bluuebunny/crossref_metadata_2025 huggingface dataset:
Donor T cells can be induced to grow and survive long term in vivo without previous host immunosuppression.
Authors: Chen W , Cheever M A
Published: December 2022

To examine whether donor T cells can grow and survive long term in hosts without previous immunosuppression, cultured T cells (B6/Thy 1.1) specific to FBL-3 tumor were adoptively transferred into normal B6/Thy 1.2 mice and induced to proliferate in vivo by specific stimulation with irradiated FBL-3. Donor T cells residing in the spleen and ascites of hosts were identified and quantified by use of Ab to the Thy-1 allele. The results demonstrated that on day 7 after transfer, donor T cells in greater numbers than input (1.2-fold) could be recovered. By day 35, donor T cells had decreased to approximately 10% of T cells input. Administration of IL-2 at doses of 2,500 or 25,000 U/day for 7 days preferentially increased the growth of Ag-activated donor T cells rather than host lymphocytes, and increased both the short term growth on day 7 (6.6-fold and 14.5-fold greater than input, respectively) and the long term survival on day 35 (0.75-fold and 3.15-fold of input, respectively) of donor T cells. The combination of CY pretreatment plus low dose IL-2 (2,500 U/day) increased donor T cell growth over and above that of either manipulation alone. However, with higher dose IL-2 to 25,000 U/day, donor T cell growth was equivalent in CY-pretreated and normal hosts (18-fold vs 16-fold increase, respectively; p > 0.05). With either dose of IL-2 there was no significant difference in the survival of donor T cells on day 35 in CY-pretreated vs normal hosts. Functional assay confirmed that specific cytolytic function of donor T cells could be maintained in hosts without previous immunosuppression. Accordingly, established disseminated FBL-3 leukemia could be cured without CY treatment, in a regimen using 10(7) cultured immune T cells plus IL-2 (25,000 U/day x 7 days). Thus, adoptively transferred donor T cells can be grown to large numbers and survive long term in vivo with maintenance of substantial function without the necessity of previous host immunosuppression.
Onset of TCR-beta gene rearrangement and role of TCR-beta expression during CD3-CD4-CD8- thymocyte differentiation.
Authors: Godfrey D I , Kennedy J , Mombaerts P , Tonegawa S , Zlotnik A
Published: December 2022

TCR-beta gene rearrangement or expression is necessary and sufficient for the progression of early alpha beta thymocyte differentiation from the CD3-CD4-CD8- triple negative (TN)3 to the CD4+CD8+ double positive stage. The onset of TCR-beta rearrangement is currently thought to occur gradually. Some thymocytes were reported to be rearranged at the earliest (CD44+CD25-) TN stage, whereas other thymocytes did not initiate TCR-beta rearrangement until the latest (CD44-CD25-) TN stage. Here, we have isolated subsets of TN thymocytes on the basis of surface expression of CD44 and CD25, with c-kit as an additional marker. We present a revised model of early T cell development in which TCR-beta and TCR-gamma rearrangements occur abruptly, at the CD44lowCD25+ c-kitlowTN stage. A high level of c-kit expression defines pro-T cells which have not yet rearranged their TCR genes. Germ-line TCR-beta transcripts, and transcripts of recombination activating genes (RAG)-1 and 2, are detected before TCR-beta gene rearrangement. Analyses of TN thymocytes of RAG-1 mutant mice, and of various TCR mutant and TCR transgenic RAG-1 mutant mice, indicate the existence of a control point at the CD44-CD25+TN stage at which cells expressing a productively rearranged TCR-beta chain are selected for further differentiation.
Absence of MHC class II expression distinguishes fetal from adult B lymphopoiesis in mice.
Authors: Hayakawa K , Tarlinton D , Hardy R R
Published: December 2022

Early B-lineage progenitor cells (Pro-B) isolated from murine fetal liver and adult bone marrow can differentiate to the immature B cell stage in a stromal cell-dependent culture system and to mature B cells upon transfer into immunodeficient SCID mice. By using immunofluorescence analysis, we found that progenitor cells from day 16 fetus differentiating in culture lacked MHC class II expression during the Pre-B and immature B cell stages, whereas such expression was readily apparent on the surface of corresponding adult-derived populations. RT-PCR analysis of RNA message levels for the four class II genes (A alpha, A beta, E alpha, E beta) yielded completely concordant results. B cells of fetal progeny did eventually express class II upon further maturation in vivo. Thus, the onset of class II expression is uniquely delayed during fetal B cell differentiation. This result explains an apparent paradox, i.e., that class II expression is absent from B cells in neonatal spleen but present as early as the Pre-B cell stage in adult bone marrow. Furthermore, we suggest that such distinct programs of class II expression during fetal and adult lymphopoiesis could result in differences in susceptibility to tolerance.
Signaling events during helper T cell-dependent B cell activation. I. Analysis of the signal transduction pathways triggered by activated helper T cell in resting B cells.
Authors: Marshall L S , Shepherd D M , Ledbetter J A , Aruffo A , Noelle R J
Published: December 2022

gp39 is expressed on anti-CD3-activated Th, and binds CD40 on the B cell, driving B cell cycle entry. In this study, the signal-transduction pathway initiated in B cells as a consequence of interacting with activated Th is examined. Unlike anti-membrane Ig (anti-mlg) or anti-MHC class II, plasma membranes (PM) isolated from anti-CD3-activated Th, PMAct did not trigger an increase in the B cell intracellular concentrations of cAMP or calcium. In addition, PMAct did not stimulate protein kinase C activation as measured by myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation and protein kinase C translocation. The failure to detect these biochemical events may be caused by the asynchrony with which PMAct induce these normally transient biochemical changes. Alternatively, PMAct may not trigger these events. PMAct did induce the tyrosine phosphorylation of several B cell substrates. Neutralizing Abs directed against gp39 inhibited PMAct-induced protein tyrosine phosphorylation of B cell substrates. These results suggest that cognate interactions in B cells initiate a signal-transduction pathway that is different from the pathway initiated by cross-linking of mlg or MHC class II.
Differential expression of Mtv loci in MHC class II-positive thymic stromal cells.
Authors: Moore N C , Anderson G , McLoughlin D E , Owen J J , Jenkinson E J
Published: December 2022

