashley , Colonna Marco , Trowsdale John , Wilson Michael J.
Published: December 2022

Transmembrane adapter proteins are a class of molecules that mediate signals from an extracellular receptor to the cytoplasm of the cell. We have cloned a novel transmembrane adapter protein called KAP10, a ∼10-kDa protein that is encoded within 100 bp of the DAP12 locus on human chromosome 19. KAP10 is predominantly expressed in immune cells, including NK cells, T cells, and monocytes. We show that KAP10, unlike other transmembrane adapter proteins, binds phosphatidylinositol-3 kinase following phosphorylation of a cytoplasmic YINM motif, which results in activation of Akt. In addition, we identify KAP10 as being able to bind the adapter protein Grb2. Based on our data, we suggest that this molecule is involved in stimulation and costimulation in cells of both myeloid and lymphoid origin.
Granzyme B-Induced Loss of Mitochondrial Inner Membrane Potential (ΔΨm) and Cytochrome c Release Are Caspase Independent
Authors: Heibein Jeffrey A. , Barry Michele , Motyka Bruce , Bleackley R. Chris
Published: December 2022

CTLs kill targets by inducing them to die through apoptosis. A number of morphological and biochemical events are now recognized as characteristic features of the apoptotic program. Among these, the disruption of the inner mitochondrial transmembrane potential (ΔΨm) and the release of cytochrome c into the cytoplasm appear to be early events in many systems, leading to the activation of caspase-3 and, subsequently, nuclear apoptosis. We show here that, in Jurkat targets treated in vitro with purified granzyme B and perforin or granzyme B and adenovirus, ΔΨm collapse, reactive oxygen species production, and cytochrome c release from mitochondria were observed. Loss of ΔΨm was also detected in an in vivo system where green fluorescent protein-expressing targets were attacked by a cytotoxic T cell line that kills predominantly through the granzyme pathway. DNA fragmentation, phosphatidylserine externalization, and reactive oxygen species production were inhibited in the presence of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk) in our in vitro system. Importantly, in either the in vitro or in vivo systems, these inhibitors at concentrations up to 100 μM did not prevent ΔΨm collapse. In addition, cytochrome c release was observed in the in vitro system in the absence or presence of zVAD-fmk. Thus the granzyme B-dependent killing pathway in Jurkat targets involves mitochondrial alterations that occur independently of caspases.
Differential Requirements for CD4 in TCR-Ligand Interactions
Authors: Vidal Karine , Daniel Claude , Hill Mark , Littman Dan R. , Allen Paul M.
Published: December 2022

The coreceptor molecule, CD4, plays an integral part in T cell activation; it is involved in both extracellular Ag recognition and intracellular signaling. We wanted to examine the functional role of CD4 in the recognition of agonist and altered peptide ligands (APLs). We generated two CD4-deficient T cell lines expressing well-characterized TCRs specific for Hb(64–76)/I-Ek. Although the responsiveness of the T cell lines to the agonist peptide was differently affected by the loss of CD4 expression, the recognition of APLs was in both cases dramatically reduced. Nearly full responsiveness to the agonist peptide was achieved by expression of a CD4 variant that did not associate with p56lck; however, the stimulation by APLs was only partially restored. Importantly, the expression of a CD4 variant in which domains interacting with MHC class II molecules have been mutated failed to restore the reactivity to all ligands. CD4-deficient T cells were able to be antagonized by APLs, indicating that CD4 was not required for antagonism. Overall, these findings support the concepts that CD4 is an integral part of the initial formation of the immunological synapse, and that the requirement for different CD4 functions in T cell activation varies depending upon the potency of the ligand.
The Th1/Th2 Nature of Concurrent Immune Responses to Unrelated Antigens Can Be Independent
Authors: Ismail Nahed , Bretscher Peter A.
Published: December 2022

