bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectratyping of TCRs expressed by CTL-infiltrating minor histocompatibility antigen-disparate allografts.
Authors: Johnston S L , Borson N D , Wettstein P J
Published: December 2022

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this process. However, there is no information on the diversity of TCRs that are specific for single minor histocompatibility Ag peptides and expressed by CTLs in vivo. We have used the technique of spectratyping to study the diversity of Vbeta usage and beta complementarity-determining region 3 (CDR3) length of TCRs expressed by CTL-infiltrating skin allografts expressing the immunogenic H4 peptide during the process of rejection. Spectratyping revealed overall reduction in diversity of both Vbeta usage and CDR3 length, with sequential application of primary, second-set, and third-set H4-incompatible grafts. This dissection of the array of beta-chains expressed by graft-infiltrating CTLs allowed the direct sequencing of individual beta-chain PCR products. Beta CDR3s were characterized by a net negative charge, as we have observed previously with CDR3s expressed by H4-specific CTL clones selected in vitro. Identical and closely related CDR3 amino acid sequences could be identified that were shared by TCRs that 1) utilized different Vbeta genes, 2) derived from different mice, or 3) derived from different sequential sets of allografts on individual mice. Furthermore, a number of CDR3 sequences expressed by graft-infiltrating CTLs were identical or closely related to sequences we have identified previously in in vitro selected CTL clones that were specific for the H4 peptide.
The p38 mitogen-activated protein kinase is activated by ligation of the T or B lymphocyte antigen receptors, Fas or CD40, but suppression of kinase activity does not inhibit apoptosis induced by antigen receptors.
Authors: Salmon R A , Foltz I N , Young P R , Schrader J W
Published: December 2022

We have investigated the activation of the p38 mitogen-activated protein kinase (MAPK) in normal mouse T and B cells and its role in apoptosis. Cross-linking of the CD3 chains of the TCR complex on proliferating T cells resulted in activation of p38 MAPK and MAPKAP kinase-2. Cross-linking of CD28 failed to activate p38 MAPK or MAPKAP kinase-2, but synergized strongly with low doses of anti-CD3. Cross-linking of Fas on T cells also induced rapid activation of p38 MAPK and MAPKAP kinase-2. The in vivo activation of MAPKAP kinase-2 in response to cross-linking of CD3, Fas, or CD3 and CD28 was shown to be dependent on p38 MAPK activity using a specific inhibitor, SB 203580. SB 203580 did not inhibit activation-induced cell death in T cells when used at concentrations that suppressed activation of MAPKAP kinase-2 in vivo. Cross-linking of the B cell Ag receptor (BCR) or CD40 on freshly isolated or LPS-activated splenic B cells or the immature B lymphoma, WEHI 231, resulted in activation of p38 MAPK and MAPKAP kinase-2. In vivo inhibition of p38 MAPK activity in WEHI 231 cells by SB 203580 had no effect on either BCR-induced apoptosis or anti-CD40-mediated suppression of apoptosis. We conclude that the activation of p38 MAPK and MAPKAP kinase-2 by cross-linking of the TCR, BCR, Fas, or CD40 was not correlated with their roles in regulating lymphocyte survival, and that suppression of kinase activity did not inhibit the induction of apoptosis.
T cell stimulation via CD47: agonistic and antagonistic effects of CD47 monoclonal antibody 1/1A4.
Authors: Waclavicek M , Majdic O , Stulnig T , Berger M , Baumruker T , Knapp W , Pickl W F
Published: December 2022

