bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and presentation of endogenous mouse mammary tumor virus superantigens by thymic and splenic dendritic cells and B cells.
Authors: Ardavín C , Waanders G , Ferrero I , Anjuère F , Acha-Orbea H , MacDonald H R
Published: December 2022

Tolerance against superantigens (SAgs) encoded by endogenous mouse mammary tumor virus (Mtv) loci involves the intrathymic deletion of SAg-reactive T cells expressing a particular TCR V beta-chain, presumably upon presentation of the SAg by specialized APC. However, although the role of dendritic cells (DC) in the induction of tolerance against conventional Ags has been demonstrated, little is known about the role played by DC in tolerance induction against Mtv SAgs. Moreover, there is conflicting evidence concerning the capacity of DC to express and present Mtv SAgs. In this report we have analyzed the expression of Mtv SAgs in highly purified thymic and splenic DC and B cells by reverse transcriptase-PCR, using primers amplifying Mtv SAg-specific spliced mRNAs. DC express Mtv SAgs at levels comparable to B cells, but display a differential expression pattern of the various Mtv loci compared with B cells. Furthermore, our results show that DC are able to induce the deletion of SAg-reactive thymocytes in an in vitro assay, indicating that Mtv SAgs are functionally expressed on the DC surface. Collectively, our data are consistent with the hypothesis that DC play a role in the induction of intrathymic tolerance to Mtv SAgs.
Durable mixed allogeneic chimerism and tolerance by a nonlethal radiation-based cytoreductive approach.
Authors: Colson Y L , Li H , Boggs S S , Patrene K D , Johnson P C , Ildstad S T
Published: December 2022

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.
Conversion of low antibody responders into high responders by up-regulating the T cell response to a selective epitope.
Authors: Mikszta J A , Kim B S
Published: December 2022

Our previous studies have shown that hen egg-white lysozyme (HEL), structurally modified by diazonium-linked conjugation with a simple hapten such as phosphorylcholine (PC), induces more efficient T cell stimulation than the native Ag in low/non-responder C57BL/6 mice. In the present study, we examined whether such a structural modification could have any effect on the Ab response to native HEL in low- and high-responder mice. Surprisingly, C57BL/6 mice immunized with PC-HEL generated a markedly enhanced anti-HEL Ab response, while high responders failed to exhibit such an enhancement. T cell-proliferative responses to a single epitope, HEL47-61, were enhanced exclusively in the PC-HEL-immunized low/non-responder strain. To directly investigate whether a T cell response to this particular epitope is sufficient to induce anti-HEL Ab, peptide priming experiments were performed. Interestingly, C57BL/6 mice directly primed with HEL47-61 generated strong Ab responses against HEL upon immunization with non-immunogenic native HEL, whereas a different HEL epitope (HEL74-88) failed to induce such responses. Further analyses indicated that HEL47-61 induces strong Th2 (in addition to Th1)-type priming required for the IgG1 Ab response, whereas HEL74-88 stimulates only Th1. These results strongly suggest that individual T cell epitopes within the same protein Ag can induce qualitatively different types of T cell responses and that the up-regulated T cell response to a selective epitope, through altered Ag processing or direct priming, can lead to the conversion of low Ab responders into high responders.
Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells.
Authors: Oshimi Y , Oda S , Honda Y , Nagata S , Miyazaki S
Published: December 2022

The cytotoxicity of NK cells has been thought to be mediated mainly by the perforin-dependent pathway. We investigated the involvement of Fas-mediated pathway in the killing activity of purified human CD3-, CD16+ NK cells. Fas ligand mRNA was expressed in freshly isolated NK cells. Apoptosis, which was identified by the fragmented chromatin in individual cells, was induced in the cells that expressed high levels of Fas via direct NK-to-target cell interaction or Ab-dependent cell-mediated cytotoxicity, even in Ca(2+)-free medium, in which perforin pores are known not to be formed. Apoptosis in both the presence and absence of external Ca2+ was inhibited by Fab of an anti-Fas mAb. Transfection of the Fas gene in target cells facilitated the induction of apoptosis, compared with the parental cell line. The function of the Fas-mediated pathway in the coexistence of the perforin-dependent pathway was examined in 10 cell lines expressing different levels of Fas by Ca2+ imaging and morphologic observation of single cells. With a certain boundary level, low or high levels of Fas expression in target cells were correlated to a great degree with either acute necrosis due to severe membrane damage after NK-target cell contact or apoptosis at a later period, respectively. We concluded that Fas ligand/Fas interaction is present and plays a significant role in the human NK cell-induced apoptosis.
B7 costimulation and autoantigen specificity enable B cells to activate autoreactive T cells.
Authors: Roth R , Nakamura T , Mamula M J
Published: December 2022

