bluuebunny/crossref_metadata_2025 huggingface dataset:
Bi-Directional Induction of Matrix Metalloproteinase-9 and Tissue Inhibitor of Matrix Metalloproteinase-1 During T Lymphoma/Endothelial Cell Contact: Implication of ICAM-1
Authors: Aoudjit Fawzi , Potworowski Edouard F. , St-Pierre Yves
Published: December 2022

The mechanisms that lead to the expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMPs) during the invasive process of normal and transformed T cells remain largely unknown. Since vascular cells form a dynamic tissue capable of responding to local stimuli and activating cells through the expression of cytokine receptors and specific cell adhesion molecules, we hypothesized that the firm adhesion of T lymphoma cells to endothelial cells is a critical event in the local production of MMP and TIMP. In the present work, we show that adhesion of lymphoma cells to endothelial cells induced a transient and reciprocal de novo expression of MMP-9 mRNA and enzymatic activity by both cell types. Up-regulation of MMP-9 in T lymphoma cells was concomitant to that of TIMP-1, and required direct contact with endothelial cells. Induction of MMP-9, but not of TIMP-1, was blocked by anti-LFA-1 and anti-intercellular adhesion molecule-1 Abs, indicating that induction of MMP-9 and TIMP-1 in lymphoma cells required direct, yet distinct, intercellular contact. In contrast, the induction of MMP-9 in endothelial cells by T lymphoma cells did not necessitate direct contact and could be achieved by exposure to IL-1 and TNF, or to the supernatant of T lymphoma cell culture. Together, these results demonstrate that firm adhesion of T lymphoma cells to endothelial cells participates in the production of MMP-9 in both cell types through bi-directional signaling pathways, and identify intercellular adhesion molecule-1/LFA-1 as a key interaction in the up-regulation of MMP-9 in T lymphoma cells.
Segregated Coupling of Phospholipases A2, Cyclooxygenases, and Terminal Prostanoid Synthases in Different Phases of Prostanoid Biosynthesis in Rat Peritoneal Macrophages
Authors: Naraba Hiroaki , Murakami Makoto , Matsumoto Hideki , Shimbara Satoko , Ueno Akinori , Kudo Ichiro , Oh-ishi Sachiko
Published: December 2022

We examined herein the functional linkage of enzymes regulating the initial, intermediate, and terminal steps of PG biosynthesis to provide PGs in rat peritoneal macrophages stimulated with LPS and/or A23187. Quiescent cells stimulated with A23187 produced thromboxane B2 (TXB2) in marked preference to PGE2 within 30 to 60 min (constitutive immediate response), which was mediated by preexisting cytosolic phospholipase A2 (cPLA2), cyclooxygenase-1 (COX-1), and TX synthase. Cells treated with LPS predominantly produced PGE2 during culture for 3 to 24 h (delayed response), where cPLA2 and secretory PLA2 functioned cooperatively with inducible COX-2, which was, in turn, coupled with inducible PGE2 synthase. Cells primed for 12 h with LPS and stimulated for 30 min with A23187 produced PGE2 in marked preference to TXB2 (induced immediate response), in which three inducible enzymes, cPLA2, COX-2, and PGE2 synthase, were functionally linked. Preferred coupling of the two inducible enzymes, COX-2 and PGE2 synthase, was further confirmed by the ability of LPS-treated cells to convert exogenous arachidonic acid to PGE2 optimally at a time when both enzymes were simultaneously induced. These results suggest that distinct PG biosynthetic enzymes display segregated functional coupling following different transmembrane stimulation events even when enzymes that catalyze similar reactions in vitro coexist in the same cells.
Cutting Edge: Multiple Mechanisms of Peripheral T Cell Tolerance to the Fetal “Allograft”
Authors: Jiang Shi-Ping , Vacchio Melanie S.
Published: December 2022

The fetus represents a foreign entity to the maternal immune system, yet this “natural” allograft is not normally rejected. This unique situation provides a physiologic system to evaluate peripheral tolerance in which the maternal immune system is challenged with relatively rare Ags not previously encountered in the thymus. Using H-Y-specific TCR transgenic mice, we demonstrate that T cells specific for fetal Ags decrease in an Ag-specific manner during pregnancy and remain low postpartum, the result of an encounter with fetal cells expressing the appropriate MHC/peptide complexes. The finding that placental trophoblasts can induce Fas-mediated death of T cells is consistent with peripheral clonal deletion as one mechanism of tolerance. The remaining clonotypic T cells are unresponsive to antigenic stimulation, although neither TCR nor coreceptor is down-regulated. Our study demonstrates that specific recognition of fetal allogeneic Ags by maternal T cells results in tolerance induction of reactive T cells via multiple mechanisms.
Intercellular and Intracellular Events Following the MHC-Unrestricted TCR Recognition of a Tumor-Specific Peptide Epitope on the Epithelial Antigen MUC1
Authors: Magarian-Blander Julie , Ciborowski Pawel , Hsia Shyuan , Watkins Simon C. , Finn Olivera J.
Published: December 2022