We tested the independence hypothesis, namely that the Th1/Th2 nature of concurrent immune responses, generated in the same secondary lymphoid organ to non-cross-reacting Ags, can be independently determined. Some infectious agents and some adjuvants contain modulatory molecules that affect the Th1/Th2 nature of immune responses in a non-Ag-specific manner. We therefore excluded infectious agents as Ags and the use of adjuvants to generate immune responses. We first show that the dose of xenogeneic RBC administered i.v. determines the Th1/Th2 nature of the splenic immune response. Low doses generate a virtually exclusive Th1 response, whereas a higher dose induces either a mixed Th1/Th2 or a predominantly Th2 response, and stimulates the production of specific Abs. We immunized individual mice simultaneously with a low dose of one kind of xenogeneic RBC and with a higher dose of another non-cross-reacting xenogeneic RBC and assessed the Th1/Th2 nature of the immune responses generated in the spleen to each kind of RBC. The Th1/Th2 nature of the response to each RBC in doubly immunized mice was indistinguishable from that of the corresponding immune response in singly immunized mice. We discuss the significance of our findings for understanding immune class regulation, and the possible reasons why such independence is not always seen.
Thymocyte Antigens Do Not Induce Tolerance in the CD4+ T Cell Compartment
Authors: Jhaver Kanchan G. , Chandler Phillip , Simpson Elizabeth , Mellor Andrew L.
Published: December 2022

Thymocytes fail to tolerize the developing T cell repertoire to self MHC class I (MHC I) Ags because transgenic (CD2Kb) mice expressing H-2Kb solely in lymphoid cell lineages reject skin grafts mismatched only for H-2Kb. In this study, we examined why thymocytes fail to tolerize the T cell repertoire to self MHC I Ags. The ability of CD2Kb mice to reject H-2Kb skin grafts was age dependent because CD2Kb mice older than 20 wk accepted skin grafts. T cells from younger CD2Kb mice proliferated, but did not develop cytotoxic functions in vitro in response to H-2Kb. Proliferative responses were dominated by H-2Kb-specific, CD4+ T cells rather than CD8+ T cells. Representative CD4+ T cell clones from CD2Kb mice were MHC II restricted and recognized processed H-2Kb. TCR transgenic mice were generated from one CD4+ T cell clone (361) to monitor development of H-2Kb-specific immature thymocytes when all thymic cells or lymphoid cell lineages only expressed H-2Kb. Thymocyte precursors were not eliminated and mice were not tolerant to H-2Kb when Tg361 TCR transgenic mice were intercrossed with CD2Kb mice. In contrast, all thymocyte precursors were eliminated efficiently in thymic microenvironments in which all cells expressed H-2Kb. We conclude that self MHC I Ags expressed exclusively in thymocytes do not induce T cell tolerance because presentation of processed self MHC I Ags on self MHC II molecules fails to induce negative selection of CD4+ T cell precursors. This suggests that some self Ags are effectively compartmentalized and cannot induce self-tolerance in the T cell repertoire.
T Cell Infiltration into Class II MHC-Disparate Allografts and Acute Rejection Is Dependent on the IFN-γ-Induced Chemokine Mig
Authors: Koga Shoji , Auerbach Michael B. , Engeman Tara M. , Novick Andrew C. , Toma Hiroshi , Fairchild Robert L.
Published: December 2022

Direct evidence that cytokines with chemoattractant properties for leukocytes, chemokines, recruit alloantigen-primed T cells into transplanted allografts has been lacking. We present evidence that neutralization of a single chemokine inhibits T cell infiltration into class II MHC-disparate murine allografts and acute rejection. The chemokines IFN-γ-inducible protein-10 and monokine induced by IFN-γ (Mig) are expressed in allogeneic skin grafts during the late stages of acute rejection. Survival of class II MHC-disparate B6.H-2bm12 allografts is prolonged from day 14 to day 55 posttransplant when C57BL/6 recipients are given a short course treatment with an antiserum to Mig. This treatment also inhibits T cell and macrophage infiltration into the allografts. B6.H-2bm12 allografts are also not rejected by IFN-γ−/− C57BL/6 recipients. Injection of Mig directly into B6.H-2bm12 grafts on IFN-γ-deficient recipients restores T cell infiltration and rejection. Therefore, the inability of IFN-γ-deficient recipients to reject the class II MHC-disparate allografts is due to the lack of intraallograft Mig production and alloantigen-primed T cell recruitment to the graft. These results indicate for the first time the potential utility of chemokine neutralization strategies in preventing T cell infiltration into allografts and abrogating acute rejection.
In Vivo Evidence That Caspase-3 Is Required for Fas-Mediated Apoptosis of Hepatocytes
Authors: Woo Minna , Hakem Anne , Elia Andrew J. , Hakem Razqallah , Duncan Gordon S. , Patterson Bruce J. , Mak Tak W.
Published: December 2022