This study examines the role of B cells as auto-APCs in activating autoimmune T cells responses. Mice immunized with their own cytochrome c (cyt c) elicit no detectable B or T cell responses. However, mice first primed with a cryptic self peptide, mouse cyt c 81-104, followed at 3 wk with a boost of whole cyt c, elicit autoreactive T cells specific to self cyt c. T cell autoimmunity is not elicited in similarly immunized B cell-deficient (mu MT) mice. The expression of the B7-2 and/or B7-1 costimulatory molecules, as well as specificity to a self Ag, cyt c, enabled B cells to activate T cells to proliferate and to express IFN-gamma, IL-4, IL-5, and IL-10 cytokine mRNAs. In contrast, neither adoptively transferred B7- B cells nor nonspecific B7+ B cells were able to activate naive T cells. Moreover, anti-B7-2 treatment of mice prevented the in vivo expression of the IL-4, IL-5, and IFN-gamma cytokine mRNA responses. Our results suggest a major role of autoantigen-specific, B7-bearing B cells in breaking T cell tolerance to self Ag.
Quantitative analysis of the influenza virus-specific CD4+ T cell memory in the absence of B cells and Ig.
Authors: Topham D J , Tripp R A , Hamilton-Easton A M , Sarawar S R , Doherty P C
Published: December 2022

The role of B lymphocytes and their Ig product in the development and maintenance of virus-specific CD4+ T cells has been analyzed in mice homozygous for disruption of the Ig mu gene (mu MT). These mice lack mature B220+ B cells and do not secrete Ig, but generate normal CD8+ cytotoxic T lymphocyte responses and have no difficulty clearing the HKx31 influenza A virus from the infected respiratory tract. Sequential limiting dilution analysis of virus-specific CD4+ T cells established that the frequencies of IL-2-producing T helper cell precursors in the draining lymph nodes and/or spleen from 7 days to 6 mo after infection were essentially similar in mu MT and C57BL/6 (B6) mice. Ag presentation and processing mechanisms involving Ig or B cells are apparently not required to generate virus-specific T helper cell precursors, and Ag-Ig complexes on follicular dendritic cells are not essential for the persistence of virus-specific CD4+ T cell memory. The main difference was that the spleens of the mu MT mice were much smaller than those of the B6 controls, and greater numbers of CD4+ T cells were found consistently in the regional mediastinal lymph nodes. This could be the result of abnormal expression of the lymph node homing receptor (CD62L) on the mu MT CD4+ T cells. However, the profiles of CD62L expression over the long term were comparable for both total and virus-specific CD4+ T cells from the two groups. The diminished role of the mu MT spleen is thus more likely to reflect the absence of germinal centers and/or Ig rather than a disruption of CD62L-mediated T cell trafficking.
Ig light chains are secreted predominantly as monomers.
Authors: Dul J L , Aviel S , Melnick J , Argon Y
Published: December 2022

Ig light (L) chains are secreted not only as part of assembled Ab molecules, but also as free L chains, the latter process being involved in the pathology of several diseases. The secretion competence of free L chains distinguishes them from free subunits of other oligomeric proteins, which are usually retained intracellularly. We used several techniques to test the idea that secretion of free L chains is dependent on dimerization. Coexpression of pairs of L chains, differing in only one amino acid, which alters the secretory phenotype, shows that these L chains behave independently: the wild-type chains are secreted, whereas the mutants are retained intracellularly. A survey of kappa- or lambda-producing cell lines by nonreducing gel electrophoresis shows that a negligible fraction of these L chains exists as disulfide-bonded dimers. Moreover, chemical cross-linking and density gradient centrifugation demonstrate that there is no significant pool of noncovalent L chain dimers. Noncovalent heterodimers can be detected readily between a kappa-chain and a chimera consisting of a heavy chain variable domain linked to the kappa-chain constant domain. This confirms that noncovalent L chain homodimers would have been detected if they were present. These findings about the association state of free L chains are independent of the host cell, as they are observed in both myeloma cells and COS fibroblasts. We conclude that L chain dimerization is a rare event that neither facilitates secretion nor is required for it.
Staphylococcal protein A simultaneously interacts with framework region 1, complementarity-determining region 2, and framework region 3 on human VH3-encoded Igs.
Authors: Potter K N , Li Y , Capra J D
Published: December 2022

Staphylococcal protein A (SPA) is a B cell superantigen that binds to human VH3-encoded Igs independently of the D- and JH-encoded regions and light chain sequences. The SPA-binding structure formed by VH3-encoded Igs remains controversial. We localized the regions in a VH3-encoded Ab required for SPA binding by producing mutant Abs in the baculovirus expression system in which regions of a human-derived Ab known to bind SPA were exchanged with those from a mouse Ab of the J558 family, a family not associated with SPA binding. The pattern of SPA binding indicates not only that residues in FR1, CDR2, and FR3 are involved but also that the three regions are required to interact simultaneously with SPA for binding to occur. When any one of the three regions was replaced with the corresponding region from the nonbinding Ab, SPA binding was severely disrupted. These data indicate that SPA requires simultaneous interaction with three distinct regions of a VH3 structure, which together in three-dimensional space form an extended solvent-exposed surface. These studies more precisely define the genetic requirements for VH3-encoded Ig binding to SPA.
Human dendritic cells phagocytose and process Borrelia burgdorferi.
Authors: Filgueira L , Nestlé F O , Rittig M , Joller H I , Groscurth P
Published: December 2022