We examined the functional and molecular parameters involved in direct TCR recognition of a tumor-specific peptide epitope on the tumor Ag MUC1. This peptide epitope is tandemly repeated and recognized on the native molecule rather than processed and bound to the MHC. Even though the TCR was not MHC restricted, intercellular interactions found to facilitate this recognition included intercellular adhesion molecule-1/LFA-1, LFA-3/CD2, and class I/CD8. Intracellular parameters of MHC-unrestricted CTL activation were examined to compare the recognition of the MUC1 epitope presented on synthetic microspheres, with the recognition of the native epitope in the context of other molecules on the target cells. The epitope on microspheres induced a transient influx of Ca2+ that was not accompanied by detectable tyrosine phosphorylation of the ζ-associated protein ZAP-70, whereas recognition of MUC1 epitopes on tumor cells caused a sustained Ca2+ influx and ZAP-70 phosphorylation. The transient influx of Ca2+ was not sufficient to cause translocation of the nuclear factor of activated T cells (NF-AT) into the nucleus or CTL proliferation. In contrast, recognition of the MUC1 epitope on tumor cells resulted in full activation of the CTL, nuclear translocation of NF-AT, and proliferation. MHC-unrestricted TCR triggering, therefore, involves similar intercellular and intracellular events that participate in the conventional, MHC-restricted Ag recognition. Direct recognition of the MUC1 peptide epitope by the TCR in the absence of presentation by the MHC induces a partial signal that is completed by further interactions of other receptor/ligand pairs on the surface of the CTL and their target cells.
Inhibitory and Stimulatory Effects of IL-10 on Human CD8+ T Cells
Authors: Groux Hervé , Bigler Mike , de Vries Jan E. , Roncarolo Maria-Grazia
Published: December 2022

IL-10 is a well-documented immunosuppressant that inhibits macrophage-dependent Ag presentation and CD4+ T cell proliferation in vitro. We report that IL-10 inhibits alloantigen-specific proliferative responses and induces a long lasting anergic state in human purified CD8+ T cells when added concomitantly with the Ag in the presence of APC. Moreover, the generation of allospecific cytotoxic activity is inhibited by IL-10. These effects are indirect and are mediated through inhibition of the costimulatory functions of APC. In contrast, IL-10 has no direct inhibitory effects on the proliferation of purified CD8+ T cells activated by anti-CD3 mAb and promotes the growth of activated CD8+ T cells in combination with low doses of IL-2. Taken together, these results indicate that IL-10 has differential effects on CD8+ T cells depending on their state of activation, which may explain both the enhancing and inhibitory effects observed after IL-10 treatment in different in vivo experimental models.
CD34+CD38−lin− Cord Blood Cells Develop into Dendritic Cells in Human Thymic Stromal Monolayers and Thymic Nodules
Authors: Miralles G. Diego , Smith Clayton A. , Whichard Leona P. , Morse Michael A. , Haynes Barton F. , Patel Dhavalkumar D.
Published: December 2022

Thymic dendritic cells (DCs) appear to have distinct biologic and functional properties compared with DCs in other tissues. Currently, little is known about human thymic DCs because they have been difficult to isolate and culture in vitro. Here, we report that human thymic stroma can support the development of primitive human hemopoietic stem cells into mature DCs without cytokine or serum supplementation. Coculture of CD34+CD38−lineage (lin)− and CD34+CD38+lin− umbilical cord blood cells with thymic stromal monolayers induced 43 ± 17-fold and 32 ± 16-fold expansions, respectively, of umbilical cord blood progenitors and also generated large numbers of cells with the morphologic, phenotypic, and functional characteristics of mature DCs. These cells expressed class I and class II MHC, CD1a, CD2, CD4, CD11c, CD40, CD45, CD80, CD83, and CD86 and were potent stimulators of allogeneic T cell activation. Primitive hemopoietic progenitors also developed into mature DCs in a novel tissue culture system of thymic nodules wherein thymic epithelial cells and fibroblasts were grown in nodular aggregates in vitro. These results demonstrate that human thymic stroma efficiently supports the development of CD34+CD38−lin− cord blood cells into mature DCs. In addition, the culture conditions described in this report are useful systems for studying the ontogeny of human DCs in thymic microenvironments.
Activation of Lyn, Blk, and Btk But Not Syk in CD72-Stimulated B Lymphocytes
Authors: Venkataraman Chandrasekar , Muthusamy Natarajan , Muthukkumar Subramanian , Bondada Subbarao
Published: December 2022