Despite the important role played by endogenous superantigen in shaping the T cell repertoire, little is known concerning the expression of the different Mtv loci in cells of the thymic microenvironment involved in repertoire selection. Here we have examined the expression of a panel of Mtv Ags by different MHC class II+ stromal cel types using reverse transcriptase-PCR and monitored the effects of these stromal cells on the development of cells expressing Mtv-reactive TCR V beta elements in closed thymic organ culture systems. Although Mtv-6 and Mtv-8/9 mRNAs are expressed in normal thymus lobe organ cultures, no Mtv expression was detected in MHC class II+ thymic epithelial cells. In contrast a striking pattern of differential expression was observed in dendritic cells of thymic origin that were devoid of Mtv-8/9 but expressed readily detectable levels of Mtv-6. This pattern of Mtv gene expression correlated well with TCR V beta repertoire development. TCRV beta 3+ T cells, normally deleted in response to Mtv-6, were virtually absent from the single positive thymocyte compartment in thymic organ cultures where dendritic cells are present but were present in reaggregate cultures where the only MHC class II-positive cells were thymic epithelial cells. On the other hand, V beta 11+ T-cells were not deleted in organ cultures, possibly reflecting the absence of Mtv-8/9 expression in dendritic cells. Our studies suggest that the influence Mtvs have on shaping the T cell repertoire not only depends on their expression within a particular strain but also on their tissue specific expression in relation to MHC class II, which is necessary for their presentation.
An in vitro model for T cell-independent induction of humoral immunity. A requirement for NK cells.
Authors: Snapper C M , Yamaguchi H , Moorman M A , Mond J J
Published: December 2022

We previously established an in vitro polyclonal model for membrane Ig-mediated Ig secretion by B cells in response to T cell-independent type 2 (TI-2) Ags, by using dextran-conjugated anti-IgD Abs (alpha delta-dex). We demonstrated that resting B cells activated with alpha delta-dex plus IL-2 secreted large amounts of Ig only in the presence of NK cells. In this report we show that, in contrast to small B cell-enriched spleen cells that require activation with the combination of alpha delta-dex and IL-2 for induction of Ig secretion, large B cells were induced to secrete Ig in response to alpha delta-dex, alone. These responses were inhibited by previous depletion of asialo-Gm-1+ cells from the B cell-enriched population, and restored both by the addition of freshly explanted NK cells and by in vitro-activated NK cells, suggesting a requirement for NK cells. Small alpha delta-dex-activated B cells could, however, also be stimulated to secrete Ig in the absence of added cytokines but only after the addition of in vitro-activated NK cells and not freshly explanted splenic NK cells. When highly purified large B cells, obtained by electronic cell sorting, were cultured with in vitro-activated NK cells, Ig secretion was induced even in the absence of any other added B cell stimulus. Because the splenic marginal zone has been implicated in humoral immune responses to TI-2 Ags, we further fractionated large B cells into marginal zone (MZB) and follicular (FB) B cells by electronic cell sorting. In vitro-activated NK cells stimulated large MZB, but not FB, cells to secrete Ig in the absence of exogenous stimuli. These data establish a T cell-independent model for induction of Ig synthesis in the absence of any added cytokine and demonstrate a role for freshly explanted NK cells in stimulating Ab production in response to TI-2 Ags. Further, they show that pre-activated NK cells can induce Ig secretion from pre-activated B cells even in the absence of any added stimuli. These data also underscore a special role for the MZB cell in these responses.
MHC-specific recognition of a bacterial superantigen by weakly reactive T cells.
Authors: Surman S , Deckhut A M , Blackman M A , Woodland D L
Published: December 2022

Previous studies have suggested that MHC class II polymorphism can influence the recognition of retroviral superantigen by murine T cells that have an intrinsically weak avidity for the superantigen. The aim of the present study was to determine whether bacterial superantigen recognition also is influenced by MHC polymorphism. Therefore, we screened for TCR with a low avidity for the bacterial superantigen SEB, and identified two V beta elements (V beta 14 and V beta 16) that had not been associated previously with SEB recognition. This finding extends the number of previously identified SEB-reactive V beta elements (V beta 6, V beta 7, V beta 8.1, V beta 8.2, and V beta 8.3) to at least seven. A detailed comparison of SEB recognition by V beta 14+ and V beta 8.2+ T cell hybridomas revealed two interesting features. First, SEB recognition by V beta 14+ hybridomas was relatively weak compared with V beta 8.2+ hybridomas. Second, in contrast to V beta 8.2+ hybridomas, individual V beta 14+ hybridomas responded differentially to SEB presented by either I-Ed or I-Ek molecules on the surface of L cell transfectants, indicating a role for polymorphic residues of the MHC in superantigen presentation. These findings demonstrate that T cell recognition of bacterial superantigens can be influenced by MHC polymorphism in a manner analogous to that of retroviral superantigen recognition, and that this characteristic is a feature of low avidity T cells. Taken together, these data support the hypothesis that there is a direct interaction between the TCR and MHC molecules during superantigen recognition.
Preferential adherence of human gamma delta, CD8+, and memory T cells to fibroblasts.
Authors: White B , Korn J H , Piela-Smith T H
Published: December 2022