There is strong evidence that the immune response to Borrelia burgdorferi (Bb) contributes to the pathogenesis of Lyme disease. Bb are transmitted by ticks to the skin, which is particularly rich in dendritic cells (DC). The initial reaction of these APCs may already set the course to immune pathogenesis. To study the role of DC, biopsies from human skin were incubated with Bb and investigated in toto with electron microscopy. In addition, DC freshly isolated from dermis (DDC) and epidermis (Langerhans cells) were compared with blood-derived DC (BDC). In situ, Bb were found in the dermal layer of the skin only, occasionally cleared by DDC. Isolated DDC and BDC, but not Langerhans cells, readily engulfed Bb preferentially using coiling phagocytosis. Internalized Bb were located free in the cytosol and inside of phagolysosomes of DDC and BDC. Intravesicular Bb Ags were colocalized with MHC class II molecules. In addition, live Bb induced IL-12 production in BDC. Bb-pulsed BDC activated naive and primed autologous Bb-specific T cells, as measured by detection of granulocyte-macrophage CSF gene transcription and proliferative response, respectively. These data indicate that human DC phagocytose, process, and present Bb Ags. The way in which DC may influence the immune response in Lyme disease, however, remains to be evaluated.
Protection against lethal viral infection by vaccination with nonimmunodominant peptides.
Authors: Oukka M , Manuguerra J C , Livaditis N , Tourdot S , Riche N , Vergnon I , Cordopatis P , Kosmatopoulos K
Published: December 2022

CTL response of H-2b mice to influenza PR8 virus is directed against the nucleoprotein (NP)-derived immunodominant 366-374 (NP366PR8) peptide presented by the Db molecule. However, NP has three nonimmunodominant peptides corresponding to the 17-25 (NP17), 55-63 (NP55), and 97-105 (NP97) sequences that have the Db consensus motifs and bind to the Db molecule with an intermediate (NP55) or low (NP17 and NP97) affinity. In a previous report, we have shown that NP55 peptide is naturally processed by infected cells. In the present work, we studied whether nonimmunodominant peptides can protect mice against viral infection. Antiviral protection was evaluated by measuring three parameters: survival after inoculation of a lethal dose of mouse-adapted PR8 virus, percentage of pulmonary lesions in surviving mice, and virus clearance from lungs of infected mice. Our results showed that immunization of B6 mice with nonimmunodominant peptides protected from PR8 virus infection, although less efficiently than immunization with the immunodominant NP366PR8 peptide. Protection was mediated by CD8 T cells. The efficacy of nonimmunodominant peptides correlated with their Db binding affinity; the low affinity binders NP17 and NP97 induced a weaker protection than the intermediate affinity binder NP55. A mixture of NP366PR8 and nonimmunodominant peptides gave a higher protection than NP366PR8 peptide alone. In conclusion, nonimmunodominant peptides protect against a viral infection with an efficacy that is proportional to their affinity for the restricting class I molecule.
Exhaustion of CTL memory and recrudescence of viremia in lymphocytic choriomeningitis virus-infected MHC class II-deficient mice and B cell-deficient mice.
Authors: Thomsen A R , Johansen J , Marker O , Christensen J P
Published: December 2022

To study the contribution of CD4+ T cells and B cells to antiviral immunity and long term virus control, MHC class II-deficient and B cell-deficient mice were infected with lymphocytic choriomeningitis virus. In class II-deficient mice, which lack CD4+ T cells, the primary CTL response is virtually intact, and the infection is initially controlled in most organ sites including the blood. However, approximately 2 mo postinfection, CTL memory cannot be detected, and recrudescence of viremia to titers as high as seen during the acute phase of the infection is observed. To investigate whether this phenomenon could reflect participation of B cells and/or Abs in long term virus control, similar experiments were performed with mice that do not have mature B cells because of a disrupted membrane exon of the mu chain gene. In these mice, the cell-mediated immune response was slightly delayed, but transient virus control was observed in most mice. However, approximately 3 mo postinfection, blood virus titers were as high as observed during the acute infection, and no CTL memory could be demonstrated. These results indicate that Abs and/or B cells are required for permanent virus control and that in their absence, the virus-specific CTL potential becomes exhausted. Together our results indicate that while CD8+ cells play a dominant role in acute virus control, all three major components of the immune system are required for long term virus control.
Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).
Authors: Memar O , Christensen B , Rajaraman S , Goldblum R , Tyring S K , Brysk M M , McCormick D J , el-Zaim H , Fan J L , Prabhakar B S
Published: December 2022

