bluuebunny/crossref_metadata_2025 huggingface dataset:
Class I-Restricted Alloreactive Cytotoxic T Lymphocytes Recognize a Complex Array of Specific MHC-Associated Peptides
Authors: Wang Wei , Man Stephen , Gulden Pamela H. , Hunt Donald F. , Engelhard Victor H.
Published: December 2022

A major issue in understanding alloreactive T cell responses is whether the Ags recognized reside in allogeneic MHC proteins themselves regardless of the structure of the associated peptides or whether specific peptides presented by allogeneic MHC proteins determine each epitope. We developed HLA-A*0201-specific alloreactive human CD8+ CTL lines and clones to address this issue. Acid treatment of HLA-A*0201+ target cells resulted in the loss of Ab-defined epitopes as well as recognition by all alloreactive CTL. In the presence of brefeldin A, no class I molecules were re-expressed at the surface of the acid-treated cells. Addition of a mixture of synthetic peptides corresponding to known, naturally processed, HLA-A*0201-associated peptides together with exogenous human β2m restored binding by specific Ab but not recognition by alloreactive CTL. However, addition of a more complex mixture of peptides directly extracted from HLA-A*0201 reconstituted CTL recognition. This demonstrates that these alloreactive CTL recognize specific peptides and not a common peptide-dependent conformation of HLA-A*0201. Reverse phase HPLC fractionation of the extracted peptides resulted in the loss of recognition by CTL lines from three individuals. This was not due to the loss of specific peptide species because repooling of the HPLC fractions led to a recovery of recognition. Furthermore, three HLA-A*0201-alloreactive CTL clones recognized single distinct peptide peaks from the same HPLC fractionation. These data suggest that the epitopes recognized in allogeneic responses to HLA-A*0201 are complex, and the response is a result of recognition of multiple unique peptide-MHC complexes.
Role of CD38 and Its Ligand in the Regulation of MHC-Nonrestricted Cytotoxic T Cells
Authors: Cesano Alessandra , Visonneau Sophie , Deaglio Silvia , Malavasi Fabio , Santoli Daniela
Published: December 2022

Human CD38 is a type II transmembrane glycoprotein that regulates lymphocyte adhesion, proliferation, and cytokine production. The mAb Moon-1 recognizes a ligand for CD38 (CD38L) and specifically inhibits CD38-mediated cell adhesion. To analyze the role of CD38 and its ligand in MHC-nonrestricted T cell activation, we examined the effects of Moon-1 and the anti-CD38 mAb IB4 on the effector functions of the IL-2-dependent T cell line TALL-104 (CD3/TCR-αβ+, CD8+, CD56+) and of LAK cells (90% CD3+). TALL-104 cells were almost 100% reactive with both mAbs, whereas the reactivity of LAK cells for IB4 and Moon-1 ranged from 10 to 60% among different donors. From 78 to 94% of the cytotoxic CD8+/CD56+ LAK subset was CD38L+. Like mAb OKT3 (anti-CD3), and at variance with IB4, Moon-1 drastically enhanced the cytotoxicity of TALL-104 and CD8+ LAK cells against a resistant tumor target. Granule exocytosis did not appear to play a role in Moon-1-induced cytotoxicity. Moreover, neither IB4 nor Moon-1 induced [Ca2+]i mobilization in LAK and TALL-104 cells. Whereas stimulation of CD3 and CD38 resulted in a dramatic induction of cytokine (granulocyte-macrophage-CSF, IFN-γ, TNF-α, and TNF-β) release by both TALL-104 and LAK cells, ligation of CD38L was not followed by cytokine production in TALL-104 cells. Thus, cytotoxicity and cytokine release are independently regulated, at least in this system. These data demonstrate that CD38 and its ligand can regulate some T cell functions using signaling pathways distinct from those of CD3.
Differential Expression of the Melatonin Receptor in Human Monocytes
Authors: Barjavel Marc J. , Mamdouh Zahra , Raghbate Nadjibe , Bakouche Ouahid
Published: December 2022

Earlier studies have shown that the pineal hormone melatonin activates human monocytes. It is reported here that melatonin induces the secretion of IL-1, IL-6, and TNF in fresh and 1-day in vitro cultured monocytes that also express the melatonin receptor (Kd = 270 ± 60 pM; 42,000–48,000 receptors/cell). However, when monocytes were cultured in vitro for 2 days, the number of receptors decreased to 11,000 receptors/cell, with the same Kd. LPS activation of fresh or 1-day cultured monocytes did not result in any increase in melatonin receptor number. LPS activation of 2-day cultured monocytes led to an increase in the number of melatonin receptors, from 11,000 receptors/cell to the plateau of 42,000 to 48,000 receptors/cell. The loss of receptors by 2-day cultured monocytes was not irreversible. Melatonin did not induce the release of IL-1, TNF, or IL-6 in monocytes cultured in vitro for 3 days and for up to 15 days, and these long time cultured monocytes did not express the melatonin receptors even after activation by LPS. The loss of melatonin receptors by monocytes cultured in vitro for 3 days and for up to 15 days was irreversible. Therefore, it is shown for the first time that human monocytes express melatonin receptors. Furthermore, human monocytes express melatonin receptors differentially depending on their state of maturation, and it appears that in vitro monocyte differentiation and maturation negatively affect human monocyte melatonin receptor expression.
VH Mutant Rabbits Lacking the VH1a2 Gene Develop a2+ B Cells in the Appendix by Gene Conversion-Like Alteration of a Rearranged VH4 Gene
Authors: Sehgal Devinder , Mage Rose G. , Schiaffella Enrico
Published: December 2022

