bluuebunny/crossref_metadata_2025 huggingface dataset:
The requirement of intestinal bacterial flora for the development of an IgE production system fully susceptible to oral tolerance induction.
Authors: Sudo N , Sawamura S , Tanaka K , Aiba Y , Kubo C , Koga Y
Published: January 2023

The role of intestinal bacterial flora in oral tolerance induction to the IgE response was investigated using germfree (GF) mice. When GF mice were orally administered 20 mg of OVA as tolerogen before a systemic challenge with OVA, the Th1-mediated responses, such as the production of IgG2a and IFN-gamma, were abrogated, while the Th2-mediated immune responses, such as the production of IgE, IgG1, and IL-4, were maintained. Moreover, the basal level of IL-4 production in vitro was significantly higher in the GF mice than that of IL-4 in specific pathogen-free mice when challenged systemically with OVA. On the other hand, both Th1 and Th2 responses were fully sensitive to such tolerance induction in specific pathogen-free mice. The reconstitution of intestinal flora of GF mice with Bifidobacterium infantis, one of the predominant bacteria in the intestinal flora, restored the susceptibility of these Th2 responses to oral tolerance induction; however, this was only effective when such reconstitution was performed in neonates, but not in mice at an older age. These results thus suggested that intestinal bacterial flora play a crucial role in generating a Th2 cell population whose size and response are adequately regulated and, consequently, fully susceptible to oral tolerance induction, probably by affecting the development of gut-associated lymphoid tissue at the neonatal stage.
T cell receptor engagement induces tyrosine phosphorylation of FAK and Pyk2 and their association with Lck.
Authors: Berg N N , Ostergaard H L
Published: January 2023

Stimulation through the TCR is known to induce tyrosine phosphorylation of a number of proteins, which leads to functional activation of T cells. Identification of the substrates that become phosphorylated and defining their interactions with other signaling molecules will provide insight into the mechanisms controlling T cell activation. Focal adhesion kinase (FAK) and the recently described Pyk2 kinase are homologous members of a non-receptor protein tyrosine kinase family. FAK has been shown to become phosphorylated upon TCR stimulation, but its role, if any, in T cell activation remains to be defined. Although Pyk2 has been shown to play a role in neuronal cell activation stimulated through G-protein-coupled receptors, a role in T cell activation has not been described. In this study we show that FAK and Pyk2 are two of the major 115-to-120-kDa proteins that become tyrosine phosphorylated in T cells following TCR complex stimulation. Furthermore, coincident with the increase in tyrosine phosphorylation, we show an association of these kinases with the SH2 domain of the tyrosine kinase Lck in vivo. The increase in tyrosine phosphorylation of both FAK and Pyk2, however, occurs in Lck-deficient cells suggesting that phosphorylation of both of these kinases does not require Lck. Taken together, these results suggest that FAK and Pyk2, perhaps in coordination with Lck, play a role in T cell activation.
TCR expression in human fetal intestine and identification of an early T cell receptor beta-chain transcript.
Authors: Koningsberger J C , Chott A , Logtenberg T , Wiegman L J , Blumberg R S , van Berge Henegouwen G P , Balk S P
Published: January 2023

TCR expression by human fetal intestinal intraepithelial lymphocytes (ilELs) and intestinal lamina propria lymphocytes was analyzed to address whether T cell development occurs in human fetal intestine, the diversity of human fetal iIELs, and whether human fetal iIELs may contribute to the adult iIEL repertoire. ilELs and intestinal lamina propria lymphocytes from second trimester human fetal intestine were analyzed for TCR-alphabeta transcripts. Rearranged TCR-alpha transcripts were undetectable at 14 wk in the intraepithelial lymphocytes (IELs), whereas multiple TCR-beta transcripts were found at this stage. The TCR-alpha repertoire remained restricted relative to TCR-beta at later stages, and the IEL repertoire was restricted relative to the lamina propria lymphocytes at all stages. A previously reported T early alpha message was the major transcript from the TCR-alpha locus early in gestation. A previously undescribed TCR-beta transcript initiating upstream of the Dbeta1 locus and spliced to Cbeta1 or Cbeta2 was also identified and may represent a T early beta message. These results provide evidence for ongoing TCR gene rearrangement in human fetal intestine and suggest that transcription from the TCR-beta locus initiates with a T early beta transcript. The TCR-alpha repertoire (and hence the repertoire of potentially functional IELs) was limited through the second trimester.
B cell responses to a peptide epitope: IV. Subtle sequence changes in flanking residues modulate immunogenicity.
Authors: Vijayakrishnan L , Sarkar S , Roy R P , Rao K V
Published: January 2023