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.
B-cell activation in vitro by helper T cells specific to region alpha 146-162 of Torpedo californica nicotinic acetylcholine receptor.
Authors: Rosenberg J S , Oshima M , Atassi M Z
Published: December 2022

We have previously mapped the T and B cell epitopes on the alpha-subunit of acetylcholine receptor (AChR) in human myasthenia gravis (MG) and in experimental autoimmune MG-susceptible (C57BL/6 (B6)) and nonsusceptible mouse strains. In addition to regions recognized by both T and B cells, the AChR alpha-subunit has regions that are recognized solely by T cells. An exclusive T cell epitope within residues alpha 146-162 of Torpedo californica (t), tAChR, plays an important role in experimental autoimmune MG pathogenesis in B6 mice. To study its function, we established, from tAChR-primed B6 mice, two t alpha 146-162-specific T cell lines (P14Th) which comprised Th2-type cells because they secreted IL-4 but not IL-2. P14Th did not recognize the corresponding region on mouse (m) AChR (m alpha 146-162). They caused in vitro differentiation of tAChR-primed B cells into plasma cells that secreted anti-AChR Abs directed, in decreasing order, against the following tAChR alpha regions: t alpha 122-138 > t alpha 134-150 > t alpha 45-60 > t alpha 170-186 > t alpha 56-71. Little or no Ab response could be detected against peptides t alpha 182-198 or t alpha 146-162 itself. The major enhancement was in the Abs against region t alpha 122-150 (spanning the t alpha 122-138/t alpha 134-150 overlap) that is involved in ACh binding. These Abs cross-reacted completely with m alpha 122-150, the corresponding region on mAChR. Therefore, t alpha 146-162-specific T cells, although unable to recognize m alpha 146-162, are nevertheless pathogenic because they help B cells responding to a tAChR region that is conserved in mAChR and involved in ACh binding. These Abs cross-react with the corresponding effector-binding region of mAChR, thereby disrupting the normal physiologic function of the mouse receptor.
Autoantibodies to ribosomal P antigens with immune complex glomerulonephritis in SJL mice treated with pristane.
Authors: Satoh M , Hamilton K J , Ajmani A K , Dong X , Wang J , Kanwar Y S , Reeves W H
Published: December 2022

BALB/c ByJ mice develop a lupus-like syndrome characterized by anti-nRNP/Sm and Su autoantibodies and immune complex glomerulonephritis after a single i.p. pristane injection. In contrast, mercuric chloride induces anti-fibrillarin Abs only in SJL and other H-2s mice, and not in BALB/c (H-2d) mice. In the present study, the specificities of autoantibodies induced by pristane and HgCl2 were compared in SJL and BALB/c mice to examine whether these strains are "programmed" to make different sets of autoantibodies in response to nonspecific immune stimulation. Unexpectedly, the predominant autoantibodies induced by pristane in SJL mice were neither those characteristic of HgCl2-treated SJL mice nor those associated with pristane-induced disease in BALB/c mice but, rather, anti-ribosomal P, another lupus-related specificity. The autoantibodies were strongly reactive with the C-terminal 22 amino acids of the ribosomal P2 protein, indicating that they exhibited similar fine specificities to anti-P Abs in human SLE and MRL/Ipr mice. Like BALB/c mice, pristane-treated SJL mice developed severe glomerulonephritis characterized by proteinuria, mesangial proliferation, and glomerular immune complex deposits. This is the first evidence that the induction of a lupus-like syndrome by pristane is not restricted to BALB/c mice. The predominance of anti-P Abs in SJL mice contrasts sharply with the predominance of anti-nRNP/Sm and Su, in pristane-treated BALB/c mice, even though the renal lesions were similar in both strains. The data suggest that H-2s does not program mice to produce anti-fibrillarin Abs in response to nonspecific immune stimulation, arguing that autoantibody induction by pristane involves Ag-specific mechanisms.
The catalytic domain of pp56(lck), but not its regulatory domain, is sufficient for inducing IL-2 production.
Authors: Carrera A C , Calvo V , Borlado L R , Paradis H , Alemany S , Roberts T M , Martinez-A C
Published: December 2022

The lymphoid src kinase pp56(lck) has been shown to be essential for the induction of different T lymphocyte responses, including CD4-mediated enhancement of Ag-induced T cell activation, early T cell differentiation, induction of IL-2 production, and cytotoxicity. It is assumed that pp56(lck) acts on these processes by phosphorylating substrates. However, it has been recently reported that the NH2 regulatory domain is sufficient to mediate CD4 accessory function. In this report we address the contribution of the regulatory and catalytic domains of pp56(lck) to another function of this enzyme independent of CD4: TCR-induced IL-2 production. Two pp56(lck) mutants lacking either the entire catalytic domain or the entire NH2 regulatory domain were generated, and their abilities to trigger transactivation of the TCR-regulated nuclear factor of activated T cells (NF-AT) region of the IL-2 promoter were compared. Only the catalytic, but not the NH2 regulatory, domain of pp56(lck) was able to induce NF-AT region transactivation on its own and to cooperate with other intracellular signals to trigger this response. Moreover, the catalytic domain of pp56(lck) was able to induce IL-2 cytokine production to an extent similar to that of wild-type pp56(lck). We conclude that different domains of the pp56(lck) molecule contribute to regulate distinct biologic functions. In fact, while the NH2 regulatory domain is sufficient to mediate CD4 accessory function, we show here that the catalytic domain of pp56(lck) is sufficient for induction of IL-2 production, mimicking TCR ligation.
Overexpression of c-Fos induces apoptosis of CD43+ pro-B cells.
Authors: Hu L , Hatano M , Rüther U , Tokuhisa T
Published: December 2022