CD72 is a B cell-specific glycoprotein that has been shown to be important for activation of mature B cells. Previously we showed that some of the early signaling events, such as calcium mobilization and phospholipase-γ activation, were similar in B cell Ag receptor (BCR)- and CD72-stimulated B cells and that BCR- but not CD72-mediated early signaling events were blocked by protein kinase A activation. The present report shows that CD72 ligation induces a variety of tyrosine-phosphorylated proteins, most of which were of the same molecular mass as those seen in anti-IgM-treated B cells, except for a 72-kDa protein. Further analysis showed that the tyrosine kinases lyn and blk were activated in CD72-ligated B cells. Interestingly, the non-src kinase syk was not activated in CD72-stimulated cells whereas the tec family kinase btk was activated in both CD72- and BCR-stimulated B cells. Furthermore, B cells from xid mice were unresponsive to CD72-induced proliferation, indicating an essential role for btk in CD72-induced signaling events. Surprisingly, tyrosine phosphorylation of phospholipase C-γ2 was normal in CD72-stimulated cells in spite of a lack of activation of syk. Furthermore, B cell proliferation through CD72 was blocked by the immunosuppressive agents cyclosporin A and FK506, indicating the important role for Ca2+-regulated activation events similar to BCR-stimulated cells. We propose that btk can substitute for syk in inducing phospholipase C-γ2 tyrosine phosphorylation and initiating calcium mobilization in CD72-stimulated B lymphocytes.
Effect of C2-Associated Carbohydrate Structure on Ig Effector Function: Studies with Chimeric Mouse-Human IgG1 Antibodies in Glycosylation Mutants of Chinese Hamster Ovary Cells
Authors: Wright Ann , Morrison Sherie L.
Published: December 2022

The complex biantennary oligosaccharide at Asn297 of IgG is essential for some effector functions. To investigate the effect of carbohydrate structure on Ab function, we have now expressed mouse-human chimeric IgG1 Abs in Chinese hamster ovary (CHO) cells with defined defects in carbohydrate biosynthesis. We had previously shown that IgG1 Abs produced in the cell line Lec 1, which attaches a high-mannose intermediate carbohydrate, were severely deficient in complement activation, showed a slightly reduced affinity for FcγRI, and had a reduced in vivo half-life. We have extended these studies by producing the same dansyl-specific IgG1 in cell lines deficient in attachment of sialic acid (Lec 2) and galactose (Lec 8). IgG1-Lec 1, IgG1-Lec 2, and IgG1-Lec 8 all showed varying reactivity with a mAb specific for an epitope in the amino terminal region of CH2, suggesting that the conformations of these proteins were altered by the different carbohydrate structures. Functionally, IgG1-Lec 2 and IgG1-Lec 8 were comparable to wild type with respect to in vivo half-life, affinity for FcγRI, and capacity for complement-mediated hemolysis. While IgG1-Lec 2 was essentially identical to wild type in its capacity to interact with individual components of the classical complement activation pathway, IgG1-Lec 8 demonstrated equivalent maximal binding at lower concentrations and was preferentially bound by mannose-binding protein. Although IgG1-Lec 1 was deficient in activation of the classical pathway, it had a superior capacity to activate the alternative pathway. These studies demonstrate that Abs bearing CH2-linked carbohydrate of differing structures have different functional properties.
Trypanosoma cruzi: Tc52 Released Protein-Induced Increased Expression of Nitric Oxide Synthase and Nitric Oxide Production by Macrophages
Authors: Fernandez-Gomez Rodolfo , Esteban Serra , Gomez-Corvera Rosalia , Zoulika Kherrouche , Ouaissi Ali
Published: December 2022

Trypanosoma cruzi target molecules that might regulate the host immune responses have not yet been fully identified. In the present study, we demonstrate that the parasite-released molecule (Tc52) was able to synergize with IFN-γ to stimulate nitric oxide production by macrophages. This synergistic effect was also observed at the level of inducible nitric oxide synthase gene expression. Furthermore, Tc52 was also shown to induce gene expression for IL-1α, IL-12, and IL-10. Moreover, the combination of Tc52 and IFN-γ down-regulates IL-1α and IL-10 gene expression, but not IL-12. Isotype profiles and Tc52 or anti-CD3-induced T cell proliferation were also analyzed, indicating that active immunization with Tc52 partially relieves the immunosuppression observed during the acute phase of the disease. Moreover, under conditions of experimental infection, the Tc52 appears immunologically silent, failing to elicit Ab response and lymphocyte proliferation during the initial acute phase infection. Following active immunization, Tc52 was capable of stimulating T cell proliferation and Ab production with a predominance of IgG1, IgG2a, IgG2b, IgG3, and to a lesser extent IgA. Taken together, these results demonstrate that T. cruzi Tc52-released Ag could be involved in the immunoregulatory processes. The immune response against Tc52 that appears late in the T. cruzi infection may play a role in the modulation of its biological function(s).
Dual Role of Ceramide in the Control of Apoptosis Following IL-2 Withdrawal
Authors: Flores Ignacio , Martinez-A Carlos , Hannun Yusuf A. , Mérida Isabel
Published: December 2022

Ceramide is largely known as a lipid second messenger with pleiotropic effects. Increases in ceramide levels have been related to the onset of apoptosis, terminal differentiation, or growth suppression. In this study, addition of exogenous C2-ceramide to CTLL-2 cells is found to block IL-2-induced cell cycle entry, as well as the apoptosis triggered by IL-2 deprivation. The protective effect of C2-ceramide is achieved only in the early stages following cytokine deprivation and is related to the inhibition of bcl-xL degradation and the induction of a G0 arrest of cells. The same treatment over a longer time when, as we demonstrate, ceramide is produced physiologically, enhances cell death by apoptosis. The dual effect of ceramide both in protecting from or inducing apoptosis is discussed further.
Clonal Analysis of Intrahepatic B Cells from HCV-Infected Patients With and Without Mixed Cryoglobulinemia
Authors: Sansonno Domenico , De Vita Salvatore , Iacobelli Anna Rina , Cornacchiulo Vito , Boiocchi Mauro , Dammacco Franco
Published: December 2022