We examined modulation of primary humoral responses to a model synthetic peptide immunogen, peptide PS1CT3, as a consequence of single amino acid substitutions. Two analogues were employed, one in which the amino-terminal histidine (His1, peptide G28CT3) and another in which an internal proline (Pro14, peptide G41CT3) were replaced with glycine residues. Peptide G28CT3 displayed markedly enhanced immunogenicity relative to peptide PS1CT3 in BALB/c mice, whereas peptide G41CT3 was only poorly immunogenic. Nevertheless, in all three cases the mature polyclonal IgG response was predominantly directed against a tetrapeptide segment of sequence Asp-Pro-Ala-Phe between positions 4 and 7 of the sequence. While all three peptides proved equally capable of priming Ag-specific Th cells, they, however, displayed significant differences in their abilities to recall T cell responses. Regardless of the priming immunogen, in vitro challenge with either PS1CT3 or its analogues consistently gave a hierarchy of potencies as G28CT3 > PS1CT3 > G41CT3. This could also be correlated with B cell recall responses in which an identical hierarchy was obtained on restimulation of G41CT3-primed B cells in adoptive transfer experiments. Subsequent studies revealed that peptide-mediated modulation of Th cell recruitment by Ag-primed B cells was probably due to differences in on-rates for engagement of B cell Ag receptor by these analogues. This was despite the fact that all three peptides displayed equally randomized conformations in solution. These studies indicate that even subtle variations in the flanking sequences can markedly influence the immunogenicity of B cell epitopes.
Salmonella typhimurium infections in mice deficient in interleukin-4 production: role of IL-4 in infection-associated pathology.
Authors: Everest P , Allen J , Papakonstantinopoulou A , Mastroeni P , Roberts M , Dougan G
Published: January 2023

Mice harboring mutations in the IL-4 gene (IL-4(-/-)) were infected with a range of Salmonella typhimurium HWSH derivatives using different routes of infection. Compared with IL-4(+/+) mice, IL-4(-/-) mice exhibited a delayed time to death following infection with wild-type S. typhimurium HWSH. Groups of IL-4(+/+) mice infected with S. typhimurium HWSH purE, a less virulent derivative, showed sporadic deaths and harbored micro- or macroabscesses in their tissues, particularly associated with the liver. However, IL-4(-/-) mice infected with similar doses of S. typhimurium HWSH purE bacteria were resistant to killing and failed to develop detectable abscesses. Abscess formation in IL-4(-/-) mice could be induced by i.v. administration of rIL-4 during the S. typhimurium HWSH purE infection. The immune response in both IL-4(-/-) and IL-4(+/+) mice was of the Th1-type. Viable salmonella bacteria could be found associated with abscesses. Both IL-4(-/-) and IL-4(+/+) mice were resistant to killing by S. typhimurium aroA.
Presentation via the class I pathway by Leishmania amazonensis-infected macrophages of an endogenous leishmanial antigen to CD8+ T cells.
Authors: Kima P E , Ruddle N H , McMahon-Pratt D
Published: January 2023

CD8+ T cells play a protective role in immunity to cutaneous leishmaniasis. However, it has been unclear how these cells execute this function, since results from several investigations attempting to demonstrate recognition of Leishmania-infected macrophages by CD8+ T cells have been contradictory. In this study, we report the generation of CD8+ T cell lines specific for GP46/M-2, a leishmanial Ag, previously shown to protectively immunize mice against a Leishmania amazonensis challenge. Using T cell cytolysis and IFN-gamma production to assess CD8+ T cell activation, we show that in addition to recognizing mammalian cells transfected with GP46/M-2, these CD8+ T cell lines also recognize macrophages infected with Leishmania amazonensis. MHC class I presentation of GP46/M-2 by infected macrophages can be blocked by treatment with brefeldin A and also by inhibitors of the cytosolic multicatalytic proteasome, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal and N-acetyl-L-leucinyl-L-leucinylmethional. These results suggest that this leishmanial Ag is processed in the macrophage cytoplasm and is presented to CD8+ T cells via the classical pathway of MHC class I presentation. The relevance of these findings as they impact on our understanding of the biology of the parasite within the macrophage is discussed.
Virus dissemination through the brain parenchyma without immunologic control.
Authors: Stevenson P G , Freeman S , Bangham C R , Hawke S
Published: January 2023

After inoculation into the cerebrospinal fluid, the neurovirulent influenza virus A/WSN caused a rapidly progressive encephalitis that was uniformly fatal within 8 days. After inoculation into the brain parenchyma, the same virus replicated for 7 to 20 days without causing clinical illness, but when infection reached the cerebrospinal fluid, encephalitis was lethal within a further 6 days. As the virus spread through the brain parenchyma, there was intense intracerebral inflammation, with up-regulation of MHC class I and MHC class II expression and recruitment of CD44(high) CD49d(high) T cells. However, this was not associated with antiviral Ab production, and the infiltrating cells, unlike primed A/WSN-specific T cells, did not eliminate the virus in vivo or show evidence of virus recognition in vitro. Thus, a neurovirulent virus was able to disseminate widely through the brain parenchyma and induce considerable intracerebral inflammation without eliciting protective immunity.
Matching antibody class with pathogen type and portal of entry: cognate mechanisms regulate local isotype expression patterns in lymph nodes draining the respiratory tract of mice inoculated with respiratory viruses, according to virus replication competence and site of inoculation.
Authors: Sangster M Y , Mo X Y , Sealy R , Coleclough C
Published: January 2023