The proto-oncogene product c-Fos, a component of the transcription factor AP-1, is induced in early B lineage cells. To investigate a role of c-Fos in early B cell development, fetal liver (FL) cells from transgenic mice carrying an IFN-alphabeta (IFN)-inducible c-fos gene (Mx-c-fosD) were cultured on a stromal cell layer with IL-7. Although B lineage cells normally developed in the Mx-c-fosD FL cell culture, the development was perturbed by the addition of IFN at the beginning of culture. When IFN was added in the FL culture after B lineage cells developed, pro-B (B220+,CD43+) cells were selectively dying by apoptosis within 48 h after IFN stimulation. This apoptosis was intrinsically induced in the pro-B cells that overexpressed c-fos when the Mx-c-fosD FL (H-2Kb) cells were cocultured with the normal C3H FL (H-2Kk) cells. The molecular basis of the apoptosis was investigated by examining expression of the genes that regulate apoptosis. The IFN stimulation did not modulate expression of Bcl-2 and Fas in early B lineage cells from the Mx-c-fosD FL culture. However, Rag-2 was down-regulated in these cells within 12 h after IFN stimulation. These results suggest that the c-Fos plays a causal role in deletion of pro-B cells with nonfunctional Ag receptor.
Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity.
Authors: Moreno M B , Memon S A , Zacharchuk C M
Published: December 2022

Fas-mediated apoptosis plays an important role in regulating the immune response in peripheral T cells. Restimulation of T cell blasts up-regulates Fas and Fas ligand expression, with subsequent interaction leading to cell death. Overexpression of Bcl-2 in tumor cells blocks apoptosis induced by many stimuli, but inhibition of Fas-mediated killing has not been consistently observed. To examine the behavior of Bcl-2 in normal cells, T cell blasts were transiently transfected with Bcl-2 and related gene products to determine the effect on apoptotic signaling. Transient overexpression of Bcl-2 in mouse and human T cell blasts did not block Fas-mediated apoptosis, whereas etoposide- and glucocorticoid-induced cytotoxicity was potently inhibited. Expression of Bcl-xL and adenovirus E1B 19K did not interfere with anti-Fas killing. In contrast, interleukin-1beta-converting enzyme family protease inhibitors Ac-DEVD-CHO and CrmA blocked Fas-mediated apoptosis. These results suggest that peripheral T cells use distinct apoptosis signaling pathways with differential sensitivity to Bcl-2 and interleukin-1beta-converting enzyme family protease inhibitors. Since T cells normally express Bcl-2 and Bcl-xL following activation, their inability to block Fas-mediated apoptosis may allow for the elimination of self-reactive cells and the appropriate regulation of immune responses.
Cytotoxic gammadelta or alphabeta T cells with a natural killer cell marker, CD56, induced from human peripheral blood lymphocytes by a combination of IL-12 and IL-2.
Authors: Satoh M , Seki S , Hashimoto W , Ogasawara K , Kobayashi T , Kumagai K , Matsuno S , Takeda K
Published: December 2022

Populations of cytotoxic CD3+CD56+ cells were selectively expanded when monocyte-depleted human PBL (M(-) PBL) were cultured with 20 U/ml IL-12 and 100 U/ml IL-2. In a majority of cases, CD4-CD8- as well as CD8+ gammadelta T cells with CD56 Ag were induced, but in some cases CD4+CD56+ alphabeta T cells were selectively increased. Determination of which will increase, gammadelta or alphabeta T cells, apparently is dependent upon individuals. When M(-) PBL were stimulated with IL-12 and IL-2 in the presence of immobilized anti-CD3 Ab, CD8+CD56+ alphabeta T cells increased exclusively. When M(-) PBL were cultured by IL-2 alone, CD3+CD56- cell expansion was predominant while CD3+CD56+ cells remained a minor population. Cytotoxic activity of M(-) PBL cultured with a combination of IL-12 and IL-2 is much greater than when cultured with IL-2 alone. The cytotoxicity of activated M(-) PBL was abrogated by the depletion of either CD3+ or CD56+ cells irrespective of their gammadelta or alphabeta phenotypes. These types of cells are preferentially present in the livers of humans. The results revealed that, under certain conditions, IL-12 synergizes with IL-2 to induce potent cytotoxic T cells with CD56 Ag of humans, and suggest that these cells in the human liver are functionally similar to NK1+ alphabeta T cells in murine liver.
A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells.
Authors: Zhang C , Ao Z , Seth A , Schlossman S F
Published: December 2022