Caspase-3 is essential for Fas-mediated apoptosis in vitro. We investigated the role of caspase-3 in Fas-mediated cell death in vivo by injecting caspase-3-deficient mice with agonistic anti-Fas Ab. Wild-type controls died rapidly of fulminant hepatitis, whereas the survival of caspase-3−/− mice was increased due to a delay in hepatocyte cell death. Bcl-2 expression in the liver was dramatically decreased in wild-type mice following anti-Fas injection, but was unchanged in caspase-3−/− mice. Hepatocytes from anti-Fas-injected wild-type, but not caspase-3−/−, mice released cytochrome c into the cytoplasm. Western blotting confirmed the lack of caspase-3-mediated cleavage of Bcl-2. Presumably the presence of intact Bcl-2 in caspase-3−/− hepatocytes prevents the release of cytochrome c from the mitochondria, a required step for the mitochondrial death pathway. We also show by Western blot that Bcl-xL, caspase-9, caspase-8, and Bid are processed by caspase-3 in injected wild-type mice but that this processing does not occur in caspase-3−/− mice. This study thus provides novel in vivo evidence that caspase-3, conventionally known for its downstream effector function in apoptosis, also modifies Bcl-2 and other upstream proteins involved in the regulation of Fas-mediated apoptosis.
Systematic Mutagenesis of TCR Complementarity-Determining Region 3 Residues: A Single Conservative Substitution Dramatically Improves Response to Both Multiple HLA-DR Alleles and Peptide Variants
Authors: Brawley James V. , Concannon Patrick
Published: December 2022

To define the relative contributions of HLA and peptide contacts with TCR complementarity-determining region (CDR) 3 residues in T cell recognition, systematic mutagenesis and domain swapping was conducted on two highly similar TCRs that both respond to the influenza hemagglutinin (HA) peptide, HA307–319, but with different HLA restrictions. Despite the primary sequence similarity of these TCRs, exchange of as little as two CDR3 residues between them completely abrogated responsiveness. At position 95 within CDR3α, various substitutions still allowed for some degree of recognition. One modest substitution, alanine for glycine (essentially the addition of a methyl group), significantly broadened the specificity of the TCR. Transfectants expressing this mutant TCR responded strongly in the context of multiple HLA-DR alleles and to HA peptide variants with substitutions at each TCR contact residue. These results suggest that the conformations of CDR3 loops are crucial to TCR specificity and that it may not be reliable to extrapolate from primary sequence similarities in TCRs to similarities in specificity. The ease with which a broad specificity is induced in this mutant TCR has implications for the mechanisms and frequency of alloreactivity and promiscuity in T cell responses.
Neutrophil Dysfunctions, IL-8, and Soluble L-Selectin Plasma Levels in Rapidly Progressive Versus Adult and Localized Juvenile Periodontitis: Variations According to Disease Severity and Microbial Flora
Authors: Gainet Jean , Dang Pham M. C. , Chollet-Martin Sylvie , Brion Monique , Sixou Michel , Hakim Jacques , Gougerot-Pocidalo Marie-Anne , Elbim Carole
Published: December 2022