We investigated the molecular basis for the appearance of VHa2 allotype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of VH genes because of a small deletion encompassing VH1a2, the 3′-most gene in the VH locus. The VH1 gene is the major source of VHa allotype because this gene is preferentially rearranged in normal rabbits. In young homozygous ali/ali animals, the levels of a2 molecules found in the serum increase with age. In adult ali/ali rabbits, 20 to 50% of serum Igs and B cells bear a2 allotypic determinants. Previous studies suggested that positive selection results in expansion of a2 allotype-bearing B cells in the appendix of young mutant ali/ali rabbits. We separated appendix cells from a 6-wk-old Alicia rabbit by FACS based on the expression of surface IgM and a2 allotype. The VDJ portion of the expressed Ig mRNA was amplified from the IgM+ a2+ and IgM+ a2− populations by reverse transcriptase-PCR. The cDNAs from both populations were cloned and sequenced. Analysis of these sequences suggested that, in a2+ B cells, the first D proximal functional gene in Alicia rabbits, VH4a2, rearranged and was altered further by a gene conversion-like mechanism. Upstream VH genes were identified as potential gene sequence donors; VH9 was found to be the most frequently used gene donor. Among the a2− B cells, y33 was the most frequently rearranged gene.
Activation-Induced T Cell Death ExacerbatesTrypanosoma cruziReplication in Macrophages Cocultured with CD4+ T Lymphocytes from Infected Hosts
Authors: Nunes Marise P. , Andrade Regis M. , Lopes Marcela F. , DosReis George A.
Published: December 2022

Activation-induced cell death (AICD) of CD4+ T lymphocytes was described in infection with Trypanosoma cruzi, but a role for AICD in modulating parasite spread in host cells has not been investigated. In this study, replication of T. cruzi in vitro in murine macrophage (Mφ) monolayers was investigated. Long term (5 to 13 day) replication of infective (trypomastigote) T. cruzi forms was blocked by supernatants from activated (anti-TCR) CD4+ T cells of infected mice or by rIFN-γ. However, when CD4+ T cells from infected mice were cocultured with Mφ and activated by anti-TCR, marked exacerbation of trypomastigote growth in Mφ ensued. The deleterious effect required contact between T cells and infected Mφ. Both anti-Fas and TCR activation killed a proportion of CD4+ T cells. Ly-6 activation did not induce AICD and did not exacerbate parasite growth. However, Fas-mediated killing of T cells before Ly-6 activation led to exacerbated parasite growth. Although a minor population, Fas-susceptible cells were the major source of IFN-γ production by activated T cells. Addition of a neutralizing anti-Fas ligand antibody blocked 50 to 60% of CD4+ T cell AICD and reduced trypomastigote growth in T/Mφ cocultures stimulated by anti-TCR. The results demonstrate that in CD4+ T cells from infected mice, the onset of AICD selectively ablates IFN-γ production and up-regulates parasite replication in Mφ in vitro. These findings suggest a deleterious role for AICD in T. cruzi infection.
Pre-Exposure to Oxidative Stress Decreases the Nuclear Factor-κB-Dependent Transcription in T Lymphocytes
Authors: Lahdenpohja Nina , Savinainen Kimmo , Hurme Mikko
Published: December 2022

Reactive oxygen species (ROS) are used as signaling molecules in T cell activation. One of the main targets of ROS is the transcription factor nuclear factor-κB (NF-κB). NF-κB-dependent transcription is inhibited by antioxidants, and the activation is induced or potentiated by ROS. However, chronic oxidative stress is known to reduce the activation of T cells and NF-κB. To analyze these phenomena in more detail, we have exposed Jurkat T cells in vitro to oxidative stress (H2O2) at various times before or simultaneously with signals known to activate NF-κB (phorbol dibutyrate (PDBu) and TNF). Simultaneously applied H2O2 strongly potentiated the PDBu- or TNF-induced transcriptional activity of NF-κB. In contrast to this, H2O2 given 3 to 20 h before the activating signal reduced NF-κB-dependent transcriptional activity. This was not due to the oxidation-induced modification of NF-κB; cytoplasmic NF-κB was able to bind to DNA after dissociation from IκBα by detergent treatment. H2O2 pre-exposure effectively inhibited the PDBu- or TNF-induced phosphorylation and degradation of IκBα, but H2O2 given simultaneously with PDBu or TNF enhanced the degradation. Oxidative stress was also followed by a strongly decreased ability to form intracellular ROS. Taken together, these data indicate that IκBα phosphorylation is the target of action of ROS, and as the ROS-forming capacity is weaker after chronic oxidative stress, IκBα is not effectively phosphorylated and degraded, thus leading to decreased NF-κB-dependent transcription.
Factors Involved in Regulating Primary and Secondary Immunity to Infection withHistoplasma capsulatum: TNF-α Plays a Critical Role in Maintaining Secondary Immunity in the Absence of IFN-γ
Authors: Zhou Ping , Miller Giorgina , Seder Robert A.
Published: December 2022

Primary infection to Histoplasma capsulatum often results in a self-limited upper respiratory infection in humans; however, in immunocompromised hosts, disseminated infection can occur through reactivation of a previous infection. Since disseminated histoplasmosis has emerged as a difficult clinical entity to treat in individuals infected with HIV, it was of interest to study the mechanisms involved in maintaining an effective memory immune response. It has been previously shown in a murine model of disseminated histoplasmosis that IL-12, IFN-γ, and TNF-α were important factors in mediating primary protection. To study whether these and other factors were involved in maintaining a protective immune response following secondary infection, normal C57BL/6 mice were first infected with a sublethal dose of H. capsulatum (1 × 105) and then reinfected 3 wk later with a lethal dose of H. capsulatum (6 × 105). Under these conditions, all mice developed an effective immune response with sterilizing immunity. Moreover, normal C57BL/6 mice treated with neutralizing Abs against either IL-12, TNF-α, or IFN-γ, depleted of neutrophils or treated with aminoguanidine at the time of reinfection, maintained an effective immune response. The ability of animals to survive a secondary infection in the absence of IFN-γ was verified by showing that IFN-γ−/− mice previously immunized with H. capsulatum and treated with amphotericin B at the time of primary infection had prolonged survival following reinfection with a normally lethal dose. It was further shown that enhancement of TNF-α production in IFN-γ−/− mice was the major mechanism by which these mice were effective in controlling secondary infection.
Immune Response Against Large Tumors Eradicated by Treatment with Cyclophosphamide and IL-12
Authors: Tsung Kangla , Meko Jennifer B. , Tsung Ying L. , Peplinski Gary R. , Norton Jeffrey A.
Published: December 2022