Fibroblasts and extracellular matrix produced by fibroblasts provide a framework within solid tissues to which lymphocytes can adhere. Lymphocyte adherence to this framework under basal and inflammatory conditions should contribute to retention of cells involved in innate and specific immunity. The purpose of this work was to determine whether all T cell subsets bind equally well to fibroblast cultures. T cells from healthy humans were co-cultured with autologous or allogenic dermal fibroblast cultures and adherent cells evaluated for surface markers. T cells adherent to untreated autologous fibroblast cultures were enriched for gamma delta T cells, with a striking increase in the V delta 1+ subpopulation. Exposure of the fibroblast cultures to rhIFN-gamma increased the number of adherent T cells and changed the pattern of adherence. Adherent T cells were further enriched for gamma delta T cells but not the V delta 1+ subset, and enriched for CD8+ and memory T cells. These findings suggest that T cell populations that bind to basal and stimulated fibroblast cultures are distinct. There were no qualitative differences in the binding of T cells to autologous and allogeneic fibroblast cultures.
Intron-exon organization and chromosomal localization of the human TIA-1 gene.
Authors: Kawakami A , Tian Q , Streuli M , Poe M , Edelhoff S , Disteche C M , Anderson P
Published: December 2022

TIA-1 is a 40-kDa cytotoxic granule-associated RNA-binding protein (p40-TIA-1), the expression of which is restricted to cytolytic lymphocytes. The major granule-associated species is a 15-kDa protein (p15-TIA-1) that seems to be derived from the carboxyl terminus of p40-TIA-1. Although some evidence suggests that p15-TIA-1 is derived from p40-TIA-1 by proteolytic processing, it is also possible that each isoform is derived from discrete mRNA initiated from alternative promoters within the TIA-1 gene. To learn more about the relationship between p15-TIA-1 and p40-TIA-1, we have determined the complete intron-exon organization of the TIA-1 gene. The gene consists of 13 exons separated by 12 intervening sequences and spans greater than 46 kb of DNA located on chromosome 2, band p13. The transcription start site of the mRNA transcript encoding p40-TIA-1 was identified by primer extension and S1 mapping analysis. The putative promoter region preceding this transcription start site stimulated the expression of a reporter gene in transfected Jurkat and YT cells. Attempts to identify a second promoter capable of initiating an mRNA encoding p15-TIA-1 were unsuccessful, supporting the possibility that p15-TIA-1 can be derived from p40-TIA-1 proteolytically. Granule-associated serine proteases that have been implicated in cytolytic lymphocyte killing (granzyme A and granzyme B) were unable to cleave TIA-1, suggesting that processing may occur before TIA-1 enters the cytotoxic granule.
Identification of a new human V lambda gene family--V lambda X.
Authors: Stiernholm N B , Kuzniar B , Berinstein N L
Published: December 2022

Although approximately 40% of Ig light chains in human sera are of the Ig lambda isotype, until recently very little was known about the human Ig lambda gene locus. The genomic structure of the Ig lambda C region has been determined and consists of seven tandemly organized J-C lambda segments. The structure of the region containing the Ig lambda V gene segments is less well defined. Based on amino acid and nucleotide sequence homologies, the V lambda gene segments isolated have been grouped into nine families. Here we report on the isolation of a V lambda gene from the functional rearrangement of a human B lymphoma cell line that has less than 71% nucleotide sequence and 57% deduced amino acid sequence homology to any known V lambda gene. According to the current classification schemes, this gene does not belong to any existing V lambda family. Thus we suggest that this gene belongs to a new V lambda family, V lambda X. The genomic counterpart of the rearranged V lambda gene, and a second member of the family were isolated. The second member is a pseudogene. Southern analysis suggests that the V lambda X family is a small family. The functional V lambda X gene has a consensus heptamer and a nonamer that differs at two sites from the consensus recombination signal sequence. The promoter region contains a consensus TATA box and an octamer that diverge from the consensus sequence by two base pairs.
Elimination of mouse splenic macrophages correlates with increased susceptibility to experimental disseminated candidiasis.
Authors: Qian Q , Jutila M A , Van Rooijen N , Cutler J E
Published: December 2022