CD47/integrin-associated protein has been extensively studied on various cell types. The function of CD47 on T cells, however, remained poorly understood. We demonstrate here that our CD47 mAb 1/1A4 has both inhibitory as well as costimulatory effects in terms of T cell activation. Soluble, not cross-linked, CD47 mAb 1/1A4 blocks allogeneic MLRs. This effect is predominantly observed with suboptimal numbers of stimulator cells. In contrast, cross-linking of CD47 in the presence of CD28 mAb or phorbol ester induces vigorous T cell proliferation that is sensitive to cyclosporin A. Cross-linking, but not immobilization, of the CD47 mAb 1/1A4 is an essential requirement for the CD28- or phorbol ester-dependent induction of T cell mitogenesis. Furthermore, CD47 mAb 1/1A4 induces T cell proliferation when coimmobilized with a CD3 mAb to the same surface. Ligation with cross-linked 1/1A4 mAb induces an increase in intracellular free calcium levels and leads to tyrosine phosphorylation of various cellular proteins and, in the presence of suboptimal concentrations of plate-bound CD3 mAb, to enhanced IL-2 promotor activity in T cells. Furthermore, we present evidence that upon cross-linking of CD47 with mAb 1/1A4, purified T cells acquire responsiveness for several T cell growth factors. IL-1beta and/or IL-6 dramatically augment this CD47-induced cytokine responsiveness. Our results suggest that the novel activation pathway via CD47 might be critically involved in Ag-dependent and Ag-independent T cell functions.
Adoptive transfer of in vitro-targeted, activated T lymphocytes results in total tumor regression.
Authors: Altenschmidt U , Klundt E , Groner B
Published: December 2022

Adoptive immunotherapy of cancer uses the transfer of tumor-reactive immune cells. The success of this procedure is dependent upon the specificity of the transferred immune cells, their number, and their ability to reach their target cells. We genetically modified T lymphocytes and equipped them with the ability to specifically recognize tumor cells. Tumor cells overexpressing the ErbB-2 receptor served as a model. The target cell recognition specificity is conferred to T lymphocytes by transduction of a chimeric gene encoding the zeta-chain of the TCR and a single chain antibody (scFv(FRP5)) directed against the human ErbB-2 receptor. The chimeric scFv(FRP5)-zeta gene was introduced into primary mouse T lymphocytes via retroviral gene transfer. Naive T lymphocytes were activated and infected by cocultivation with a retrovirus-producing packaging cell line. The scFv(FRP5)-zeta fusion gene was expressed in >75% of the T cells. These T cells lysed ErbB-2-expressing target cells in vitro with high specificity. We tested the antitumor efficacy of scFv(FRP5)-zeta-expressing T cells in a syngeneic BALB/c model. The mice were treated with autologous, transduced T cells. The adoptively transferred scFv(FRP5)-zeta-expressing T cells caused total regression of ErbB-2-expressing tumors. The presence of the transduced T lymphocytes in the tumor tissue was monitored. No humoral response directed against the transduced T cells was observed. Abs directed against the ErbB-2 receptor were detected upon tumor lysis.
TCR vaccines for active immunotherapy of T cell malignancies.
Authors: Okada C Y , Wong C P , Denney D W , Levy R
Published: December 2022

We have developed a TCR-based vaccine approach for the treatment of T cell malignancies. TCR genes were isolated from C6VL, a T cell tumor of C57BL/Ka origin. The transmembrane encoding domains of the TCR genes were replaced by sequences encoding for phosphatidylinositol-linked cell surface expression. A high expressing cell line was produced by transfection and amplification of the TCR genes. Large quantities of soluble native C6VL TCR-alphabeta protein was obtained by treating the high-expressing cells with a specific phospholipase and purifying the released TCR by affinity chromatography. Following vaccination with the TCR linked to keyhole limpet hemocyanin, specific anti-TCR humoral responses were induced. Both the carrier protein and an adjuvant were required for optimal responses. Hyperimmune serum from vaccinated mice reacted specifically with C6VL cells, and the immunizations did not affect the TCR repertoire, which suggested that the immune response was Id specific. The TCR-vaccinated mice were specifically protected from a lethal number of C6VL tumor cells. B cell-deficient mice were not protected by TCR vaccinations. Similarly, TCR-immunized mice depleted of CD8+ cells prior to tumor challenge were not protected. Thus, C6VL TCR vaccine effectively stimulated tumor protection, which depends on the presence of both B cells and CD8+ T cells.
Beta2 integrins are not required for tyrosine phosphorylation of paxillin in human neutrophils.
Authors: Fernandez R , Boxer L A , Suchard S J
Published: December 2022