We used flow cytometry to analyze the expression of adhesion molecules and the oxidative burst of whole-blood polymorphonuclear neutrophils (PMN) from 26 patients with periodontitis. Three different clinical entities were studied: adult periodontitis (AP), localized juvenile periodontitis (LJP), and rapidly progressive periodontitis (RPP). Unstimulated PMN from the patients showed reduced Lewis x, sialyl-Lewis x, and L-selectin expression relative to those from healthy control subjects. These alterations were present whatever the severity of periodontal disease. However, PMN from RPP patients showed increased basal H2O2 production and decreased L-selectin shedding. These latter impairments, which correlated with increased IL-8 plasma levels, could contribute to initial vascular damage. In addition, decreased IL-8 priming of H2O2 production by PMN from RPP patients could account for a lower bactericidal capacity of PMN, leading to the large number of bacteria in the subgingival region of RPP patients. Soluble L-selectin plasma levels were also decreased in the RPP group, indicating more severe or diffuse endothelial damage. These abnormalities were not found in the patients with less destructive forms of periodontitis (AP and LJP). Porphyromonas gingivalis, a bacterial pathogen known to increase IL-8 production by PMN, was found in the periodontal pockets of RPP patients only. These results show links among PMN abnormalities, the clinical form of periodontitis, and the gingival bacterial flora.
Tissue Specificity of E- and P-Selectin Ligands in Th1-Mediated Chronic Inflammation
Authors: Chu Alvina , Hong Kenneth , Berg Ellen L. , Ehrhardt Rolf O.
Published: December 2022

The demonstrated role of E- and P-selectin ligands in the recruitment of Th1 cells raises the question of tissue specificity determination by pathogenic T cells. We took advantage of the fact that chronic Th1-mediated inflammation in the scid/scid CD4+CD45RBhigh T cell transfer model can occur at multiple tissue sites, resembling inflammatory bowel disease in the colon and psoriasis in the skin. We show that the majority of infiltrating effector T cells from psoriatic skin expresses high levels of functional P-selectin ligand (87 ± 3%), detected by P-selectin-Ig (PIg), while a significantly smaller subset of T cells from colitic lesions expresses this ligand (24 ± 2%). Similarly, E-selectin ligand is preferentially expressed on CD4+ T cells infiltrating the skin (24 ± 2%), but only on very few CD4+ T cells infiltrating the colon (CIT; 1.3 ± 0.8%). In contrast, CD4+ T cells infiltrating the skin express α4β7 at a significantly lower level than CIT (mean fluorescence intensity, 28 vs 61, respectively), although, interestingly, αEβ7 was expressed at high levels on both populations. Analysis of the disease-inducing potential of PIg+ and PIg− CD4+ CIT cells revealed that both populations not only express similar levels of the gut-homing molecule α4β7 (mean fluorescence intensity, 50 vs 56, respectively), but do not differ in their capacity to express IFN-γ. Furthermore, CIT depleted of cells expressing functional P-selectin ligand were able to induce colitis upon transfer, suggesting that induction of colitis in this model may be independent of E- and P-selectin. These results indicate that adhesion molecule expression and the homing pattern of inflammatory T cells are regulated by the local environment independently of their inflammatory capacity.
Photochemical Treatment with S-59 Psoralen and Ultraviolet A Light to Control the Fate of Naive or Primed T Lymphocytes In Vivo After Allogeneic Bone Marrow Transplantation
Authors: Truitt Robert L. , Johnson Bryon D. , Hanke Carrie , Talib Sohel , Hearst John E.
Published: December 2022

Donor leukocyte infusions after allogeneic bone marrow transplantation can provide a curative graft-vs-leukemia (GVL) effect, but there is a significant risk of graft-vs-host (GVH) disease. A simple and effective method for controlling the fate of naive or primed T-lymphocytes in vivo without eliminating their beneficial properties is needed. In this report, photochemical treatment (PCT) ex vivo with a synthetic psoralen (S-59) and UVA light was evaluated as a pharmacological approach to limiting the proliferation and GVH potential of naive and primed donor T cells in vivo. S-59 rapidly intercalates into and cross-links DNA on UVA illumination. The effects of PCT on T cells were found to be both S-59 and UVA dose dependent. With selected PCT regimens, treated T cells still expressed activation markers (CD25 and CD69) and secreted IL-2 on activation, but they showed limited proliferative capacity in vitro and in vivo. Clonal expansion of CTL in MLR was reduced after PCT, but short term lytic activity of primed CTL was not affected. In a murine model of MHC-mismatched bone marrow transplantation, the addition of PCT-treated T cells to T-depleted bone marrow facilitated donor engraftment and complete chimerism without causing acute or chronic graft-vs-host disease. Allospecific GVL reactivity was reduced but not eliminated after PCT treatment. In an MHC-matched model using host-presensitized donor T cells, PCT significantly reduced GVH-associated mortality without eliminating GVL reactivity. Thus, PCT ex vivo offers a simple, rapid, and inexpensive method by which to control the fate of naive and primed T cells in vivo.
The Reticulo-Endothelial System and Antibody Production
Authors: Roberts Edward F.
Published: December 2022