To evaluate the role of macrophages in experimental disseminated candidiasis, mouse splenic macrophages were eliminated by i.v. delivery of liposome-entrapped dichloromethylene diphosphonate (L-Cl2MDP). Splenic tissue sections that were immunoperoxidase-stained with mAbs against marginal zone macrophages (MONTS-4), red pulp macrophages (SK39), and neutrophils (SK208) showed that 3 days after L-Cl2MDP treatment, macrophages but not neutrophils were depleted, and circulating neutrophils responded normally to an irritated peritoneum and showed normal phagocytic ability. That is, in response to thioglycollate in the peritoneum, neutrophils migrated in normal numbers to the peritoneal cavity and expressed the normal activation phenotype of high Mac-1 and low Mel-14 Ag levels. These neutrophils also showed normal ability to ingest Candida albicans yeast cells in both in vitro and in vivo assays. However, the spleens from L-Cl2MDP-treated mice lost their ability to bind yeast, which agrees with our previous findings that yeast cells bind specifically to marginal zone macrophages. When macrophage-depleted were systemically challenged with C. albicans, clearance of viable fungal elements from blood was slower, their kidneys had higher recoverable CFU, and both BALB/cByJ and congenitally thymic-deficient (nude) mouse strains did not survive as long as control mice. Mice given L-Cl2MDP recovered most of their macrophage function by 56 days and became normal in their resistance to C. albicans. These results indicate that macrophages play an important role in host resistance to experimental disseminated candidiasis, but the mechanism does not appear to involve T cell functions.
Sepsis-induced apoptosis of the thymocytes in mice.
Authors: Wang S D , Huang K J , Lin Y S , Lei H Y
Published: December 2022

Intraperitoneal injection of Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa) induces thymic atrophy in mice. The thymus weight, cell number, and viability began to decrease at 3 h, and reached their lowest level at 72 h. The thymocyte death was associated with DNA fragmentation of approximately 200 base pairs in ladder form. The kinetic study on histopathology revealed the process of thymocyte death and thymic atrophy. Flow-cytometric analysis showed that CD4+CD8+ thymocytes decreased predominantly. LPS caused thymocyte apoptosis, but only in LPS-responder mice, unlike Gram-negative bacteria that induced apoptosis in both LPS-responder (C3H/HeN) and LPS-nonresponder (C3H/HeJ). Gram-positive bacteria Streptococcus pneumoniae also caused apoptosis in LPS-nonresponder (C3H/HeJ) and LPS-responder mice (B6). The kinetics of serum TNF-alpha production after Gram-negative or Gram-positive bacteria injection was slightly different. E. coli induced serum TNF-alpha peak at 1 h in B6 mice, whereas S. pneumoniae induced a peak at 6 h in C3H/HeJ and at 9 h in B6 mice. Similarly, S. pneumoniae induced thymocyte apoptosis around 9 to 12 h, which was 6 to 9 h later than that observed with E. coli in B6 mice. Anti-TNF-alpha Ab completely blocked the E. coli-induced thymocyte apoptosis, but was only partially inhibitory on the S. pneumoniae-induced thymocyte apoptosis. Furthermore, thymocyte apoptosis induced by E. coli was inhibited by cycloheximide or actinomycin D. These data indicate that both Gram-negative and Gram-positive bacteria could induce thymus atrophy via apoptosis, and that TNF-alpha is a common denominator released and might be responsible for the thymocyte apoptosis.
Nitric oxide involvement in tumor-induced immunosuppression.
Authors: Lejeune P , Lagadec P , Onier N , Pinard D , Ohshima H , Jeannin J F
Published: December 2022

The mechanisms of immunosuppression induced by colon cancer in rats were investigated at the systemic and tumor levels. During tumor growth (after i.p. injection of rat colon adenocarcinoma cells in syngeneic BD IX rats), Con A-induced proliferation of splenic mononuclear cells decreased and nitric oxide (NO) production by splenic macrophages increased concomitantly. Incubating splenic mononuclear cells with an inhibitor of NO synthase, NG-monomethyl-L-arginine, restored lymphocyte proliferation. A low level of inducible NO synthase mRNA was detectable in tumors by Northern blotting, with a weak increase during tumor growth. The NO concentration measured in the tumor nodules increased weakly parallel to the tumor growth. Five and six weeks after tumor cell injection, tumor-infiltrating lymphocytes from disaggregated tumors did not proliferate in the presence of Con A. Addition of NG-monomethyl-L-arginine inhibited the production of NO in tumor dissociations and enhanced tumor-infiltrating lymphocyte proliferation. Glyceryl trinitrate (a NO-releasing compound) totally inhibited the lymphocyte proliferation in vitro while it slightly reduced the tumor cell proliferation. T lymphocytes were therefore more sensitive to NO than were tumor cells. Culture medium from tumor cells induced NO production by splenic macrophages, although the factor involved has not yet been identified. Furthermore, tumor cells could also play a part in NO production by tumors because the tumor cells were induced to produce NO by IFN-gamma plus IL-1. These results strongly suggest the participation of NO in the tumor-induced immunosuppression in rats.
Heightened intragraft CTL gene expression in acutely rejecting renal allografts.
Authors: Lipman M L , Stevens A C , Strom T B
Published: December 2022

The role of CTL in the immunopathogenesis of acute cellular rejection is controversial. To further define the relationship of activated CTLs to rejection, we analyzed gene expression of three CTL-derived effector molecules in renal allograft biopsies. CTLs are endowed with the ability to promote allograft damage through the elaboration of these highly cytopathic molecules. Intragraft gene transcript levels were determined for granzyme B, perforin, and TIA-1 and correlated with the immunologic status of the allograft as categorized by conventional clinical and histologic criteria. The categories were acute cellular rejection, chronic rejection, elements of both acute and chronic rejection, and no evidence of rejection. Biopsies were snap-frozen, total RNA extracted, and the mRNA converted to cDNA by reverse transcription. Levels were quantitated by competitive template PCR techniques. Intragraft granzyme B and perforin transcripts were highly restricted to biopsies in the acute cellular rejection category. TIA-1 expression was more ubiquitous but significantly higher transcript levels were found in the acute rejection category. The presence of these transcripts in acute cellular rejection samples implicates CTL in the pathogenesis. Moreover, intragraft CTL-specific transcript levels may serve as markers of rejection.
Engagement of the CD45 molecule induces homotypic adhesion of human thymocytes through a LFA-1/ICAM-3-dependent pathway.
Authors: Bernard G , Zoccola D , Ticchioni M , Breittmayer J P , Aussel C , Bernard A
Published: December 2022