Intranasal deposition of Sendai virus (SV) in C57BL/6 mice provokes an Ab-forming cell (AFC) reaction in mediastinal (MLN) and cervical lymph nodes (CLN), which drain the lungs and upper respiratory tract, respectively. While the majority of AFC elicited by infectious SV at both sites produced IgG, the CLN response to SV rendered inactive in replication was restricted almost entirely to IgA, although isotype switching in mediastinal continued to be skewed heavily to IgG. However, in vitro restimulation of the accompanying virus-specific T cell populations from the two sites did not reveal any significant difference in lymphokine output, and isotype expression was not altered substantially in mice lacking IL-4 or IL-6 genes. To dissociate the response to specific Ags from the inflammatory reaction to viral infection, we examined the response to inactivated SV in the face of infection with influenza virus A/HKx31. The magnitude and IgA dominance of the anti-SV AFC population in the CLN were unaffected by a simultaneous, vigorous, IgG-dominated CLN anti-influenza reaction. Evidently, the characteristics of this antiviral response are determined primarily by cognate interactions. Moreover, the IgA bias of the CLN AFC response to inactivated SV was observed only when the virus was delivered intranasally: injection under the epidermis of the cheek, a site that has a lymphatic drainage into the CLN, resulted in an IgG-dominated CLN AFC reaction, lacking IgA. The site of deposition of a vaccine can thus have more influence on the pattern of isotypes induced than the site at which the immune response is initiated.
Perforin-mediated cytolysis plays a limited role in host resistance to Toxoplasma gondii.
Authors: Denkers E Y , Yap G , Scharton-Kersten T , Charest H , Butcher B A , Caspar P , Heiny S , Sher A
Published: January 2023

Resistance of perforin knockout (PKO) mice to infection with Toxoplasma gondii was assessed in models of acute infection and during chronic disease. PKO mice vaccinated with the attenuated mutant, ts-4, displayed severely defective CTL responses against tachyzoite-infected targets. Lysis of the NK target, YAC-1, was also severely impaired in PKO mice following ts-4 vaccination. In contrast, wild-type mice developed high levels of CTL and NK lytic activity after ts-4 vaccination. Despite severely defective lytic activity, vaccinated PKO animals were completely resistant to challenge with the virulent strain RH, which normally causes a lethal acute infection. Resistance was attributable to production of IFN-gamma, which remained unimpaired in the PKO animals. In contrast, when PKO mice were infected with low virulence parasite strain ME49, which progresses to the cyst-forming stage after passage through an acute phase, accelerated mortality was observed beginning at 75 days postinfection. A three- to fourfold increase in brain cyst numbers was also found by day 30 in infected PKO animals. Nevertheless, the PKO strain produced normal levels of IFN-gamma after ME49 infection, ruling out impaired production of the latter cytokine as a cause of increased susceptibility. Together, these results show that perforin-dependent cytolytic function is not required for host resistance to lethal acute infection in preimmunized animals, but that the latter activity contributes to the control of infection during the chronic stage.
Expression of leukocyte fucosyltransferases regulates binding to E-selectin: relationship to previously implicated carbohydrate epitopes.
Authors: Wagers A J , Stoolman L M , Kannagi R , Craig R , Kansas G S
Published: January 2023

E-selectin is a carbohydrate-binding endothelial cell adhesion molecule that reportedly interacts with several related sialylated and fucosylated carbohydrates. The activity of leukocyte alpha1,3-fucosyltransferases (FucT-IV or FucT-VII) is an essential step in the synthesis of E-selectin ligands. Using a panel of stably transfected hemopoietic cell lines, we have investigated the role of alpha1,3-fucosyltransferases in generating E-selectin ligands, and the relationship between adhesion to E-selectin and expression of mAb-defined carbohydrates. Expression of FucT-VII was always sufficient for binding to E- and P-selectin, while the ability of FucT-IV to construct E-selectin ligands varied among different cell types. Furthermore, FucT-IV was unable to support any binding to P-selectin in a lymphoid cell line, even when expressed at levels equivalent to those in myeloid cells. FucT-IV expression generated high levels of surface Le(x)/CD15 and CDw65, whereas expression of FucT-VII correlated with a subset of mAb-defined sialyl Lewis X (sLex)-like structures. FucT-IV-associated epitopes were present on both binding and nonbinding cells, whereas all cells that expressed FucT-VII-associated epitopes bound E-selectin. However, treatment of HL60 cells with neuraminidase destroyed FucT-VII-associated epitopes at a faster rate than E-selectin binding sites. Surface expression of a subset of mAb-defined sLex-like carbohydrates is therefore a good marker for high levels of FucT-VII activity, but these carbohydrates are not themselves required for recognition of E-selectin.
Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells.
Authors: Mazumder B , Mukhopadhyay C K , Prok A , Cathcart M K , Fox P L
Published: January 2023

Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.
Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10.
Authors: Daheshia M , Kuklin N , Kanangat S , Manickan E , Rouse B T
Published: January 2023

Ocular infection with herpes simplex virus leads to an inflammatory lesion in the cornea orchestrated by CD4+ Th1 lymphocytes. This immunopathologic disease, called herpetic stromal keratitis, is an important cause of impaired vision. In this study, we set out to determine whether established lesions of herpetic stromal keratitis could be controlled by topically administering naked plasmid DNA encoding cytokines to the corneal surface. A single topical administration of DNA encoding IL-10 was beneficial to the majority (75%) of treated animals, and 50% (vs 10% in controls) resolved their lesions completely over a 23-day observation period. Topical ocular application of DNA encoding foreign proteins was also shown to be an effective means of inducing systemic and mucosal immune responses. The direct application of DNA encoding cytokines may represent an additional therapeutic option for the management of immunoinflammatory disease.
Listeria monocytogenes potently induces up-regulation of endothelial adhesion molecules and neutrophil adhesion to cultured human endothelial cells.
Authors: Krüll M , Nöst R , Hippenstiel S , Domann E , Chakraborty T , Suttorp N
Published: January 2023