Previous studies have demonstrated eradication of small (4–8 mm) established murine MCA207 sarcomas by treatment with systemic IL-12. Analysis of the mechanism has revealed a cellular and molecular immune response at the tumor typical of a Th1 cell-mediated, macrophage-effected, delayed-type hypersensitivity (DTH) response. In the current study we investigate the immune response against long term established, large MCA207 tumors induced by combined treatment with IL-12 and cyclophosphamide (Cy), an agent known to potentiate the DTH response. Our results demonstrate that s.c. large MCA207 tumors (15–20 mm) that are refractory to treatment by either IL-12 or Cy alone can be completely eradicated by the combination of Cy and IL-12. IL-12 is apparently the only cytokine capable of mediating tumor eradication, and the effect is dependent on IFN-γ. The contribution of Cy is probably due to immunopotentiation of DTH rather than to direct cytotoxicity to the tumor. The regression of these large tumors takes >4 wk and, in many cases, is self-sustained, in that little or no additional IL-12 is needed beyond the initial week of administration. Analysis of the cellular and molecular events at the tumor site suggests that the mechanism is a Th1-mediated antitumor immune response.
Novel Immunoregulatory B Cell Pathways Revealed bylpr-+ Mixed Chimeras
Authors: Sobel Eric S. , Kakkanaiah Vellalore N. , Schiffenbauer Joel , Reap Elizabeth A. , Cohen Philip L. , Eisenberg Robert A.
Published: December 2022

lpr, a murine mutation of the Fas apoptosis receptor, causes lymphadenopathy and autoantibody production, with lymphadenopathy primarily due to a population of CD4−CD8−B220+ T cells. Previous in vivo experiments, in which lpr and normal bone marrow cells were coinfused into lpr hosts, have demonstrated that only T cells of lpr origin accumulated abnormally and only B cells of lpr origin produced autoantibodies. Moreover, in these chimeras, B cells of normal origin were unable to respond to conventional, T cell-dependent exogenous Ag. To address the role of lpr B cells in regulation of lpr autoimmunity, we have prepared lpr-+ mixed chimeras and selectively eliminated lpr B cells using allele-specific, mAb treatment, thus allowing normal B cells to develop in an environment with lpr T cells. From these data, we arrived at four major conclusions: 1) Compared with control-treated chimeric mice, lpr B cell-depleted mice had greatly reduced total lymph node cell counts; 2) the T cells were derived equally from normal and lpr donors, and the percentage of lpr-derived CD4−CD8− T cells was greatly reduced; 3) despite the presence of the remaining lpr T cells, no autoantibodies were produced by the normal derived B cells; and 4) lpr T cells without lpr B cells were unable to prevent a normal B cell response to conventional Ag. These data demonstrate that B cells can play a critical and expansive regulatory role, not only for T cells, but for other B cells as well.
Mechanisms of Acquired Thymic Tolerance In Vivo: Intrathymic Injection of Antigen Induces Apoptosis of Thymocytes and Peripheral T Cell Anergy
Authors: Chen Wanjun , Sayegh Mohamed H. , Khoury Samia J.
Published: December 2022

Intrathymic injection of Ag induces Ag-specific tolerance in several clinically relevant experimental autoimmune and transplantation models. However, the exact mechanisms of acquired thymic tolerance in vivo remain unclear. We investigated the mechanisms of acquired thymic tolerance in mice that are transgenic for the TCR specific for peptide 323-339 of OVA. Intrathymic injection of OVA leads to apoptosis of thymocytes starting as early as 3 h after injection and persisting up to 7 days. Double positive thymocytes undergo apoptosis earlier than single positive thymocytes, and significantly higher percentages of double positive thymocytes ultimately die as compared with single positive cells. Apoptotic cells show decreased surface expression of CD4. In the periphery, T cells from intrathymically injected animals had suppressed proliferation and IL-2 production to OVA compared with T cells from control Ag-injected mice. We conclude that intrathymic injection of Ag induces apoptosis of immature thymocytes and a subpopulation of mature thymocytes and induces prolonged anergy in peripheral T cells in vivo. Understanding the mechanisms of acquired thymic tolerance may lead to development of novel clinical strategies to prevent autoimmune disease and transplant rejection.
A Physiologic Role of Bcl-xL Induced in Activated Macrophages
Authors: Okada Seiji , Zhang Hong , Hatano Masahiko , Tokuhisa Takeshi
Published: December 2022