Although many of the receptors mediating neutrophil (polymorphonuclear leukocyte (PMN)) adhesion to extracellular matrix (ECM) proteins have been identified, the signal transduction pathways leading to cell spreading are not clearly defined. Studies showing that protein tyrosine phosphorylation increases with PMN adhesion suggest that phosphorylation is critical for adhesion-dependent processes. In other cells, increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin are thought to play pivotal roles in ECM-mediated signaling. In the present study we compared the tyrosine phosphorylation of FAK and paxillin in PMNs plated onto thrombospondin (TSP) or laminin (LN). Increased tyrosine phosphorylation of paxillin correlated with adhesion and spreading of PMNs plated onto LN or TSP. In PMNs adherent to LN, the kinetics of FAK phosphorylation preceded the kinetics of paxillin phosphorylation, suggesting that paxillin may be a downstream target of FAK. In contrast, FAK was not phosphorylated in PMNs spread on TSP. These findings suggested activation of different signaling pathways in TSP- vs LN-adherent PMNs. Others have proposed that paxillin phosphorylation requires beta2 integrins. Therefore, we used PMNs from a patient with leukocyte adhesion deficiency (LAD), lacking beta2 integrins, to determine whether these receptors were necessary for paxillin phosphorylation. LAD PMNs did not adhere to LN, and consequently, there was no tyrosine phosphorylation of FAK or paxillin. In contrast, paxillin, but not FAK, was phosphorylated in LAD PMNs adherent and spread on TSP. These results indicate that ECM-beta2 integrin-mediated signaling initiates the FAK/paxillin signaling pathway(s), while TSP-mediated signaling results in paxillin phosphorylation independent of FAK phosphorylation.
Characterization of the CCR2 chemokine receptor: functional CCR2 receptor expression in B cells.
Authors: Frade J M , Mellado M , del Real G , Gutierrez-Ramos J C , Lind P , Martinez-A C
Published: December 2022

We have derived anti-human CCR2-specific mAbs by immunization with synthetic peptides corresponding to CCR2 sequences presumably involved in the interaction with its ligand(s). The characterization of these mAbs includes the ability to recognize the CCR2 receptor specifically, as well as the function based on their ability to promote Ca2+ influx or to block MCP-1-induced Ca2+ influx and chemotaxis. One mAb (MCP-1 R02) that is directed to the NH2 terminal domain of the CCR2 receptor has MCP-1 agonist activity, and two that recognize the third extracellular domain (MCP-1R04 and MCP-1 R05) have MCP-1 antagonist activity. We analyzed the presence of CCR2 in several PBL and tonsil-derived leukocyte populations and found expression of this receptor in monocytes, activated T cells, and, surprisingly, in B cells. CCR2 receptor expression in B cells was further corroborated in Southern blot using CCR2-specific probes. Moreover, both MCP-1 and the agonist mAb trigger specific B cell migration via a PTX-sensitive mechanism, indicating the presence of a functional CCR2 receptor in these cells.
Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies.
Authors: Heijnen I A , Rijks L J , Schiel A , Stockmeyer B , van Ojik H H , Dechant M , Valerius T , Keler T , Tutt A L , Glennie M J , van Royen E A , Capel P J , van de Winkel J G
Published: December 2022