In a previous study (5) consideration was given to the influence of a reticulo-endothelial blockade upon the appearance of antibody in the circulating blood of the rabbit injected with antigens of various types. India ink, diluted in distilled H2O and filtered, was found to qualify as a suitable blockading agent since the carbon particles were intensely phagocytized and the mechanical barrier thus effected persisted for a considerable length of time. Various types of antigen were employed singly and in combination. Most significant of all, the study revealed that the results derived from a given experiment of this kind might suggest an interpretation in a definite direction whereas the results secured in a second experiment, carried out under almost identical conditions, could only be interpreted in an entirely different way. Moreover, the disagreement in results of duplicate experiments was not surpassed by the varied observations of workers who, compared one with the other, conducted their experiments under far more dissimilar conditions.
Schick Test as Applied to the Selection of Horses for the Production of Diphtheria Antitoxin
Authors: Nishimura H.
Published: December 2022

That horses possessing natural antitoxin in their blood produce antitoxin readily is well known by workers (Hitchens and Tingley, Hida, Glenny, Wadsworth, Sordelli, Ramon, etc.) in this field. Park and Zingher pointed out that persons yielding negative Schick tests respond to toxin-antitoxin injections with the production of antitoxin more readily than those who react in a positive manner.

In the selection of such naturally immune horses for the purpose of immunization a trial bleeding is usually taken and the serum is tested for natural antitoxin by Römer's cutaneous method or Ehrlich's subcutaneous method. Hitchens and Tingley injected 0.2 cc. diphtheria toxin, equaling 3 m.l.d.'s for 250 gram guinea pigs, into the conjunctiva of one eye of horses under examination for the purposes of immunization, and found that many yielded negative tests read at the end of forty-eight hours, indicating the presence of natural diphtheria-antitoxin in the blood of normal horses.
On the Action of Serum on the Fibrins of Various Species
Authors: Van Der Scheer J.
Published: December 2022

Summary and Conclusions

The author tried without success to duplicate the experiments by Fuchs and von Falkenhausen, from which it would follow that the distinction of proteins (fibrin) of various species and races can be made by means of serum ferments.
The Toxicity of Human Serum for the Guinea Pig As Affected by Absorption of Agglutinins
Authors: Davidsohn I. , Ramsdell Susan Griffith
Published: December 2022

Sera from persons treated with immune sera and showing a toxicity of the heterophilic type for the guinea pig, when treated with the red cells of this animal for the removal of agglutinins do not lose such toxicity. When the same sera are absorbed in the same way with sheep cells, as a heterophilic antigen, the toxicity is usually but not always completely, removed thereby. The experiments further indicate the heterophilic nature of the antibodies concerned in the toxicity. But there is also the possibility that, in these cases, sheep cells constitute an incomplete antigen; or else that another factor, as yet unidentified, is contained in such sera.
On the Anticomplementary Action of Pancreatic Extracts
Authors: Grove Ella F.
Published: December 2022

The anticomplementary action of the pancreatic extract known as pangestin is exhibited in a narrow quantitative zone, the larger quantities causing progressively less inhibition. This zone phenomenon is not present in the digestive activity of this preparation. This finding shows that the inhibition of complement by pancreatic extracts is not due to digestion of a component of complement.