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. Listeriolysin (Hly) is one of its major virulence factors. In the early phase of the disease polymorphonuclear leukocytes (PMN) substantially contribute to the nonspecific anti-listerial resistance. We characterized the effects of L. monocytogenes on the expression of endothelial adhesion molecules and on subsequent PMN adhesion to cultured HUVEC. P-selectin, E-selectin, intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) were up-regulated in HUVEC after cell incubation with L. monocytogenes (wild type), but not with the nonpathogenic Listeria innocua strain. P-selectin expression peaked after 30 min and could be mimicked with similar kinetics by exposure to L. innocua engineered to produce large amounts of Hly or by addition of purified Hly. Listeriolysin production, however, was not necessary for an up-regulation of E-selectin after 6 h or of ICAM-1 and VCAM-1 after 18 to 24 h in HUVEC, because L. monocytogenes defective for Hly synthesis was almost as effective as the wild type. Listeria-induced up-regulation of endothelial adhesion molecules was accompanied by an increased binding of PMN to infected HUVEC. PMN adhesion was significantly reduced in the presence of anti-beta2 integrin, anti-E-selectin, and anti-ICAM-1, but not anti-VCAM-1 Abs. Our data indicate that infection of endothelial cells with L. monocytogenes induced up-regulation of adhesion molecules by two different mechanisms: a Hly-dependent up-regulation of P-selectin and a Hly-independent expression of E-selectin, ICAM-1, and VCAM-1. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.
P- and L-selectin mediate distinct but overlapping functions in endotoxin-induced leukocyte-endothelial interactions in the rat mesenteric microcirculation.
Authors: Davenpeck K L , Steeber D A , Tedder T F , Bochner B S
Published: January 2023

Endotoxin is a potent stimulus of leukocyte infiltration, but the adhesion-related mechanisms responsible for LPS-induced cell recruitment events in vivo remain poorly characterized. Utilizing intravital microscopy, we examined the role of P- and L-selectin in LPS-induced inflammation. We demonstrated that superfusion of rat mesentery with LPS resulted in significant increases in both leukocyte rolling and adherence, which were maintained for at least 2 h. Pretreatment with a P-selectin neutralizing mAb only partially inhibited LPS-induced leukocyte rolling, but completely inhibited LPS-induced leukocyte adherence throughout the 2-h observation period. Pretreatment with an L-selectin neutralizing mAb dramatically inhibited LPS-induced increases in leukocyte rolling, but unlike the P-selectin mAb did not inhibit leukocyte adhesion. Fucoidin, which blocks both P- and L-selectin function, completely inhibited LPS-induced leukocyte rolling and adhesion. Consistent with previous studies, leukocyte rolling velocities on P-selectin were observed to be far less than velocities observed for leukocytes rolling on L-selectin in vivo. These data suggest that P-selectin plays a role in LPS-induced rolling and is essential for LPS-induced leukocyte adherence, while L-selectin functions in LPS-induced rolling, but not in adhesion.
Human T cell leukemia virus type I (HTLV-I)-specific CD8+ CTL clones from patients with HTLV-I-associated neurologic disease secrete proinflammatory cytokines, chemokines, and matrix metalloproteinase.
Authors: Biddison W E , Kubota R , Kawanishi T , Taub D D , Cruikshank W W , Center D M , Connor E W , Utz U , Jacobson S
Published: January 2023

Human T cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurologic disease characterized by marked degeneration of the spinal cord and the presence of infiltrating CD8+ T cells and macrophages. HAM/TSP patients have very high frequencies of HTLV-I-specific CD8+ CTL in peripheral blood and in cerebrospinal fluid. In this study, we show that HAM/TSP patients also have elevated levels of peripheral blood CD8+ T cells that produce intracellular IFN-gamma. To address the potential role of soluble mediators secreted by CD8+ T cells in the pathogenesis of HAM/TSP, we have analyzed the capacity of a panel of nine HTLV-I-specific CD8+ CTL clones derived from three HAM/TSP patients to secrete cytokines, chemokines, and matrix metalloproteinases. The results demonstrate that the majority of these CTL clones secrete IFN-gamma, TNF-alpha, macrophage-inflammatory protein-1alpha and -1beta, IL-16, and matrix metalloproteinase-9. These findings indicate that HTLV-I-specific CD8+ CTL are an important source of proinflammatory soluble mediators that may contribute significantly to the pathogenesis of HAM/TSP.
Accumulation of human T lymphotropic virus (HTLV)-I-specific T cell clones in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.
Authors: Höger T A , Jacobson S , Kawanishi T , Kato T , Nishioka K , Yamamoto K
Published: January 2023