Studies toward the biologic and molecular understanding of programmed cell death have been stimulated by the recent identification of genes and their products that regulate apoptosis. A panel of mAbs has been raised against dying cells in the present study by immunizing mice with apoptotic Jurkat cells. One of these Abs, anti-7A6, was found to react with apoptotic cells. Using ELISA or flow cytometry, little reactivity of anti-7A6 was observed in normal or digitonin-permeabilized human peripheral blood lymphocytes and a number of hemopoietic cell lines tested. The Ab, however, strongly reacted with these cells when they were induced to undergo apoptosis by irradiation or treatment with apoptosis-inducing agents. Cell sorting and DNA fragmentation experiments revealed that 7A6-positive cells, but not 7A6-negative cells, had apparent DNA fragments characteristic of cells undergoing apoptosis. By immunoblot, under reducing conditions, anti-7A6 detected a 38-kDa protein band in the cell lysates prepared from apoptotic cells. Immunoelectron microscopy showed the 7A6 Ag to be localized to the membrane of mitochondria in apoptotic Jurkat cells. These results indicate that anti-7A6 defines a novel epitope on the mitochondrial membrane protein that appears to be exposed on cells undergoing apoptosis, suggesting that the 7A6 molecule may be involved in the molecular cascade of apoptotic cell death.
L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity.
Authors: Tu L , Chen A , Delahunty M D , Moore K L , Watson S R , McEver R P , Tedder T F
Published: December 2022

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.
In the absence of endogenous IFN-gamma, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection.
Authors: Scharton-Kersten T M , Wynn T A , Denkers E Y , Bala S , Grunvald E , Hieny S , Gazzinelli R T , Sher A
Published: December 2022

The relationship between IFN-gamma and IL-12 in generating innate immune responses and resistance to acute Toxoplasma gondii infection was assessed in T. gondii-exposed IFN-gamma knockout (gko) mice. Gko mice, in contrast to wild-type (wt) animals, rapidly succumbed to infection with either the avirulent ME49 strain or, surprisingly, an attenuated temperature-sensitive mutant strain, ts4. Microscopic examination of peritoneal exudates from infected gko mice demonstrated that mortality is associated with unchecked tachyzoite replication. Nevertheless, both wt and gko animals developed a peritoneal inflammatory response that in gko animals was greater due to a 5- to 10-fold increase in the number of granulocytes recruited to the site of infection. In addition, IL-12 production in gko mice was both unimpaired and functional since a significant, albeit lower than wt, IL-12-dependent NK cell response developed in these animals. Regardless, no evidence for an IFN-gamma-independent protective function for IL-12 or NK cells was apparent since in vivo treatment of gko mice with an IL-12-neutralizing mAb ablated the NK cell response, but did not decrease survival. Together, these data identify distinct functions for IL-12 and IFN-gamma in host resistance to T. gondii: IL-12 precedes and initiates synthesis of IFN-gamma, while the latter lymphokine directly controls parasite growth and diminishes the contribution of IL-4- and IL-5-producing T cell subsets.
Constitutive and inducible mechanisms for synthesis and release of cytokines in immune cell lines.
Authors: Baumgartner R A , Deramo V A , Beaven M A
Published: December 2022

We found that production and release of two functionally antagonistic cytokines, TGF-beta and TNF-alpha, were regulated differently in the mast cell, T cell, and macrophage cell lines RBL-2H3, MLA-144, and U-937, respectively. TGF-beta was produced and released constitutively in all three cell lines. When, however, the cell lines were stimulated with Ag, LPS, or calcium ionophore plus PMA, acceleration of release and some additional production of TGF-beta were apparent. In contrast, TNF-alpha was produced and released only when these lines were stimulated. Although neither the glucocorticoid, dexamethasone, nor the protein kinase C inhibitor, Ro31-7549, suppressed constitutive production or release of TGF-beta, these agents inhibited TNF-alpha production and the inducible component of TGF-beta production noted above. The release of these cytokines, whether constitutive or inducible, was dependent on Golgi-processing as indicated by inhibition with brefeldin A. Therefore, although both types of cytokines were processed by Golgi, only TNF-alpha and the inducible component of TGF-beta production were protein kinase C or steroid-regulated processes. These findings suggested that constitutive and inducible pathways exist for production and release of cytokines and that the inducible pathways can be selectively suppressed by pharmacologic agents.
B cells regulate CD40 ligand-induced IL-12 production in antigen-presenting cells (APC) during T cell/APC interactions.
Authors: Maruo S , Oh-hora M , Ahn H J , Ono S , Wysocka M , Kaneko Y , Yagita H , Okumura K , Kikutani H , Kishimoto T , Kobayashi M , Hamaoka T , Trinchieri G , Fujiwara H
Published: December 2022