Cell-cell interactions play a central role during differentiation and development of the immune system. T or B lymphocyte homotypic adhesions can be induced via several surface molecules which, in some cases, are known to trigger the LFA-1/ICAM-1 adhesion pathway. We show here that mAbs reacting with the CD45 common epitopes or restricted RO epitope lead to a strong and rapid aggregation of all human thymocytes, and of T cell lines with an immature phenotype, but not of peripheral T lymphocytes. Aggregation requires energy, a physiologic temperature, Mg2+ divalent cations, and an intact cytoskeleton. It is LFA-1 dependent because CD11a and CD18 mAbs inhibit homotypic aggregation, whereas CD11b and CD11c mAbs do not. Homotypic thymocyte adhesion, however, is not decreased by CD54 mAb (anti-ICAM-1) but is inhibited by CDw50 mAb (anti-ICAM-3). Soluble CD22 fails to induce thymocyte adhesion, suggesting that CD45-induced aggregation is triggered by another ligand. Finally, because inhibitions observed with mAbs against LFA-1 and ICAM-3 are only partial, it can be assumed that another adhesion pathway is involved in thymocyte adhesion. The adhesion event specific for thymocytes we describe here is likely to play an important role in T cell differentiation.
A minority subpopulation of CD4+ T cells directs the development of naive CD4+ T cells into IL-4-secreting cells.
Authors: Gollob K J , Coffman R L
Published: December 2022

The culture of CD4+ T cells with immobilized anti-CD3 and IL-2 generated a population of cells that produced both IL-4 and IFN-gamma on restimulation. In contrast, CD4+ T cells stimulated with immobilized anti-V beta 6 under otherwise identical culture conditions generated cells that produced IFN-gamma but not IL-4 on restimulation. This difference was likely a result of quantitative differences in the concentration of responding T cells in the two cultures rather than to qualitative differences between the two Abs. Anti-CD3 induced development of IL-4 secreting cells required IL-4 during the primary stimulation. This endogenous IL-4 in primary cultures was produced by cells with a Mel-14low, memory/activated phenotype but not by cells expressing the Mel-14high, naive phenotype. However, co-cultures of Mel-14high and Mel-14low populations marked with different Ly-5 allotypes demonstrated that nearly all of the IL-4-secreting cells that developed from primary cultures were generated from the Mel-14high population. Moreover, Mel-14high T cells could generate IL-4-secreting cells only in the presence of Mel-14low T cells or rIL-4. In addition, co-culture of CD4+, Mel-14low T cells from IL-4-deficient mice with CD4+, Mel-14high T cells from wild-type mice did not lead to the development of IL-4-secreting cells. Thus, IL-4, made by a minority population of previously differentiated CD4+ T cells, can induce the development of IL-4-secreting cells from the naive T cells but naive CD4+ T cells themselves do not develop into IL-4-secreting cells.
Costimulator dependence of lymphokine secretion by naive and activated CD4+ T lymphocytes from TCR transgenic mice.
Authors: McKnight A J , Perez V L , Shea C M , Gray G S , Abbas A K
Published: December 2022

The role of costimulation in Ag-induced responses of naive and differentiated IL-2 and IL-4-producing CD4+ T cells has been examined using cells from mice expressing a transgenic TCR specific for cytochrome c (81-104) + I-Ek. IL-2 secretion by naive and pre-activated T cells is dependent on costimulation, because it is not induced with chemically fixed APCs and is significantly inhibited by the B7 antagonist, CTLA4-Ig. In contrast, IL-4 secretion by in vitro differentiated Th2-like cells is relatively independent of costimulators. Thus, the requirement for costimulation is related more to lymphokine secretion profiles than to the previous activation status of CD4+ T lymphocytes.
Ligation of MHC class I and class II molecules can lead to heterologous desensitization of signal transduction pathways that regulate homotypic adhesion in human lymphocytes.
Authors: Wagner N , Engel P , Vega M , Tedder T F
Published: December 2022

Engagement of lymphocyte MHC class I and class II Ags activates an array of intracellular signal transduction pathways that up-regulates the activity of cell-surface adhesion receptors, resulting in homotypic cell-cell aggregation. In this study, engagement of MHC class I and class II molecules with specific mAbs was shown to also inhibit lymphocyte homotypic adhesion. Two mAbs reactive with class II Ag, homotypic adhesion blocking mAb (HAB)-2, and HAB-3, and one mAb reactive with class I Ag, HAB-4, were generated that inhibited homotypic adhesion of activated lymphocytes and B and T cell lines at concentrations as low as 0.1 microgram/ml. Binding of these mAbs resulted in heterologous desensitization of other surface signal transduction molecules as homotypic adhesion induced through class I, class II, CD19, CD20, CD39, CD40, Leu-13, and PMA was also inhibited. The spontaneous adhesion exhibited by some cell lines was also abrogated by binding of these mAbs. Abs that either induced, blocked, or had no effect on adhesion bound to distinct epitopes on class I, whereas the anti-class II mAbs recognized either distinct or overlapping epitopes. Thus, engagement of distinct epitopes on MHC molecules can result in homologous or heterologous desensitization of cell-surface signaling molecules. The induction or inhibition of homotypic adhesion through class I molecules did not require the presence of the cytoplasmic domain, as deletion of this portion of the class I molecule had no effect. In contrast, the transmembrane region was essential for signal transduction as the mAbs binding to a chimeric molecule in which the transmembrane and cytoplasmic domains of class I were exchanged with those of the HB15 molecule did not induce or inhibit homotypic adhesion. Although this report is the first demonstration that homotypic adhesion can be influenced in a negative manner through MHC molecules, these findings demonstrate a considerable level of cross-talk between MHC molecules and other cell-surface receptor systems and their signal transduction pathways, and suggests that MHC class I and class II molecules may serve important roles in the regulation of adhesive events during lymphocyte activation.
Critical binding site amino acids of anti-Z-DNA single chain Fv molecules. Role of heavy and light chain CDR3 and relationship to autoantibody activity.
Authors: Polymenis M , Stollar B D
Published: December 2022