Activated macrophages produce nitric oxide (NO) that is an important effector molecule for their antimicrobial and antitumor activities. Since this NO is also toxic for themselves, they have self-defense mechanisms. To elucidate the mechanisms in a physiologic condition, expression of bcl-2 family genes were examined in peritoneal macrophages and RAW264 macrophage cell line activated with IFN-γ and LPS. Bcl-xL, but not bcl-2 and bax mRNA, was highly inducible within 3 h after stimulation. The induction required new protein synthesis, but was independent of effects of synthesized NO. Since activated RAW264 were more resistant to NO-induced apoptosis mediated by the exposure to S-nitroso-N-acetyl-penicillamine (SNAP) than nonactivated RAW264, the inducible Bcl-xL may play a role in the protection from NO toxicity. To confirm the protective function, RAW264 were stably transfected with bcl-xL. Those transfectants activated with IFN-γ and LPS appeared highly resistant to NO-induced cell death detected within 24 h after stimulation, although their NO production was similar to those of parental RAW264 and neomycin control-transfected cells. Furthermore, bcl-xL transfectants displayed substantial protection from SNAP-induced apoptosis. These results establish a link between self-defense to the synthesized NO and the induction of Bcl-xL in activated macrophages.
Suppression of Fas/APO-1-Mediated Apoptosis by Mitogen-Activated Kinase Signaling
Authors: Holmström Tim H. , Chow Sek C. , Elo Iina , Coffey Eleanor T. , Orrenius Sten , Sistonen Lea , Eriksson John E.
Published: December 2022

Jurkat T cells undergo rapid apoptosis upon stimulation of the Fas/APO-1 (CD95) receptor. We examined the role of the mitogen-activated protein kinase (MAPK) cascade as a negative regulator of Fas-mediated apoptosis. To this end, we used both physiologic and artificial activators of MAPK, all of which activate MAPK by distinct routes. MAPK activity could be efficiently elevated by two T cell mitogens, the lectin PHA and an agonistic Ab to the T cell receptor complex as well as by the type 1 and 2A phosphatase inhibitor, calyculin A, and the protein kinase C-activating phorbol ester, tetradecanoyl phorbol acetate. All these treatments were effective in preventing the characteristic early and late features of Fas-mediated apoptosis, including activation of caspases. Our results indicate that the elevated MAPK activities intervene upstream of caspase activation. The degree of MAPK activation by the different stimuli used in our study corresponds well to their potency to inhibit apoptosis, indicating that MAPK activation serves as an efficient modulator of Fas-mediated apoptosis. The role of MAPK in modulation of Fas-mediated apoptosis was further corroborated by transient transfection with constitutively active MAPK kinase, resulting in complete inhibition of the Fas response, whereas transfection with a dominant negative form of MAPK kinase had no effect. Furthermore, the apoptosis inhibitory effect of the MAPK activators could be abolished by the specific MAPK kinase inhibitor PD 098059. Modulation of Fas responses by MAPK signaling may determine the persistence of an immune response and may explain the insensitivity of recently activated T cells to Fas receptor stimulation.
Age-Dependent Appearance of NK1.1+ T Cells in the Livers of β2-Microglobulin Knockout and SJL Mice
Authors: Murakami Masao , Paul William E.
Published: December 2022

NK1.1+ T cells, a specialized set of T cells that recognize CD1, are reportedly absent in young β2-microglobulin-deficient (β2m-knockout (KO)) and SJL mice. In this study, we show that a significant number of NK1.1+ T cells exist in the livers of older β2m-KO and SJL mice, and that the number of liver NK1.1+ T cells increases as the animals age. The surface phenotypes of liver NK1.1+ T cells from β2m-KO and SJL mice were similar to NK1.1+ T cells from C57BL/6 mice, except that the bulk of these cells were CD4−CD8−. After anti-CD3 injection in vivo, the cells promptly expressed IL-4 mRNA just as NK1.1+ T cells did in normal mice. Using L cells expressing CD1, liver NK1.1+ T cells from both β2m-KO and SJL mice were stimulated to proliferate, although to a lesser degree than were such cells from C57BL/6 mice. Our studies show that some NK1.1+ T cells accumulate in the livers of older β2m-KO and SJL mice, and that they appear to have functional properties similar to “normal” NK1.1+ T cells.
The Primary Site of CD4−8−B220+ αβ T Cells inlprMice: The Appendix in Normal Mice
Authors: Yamagiwa Satoshi , Sugahara Satoshi , Shimizu Takao , Iwanaga Toshihiko , Yoshida Yuhei , Honda Shigeru , Watanabe Hisami , Suzuki Kenji , Asakura Hitoshi , Abo Toru
Published: December 2022

There have been no reports on an abundance of CD4−8−B220+ αβ T cells, seen in autoimmune mice carrying the lpr gene (abnormal Fas gene), in any immune organs of normal mice. We herein report, however, that such αβ T cells were abundant at intraepithelial sites of the appendix in normal mice. They lacked the expression of NK1.1 Ags (C57BL/6 mice), but had the morphology of granular lymphocytes and contained forbidden T cell clones in the minor lymphocyte-stimulating antigen (Mls) system (C3H/He mice with Mls-1b2a). In other words, many properties of intraepithelial T cells in the appendix resembled those ascribed to abnormal αβ T cells, which expand in the lymph nodes and spleen of lpr mice. In the case of lpr mice, CD4−8−B220+ αβ T cells first expanded in the appendix and then extended to other organs. CD4−8−B220+ αβ T cells seemed to originate in situ from c-kit+ stem cells in the appendix. These results suggest that the appendix is one of the primary sites in which CD4−8−B220+ αβ T cells exist, and that these cells carry many primordial properties as prototype T cells.
Dependence of Antibody Somatic Diversification on Gut-Associated Lymphoid Tissue in Rabbits
Authors: Vajdy Michael , Sethupathi Periannan , Knight Katherine L.
Published: December 2022