Clonal rearrangements of Ig heavy chain (IgH) genes and hepatitis C virus (HCV) genomic sequences were assayed on intrahepatic B lymphocytes isolated from HCV chronically infected patients with and without type II mixed cryoglobulinemia (MC). Liver tissue samples from eight patients with and nine without MC were subjected to routine histologic studies, immunophenotyping, and genotypic analysis including IgH V-D-J region gene rearrangements by PCR. RT-PCR, signal amplification by branched DNA assay, and in situ hybridization technique were used to detect and quantitate HCV RNA genomic sequences in selected B cells purified from each tissue sample. Although HCV infection of intrahepatic B cells was shown in all patients both with and without MC, frank B cell monoclonal and oligoclonal patterns were found in only three and four patients with MC, respectively. No monoclonal profile was seen in the noncryoglobulinemic patients, whereas an oligoclonal profile was demonstrated in four of them. No clonalities were shown in HCV-unrelated patients matched for age and severity of liver disease. No obvious difference in HCV genotype distribution was found in relation to the clonal expansion profile. Noncryoglobulinemic patients showing clonal expansion in liver tissue had higher titers of serum rheumatoid factor (RF). Spontaneous production of RF was shown in cell cultures of intrahepatic B cells, suggesting their persistent stimulation in vivo. These data indicate that HCV infection of B cells and B cell clonal expansions occur in the liver microenvironment and preferentially involve RF-producing cells.
Active Wegener’s Granulomatosis Is Associated with HLA-DR+ CD4+ T Cells Exhibiting an Unbalanced Th1-Type T Cell Cytokine Pattern: Reversal with IL-10
Authors: Lúdvíksson Björn R. , Sneller Michael C. , Chua Kevin S. , Talar-Williams Cheryl , Langford Carol A. , Ehrhardt Rolf O. , Fauci Anthony S. , Strober Warren
Published: December 2022

Wegener’s granulomatosis (WG) is a granulomatous vasculitis that affects the upper respiratory tract, lung, and kidney. Since T cells make up a significant proportion of cells infiltrating granulomatous lesions in WG, we investigated the proliferative response and cytokine profile of T cells from these patients. PBMCs were isolated from 12 patients with active WG, 7 patients with inactive disease, and 12 healthy normal donors. PBMCs from clinically active WG patients exhibited increased proliferation following stimulation with either PMA/ionomycin or anti-CD2 and anti-CD28, when compared with normal donors. In addition, these PBMCs exhibited increased secretion of IFN-γ, but not of IL-4, IL-5, or IL-10. Furthermore, TNF-α production from PBMCs and CD4+ T cells isolated from patients with WG was elevated, when compared with healthy donors. In further studies, we investigated the ability of WG patients’ monocytes to produce IL-12 and showed that both inactive and active patients produced increased amounts of IL-12. Finally, the in vitro IFN-γ production by WG PBMC is inhibited in a dose-dependent manner by exogenous IL-10. These data suggest that T cells from WG patients overproduce IFN-γ and TNF-α, probably due to dysregulated IL-12 secretion, and that IL-10 may therefore have therapeutic implications for this disease.
Extracellular Granzymes A and B in Humans: Detection of Native Species During CTL Responses In Vitro and In Vivo
Authors: Spaeny-Dekking Elisabeth H. A. , Hanna William L. , Wolbink Angela M. , Wever Peter C. , Kummer Alain J. , Swaak Anton J. G. , Middeldorp Jaap M. , Huisman Han G. , Froelich Christopher J. , Hack C. Erik
Published: December 2022

Activated CTLs and NK cells induce apoptosis via multiple mechanisms, including that termed granule exocytosis. The latter pathway consists of vectorial secretion of perforin and a family of granule-associated serine proteases (granzymes) to the target cell. To establish whether granzymes are released extracellularly during cytolytic reactions in vivo, ELISAs that measure the native enzymes were developed and were found to specifically detect granzyme A (GrA) and granzyme B (GrB) at picogram concentrations. Low levels of GrA and GrB were present in plasma of healthy individuals (GrA, 33.5 pg/ml (median); GrB, 11.5 pg/ml (median)), whereas significantly higher levels were present in patients with ongoing CTL response, i.e., patients suffering from infections by EBV or HIV type 1. Markedly elevated levels were also noted in synovial fluid of patients with active rheumatoid arthritis. The measurement of soluble granzymes should be useful to assess clinical disorders associated with activated CTL and NK cells. Furthermore, these results suggest that granzymes mediate biologic effects beyond their described role in apoptotic cell death.
Cutting Edge: Selective Up-Regulation of Chemokine Receptors CCR4 and CCR8 upon Activation of Polarized Human Type 2 Th Cells
Authors: D’Ambrosio Daniele , Iellem Andrea , Bonecchi Raffaella , Mazzeo Daniela , Sozzani Silvano , Mantovani Alberto , Sinigaglia Francesco
Published: December 2022