Although stimulation of freshly isolated murine spleen cells with anti-CD3 mAb or Con A failed to generate IL-12 production, the same cell preparations depleted of B cells produced IL-12. Addition of normal B cells inhibited IL-12 production in a cell number-dependent manner. IL-12 production was dependent on the presence of CD4+, but not of CD8+, T cells, and inhibited by addition of anti-CD40 ligand (CD40L) mAb. Anti-CD3 or Con A stimulation induced CD40L expression only on CD4+ T cells, which was inhibited in the presence of B cells. IL-12 production was also induced by interactions between CD40L-transfected Chinese hamster ovary cells and splenocytes depleted of T and B cells, but not of APC, indicating CD40L-induced IL-12 production by APC. The involvement of CD40 molecules was examined by comparing the ability of cells from CD40-deficient (CD40 -/-) and wild-type mice (CD40 +/+) to produce IL-12. Spleen cells from CD40 -/- and CD40 +/+ mice produced comparable amounts of IL-12 in response to bacterial stimuli. However, the B cell-depleted fraction from CD40 -/- mice failed to produce IL-12 when stimulated with anti-CD3 or Con A or when cocultured with CD40L-expressing Chinese hamster ovary cells. These results indicate that CD40L expressed on activated T cells induces APC to produce IL-12 through CD40/CD40L interaction, but this pathway is competitively inhibited by CD40+ B cells incapable of producing IL-12 upon stimulation with CD40L. Thus, this might represent a novel mechanism underlying the regulation of cell-mediated and humoral immunity.
Role of a C-terminal residue of an immunodominant epitope in T cell activation and repertoire diversity.
Authors: Mikszta J A , Jang Y S , Kim B S
Published: December 2022

In the present study, the extent of heterogeneity in the high responder T cell response to the predominant epitope region of hen egg white lysozyme (HEL46-61) was examined. Through analyses of T cell proliferation and precursor frequency, the C3H T cell response is shown not to be limited to peptides containing the previously defined minimal epitope of residues 52-61, but rather is quite heterogeneous, encompassing much of the 46-61 sequence. Further characterization using a panel of T cell hybridoma clones revealed T cell recognition of diverse minimal epitopes within this region. Interestingly, these T hybridomas could be grouped into three distinct categories based on the ability to respond to peptides with or without the native arginine residue at position 61 (61-required, 61-inhibitory, dual responders). Using analogue peptides containing single amino acid substitutions at position 61, further heterogeneity within these hybridoma groups was identified, suggesting the presence of an extremely diverse T cell repertoire for the epitope region. The charge and/or size of the C-terminal residue appears to be a critical factor for certain clones; replacement of the native arginine residue with aspartic acid or glutamic acid enabled a nonstimulatory ligand to specifically antagonize a T cell hybridoma response. Collectively, these results strongly suggest that the C-terminal residue of the predominant epitope in high responder mice plays a critical role in T cell diversity and activation.
Evidence for altered regulation of I kappa B alpha degradation in human colonic epithelial cells.
Authors: Jobin C , Haskill S , Mayer L , Panja A , Sartor R B
Published: December 2022

The nuclear factor kappaB (NF-kappaB) regulates the transcription of genes bearing the kappaB consensus motif. Transmigration of NF-kappaB from the cytoplasm to the nucleus is regulated by the IkappaB family of inhibitory NF-kappaB-binding proteins. Dissociation of the NF-kappaB-IkappaB complex requires both phosphorylation and degradation of IkappaBs. We demonstrate that IL-1beta induces complete IkappaB alpha degradation in Caco-2 cell lines but not in HT-29, T84, SW-480 transformed cell lines, or native colonic epithelial cells. A similar lack of IkappaB alpha degradation in HT-29 cells followed TNF-alpha and bacterial polymer stimulation. IL-1beta stimulated NF-kappaB nuclear translocation and NF-kappaB-dependent IL-1beta and IL-8 expression in both Caco-2 and HT-29 cells as assayed by electrophoretic mobility shift assay, immunofluorescence, kappaB-luciferase transfection, reverse transcriptase-PCR analysis and ELISA. In HT-29 cells stimulated with IL-1beta, IkappaB alpha was phosphorylated and when cycloheximide blocked new protein synthesis, IkappaB alpha was partially degraded. NF-kappaB cytoplasmic to nuclear transmigration was incomplete and preceded IkappaB alpha degradation in 9T-29 cells, in contrast to complete coordinated NF-kappaB nuclear translocation and IkappaB alpha degradation in Caco-2 cells. Greater sensitivity of HT-29 cells to a calpain inhibitor, as measured by IL-8 secretion, suggested enhanced resistance to IkappaB alpha proteolysis. These data show that IL-1beta induces NF-kappaB activity and expression of NF-kappaB-dependent genes in colonic epithelial cells and suggest altered regulation of IkappaB alpha degradation compared with other cell lineages, which may result in their increased responsiveness to therapeutic blockade.
Ig VH1 genes expressed in B cell chronic lymphocytic leukemia exhibit distinctive molecular features.
Authors: Johnson T A , Rassenti L Z , Kipps T J
Published: December 2022