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraperesis (HAM/TSP) is a slowly progressive neurologic disorder following infection with HTLV-I. It is characterized by spasticity and hyper-reflexia of the lower extremities, urinary bladder disturbance, lower extremity muscle weakness, and sensory disturbances. HTLV-I, as an inducer of a strong humoral and cytotoxic response, is a well-known pathogenic factor for the progression of HAM/TSP. Peptides derived from proviral tax and env genes provide epitopes recognized by T cells. We herein report an accumulation of distinct clonotypes of alpha/beta TCR+ peripheral blood T lymphocytes from HAM/TSP patients in comparison with that observed in both asymptomatic carriers and healthy controls, using the reverse-transcriptase PCR/single-strand conformation polymorphism method. We also found that some of the accumulated T cell clones in the peripheral blood and cerebrospinal fluid are HTLV-I Tax(11-19) peptide specific. Such clones were found to expand strongly after being cultured with an HTLV-I Tax(11-19) peptide. Moreover, the cultured samples exhibited a strong MHC class I-restricted cytotoxic activity against HTLV-I Tax(11-19) peptide-expressing targets, and therefore most likely also include the disease-associated T cell clones observed in the patients. This is the first report of a direct assessment of Ag-specific T cell responses in fresh PBL and cerebrospinal fluid.
The Chances of Establishing Non-Paternity by Determination of Blood Groups
Authors: Hooker Sanford B. , Boyd William C.
Published: January 2023

From the frequency distribution of blood groups and the laws governing their inheritance the relative usefulness of blood-grouping tests in bastardy proceedings has been calculated. The probabilities are given in table 1.
Qualitative Diversity of Agglutinin Response Among Different Rabbits Treated with the Same Complex Antigen (Streptococcus Scarlatin æ)
Authors: Hooker Sanford B.
Published: January 2023

Summary and Conclusions

The results suggest that qualitative deficiency of antibody response to scarlatinal streptococci occurs rather infrequently among rabbits. It probably explains some of the discrepant reactions obtained by different investigators who have worked with the same strains. The fact that an immune serum may not faithfully and completely reflect the antigenic features of a complex inoculum receives additional confirmation as does our previous conclusion that the injected animal's individuality may constitute an important variable in experiments of this kind.
T and B Cell Development in BP-1/6C3/Aminopeptidase A-Deficient Mice
Authors: Lin Qun , Taniuchi Ichiro , Kitamura Daisuke , Wang Jiyang , Kearney John F. , Watanabe Takeshi , Cooper Max D.
Published: January 2023

Stage-restricted expression of cell surface molecules serves to delineate B lineage cells during their progressive differentiation within the bone marrow. The BP-1/6C3 Ag, aminopeptidase A (APA), is selectively expressed by the pre-B and immature B cells. This ectoenzyme, which is also present on bone marrow-derived stromal cells, thymic cortical epithelial cells, renal proximal tubular cells, intestinal enterocytes, and endothelial cells, cleaves acidic glutamyl and aspartyl residues from the N-terminus of angiotensin and other biologically active peptides to quench their functional activity. BP-1/6C3/APA expression by early B lineage cells is up-regulated by IL-7, an important growth factor for pre-B cells and T cells. To explore the physiologic role of this peptidase, we generated a mouse model of BP-1 deficiency by gene targeting in embryonal stem cells. While mice homozygous for the BP-1 mutation did not express detectable BP-1 protein or enzyme activity, they developed normally, generated normal numbers of T and B cells, exhibited integrity of Ab responses to both thymus-dependent and -independent Ags, and produced normal serum Ig levels. Phenotypic analysis of bone marrow and thymic lymphocytes indicated a normal pattern of B and T lineage differentiation. B lymphopoiesis in fetal liver cultures and the proliferative responses of bone marrow cells to IL-7 and LPS were also unimpaired. These findings indicate that BP-1 ectoenzyme activity is not essential for normal B and T cell development.
DR/CLIP (Class II-Associated Invariant Chain Peptides) and DR/Peptide Complexes Colocalize in Prelysosomes in Human B Lymphoblastoid Cells
Authors: Stang Espen , Guerra Carolyn B. , Amaya Miguel , Paterson Yvonne , Bakke Oddmund , Mellins Elizabeth D.
Published: January 2023

In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.
In Situ Immune Response in Gut-Associated Lymphoid Tissue (GALT) Following Oral Antigen in TCR-Transgenic Mice
Authors: Gonnella Patricia A. , Chen Youhai , Inobe Jun-ichi , Komagata Yoshinori , Quartulli Marc , Weiner Howard L.
Published: January 2023

Oral administration of Ag results in systemic hyporesponsiveness termed oral tolerance. The regulatory cells induced by oral Ag are effective in the suppression of Th1-type autoimmune diseases. We examined the cytokine microenvironment in gut-associated lymphoid tissue in response to orally administered OVA in OVA TCR-transgenic mice. Mice were fed a low (0.5 mg) or high (500 mg) dose of OVA one time or five times. Immunohistochemical analysis demonstrated increased IL-4, IL-10, and TGF-β in the gut within 6 h of a low-dose feeding and after five low-dose or high-dose feedings. IFN-γ and IL-2 either decreased or showed no change, with the exception of a small transient increase in IL-2 at 6 h after a low dose. Increases in IL-4 and IL-10 were found in the dome of the Peyer’s patch, and increases in TGF-β were observed in the interfollicular region and the villi. IL-10 was also substantially increased in the villi. IL-4 and IL-10 were produced predominately by CD4+ T cells. TGF-β was found predominately in macrophages and CD4+ T cells. Peyer’s patches had a marked up-regulation of TGF-β mRNA as measured by RT-PCR. These results demonstrate the differential activation of cytokine production in discrete regions of gut-associated lymphoid tissue. The induction of cytokines known to inhibit autoimmune disease at the site of Ag absorption indicates an important role for the mucosal immune system in the establishment of oral tolerance.
Direct Evidence That Functionally Impaired CD4+ T Cells Persist In Vivo Following Induction of Peripheral Tolerance
Authors: Pape Kathryn A. , Merica Rebecca , Mondino Anna , Khoruts Alexander , Jenkins Marc K.
Published: January 2023