Polarized Th1 and Th2 cells differentially express adhesion molecules and chemokine receptors, endowing these cells with distinct tissue homing capabilities. Here we report that, in contrast to other chemokine receptors, the expression of CCR4 and CCR8 on Th2 cells is transiently increased following TCR and CD28 engagement. IL-4 is not required for this activation-induced up-regulation of CCR4 and CCR8. In accordance with receptor expression, the response of Th2 cells to I-309 (CCR8 ligand) and thymus- and activation-regulated chemokine (CCR4 and CCR8 ligand) is enhanced upon activation. Moreover, activated Th1 cells up-regulate CCR4 expression and functional responsiveness to thymus- and activation-regulated chemokine. Analysis of polarized subsets of CD8+ T cells reveals a similar pattern of chemokine receptor expression and modulation of responsiveness. Taken together, these findings suggest that an up-regulation of CCR4 and CCR8 following Ag encounter may contribute to the proper positioning of activated T cells within sites of antigenic challenge and/or specialized areas of lymphoid tissues.
Selective Induction of CD8+ Cytotoxic T Lymphocyte Effector Function by Staphylococcus Enterotoxin B
Authors: Fuller Claudette L. , Braciale Vivian Lam
Published: December 2022

Upon encounter with its antigenic stimulus, CTL characteristically proliferate, produce cytokines, and lyse the Ag-presenting cell in an attempt to impede further infection. Superantigens are extremely efficient immunostimulatory proteins that promote high levels of proliferation and massive cytokine production in reactive T cells. We compared the activation of murine influenza-specific CD8+ CTL clones stimulated with either influenza peptide or the superantigen staphylococcus enterotoxin B (SEB). We found that influenza peptide/MHC and SEB appeared equally capable of eliciting proliferation and IFN-γ production. However, while influenza peptide/MHC elicited both perforin- and Fas ligand (FasL)/Fas (CD95L/CD95)-mediated cytolytic mechanisms, SEB was unable to trigger perforin-mediated cytolysis or serine esterase release. Examination of intracellular Ca2+ mobilization events revealed that the ability to trigger intracellular Ca2+ flux was not comparable between influenza peptide and SEB. SEB stimulated only a small rise in levels of intracellular Ca2+, at times indistinguishable from background. These findings indicate that the short-term cytolytic potential of superantigen-activated CD8+ CTL clones appears to be restricted to FasL/Fas (CD95L/CD95) mediated cytolysis.
Characteristics of the Strong Antibody Response to Mycobacterial Hsp70: A Primary, T Cell-Dependent IgG Response with no Evidence of Natural Priming or γδ T Cell Involvement
Authors: Bonorino Cristina , Nardi Nance B. , Zhang Xianghua , Wysocki Lawrence J.
Published: December 2022

Despite its high degree of evolutionary conservation, hsp70 is a surprisingly robust Ag, to such a degree that it is under consideration as a potential substrate in vaccine development. The cellular basis of the strong humoral response, however, is unknown, although it is often hypothesized to derive from restimulation of memory T cells that have been primed by hsp of intestinal flora. In this study, we tested this hypothesis and performed additional studies on the immune response to hsp70 of Mycobacterium tuberculosis. Superficially, the primary Ab response to this protein resembles a T cell-dependent secondary one, constituted almost exclusively by IgG. However, there is no evidence of natural priming, as revealed both by in vitro stimulation experiments and by immunity in germfree mice. Although hsp70 stimulates γδ and αβ T cells from unprimed mice to proliferate in vitro, γδ cells are not required for the strong humoral response, which is indistinguishable in normal and γδ T cell-deficient mice. Thus, the unusual immunogenicity of this protein in eliciting a humoral response appears to be due to a strong αβ T cell response with no evidence of natural priming or a γδ T cell involvement.
Apoptotic Death of CD8+ T Lymphocytes After Immunization: Induction of a Suppressive Population of Mac-1+/Gr-1+ Cells
Authors: Bronte Vincenzo , Wang Michael , Overwijk Willem W. , Surman Deborah R. , Pericle Federica , Rosenberg Steven A. , Restifo Nicholas P.
Published: December 2022