Bacterial expression of single chain variable fragment (scFv) domains was used to assess Ag-binding contributions of specific regions and residues in a mouse mAb to Z-DNA (AbZ22). A variant scFv (Z3-3) that did not bind Z-DNA had the Z22 light chain but differed from the Z22 heavy chain at four complimentarity determining region 3 (CDR3), one FR4 and five VH segment residues. Gene segment swapping and site-directed mutagenesis indicated that the major contribution of the Z22 heavy chain is its CDR3. A scFv with the CDR3H-FR4H of Ab Z22 and the VH segment of Z3-3 had the same selective high affinity Z-DNA binding as Z22. Some Z-DNA binding was retained even when the CDR3H-FR4H of Ab Z22 was combined with a VH segment that shared only 44% sequence identity with Z22. Directed mutations indicated further that residues N99 and S98 in heavy chain CDR3 and F96 in light chain CDR3 were particularly important for Ag binding. Certain substitutions in CDR3H converted the highly selective Z22 Fv into a polyreactive Fv with autoantibody-like binding to B-DNA and denatured DNA. In a graphic molecular model, heavy chain N99 protrudes from the CDR3 loop at the base of the Ag-binding groove, and the light chain F96 is barely exposed on the base of this groove; the light chain F96 may be important in heavy chain-light chain association. Autoantibody and immunization-induced Ab to nucleic acid can be built on a very similar framework and differ by a small number of amino acid CDR3H residues.
A method for linking VL and VH region genes that allows bulk transfer between vectors for use in generating polyclonal IgG libraries.
Authors: Sarantopoulos S , Kao C Y , Den W , Sharon J
Published: December 2022

Libraries of Ab fragments have been produced by others from light and heavy chain cDNAs derived from populations of B lymphocytes and expressed in bacteria. However, such libraries have not been transferred to eukaryotic expression vectors to generate polyclonal libraries of intact glycosylated Abs that can mediate effector functions. We present a method for transferring pairs of linked VL-VH region genes between circular prokaryotic and eukaryotic vectors. The key feature of the transfer is that the VL and VH region genes are linked head to head (<-->) in opposite transcriptional orientations. To illustrate this method, a pair of VL and VH region cDNAs derived from an existing hybridoma cell line were linked head to head by PCR, transferred as a unit between vectors, and expressed as an IgG Ab with Ag binding activity. Although we tested the transfer of a single VL-VH region gene pair, this system is expected to allow the bulk transfer of physically linked VL-VH region gene combinations between different circular vectors and the expression of the same library as either Ab fragments or intact Abs.
Zymosan-induced IL-8 release from human neutrophils involves activation via the CD11b/CD18 receptor and endogenous platelet-activating factor as an autocrine modulator.
Authors: Au B T , Williams T J , Collins P D
Published: December 2022

We have investigated mechanisms that regulate the generation of IL-8 by human neutrophils on contact with zymosan particles in vitro. Zymosan stimulated IL-8 production, which increased with increasing particle numbers and was abolished by the protein synthesis inhibitor cycloheximide. IL-8 was detectable in culture supernatant at 8 h reaching a maximum at 24 h. In all further experiments IL-8 was measured at 24 h. mAbs to neutrophil CD18 (60.3 and 6.5E) caused a marked suppression of IL-8 generation, but only if added up to 2 h after zymosan stimulation. An anti-CD11b mAb (KIM 225) substantially inhibited zymosan-induced IL-8 release. We investigated whether other mediators generated during phagocytosis modulate IL-8 production. Two selective platelet-activating factor (PAF) receptor antagonists, WEB 2086 and UK 74505, produced a profound suppression of IL-8 generation, when added within 30 min to 1 h of zymosan stimulation. An IL-1R antagonist, a leukotriene B4 antagonist, and an anti-TNF-alpha Ab had no effect on IL-8 generation. FMLP, PAF, and a stable PAF agonist did not stimulate significant IL-8 production, however, a calcium ionophore (A23187) did induce IL-8 release and this was suppressed by UK 74505. We conclude that zymosan-induced IL-8 generation involves stimulation of the neutrophil via a CD11b/CD18 receptor resulting in beta 2-integrin mediated activation of signal transduction mechanisms that leads to cytokine synthesis. Furthermore, endogenously generated PAF, or a PAF, or a PAF-like molecule, appears to have an autocrine function in regulating this pathway of IL-8 production at an early stage after the interaction between the neutrophil and the particles.
Laminin peptides stimulate human neutrophil motility.
Authors: Harvath L , Brownson N E , Fields G B , Skubitz A P
Published: December 2022