A small population of CD4+ OVA-specific TCR transgenic T cells was tracked following the induction of peripheral tolerance by soluble Ag to address whether functionally unresponsive, or anergic T cells, persist in vivo for extended periods of time. Although injection of OVA peptide in the absence of adjuvant caused a transient expansion and deletion of the Ag-specific T cells, a population that showed signs of prior activation persisted in the lymphoid tissues for several months. These surviving OVA-specific T cells had long-lasting, but reversible defects in their ability to proliferate in lymph nodes and secrete IL-2 and TNF-α in vivo following an antigenic challenge. These defects were not associated with the production of Th2-type cytokines or the capacity to suppress the clonal expansion of a bystander population of T cells present in the same lymph nodes. Therefore, our results provide direct evidence that a long-lived population of functionally impaired Ag-specific CD4+ T cells is generated in vivo after exposure to soluble Ag.
CpG Motifs in Bacterial DNA Activate Leukocytes Through the pH-Dependent Generation of Reactive Oxygen Species
Authors: Yi Ae-Kyung , Tuetken Rebecca , Redford Thomas , Waldschmidt Marianella , Kirsch Jeffrey , Krieg Arthur M.
Published: January 2023

B cells and monocytes endocytose DNA into an acidified intracellular compartment. If this DNA contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs), these leukocytes are rapidly activated. We now show that both B cell and monocyte-like cell line responses to DNA containing CpG motifs (CpG DNA) are sensitive to endosomal acidification inhibitors; they are completely blocked by bafilomycin A, chloroquine, and monensin. The specificity of these inhibitors is demonstrated by their failure to prevent responses to LPS, PMA, or ligation of CD40 or IgM. Acidification of endosomal CpG DNA is coupled to the rapid generation of intracellular reactive oxygen species. The CpG DNA-induced reactive oxygen species burst is linked to the degradation of IκB and the activation of NFκB, which induces leukocyte gene transcription and cytokine secretion. These studies demonstrate a novel pathway of leukocyte activation triggered by CpG motifs.
A Critical Role for IL-4 in Regulating Disease Severity in Experimental Allergic Encephalomyelitis as Demonstrated in IL-4-Deficient C57BL/6 Mice and BALB/c Mice
Authors: Falcone Marika , Rajan Alice J. , Bloom Barry R. , Brosnan Celia F.
Published: January 2023

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system (CNS) that has served as the principal experimental model for multiple sclerosis (MS). Susceptibility to disease is thought to correlate with the ability to generate a Th1-type cytokine profile in myelin-responsive T cells, whereas T cells producing a Th2 cytokine pattern, in particular IL-4, are thought to be nonencephalitogenic and also to confer protection against a Th1-type response. However, recent studies using a variety of genetically engineered animals in which the genes for Th1-type cytokines and/or their receptors have been inactivated have called into question the Th1-Th2 paradigm in experimental allergic encephalomyelitis. In this report we have addressed the contribution of IL-4 to disease expression by studying two strains of mice, C57BL/6 and BALB/c, in which the gene for IL-4 has been inactivated. The IL-4-deficient C57BL/6 mice, and to a lesser extent the IL-4-deficient BALB/c mice, developed a more severe form of clinical disease, a more extensive pathologic involvement of the spinal cord, and an increased expression of proinflammatory cytokines in the CNS than their wild-type littermates. BALB/c and C57BL/6 mice showed a slightly different cytokine profile in the CNS. Both groups of animals recovered from the acute clinical episode in a time frame that was essentially identical to that found in the wild-type controls. We conclude that IL-4 plays an important role in modulating the severity of the encephalitogenic process, but does not by itself contribute to spontaneous remission from the disease.
Partial TCR Signals Delivered by FcR-Nonbinding Anti-CD3 Monoclonal Antibodies Differentially Regulate Individual Th Subsets
Authors: Smith Judith A. , Tang Qizhi , Bluestone Jeffrey A.
Published: January 2023