Following an infection or immunization, a primary CD8+ T cell response generally rises then falls rapidly before giving rise to a “memory” response. When we immunized mice with recombinant viral immunogens optimized to enhance the lytic capability of CD8+ T cells, we measured a profound depression in Ag-specific effector function after early restimulation. Indeed, a “mirror image” cytolytic capability was observed: the most powerful immunogens, as measured by cytolytic capacity 6 days after immunization, elicited the weakest secondary immune response when evaluated following an additional 6 days after restimulation. To understand the mechanism of this suppression, we examined the fate of splenocytes immunized with a vaccinia virus encoding Ag and IL-2 then restimulated ex vivo. We found that these splenocytes underwent an apoptotic cell death, upon early restimulation, that was not dependent on the engagement of the FasR (CD95). Unlike previously described mechanisms of “propriocidal cell death” and “clonal exhaustion,” the cell death we observed was not an inherent property of the CD8+ T cells but rather was due to a population of splenocytes that stained positive for both the Mac-1 and Gr-1 surface markers. Deletion of these cells in vitro or in vivo completely abrogated the observed suppression of cytolytic reactivity of Ag-specific CD8+ T cells. These observations could account for the apparent absence of Ag-specific immune responses after some current vaccination regimens employing powerful immunogens. Finally, our results may shed new light on a mechanism for the suppression of CD8+ T cell responses and its effect on vaccine efficacy and on immune memory.
Differentiation of Naive CTL to Effector and Memory CTL: Correlation of Effector Function with Phenotype and Cell Division
Authors: Oehen Stephan , Brduscha-Riem Karin
Published: December 2022

Phenotypically and functionally, the early steps of T cell differentiation are not well characterized. In addition, the effector T cell stage shares several phenotypic characteristics with memory T cells, which has made the analysis of T cell memory difficult. In this study, we have investigated in vitro and in vivo the differentiation of naive CTL into effector and memory CTL as a function of cell division using lymphocytic choriomeningitis virus-specific TCR-transgenic spleen cells labeled with the vital dye carboxyfluorescein diacetate, succinimidyl ester. The following major points emerged. 1) During the first nine cell divisions, the investigated cell surface markers were strongly modulated. 2) The TCR was stepwise down-regulated during viral infection. 3) Cytotoxic effector function was acquired within one cell division and was retained during the next four to five divisions. 4) In vitro, CTL reached a CD44highCD62L+ memory phenotype after 6–10 cell divisions and required restimulation to exert effector function. 5) Lymphocytic choriomeningitis virus memory mice contained two distinct memory populations: a CD44highCD62L− population, predominately located in the spleen and exerting rapid effector function, and a CD44highCD62L+ population found in the spleen and the lymph nodes, which had lost immediate effector function. This finding suggests that two types of memory CTL exist. The correlation between CD62L expression, effector function, and Ag persistence is discussed.
Characterization of a B Cell Progenitor Present in Neonatal Bone Marrow and Spleen But Not in Adult Bone Marrow and Spleen
Authors: Moscatello Kim M. , Biber Kristen L. , Dempsey Deborah C. , Chervenak Robert , Wolcott R. Michael
Published: December 2022

The neonatal period marks an important time in mammalian immunologic development, yet it is often ignored in studies of lymphocyte development. We identified a cell population with the phenotype heat stable Ag (HSA)low lin− CD43low that contained B cell progenitors at a high frequency in the neonatal bone marrow and spleen. Although cells with a similar phenotype can be identified in the bone marrow and spleen of adult animals, these populations showed a greatly reduced frequency of B cell progenitors. B lineage cells were detected after 7 days in culture at a frequency of 1:15 when HSAlow lin− CD43low cells from neonatal bone marrow were cultured on stromal cells and IL-7 under limiting dilution conditions. Under similar conditions, the equivalent population in adult bone marrow had a frequency of B cell progenitors that was less than 1:2000. The expression of terminal deoxynucleotidyl transferase in freshly sorted neonatal HSAlow lin− CD43low cells suggested that cells committed to the lymphocyte lineage were present in this population. These data suggested that the HSAlow lin− CD43low population of cells represents a pool of B lineage precursors that may be responsible for filling the immune compartment early in neonatal life.
CD11/CD18 and CD14 Share a Common Lipid A Signaling Pathway
Authors: Ingalls Robin R. , Monks Brian G. , Savedra Ricardo , Christ William J. , Delude Russell L. , Medvedev Andrei E. , Espevik Terje , Golenbock Douglas T.
Published: December 2022

The activation of phagocytes by the lipid A moiety of LPS has been implicated in the pathogenesis of Gram-negative sepsis. While two LPS receptors, CD14 and CD11/CD18, have been associated with cell signaling, details of the LPS signal transduction cascade remain obscure. CD14, which exists as a GPI-anchored and a soluble protein, lacks cytoplasmic-signaling domains, suggesting that an ancillary molecule is required to activate cells. The CD11/CD18 integrins are transmembrane proteins. Like CD14, they are capable of mediating LPS-induced cellular activation when expressed on the surface of hamster fibroblasts Chinese hamster ovary (CHO)-K1. The observation that a cytoplasmic deletion mutant is still capable of activating transfected CHO-K1 argues that CD11/CD18 also utilizes an associated signal transducer. We sought to identify further similarities between the signaling systems utilized by CD14 and CD11/CD18. LPS-binding protein, which transfers LPS to CD14, enhanced both LPS-induced cellular activation and binding of Gram-negative bacteria in CD11/CD18-transfected CHO-K1, thus implying that LPS-binding protein can also transfer LPS to CD11/CD18. When synthetic lipid A analogues were analyzed for their ability to function as LPS agonists, or antagonists, in the CHO transfectants, we found the effects were identical regardless of which LPS receptor was expressed. This supports the hypothesis that a receptor distinct from CD14 and CD11/CD18 is responsible for discriminating between the lipid A of LPS and the LPS antagonists. We propose that this receptor, which is the target of the LPS antagonists, functions as the true signal transducer in LPS-induced cellular activation for both CD14 and CD11/CD18.
Improving the Affinity and the Fine Specificity of an Anti-Cortisol Antibody by Parsimonious Mutagenesis and Phage Display
Authors: Chames Patrick , Coulon Stéphane , Baty Daniel
Published: December 2022