Prior studies revealed that the leukemic B cells of patients with chronic lymphocytic leukemia (CLL) express a restricted Ig heavy chain variable region gene (VH gene) repertoire. However, this restriction needs to be re-evaluated in light of findings that the repertoire of Ig VH genes used by primary B cells of normal adults is actually more restricted than originally assumed. Because of this, we critically examined the Ig VH1 and VH7 genes expressed by leukemia cells of a random panel of patients with B cell CLL (n = 117). Forty-one (35%) had leukemia cells with functional VH1 gene rearrangements. Of these, the large majority expressed Ig VH1-69 (n = 26; 63%). The remaining leukemia cell samples expressed VH1-2 (n = 4; 10%), VH1-3 (n = 2; 5%), VH1-8 (n = 2; 5%), VH1-18 (n = 1; 2%), VH1-45 (n = 1; 2%), and VH1-46 (n = 5; 12%). Only 1 of the 117 examined (<1%) used Ig VH genes of the VH7 subgroup. We found that certain alleles of the IgVH1-69 locus are favored in CLL. Also, leukemia B cells that express VH1-69 have a distinctive use distribution of D and JH gene segments compared with that of VH1-expressing CLL B cells that do not use VH1-69 or non-neoplastic B cells that express VH1-69. Finally, the average length of the heavy chain third complementarity-determining regions (CDR3) of VH1-69-expressing leukemia cells is significantly longer than that of normal adult B cells that also express Ig encoded by VH1-69 genes. Collectively, this study indicates that the Ig expressed in CLL have distinctive molecular features that are not representative of the Ig expressed by primary B cells of normal adults.
Schistosome egg granulomas and hepatic expression of TNF-alpha are dependent on immune priming during parasite maturation.
Authors: Leptak C L , McKerrow J H
Published: December 2022

Granulomatous inflammation is key to the pathogenesis of many infectious diseases, including hepatic schistosomiasis. The granulomas that form around schistosome eggs trapped in the liver of infected hosts were thought to be induced primarily, if not exclusively, by egg Ags. We now show that the maturation of adult worms, before the production of eggs, primes local immune responses key to granuloma formation. When parasite eggs are injected into the livers of naive animals, only a minimal, nongranulomatous, inflammatory response results. However, if eggs are injected into the livers of mice previously infected with single-sex adult worms, granuloma formation is restored. Granuloma formation is also restored if mice are presensitized with adult worm homogenates or with eggs themselves before egg injection into the liver. These sensitization studies confirm that an Ag or Ags are shared between different stages of the schistosome life cycle and that immune priming by these Ag(s) is necessary for hepatic granuloma formation. Models of infection that isolate the effects of worms from those of eggs confirm that each of these life cycle stages induces a unique pattern of cytokine expression. Hepatic expression of TNF-alpha, a major cytokine signal for granuloma formation, is a reaction to adult worms, not eggs. TNF-alpha may mediate immune priming necessary for granuloma development since injection of purified TNF-alpha alone restores formation of granulomas in the livers of naive mice injected with eggs.
Site-directed mutagenesis of the phosphocholine-binding site of human C-reactive protein: role of Thr76 and Trp67.
Authors: Agrawal A , Lee S , Carson M , Narayana S V , Greenhough T J , Volanakis J E
Published: December 2022

We have reported previously that residues Lys57, Arg58, and Trp67 of human C-reactive protein (CRP) contribute to the structure of the phosphocholine (PCh)-binding site. In this study, based on the three-dimensional structures of human CRP and serum amyloid P, we constructed an additional mutant, T76Y, to probe the structural determinants of the PCh-binding site of CRP. Binding properties of four mutant CRPs, K57Q/R58G, W67K, K57Q/R58G/W67K, and T76Y were compared. Wild-type (wt) and all mutant CRPs were purified by affinity chromatography on PCh-, pneumococcal C-polysaccharide (PnC)-, or phosphoethanolamine-conjugated agarose columns. Purified mutant CRPs, K57Q/R58G/W67K and T76Y failed to bind to solid phase, PCh-substituted BSA. They did, however, bind to immobilized PnC, although with substantially decreased avidity compared with wt CRP. W67K, K57Q/R58G/W67K, and T76Y CRP required a 10-fold higher Ca2+ concentration than wt CRP to bind PnC and exhibited decreased avidity for mAb EA4.1, which recognizes a Ca2+-dependent epitope. We conclude that Thr76 is a determinant of the PCh-binding site, probably interacting with the choline group. This conclusion is supported by recent crystallographic data indicating that this residue participates in the formation of a hydrophobic pocket that constitutes the binding site for choline. Trp67, Lys57, and Arg58 do not directly contact PCh, but appear to be required for the proper conformation of the binding site.