By ∼4 to 8 wk of age, the IgH VDJ genes of essentially all rabbit B lymphocytes have undergone somatic diversification. Some of this diversification occurs in the appendix, which is a gut-associated lymphoid tissue (GALT). To determine whether GALT is essential for somatic diversification, we surgically removed the appendix, sacculus rotundus, and Peyer’s patches from neonatal rabbits (designated GALT-less) and examined the extent to which VDJ genes were somatically diversified. We found that the IgM VDJ genes of peripheral B cells from 2- to 5-mo-old GALT-less rabbits had undergone considerably less somatic diversification than those of control rabbits. Further, the percentage of peripheral B cells in the GALT-less rabbits was generally less than that of controls. Our data suggest that, in rabbits, the primary Ab repertoire develops in GALT, and B cell expansion also occurs there. Hence, GALT may function as a mammalian bursal homologue.
Identification of an Immunodominant IgE Epitope of theParietaria judaicaMajor Allergen
Authors: Colombo Paolo , Kennedy2 Derek , Ramsdale Tracie , Costa Maria A. , Duro Giovanni , Izzo Vincenzo , Salvadori Severo , Guerrini Remo , Cocchiara Roberta , Mirisola Mario G. , Wood Stephen , Geraci Domenico
Published: December 2022

Par j 1.0101 is one of the two major allergens of the Parietaria judaica (Pj) pollen, and its three-dimensional structure was built by three-dimensional structural homology modeling. The resultant model was used to identify putative IgE binding regions. Western blot analysis of gene fragmentation products showed that the 1 to 30 region was capable of binding specific IgE from a pool of sera (n = 30) of patients allergic to Pj pollen. Using the structural model as a guide, deletion and site-directed mutagenesis of the 1 to 30 region was performed, and the amino acids involved in IgE binding were identified. In addition, a synthetic peptide covering the 1 to 30 region was capable of binding human IgE without triggering histamine release from basophils of Pj allergic patients (n = 6) and thus represents a haptenic molecule with potential use as an immunotolerant agent. This epitope is also present on the Par j 2.0101 major allergen representing a common IgE epitope. It is an immunodominant epitope, since it was capable of inhibiting 30% of all specific IgE against the Pj major allergens, and therefore, it might be a candidate for the future development of immunotherapeutics.
Relationship Among Immunodominance of Single CD8+ T Cell Epitopes, Virus Load, and Kinetics of Primary Antiviral CTL Response
Authors: Weidt Gunnar , Utermöhlen Olaf , Heukeshoven Jochen , Lehmann-Grube Fritz , Deppert Wolfgang
Published: December 2022

The primary CTL response of BALB/c mice infected with the lymphocytic choriomeningitis (LCM) virus strain WE is directed exclusively against one major epitope, n118, whereas a viral variant, ESC, that does not express n118 induces CTL against minor epitopes. We identified one minor epitope, g283, that induces primary lytic activity in ESC-infected mice. Infections of mice with WE and ESC were used to study the hierarchical control of a T cell response. Presentation of minor epitopes is not reduced in WE-infected cells. Generation of CTL against n118 does not suppress the generation of minor epitope-specific CTL systemically, as mice coinfected with WE and ESC developed CTL against n118 and g283. However, elimination of ESC and development of minor epitope-specific CTL in ESC infection were slower than elimination of WE and development of CTL against n118. CD8+ T cells against the minor epitope were activated in ESC and WE infection, but did not expand in the latter to show lytic activity in a primary response. We explain the absence of minor epitope-specific lytic activity in WE infection by the fast reduction of virus load due to the early developing n118-specific CTL. Immunodominance of CTL epitopes in primary virus infections thus can be explained as a kinetic phenomenon composed of 1) expansion of CD8+ T cells specific for individual epitopes, 2) stimulatory effect of virus load, and 3) negative feedback control on virus load by the fastest CTL population.
Functional Differences Among Multiple Isoforms of Guinea Pig Decay-Accelerating Factor
Authors: Wang Guixian , Nonaka Mayumi , He Changqing , Okada Noriko , Nakashima Izumi , Okada Hidechika
Published: December 2022

Decay-accelerating factor (DAF, CD55) is a membrane inhibitor that protects host cells from the autologous C-mediated attack. The guinea pig homologue of DAF consists of multiple isoforms generated by alternative splicing from a single copy gene. These isoforms are mainly comprised of a glycosylphosphatidylinositol (GPI)-anchored form and a transmembrane form (TM) that is not present in human DAF. Both forms occur in at least three variations that differ in the length of the Ser/Thr-rich region (termed ST-a, ST-ab, and ST-abc). We have transfected cDNAs of the six major isoforms into Chinese hamster ovary cells, and their functional differences were evaluated in inhibition of C-mediated cytolysis and C3 deposition, using the transfectants expressing DAF at the same level on cell membranes. The degree of inhibition in both the classical and alternative pathways differed according to the length of the ST region in the order of abc > ab > a in both GPI and TM forms. When GPI and TM forms were compared, those with the ab or abc variation exhibited almost the same activity, whereas a-TM was less efficient than a-GPI. Although several isoforms are expressed constitutively in most of tissues, spermatozoa preferentially express the abc-GPI isoform, suggesting that this isoform offers effective protection to spermatozoa in the female genital tract.
Role of the Rho GTPase in Bradykinin-Stimulated Nuclear Factor-κB Activation and IL-1β Gene Expression in Cultured Human Epithelial Cells
Authors: Pan Zhixing K. , Ye Richard D. , Christiansen Sandra C. , Jagels Mark A. , Bokoch Gary M. , Zuraw Bruce L.
Published: December 2022