Immunoassays are widely used to determine steroid concentrations. However, they are limited by the specificity of anti-steroid mAbs. We used the phage display system combined with molecular modeling and site-specific randomization to improve the affinity and the fine specificity of an anti-cortisol mAb. Using parsimonious mutagenesis, we have generated a library of mutant Ab fragments (scFv) derived from this Ab by randomizing five amino acids chosen by molecular modeling and Ab-hapten contact structural analysis. Anti-cortisol Ab fragments were selected from the library in the presence of steroid analogues to block cross-reacting binders. Specific elution with free cortisol allowed the recovery of clones with up to eightfold better affinity and fivefold less cross-reactivity than the wild-type scFv. This approach can be applied to any anti-hapten Ab and represents a useful approach for obtaining highly specific Abs for use in steroid immunoassays.
CD4+ Lymphocytes Provide MUC1-Specific Tumor Immunity In Vivo That Is Undetectable In Vitro and Is Absent in MUC1 Transgenic Mice
Authors: Tempero Richard M. , VanLith Michelle L. , Morikane Keita , Rowse Gerald J. , Gendler Sandra J. , Hollingsworth Michael A.
Published: December 2022

A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC1.Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.
Differential Interaction of Crkl with Cbl or C3G, Hef-1, and γ Subunit Immunoreceptor Tyrosine-Based Activation Motif in Signaling of Myeloid High Affinity Fc Receptor for IgG (FcγRI)
Authors: Kyono Wade T. , de Jong Ron , Kil Park Rae , Liu Yenbou , Heisterkamp Nora , Groffen John , Durden Donald L.
Published: December 2022

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following FcγRI aggregation in U937IF cells. FcγRI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing γ subunit of FcγRI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and FcγRIγ after FcγRI activation and the constitutive association between C3G and Crkl provide the first evidence that a FcγRIγ-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.
Phlebotomus papatasiSand Fly Salivary Gland Lysate Down-Regulates a Th1, but Up-Regulates a Th2, Response in Mice Infected withLeishmania major
Authors: Mbow M. Lamine , Bleyenberg Julie A. , Hall Laurie R. , Titus Richard G.
Published: December 2022

A vertebrate host becomes infected with Leishmania major when the sand fly vector injects parasites into skin along with saliva. Previous studies showed that salivary gland lysate of the New World sand fly Lutzomyia longipalpis markedly enhanced L. major infection in CBA mice. However, L. major is an Old World parasite transmitted in nature by the Old World sand fly Phlebotomus papatasi. Here we examine the ability of P. papatasi salivary gland lysate to enhance infection (lesion size and parasite burden) by L. major. In addition, we examine the effects of salivary gland lysate on the immune response to L. major by monitoring the levels of cytokine mRNA from the lymph nodes draining cutaneous lesions. We found that P. papatasi salivary gland lysate dramatically exacerbated lesion development in disease-resistant CBA mice. This exacerbation of disease correlated with inhibition of the production of Th1 cytokines and associated factors (IFN-γ, IL-12, and inducible nitric oxide synthase), but with enhancement of the Th2 cytokine IL-4, whereas no changes in the levels of IL-10 and TGF-β were noted. Importantly, salivary gland lysate directly up-regulated expression of IL-4 mRNA in mice in the absence of infection with L. major.
A Peptidomimetic Antagonist of TNF-α-Mediated Cytotoxicity Identified from a Phage-Displayed Random Peptide Library
Authors: Chirinos-Rojas Carlos L. , Steward Michael W. , Partidos Charalambos D.
Published: December 2022

Phage-displayed peptide libraries represent a vast collection of peptide sequences that can be used to identify novel therapeutic molecules. In this report, a 15-mer phage-displayed peptide library was used to identify potential TNF-α antagonists. After direct interaction of recombinant human TNF-α with the library, four randomly selected phage clones were shown to inhibit in a dose-dependent fashion both mouse and human TNF-α-induced cytotoxicity in vitro. DNA sequencing of the positive clones revealed a common amino acid sequence that does not bear any structural similarity to the known primary structures of the extracellular domains of either 55-kDa or 75-kDa TNF receptors. This sequence was synthesized, and the peptidomimotope was shown i) to bind to the recombinant human TNF-α using surface plasmon resonance (biosensor) technology and ii) to inhibit both recombinant mouse and human TNF-α-induced cytotoxicity in vitro in a dose-dependent fashion.