Laminin, isolated from Engelbreth-Holm-Swarm tumor, and 10 chemically synthesized peptides, corresponding to various regions of the laminin A and B1 chains, were compared for their abilities to stimulate human peripheral blood polymorphonuclear leukocyte (PMN) chemotaxis and chemokinesis through polycarbonate membrane filters in a 48-well microchemotaxis assay. Peptides F-9, F-11, F-12, and F-13 were derived from the B1 chain of laminin at the intersection of the cross, and six peptides were derived from the laminin A chain: peptide TG-1 from the amino-terminal top globule; peptides GD-1, GD-3, GD-6, and GD-7 from the carboxyl-terminal globular domain; and peptide AG-1 from above the carboxyl-terminal globular domain. Laminin and the peptides were evaluated over a concentration range of 1 to 200 micrograms/ml in motility assays. Six of the peptides, F-9, F-12, GD-1, GD-3, GD-6, and TG-1, stimulated human PMN migration in the absence of a gradient (chemokinesis). A fluorescein conjugate of the most active laminin peptide, GD-1, exhibited nonspecific, nonsaturable binding to PMN. Intact laminin and the other peptides failed to stimulate human PMN migration. In contrast, intact Engelbreth-Holm-Swarm laminin stimulated rabbit peripheral blood PMN chemokinesis. These results demonstrate that rabbit and human peripheral blood PMNs have divergent migratory responses to intact laminin. These findings suggest that intact basement membrane laminin does not directly stimulate human blood PMN motility in vivo, but that selected laminin peptide sequences, which may be generated during proteolytic digestion of laminin, can activate human PMN migration.
Soluble recombinant complement receptor 1 inhibits inflammation and demyelination in antibody-mediated demyelinating experimental allergic encephalomyelitis.
Authors: Piddlesden S J , Storch M K , Hibbs M , Freeman A M , Lassmann H , Morgan B P
Published: December 2022

Activation of complement may contribute to tissue damage in many inflammatory diseases, including those that are clearly T cell driven. We have previously provided evidence that C is involved in tissue damage in multiple sclerosis and in the animal models of this disease, experimental allergic encephalomyelitis and Ab-mediated demyelinating experimental allergic encephalomyelitis, the latter being a model more closely resembling multiple sclerosis. The development of a soluble recombinant form of human complement receptor 1 (sCR1) with potent C-inhibiting activity both in vitro and in vivo provides a potential means of preventing C-mediated tissue damage in animal models and in human disease. Here, we describe the effects of this agent on clinical disease and pathology in Ab-mediated demyelinating experimental allergic encephalomyelitis in the rat. Daily i.p. injection of sCR1 (20 mg/kg) over 6 days completely suppressed serum C activity, reduced the severity of clinical disease (clinical score 1.33 vs 2.79 in untreated animals), inhibited central nervous system inflammation (inflammatory index 2.76 vs 6.55), and almost completely blocked demyelination (average 2.43% cord cross-section vs 8.81%). Deposition of C components C1, C3, and C9 was also markedly inhibited in sCR1-treated animals. This dramatic effect on a demyelinating disease, achieved using a well-tolerated biologic reagent, offers an exciting new prospect for therapy in multiple sclerosis.
Functional cytotoxic T cells are associated with focal lesions in the brains of SJL mice with experimental herpes simplex encephalitis.
Authors: Hudson S J , Streilein J W
Published: December 2022

Using a recently developed murine model of herpes simplex encephalitis in SJL mice, we isolated and characterized brain-infiltrating mononuclear cells to identify immune effectors that might contribute to pathology in this disease. Brain infiltrating mononuclear cells obtained from temporal area lesions were found to be predominantly T cells, with CD4+ and CD8+ T cell types present in equal numbers. The herpes simplex virus type 1 (HSV-1)-specific CTL that were present within the temporal area of HSV-1 infected SJL mice were fully activated (i.e., they did not require in vitro culture to acquire cytotoxic function). These cells were Thy-1.2+, CD8+ and exhibited the classical features of CTL activity: Ag specificity and MHC restriction. To our knowledge this is the first definitive demonstration of HSV-1-specific CTL activity directly ex vivo in mice. Freshly isolated spleen or lymph node cells from the same mice failed to exhibit CTL activity. The lytic capacity of infiltrating cells appeared to be mediated exclusively by CTL because treatment with Abs to Thy-1.2, or CD8, and C completely removed cytotoxic activity. NK cell activity was not detected in spleen, lymph node, or brain-infiltrating mononuclear cells from SJL mice with HSV encephalitis. Significantly, T cells were the prominent cell type in the temporal area of HSV-1-infected SJL mice just before the development of focal lesions, with CD8+ cells being present in equal or greater numbers than CD4+ cells (a CD4:CD8 ratio of 1 < or = 1). Thus, the accumulation of functional CD8+ CTL in the temporal area of the brain correlated with the occurrence of focal entorhinal lesions.
Activation induces sensitivity toward APO-1 (CD95)-mediated apoptosis in human B cells.
Authors: Daniel P T , Krammer P H
Published: December 2022