Anti-CD3 mAbs with low FcR affinity prolong graft survival in the absence of the cytokine-mediated toxicity observed with conventional anti-CD3 treatment. Previous studies have shown that FcR-nonbinding anti-CD3 mAbs suppress immune responses, at least in part, by delivering a partial signal resulting in Th1 unresponsiveness. In this study, the biochemical and functional consequences of FcR-nonbinding anti-CD3 treatment for various activated T cell populations were examined. In contrast to Th1 cells, FcR-nonbinding anti-CD3-treated Th2 cells secreted IL-4 and proliferated. Furthermore, Th2 cells cultured with the mAb were not rendered unresponsive. Mixed “Th0” populations responded to FcR-nonbinding anti-CD3 by producing IL-4, and showed a selective decrease in IL-2 production following preculture with the mAb. The stimulation of IL-4-producing cells did not reflect a more complete TCR signal, since similar defects in ζ, ZAP-70, and MAP kinase phosphorylation were observed in Th1 and Th2 cells. Despite the proximal signaling defects, FcR-nonbinding anti-CD3 induced nuclear translocation of NF-ATc. Thus, Abs that deliver partial TCR signals may promote development of a Th2 phenotype during the course of an immune response via selective effects on different Th subsets.
Characterization of the Mouse PA28 Activator Complex Gene Family: Complete Organizations of the Three Member Genes and a Physical Map of the ∼150-kb Region Containing the α- and β-Subunit Genes
Authors: Kohda Keiko , Ishibashi Teruo , Shimbara Naoki , Tanaka Keiji , Matsuda Yoichi , Kasahara Masanori
Published: January 2023

The proteasome is a multisubunit protease responsible for the generation of peptides loaded onto MHC class I molecules. Recent evidence indicates that binding of an IFN-γ-inducible PA28 activator complex to the 20S proteasome enhances the generation of class I binding peptides. The α- and β-subunits, which constitute the PA28 activator complex in the form of an (αβ)3 heterohexamer, show significant amino acid sequence similarity to a protein, designated Ki or the γ-subunit, that is capable of binding to the 20S proteasome. In this study, we describe the complete nucleotide sequences of the mouse genes, Psme1, Psme2, and Psme3, coding for the α-, β-, and γ-subunits, respectively. The overall exon-intron organizations of the three Psme genes are virtually identical, thus providing evidence that they are descended from a single ancestral gene. The promoter regions of the Psme1 and Psme2 genes contain sequence motifs that qualify as IFN-stimulated response elements, consistent with the observation that their expression is induced strongly by IFN-γ. The Psme1 and Psme2 genes are located ∼6 kb apart with their 3′-ends pointing toward each other on bands C2 to D1 of mouse chromosome 14, supporting the idea that they emerged by tandem duplication.
Localization and Regulation of IFN-γ Production Within the Granulomas of Murine Schistosomiasis in IL-4-Deficient and Control Mice
Authors: Rakasz Eva , Blum Arthur M. , Metwali Ahmed , Elliott David E. , Li Jie , Ballas Zuhair K. , Qadir Khurram , Lynch Richard , Weinstock Joel V.
Published: January 2023

Schistosome granulomas from normal or IL-4-deficient C57BL/6 mice make little IFN-γ and show no Th1 polarization. This could signify that these granulomas have few cells capable of IFN-γ synthesis or that such cells are under tight control. Granulomas can make IL-10 and TGF-β, which can regulate IFN-γ synthesis. Using FACS analysis and ELISA, we explored the origin and regulation of IFN-γ in schistosome granulomas from both IL-4−/− and IL-4+/+ mice. FACS analysis of intracytoplasmic IFN-γ staining showed that some granuloma Thy1.2+ T cells (CD8+ and CD4+) express IFN-γ. Granulomas had NK1.1+ cells, but they appeared to produce little or no IFN-γ. Purified granuloma Thy1.2+ cells made IFN-γ in vitro, whereas isolated NK1.1+ lymphocytes secreted little even with rIL-12 stimulation. Culture of granuloma cells with blocking anti-IL-10 or anti-TGF-β mAb or with rIL-12 substantially increased T cell IFN-γ synthesis, particularly in the IL-4−/− animals. Cultured granuloma cells depleted of Thy1.2+ lymphocytes by Ab and C released no IFN-γ. It is concluded that granuloma IFN-γ comes from T cells, not NK cells. Also, this T cell-derived IFN-γ is subject to IL-10 and TGF-β regulation, which is particularly evident in IL-4−/− mice. Thus, the Th2 granuloma of schistosomiasis has large numbers of activated Th1 or Th0 lymphocytes that are under tight restraint.
CBL-GRB2 Interaction in Myeloid Immunoreceptor Tyrosine Activation Motif Signaling
Authors: Park Rae Kil , Kyono Wade T. , Liu Yenbou , Durden Donald L.
Published: January 2023

In this study, we provide the first evidence for role of the CBL adapter protein interaction in FcγRI receptor signal transduction. We study the FcγRI receptor, an immunoreceptor tyrosine activation motif (ITAM)-linked signaling pathway, using IFN-γ-differentiated U937 myeloid cells, termed U937IF cells. CBL is constitutively associated with both GRB2 and the ITAM-containing receptor subunit, FcγRIγ of FcγRI, providing direct evidence that CBL functions in myeloid ITAM signaling. FcγRI cross-linking of U937IF cells induces the tyrosine phosphorylation of CBL that is associated with an altered CBL-GRB2 interaction. Both GRB2-SH3 and SH2 domains bind CBL in resting cell lysates; upon FcγRI stimulation, phosphorylated CBL binds exclusively to the GRB2-SH2 domain. Glutathione-S-transferase fusion protein data demonstrate that the constitutive interaction of CBL with GRB2 and CRKL is mediated via two discrete regions of the CBL C terminus. The proximal C terminus (residues 461–670) binds to GRB2 constitutively, and under conditions of receptor activation binds to the tyrosine-phosphorylated SHC adapter molecule. The distal C terminus of CBL (residues 671–906) binds the CRKL adapter protein. The data demonstrate that the CBL-GRB2 and GRB2-SOS protein complexes are distinct and mutually exclusive in U937IF cells, supporting a model by which the CBL-GRB2 and GRB2-SOS complexes function in separate pathways for myeloid FcγRI signaling.
Bactericidal Monoclonal Antibodies That Define Unique Meningococcal B Polysaccharide Epitopes That Do Not Cross-React with Human Polysialic Acid
Authors: Granoff Dan M. , Bartoloni Antonella , Ricci Stefano , Gallo Eugenia , Rosa Domenico , Ravenscroft Neil , Guarnieri Valentina , Seid Robert C. , Shan Asra , Usinger William R. , Tan Siqi , McHugh Yvonne E. , Moe Gregory R.
Published: January 2023