Recent evidence suggests a novel role of bradykinin (BK) in stimulating gene transcription. This study examined the effect of BK on nuclear factor κB (NF-κB) activation and IL-1β synthesis in human epithelial cells. Stimulation of A549 cells and primary bronchial epithelial cells with BK rapidly activated NF-κB. BK also increased the level of secreted immunoreactive IL-1β in A549 culture supernatants, an effect that was blocked by actinomycin D and the B2 BK receptor antagonist HOE-140. The role of NF-κB activation in BK-induced IL-1β synthesis was demonstrated by the ability of BK to stimulate increased chloramphenicol acetyltransferase (CAT) activity in A549 cells transfected with a reporter plasmid containing three κB enhancers from the IL-1β gene, while deletion of the κB enhancer sequences eliminated BK-stimulated CAT activity. C3 transferase exoenzyme, an inhibitor of Rho, abolished BK-induced NF-κB activation at 10 μg/ml and significantly inhibited BK-stimulated IL-1β synthesis at 5 μg/ml. A dominant-negative form of RhoA (T19N) inhibited BK-stimulated reporter gene expression in a dose-dependent and κB-dependent manner. Cotransfection of A549 cells with an expression vector encoding a constitutively active form of RhoA (Q63L) along with the IL-1β promoter-CAT reporter plasmid resulted in a marked increase in NF-κB activity compared with transfection with the IL-1β promoter-CAT reporter plasmid alone. These results demonstrate that BK stimulates NF-κB activation and IL-1β synthesis in A549 cells, and that RhoA is both necessary and sufficient to mediate this effect.
Cutting Edge: Physical Interaction Between Dendritic Cells and Tumor Cells Results in an Immunogen That Induces Protective and Therapeutic Tumor Rejection
Authors: Celluzzi Christina M. , Falo Louis D.
Published: December 2022

Dendritic cells (DCs) are potent professional APCs capable of presenting Ag in the context of costimulatory signals necessary for T cell activation. Although tumor cells express target Ags, they are generally incapable of stimulating an immune response. We show that the short term physical interaction of DCs and tumor cells, with or without cell fusion, results in rapid, efficient, and stable DC-tumor cell association. Immunization of naive mice with unselected, irradiated DC-tumor cell conjugates induces tumor-specific CD8+ cytotoxic T cells and protection from lethal tumor challenge. Furthermore, the immunogenicity of this cellular vaccine is dependent on the physical interaction of DCs and tumor cells before injection. Immunization with DCs and tumor cells after physical interaction can result in the regression of established tumors and persistent antitumor immunity. These results suggest that immunization with DC-tumor cell vaccines may be a simple, rapid, and potent strategy for tumor immunotherapy.
Cutting Edge: Human Myelomonocytic Cells Express an Inhibitory Receptor for Classical and Nonclassical MHC Class I Molecules
Authors: Colonna Marco , Samaridis Jacqueline , Cella Marina , Angman Lena , L. Allen Rachel , O’Callaghan Chris A. , Dunbar Rod , S. Ogg Graham , Cerundolo Vincenzo , Rolink Antonius
Published: December 2022

Leukocyte activation can be negatively regulated by inhibitory receptors specific for MHC class I molecules. While one inhibitory receptor, Ig-like transcript 2 (ILT2), is expressed by all lymphoid and myelomonocytic cell types, other receptors display a more selective tissue distribution. Here we characterize an inhibitory receptor, termed ILT4, which is selectively expressed in monocytes, macrophages, and dendritic cells (DCs), binds classical class I molecules and the nonclassical class I molecules HLA-G, and transduces negative signals that can inhibit early signaling events triggered by stimulatory receptors. ILT4 may control inflammatory responses and cytotoxicity mediated by myelomonocytic cells and may modulate their Ag-presenting functions, focusing immune responses to microbial challenges and avoiding autoreactivity.
CD1.1 Expression by Mouse Antigen-Presenting Cells and Marginal Zone B Cells
Authors: Roark Jessica H. , Park Se-Ho , Jayawardena Jayanthi , Kavita Uma , Shannon Michele , Bendelac Albert
Published: December 2022

Mouse CD1.1 is an MHC class I-like, non-MHC-encoded, surface glycoprotein that can be recognized by T cells, in particular NK1.1+ T cells, a subset of αβ T cells with semiinvariant TCRs that promptly releases potent cytokines such as IL-4 and IFN-γ upon stimulation. To gain insight into the function of CD1.1, a panel of nine mAbs was generated and used to biochemically characterize and monitor the surface expression of CD1.1 on different cell types. CD1.1 is a heavily glycosylated, β2-microglobulin-associated surface protein. Its recognition by a panel of 12 Vα14-positive and -negative CD1-specific αβ T cell hybridomas was blocked by two groups of mAbs that bound to adjacent clusters of epitopes, indicating that different αβ TCRs bind to the same region of CD1.1, presumably above the groove. Remarkably, CD1.1 was mainly expressed by dendritic cells, B cells, and macrophages, suggesting a function in Ag presentation to Th cells. Furthermore, the cell type that expressed the highest levels of CD1.1 was the splenic marginal zone B cell, a distinct subset of B cells that also expresses CD21 (the C3d receptor) and may be involved in natural responses to bacterial Ags. Altogether, the results support the idea that CD1.1 may function in recruiting a form of innate help from specialized cytokine producer αβ T cells to APCs, a role that might be important at the preadaptive phase of immune responses to some microbial pathogens.
Characterization of Signal Transduction Through the TCR-ζ Chain Following T Cell Stimulation with Analogue Peptides of Type II Collagen 260–267
Authors: Tang Bo , Myers Linda K. , Rosloniec Edward F. , Whittington Karen B. , Stuart John M. , Kang Andrew H.
Published: December 2022