We have previously described the APO-1 (CD95) cell surface molecule, a novel member of the nerve growth factor/TNF receptor superfamily, identical with the Fas Ag. Triggering of APO-1-induced apoptosis in APO-1+, apoptosis sensitive cells. The data in this study demonstrate that human peripheral blood B cells acquire sensitivity to APO-1-mediated apoptosis on PWM activation. To also study APO-1-mediated apoptosis in an Ag-specific human B cell response, we reconstituted severe combined immunodeficient (SCID) mice i.p. with human PBMC (SCID-hu mice). SCID-hu mice were then injected i.p. with soluble, adjuvant-free tetanus toxoid or diphtheria toxoid, respectively, directly after transfer of the PBMC (day 1) and received an Ag-booster injection on day 14. Two weeks after PBMC transfer human Abs were detected and shown to be Ag specific. SCID-hu mice co-injected with monoclonal anti-APO-1 Ab (IgG3,k) showed significantly suppressed anti-tetanus toxoid or anti-diphtheria toxoid titers, respectively. Sensitivity to anti-APO-1-mediated suppression was only observed in Ag-activated cells from day 0 to day 6 after Ag immunization. Suppression of Ab titers correlated with a decrease of Ag-specific Ig-secreting cells. Re-injection of syngeneic T cells and Ag did not reverse anti-APO-1-induced suppression. These data show a direct suppressive effect of anti-APO-1 on Ag-activated, "specific" B cells. Thus, APO-1-mediated apoptosis in Ag-activated B cells may contribute to the regulation of the humoral immune response.
IL-4 treatment of small splenic B cells induces costimulatory molecules B7-1 and B7-2.
Authors: Stack R M , Lenschow D J , Gray G S , Bluestone J A , Fitch F W
Published: December 2022

IL-4 has been shown to be involved in the early stages of B cell maturation. Changes induced by IL-4 include cell enlargement, increased viability, and increased MHC class II expression. However IL-4 alone does not induce B cell activation as defined by proliferation, lymphokine production, or Ig class switching. In this study, we demonstrate that incubation with IL-4 enhances the ability of small splenic murine B cells, normally poor stimulators of murine Th1 clones, to stimulate lymphokine production and proliferation by Th1 clones. Moreover, small resting B cells induce anergy, whereas IL-4-treated B cells do not. IL-4-treated B cells were found to express both B7 (B7-1) and a second ligand for CTLA4Ig (B7-2). Although IL-4 induces both B7-1 and B7-2, the kinetics of expression of these molecules are different: B7-2 is detected by 6 h, whereas B7-1 is not detectable until 48 h. In addition, only CTLA4Ig fully blocks IL-4 induced costimulatory activity; a mAb to B7-1 does not. Thus, these results suggest that IL-4 may function indirectly as a costimulatory factor by inducing costimulatory molecules on resting B cells. Additionally, these findings support our previous findings that an alternative ligand for CD28 and CTLA4 is important in providing costimulation.
HLA-DR3 molecules can bind peptides carrying two alternative specific submotifs.
Authors: Geluk A , van Meijgaarden K E , Southwood S , Oseroff C , Drijfhout J W , de Vries R R , Ottenhoff T H , Sette A
Published: December 2022

Three different HLA-DR3-specific peptide binding motifs have been proposed. These motifs shared a major hydrophobic anchor at the N-terminus, but differed in the C-terminal anchor residues. In the present study, the structural requirements for peptide binding to HLA-DR3 were examined in further detail by using quantitative HLA-DR3-specific binding assays and sets of single substitution analogues of DR3 binding peptides (Lol pollen amino acids 171-190 and sperm whale myoglobin amino acids 132-151). We found that the requirements for binding to HLA-DR3 vary among different DR3 binding peptides; the absence of an anchor or the presence of only a weak anchor residue at either position n or n + 3 can be compensated for by the presence of a strong, positively charged anchor residue at position n + 5. These results explain several of the previously reported differences between DR3-specific peptide binding motifs. To evaluate the predictive value of the thus-refined motif, the DR3 binding capacity of an overlapping set of peptides, spanning the entire sequence of the 65-kDa heat shock protein of Mycobacterium tuberculosis was investigated and correlated with the occurrence of the different DR3 motifs. A strong correlation was found between the presence of the refined DR3 motif and peptide binding to purified HLA-DR3 molecules.
Nitric oxide production by splenic macrophages is not responsible for T cell suppression during acute infection with lactate dehydrogenase-elevating virus.
Authors: Rowland R R , Butz E A , Plagemann P G
Published: December 2022

Cellular immune responses of mice are transiently suppressed during acute infection with lactate dehydrogenase-elevating virus (LDV). Immunosuppression of mice correlated with a greatly impaired in vitro proliferative response of the majority of the T cells to Con A or anti-CD3 Abs, which could not be reversed by the addition of rIL-2. We have examined whether the T cell suppression is caused by nitric oxide (NO) produced by activated macrophages, which are observed in acutely infected mice. Spleen macrophages from 3-day LDV-infected mice exhibited a 6- to 10-fold increased potential for producing NO, measured as nitrite or nitrite plus nitrate in the culture fluid, but produced significant amounts of NO in vitro only when incubated with IFN-gamma produced by Con A-stimulated T cells in the spleen cell population. Furthermore, we found that the concentrations of NO produced by macrophages in cultures of spleen cells from LDV-infected mice in the presence of IFN-gamma were insufficient to cause a reduction in the proliferative response of T cells in the spleen cell population. An excess of activated macrophages had to be added to achieve T cell suppression. NO inhibition of Con A-induced T cell proliferation exhibited a very sharp dose-response curve. In one experiment little suppression was observed at NO concentrations equivalent to 12 microM nitrite and below, whereas almost complete inhibition was observed at twice the NO concentration. We conclude that NO is not responsible for T cell suppression in LDV-infected mice.