The poor immunogenicity of the Neisseria meningitidis group B polysaccharide capsule, a homopolymer of α(2→8) sialic acid, has been attributed to immunologic tolerance induced by prenatal exposure to host polysialyated glycoproteins. Substitution of N-propionyl (N-Pr) for N-acetyl groups on the meningococcal B polysaccharide, and conjugation of the resulting polysaccharide to a protein carrier, have been reported to yield a conjugate vaccine that elicits protective Abs with minimal autoantibody activity. To characterize the protective epitopes on the derivatized polysaccharide, we isolated 30 anti-N-Pr meningococcal B polysaccharide mAbs. These Abs were heterogeneous with respect to complement-mediated bactericidal activity, fine antigenic specificity, and autoantibody activity as defined by binding to the neuroblastoma cell line, CHP-134, which expresses long-chain α(2→8)-linked polysialic acid. Eighteen of the Abs could activate complement-mediated bacteriolysis. Seven of these 18 Abs cross-reacted with N-acetyl meningococcal B polysaccharide by ELISA and had strong autoantibody activity. Thus, N-Pr meningococcal B polysaccharide conjugate vaccine has the potential to elicit autoantibodies. However, 7 of the 18 bactericidal mAbs had no detectable autoantibody activity. These Abs may be useful for the identification of molecular mimetics capable of eliciting protective Abs specific to the bacteria, without the risk of evoking autoimmune disease.
B7 Costimulatory Requirements of T Cells at an Inflammatory Site
Authors: Chen Haixiao , Hendricks Robert L.
Published: January 2023

The requirement for T cell costimulation at sites of infection and inflammation is unresolved. Herpes stromal keratitis (HSK) is a CD4+ T cell-regulated inflammatory response to herpes simplex virus type 1 infection of the cornea. Our findings suggest that susceptibility to HSK is determined by the microenvironment of the infected cornea. The cornea is normally devoid of Langerhans cells (LC), but these APC are present in the surrounding conjunctiva, and migrate into the cornea following infection. The costimulatory molecule B7-2 was constitutively expressed on LC in conjunctiva, but B7-1 was not detectable until 3 days postinfection. LC were the only cells in the cornea that expressed B7-1 through 7 days postinfection. B7-1 was expressed on some, but not all, migrating LC, suggesting that LC migration and B7-1 expression can be independently regulated. The early LC migration and B7-1 expression was independent of T cells, but T cells were required for the massive accumulation of B7-1+ LC in the cornea at the onset of inflammation. Local inhibition of B7-1 function within the infected cornea prevented HSK. Locally blocking B7-2 function did not reduce HSK incidence, but markedly reduce HSK severity. This is the first direct demonstration that naturally expressed B7 is required within an inflammatory site.
Human Mast Cells Augment Fibroblast Proliferation by Heterotypic Cell-Cell Adhesion and Action of IL-4
Authors: Trautmann Axel , Krohne Georg , Bröcker Eva-B. , Klein C. Eberhard
Published: January 2023

Mast cells have been implicated in the pathogenesis of fibrosis because of their increased number in chronic inflammatory reactions. In a previous study, we had shown that human mast cells readily attach and form heterotypic cell-cell contacts when seeded on top of fibroblast monolayers. Here, we report that human mast cells stimulate fibroblast proliferation after cell-cell contact. Proliferation was measured by 5-bromo-2′-deoxyuridine or [3H]thymidine uptake of subconfluent fibroblast monolayers after attachment of mast cells that had been preincubated with mitomycin C. An 18-h coculture of the human mast cell line HMC-1 doubled proliferation of normal skin fibroblasts. Moreover, normal mast cells prepared from neonatal foreskin doubled fibroblast proliferation. The stimulatory effect was dependent on heterotypic cell-cell contact since it was not transferred by tissue culture supernatants from mast cells. We hypothesized that mast cell cytokines secreted after heterotypic cell-cell contact stimulate fibroblast proliferation. Several mast cell-derived cytokines were tested for effects on fibroblast proliferation. Only IL-4 was able to double fibroblast proliferation. Additional experiments revealed that: 1) the stimulatory effect of IL-4 as well as of the mast cell coculture could be completely abrogated by preincubation of fibroblasts with an anti-IL-4R mAb blocking ligand binding; 2) mast cell-derived IL-4 acts as a second signal for fibroblasts since it amplifies the action of low doses of obligatory fibroblast growth factors such as fibroblast growth factor or platelet-derived growth factor.