The immunodominant T cell determinant of type II collagen (CII) recognized by DBA/1 mice (I-Aq) is CII 260–267. The aims of this study were to determine the role of the amino acid residues within CII 245–270 in T cell signal transduction. To that end, we utilized I-Aq-restricted, CII-specific T cell hybridomas and examined tyrosine phosphorylation of TCR-ζ following stimulation with either wild-type CII 245–270 or a panel of analogue peptides. A variety of patterns occurred, ranging from increased phosphorylation of TCR-ζ to either partial or a complete abrogation of phosphorylation. Critical substitutions also completely abrogated the phosphorylation of ZAP70, a downstream molecule in TCR-ζ signaling. Evaluation of the supernatants of the T cell hybridomas for cytokine production in response to the peptides revealed a close correlation between the induction of phosphorylation of TCR-ζ and the amount of cytokine induced. Selected analogue peptides were tested as tolerogens in neonatal mice. Analogues that did not induce the phosphorylation of ζ chain, such as B3 (CII 251–270s263F→N), were completely unable to induce tolerance, while analogues that caused a partial phosphorylation, such as B6 (CII 251–270s267Q→T) and A3 (CII 245–270s269P→A), induced partial tolerance judged by intermediate degrees of suppression of arthritis. We conclude that discrete alterations in specific amino acid residues of antigenic peptides had profound effects on T cell signaling and that the signaling correlated with T cell cytokine secretion and T cell function in the induction of tolerance and suppression of arthritis.
An In Vitro Model of T Cell Activation by Autologous Cytomegalovirus (CMV)-Infected Human Adult Endothelial Cells: Contribution of CMV-Enhanced Endothelial ICAM-1
Authors: Waldman W. James , Knight Deborah A. , Huang Emina H.
Published: December 2022

Cellular immunity is strongly implicated in control of CMV disease; however, many mechanistic details remain unresolved. We previously demonstrated T cell activation responses to CMV-infected allogeneic endothelial cells (EC), suggesting EC as a mediator of CMV response in the transplant recipient. We now test the hypothesis that CMV-specific T cell responses can be directly stimulated by infected EC in an environment free of potentially confounding allogeneic factors. By isolating splenic T cells and gonadal vein endothelial cells (GVEC) from individual cadaveric organ donors, we have developed an in vitro model of T cell interaction with autologous CMV-infected EC. Proliferation assays demonstrated significantly enhanced responses by CMV-seropositive donor-derived T cells cocultured with CMV-infected GVEC, as compared with those elicited by uninfected cells. Similarly, as determined by limiting dilution analysis of IL-2-producing cells, T cell response frequencies to infected GVEC were significantly greater than to uninfected EC. In contrast, responses of CMV-seronegative donor-derived T cells were minimal, regardless of CMV status of stimulator GVEC. Intriguingly, CD4 responses were observed in spite of the fact that CMV-infected EC express no HLA class II. Finally, attenuation of CMV-stimulated T cell proliferation observed in the presence of blocking Ab specific for ICAM-1 suggests a contributing role for CMV-enhanced endothelial ICAM-1 expression in the activation response. These studies demonstrate that EC can stimulate autologous T cell responses to CMV in the absence of accessory APC and suggest potentially novel mechanisms of immune activation.
Antibodies to Domains II and III of the IL-1 Receptor Accessory Protein Inhibit IL-1β Activity But Not Binding: Regulation of IL-1 Responses Is Via Type I Receptor, Not the Accessory Protein
Authors: Yoon Do-Young , Dinarello Charles A.
Published: December 2022

The IL-1R accessory protein (IL-1RAcP) plays a role in IL-1R signaling by forming a complex with IL-1RI bound to the IL-1 ligand. We identified four hydrophilic peptide regions of the extracellular IL-1RAcP that may be available for complex formation (peptide 1, 71–83 domain I; peptide 2, 204–211 domain II; peptide 3, 282–292 domain III; and peptide 4, 304–314 domain III). These peptides were synthesized, coupled to keyhole limpet hemocyanin, and used to produce rabbit antisera. Each affinity-purified antiserum showed specificity for the respective peptide without cross-reactivity. Anti-peptide 2, 3, and 4 recognized surface expression of IL-1RAcP on the Th2 D10S cells by FACS and inhibited IL-1-driven proliferation. Anti-peptide 4 recognized intact IL-1RAcP and soluble IL-1RAcP. Anti-IL-1RAcP-peptide 4, which targets the terminal segment of domain III, inhibited 80% of IL-1β-driven proliferation of D10S cells. However, these IL-1RAcP Abs had no effect on the activity of human or mouse IL-1α. Whereas IL-1β down-regulated IL-1RI surface expression (p < 0.05), there was no change in the surface expression of IL-1RAcP. Moreover, murine IL-10 increased surface expression of IL-1RI, but did not affect expression of IL-1RAcP or the proliferation of D10S cells. Steady state levels of mRNA for IL-1RAcP and IL-1RI in D10S cells showed a similar pattern to that of surface expression of the respective receptors. We conclude that 1) blocking IL-1RAcP inhibits IL-1 signaling in D10S cells, 2) domains-II and III may be involved in complex formation with IL-1RI, 3) IL-1RAcP is not regulated as is IL-1RI in the same cells, and 4) IL-1 responsiveness is dependent on the expression of IL-1RI, not IL-1RAcP.
Purified MHC Class I and Peptide Complexes Activate Naive CD8+ T Cells Independently of the CD28/B7 and LFA-1/ICAM-1 Costimulatory Interactions
Authors: Goldstein Julia S. , Chen Trina , Brunswick Mark , Mostowsky Howard , Kozlowski Steven
Published: December 2022