These findings highlight the potential of phage-displayed random peptide libraries for the identification of novel low molecular antagonistic molecules that can block the biologic activities of TNF-α.
Regulation of T Lymphocyte Trafficking into Lymph Nodes During an Immune Response by the Chemokines Macrophage Inflammatory Protein (MIP)-1α and MIP-1β
Authors: Tedla Nicodemus , Wang Hong-Wei , McNeil H. Patrick , Di Girolamo Nick , Hampartzoumian Taline , Wakefield Denis , Lloyd Andrew
Published: December 2022

By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of inflammation. Their role in regulation of T lymphocyte traffic into lymph nodes during the development of an immune response has not previously been explored. The sensitization phase of contact hypersensitivity induced by the hapten, dinitrofluorobenzene (DNFB) in the mouse was used as a model of T lymphocyte trafficking in response to antigenic stimulation. Rapid accumulation of CD8+ and CD4+ T cells in the draining lymph nodes was closely associated with strongly enhanced expression of macrophage inflammatory protein (MIP)-1α and MIP-1β mRNAs and proteins. Mast cells accumulating in the nodes during DNFB sensitization were the predominant source of MIP-1β, whereas MIP-1α was expressed by multiple cell types. Neutralization of these chemokines profoundly inhibited T lymphocyte trafficking into lymph nodes and altered the outcome of a subsequent challenge to DNFB. Thus, β-chemokines regulate T lymphocyte emigration from the circulation into lymph nodes during an immune response and contribute significantly to the immunologic outcome.
Molecular Cloning and Functional Characterization of the Rat Analogue of Human Decay-Accelerating Factor (CD55)
Authors: Hinchliffe Stewart J. , Spiller O. Brad , Rushmere Neil K. , Morgan B. Paul
Published: December 2022

We report here the cloning of cDNAs encoding two forms of the rat analogue of human decay-accelerating factor (DAF; CD55). Screening of a rat kidney cDNA library using a mouse DAF probe identified a partial cDNA encoding the 3′ end of rat DAF. The 5′ end of the cDNA was cloned using the rapid amplification of cDNA ends (RACE) technique. A second form of rat DAF was identified using 3′RACE. Cloning and sequencing of full length cDNAs for both forms showed that they were identical up to nucleotide 1143 except for a 51-bp insert in the ST-rich region of the second form. After nucleotide 1143, the two sequences diverged; the cDNA cloned from the library encoded a unique 112-amino acid “tail,” whereas the second form, identified by 3′RACE, encoded an 18-amino acid hydrophobic stretch, which was predicted to be a glycosylphosphatidylinositol (GPI) anchor addition signal. Expression in the NIH-3T3 mouse fibroblast cell line confirmed that the short tail did encode a GPI-addition signal, whereas the longer tail caused the protein to be secreted. Northern blot analysis identified two distinct transcripts for the GPI form, as well as a variability in expression levels of the different transcripts in the panel of tissues screened. Southern blot analysis showed that both the GPI and secreted forms of rat DAF were expressed in a wide range of tissues. The GPI-linked form of rat DAF stably expressed in a murine fibroblast cell line reduced C3 deposition and conferred protection from lysis by rat serum.
The Dual Phases of the Response to Neonatal Exposure to a VH Family-Restricted Staphylococcal B Cell Superantigen
Authors: Silverman Gregg J. , Nayak Jayakar V. , Warnatz Klaus , Hajjar Fred F. , Cary Stephen , Tighe Helen , Curtiss Virginia E.
Published: December 2022

In vitro studies of several naturally occurring proteins have characterized VH family-specific B lymphocyte binding and stimulatory properties that appear analogous to those of T cell superantigens. To examine the in vivo consequences of exposure to a putative B cell superantigen, we treated neonatal BALB/c mice with a form of staphylococcal protein A (MS) devoid of Fcγ binding activity, which retains the clan VHIII Fab binding specificity. In naive adults, about 5% of peripheral B cells and >13% of splenic IgM-secreting cells display MS binding activity, in association with high IgM and low IgG circulating anti-MS Ab titers. Neonatal exposure to MS elicited two distinct temporal phases of immune responsiveness. The early phase, representing the first approximately 5 wk of life, was associated with MS-specific B cell and T cell tolerance. Microfluorometric assays revealed that exposure caused a dramatic MS-specific B cell clonal loss in bone marrow and spleen, but levels normalized by about 3 wk of life. The late phase (>6 wk of age) was associated with spontaneous priming for MS-specific T cell responses and production of MS-specific IgG1 Abs despite long term persistently depressed in vivo and in vitro MS-specific IgM responses. In vivo challenge during the late phase induced high frequencies of MS-specific IgG-secreting cells, indicating recruitment of highly focused Ab responses that were predominantly encoded by rearrangements of the S107 family, a member of the VHIII clan. These studies document the immunodominance of the VH-restricted Fab binding site on staphylococcal protein A and demonstrate the diverse effects of a B cell superantigen on the emerging peripheral B cell compartment.
Resistance to Apoptosis and Elevated Expression of Bcl-2 in Clonally Expanded CD4+CD28− T Cells from Rheumatoid Arthritis Patients
Authors: Schirmer Michael , Vallejo Abbe N. , Weyand Cornelia M. , Goronzy Jörg J.
Published: December 2022

Patients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28− T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28− T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28− T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28−CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28− T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28− T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28− T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.