T cells play a central role in the initiation, maintenance, and regulation of the immune response. Effector responses of T cells are controlled by complex combinations of lymphokines and adhesion/costimulatory molecule signals. To isolate the effects of specific adhesion/costimulatory molecules and to define the minimal molecular requirements of naive CD8+ T cell activation, we have developed an APC-free system for stimulation of naive CD8+ T cells. In this report, we demonstrate that immobilized MHC class I-peptide complexes can activate naive CD8+ T cells from TCR transgenic mice at low cell densities. The CD8+ T cells were stimulated to proliferate and secrete IL-2 independently of the molecular interactions between CD28/B7.1-B7.2 or LFA-1/ICAM-1 surface receptors. Previous reports have shown that CD28 ligation is necessary for late T cell survival of APC-stimulated naive CD8+ T cells. Our data suggest that under certain specific conditions of high intensity T cell signaling, early activation and late cell proliferation can occur independently of APC-derived costimulatory signals.
Kinetics of Multivalent Antigen DNP-BSA Binding to IgE-FcεRI in Relationship to the Stimulated Tyrosine Phosphorylation of FcεRI
Authors: Xu Keli , Goldstein Byron , Holowka David , Baird Barbara
Published: December 2022

Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to FcεRI to stimulate cellular responses, although key features of the binding process are unknown. Fluorescence quenching can be used to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number of DNP groups per BSA is less than one. The binding data are well described by a transient hapten exposure model in which most of the DNP groups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measurement. Additional experiments indicate that, for suboptimal to optimal concentrations of DNP-BSA, most of the FITC fluorescence quenching on the cell surface is due to cross-linking events. With these concentrations at 15°C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine phosphorylation of FcεRI. At 35°C, the phosphorylation kinetics are biphasic during the time period in which cross-linking continues to increase. Our results establish a quantitative relationship between the timecourse for cross-linking by multivalent Ag and FcεRI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.
Inhibition of an In Vitro CD4+ T Cell Alloresponse Using Altered Peptide Ligands
Authors: Daniel Claude , Grakoui Arash , Allen Paul M.
Published: December 2022

In this study, we explore the potential of altered peptide ligands (APLs) to modulate the alloresponse of CD4+ T cells using elements of the murine hemoglobin (Hb) Ag model. We first demonstrated that the T cell 2.102, specific for the Hb(64-76)/I-Ek complex, was alloreactive against splenocytes of the H-2p haplotype. Using Ab-blocking and transfection experiments, we further showed that this alloreactivity was restricted to the class II molecule I-Ep. We tested a panel of APLs previously shown to antagonize the Hb response of 2.102 and found that these peptides could also effectively inhibit the alloresponse to I-Ep. Importantly, these peptides were able to antagonize the alloresponse of naive T cells derived from mice transgenic for the 2.102 TCR, as well as Th1 and Th2 cell lines. The antagonism required the presence of both I-Ep and I-Ek on the same APC. Our study demonstrates the effectiveness of APLs to antagonize the primary alloresponse of specific T cells and provides a basis for the development of immunotherapeutics for use in transplantation and immune-mediated diseases.
Functional Consequences of Costimulation by ICAM-1 on IL-2 Gene Expression and T Cell Activation
Authors: Zuckerman Linda A. , Pullen Lara , Miller Jim
Published: December 2022

LFA-1 is a well-recognized adhesion molecule, but its role in providing costimulatory signals to T cells has remained controversial. We have compared the ability of class II-positive transfectants that do and do not coexpress ICAM-1 (ProAd and ProAd-ICAM) to activate Ag-specific Th1 clones and naive CD4-positive T cells isolated from TCR transgenic mice. Ag presentation by ProAd to Th1 clones can induce calcium-dependent signaling events after engagement of the TCR, as evidenced by the nuclear localization of the transcription factors NF-AT and NF-κB. Nevertheless, coexpression of ICAM-1 or B7-1 on ProAd is required to induce detectable levels of IL-2 gene expression in either Th1 clones or naive T cells. In Th1 clones, activation by ProAd-ICAM induces very transient IL-2 mRNA expression that does not result in detectable IL-2 secretion or T cell proliferation. In naive T cells, the duration of IL-2 mRNA expression is longer, allowing for a transient burst of IL-2 protein that is sufficient to drive the cells into the cell cycle. In spite of this initial response, Ag presentation by ProAd-ICAM is a tolerogenic signal to naive T cells, and responding T cells undergo apoptosis 4 to 5 days poststimulation. These data suggest that engagement of LFA-1 can provide sufficient costimulatory signals to induce T cell activation and IL-2 gene expression, but cannot protect against anergy induction or provide for T cell survival.
Differential Regulation of Translation and eIF4E Phosphorylation During Human Thymocyte Maturation
Authors: Beretta Laura , Singer Nora G. , Hinderer Robert , Gingras Anne-Claude , Richardson Bruce , Hanash Samir M. , Sonenberg Nahum
Published: December 2022

Activation of peripheral blood T cells by cross-linking of CD3 results in a rapid and substantial rise in translation rates and proliferation, which coincides with an increase in the cap-binding protein, eIF4E activity. In contrast, immature CD4+CD8+ double-positive (DP) thymocytes undergo apoptosis in response to anti-CD3 mAb. We have investigated translation initiation in the response of immature thymocytes to activating signals. Activation by anti-CD3 + anti-CD4 of immature CD4+CD8+ DP thymocytes results in a rapid decrease in protein synthesis. In contrast, similar treatment of CD4+ or CD8+ single-positive (SP) thymocytes results in an increase in protein synthesis. The rate of protein synthesis is linked to the phosphorylation status of eIF4E. Following anti-CD3 + anti-CD4 stimulation, eIF4E phosphorylation strongly decreases in immature DP thymocytes, whereas it increases in mature SP thymocytes. The expression of 4E-BP2, a specific repressor of eIF4E function, is high in DP cells but decreases during maturation, raising the possibility of a role for 4E-BP2 in repressing eIF4E phosphorylation. These data provide evidence for differential regulation of the translational machinery during T cell development.