Abs are able to induce inflammatory antitumor responses by recruiting IgG Fc receptor (FcgammaR)-bearing cytotoxic effector cells. We recently described the capacity of the high affinity FcgammaRI (CD64) to trigger cytotoxic activity of neutrophils (PMN) during granulocyte CSF (G-CSF) treatment. To take advantage of FcgammaRI as a cytotoxic trigger molecule on PMN, two Ab constructs were prepared. We show that a chimeric human IgG1 Ab (Ch520C9) and an anti-FcgammaRI bispecific Ab (BsAb; 22x520C9), both directed to the proto-oncogene product HER-2/neu, interact with FcgammaRI. In addition, both Ab constructs mediate enhanced lysis of HER-2/neu-expressing tumor cells by G-CSF-primed PMN. However, engagement of FcgammaRI by Ch520C9 was inhibited by human serum IgG, thereby abrogating the enhanced Ch520C9-mediated cytotoxicity. BsAb 22x520C9, which binds FcgammaRI outside the ligand binding domain, effectively recruits the cytotoxic potential of FcgammaRI on G-CSF-primed PMN regardless of the presence of human serum. These results indicate that under physiologic conditions, serum IgG impairs activation of FcgammaRI-mediated cytotoxicity by conventional antitumor Abs. The IgG blockade can be circumvented with anti-FcgammaRI BsAbs. Using human FcgammaRI transgenic mice we demonstrate that BsAb 22x520C9 is able to engage FcgammaRI in vivo. BsAb 22x520C9 injected i.v. was readily detected on circulating PMN of G-CSF-treated transgenic animals. In addition, we showed that PMN remain "armed" with BsAb 22x520C9 during migration to inflammatory sites, and that after isolation such PMN specifically lyse HER-2/neu-expressing tumor cells. These results point to the possibility of targeting anti-FcgammaRI BsAbs to G-CSF-primed PMN in vivo, endowing them with specific anti-tumor activity.
IL-11 regulates macrophage effector function through the inhibition of nuclear factor-kappaB.
Authors: Trepicchio W L , Wang L , Bozza M , Dorner A J
Published: December 2022

Recombinant human IL-11 (rhIL-11) is an anti-inflammatory cytokine that can reduce the production of inflammatory mediators such as TNF-alpha, IL-1beta, IL-12, IL-6, and nitric oxide. Inhibition of proinflammatory cytokine production from activated macrophages was associated with a reduction in the levels of LPS-induced TNF-alpha, IL-1beta, IL-6, and IL-12 p40 mRNA. Analysis of rhIL-11 effects on transcription factors that activate proinflammatory cytokines demonstrated that the level of LPS-induced NF-kappaB binding activity in the nucleus of rhIL-11-treated peritoneal macrophages was significantly reduced. The block to NF-kappaB nuclear translocation correlated with the ability of rhIL-11 to maintain or increase protein levels of the inhibitors of NF-kappaB, IkappaB-alpha, and IkappaB-beta following LPS treatment. Furthermore, rhIL-11-treatment of LPS macrophages resulted in significant elevation of IkappaB-alpha and IkappaB-beta mRNA levels. These results suggest that the anti-inflammatory activity of rhIL-11 is mediated in part by inhibition of NF-kappaB-dependent transcriptional activation. Furthermore, these studies demonstrate for the first time the regulation of IkappaB-beta by an anti-inflammatory cytokine. Given the finding that inappropriate activation of NF-kappaB contributes to multiple inflammatory conditions, the ability of rhIL-11 to inhibit the binding activity of this pleiotropic transcription factor indicates that rhIL-11 has therapeutic potential in a wide range of diseases.
Identification of a new mouse beta-chemokine, thymus-derived chemotactic agent 4, with activity on T lymphocytes and mesangial cells.
Authors: Tanabe S , Lu Z , Luo Y , Quackenbush E J , Berman M A , Collins-Racie L A , Mi S , Reilly C , Lo D , Jacobs K A , Dorf M E
Published: December 2022

Thymus-derived chemotactic agent 4 (TCA4), a new member of the beta-chemokine family, was cloned from a mouse thymic cDNA library. High levels of TCA4 mRNA are expressed in thymus; lower levels of message are found in spleen, heart, and kidney. Anti-TCA4 antibodies were used to localize sites of TCA4 expression within lymphoid tissues. In the thymus, UEA-1+ medullary epithelial cells, some endothelial cells, and additional undefined stromal elements were stained with anti-TCA4. TCA4 was also expressed as a meshlike network in splenic white pulp and in the medullary region of the lymph nodes. In addition, some lymph node and splenic blood vessels stained with anti-TCA4 antibodies. Rel B NFkappaB-deficient mice lack a transcription factor required for the generation of dendritic cells and the development of an organized thymic medulla. Rel B-deficient animals express very low levels of TCA4 in the thymus and little or no TCA4 in the periphery. At subnanomolar concentrations, TCA4 is a chemoattractant of mature T cells; the potential role of this novel chemokine in facilitating normal lymphocyte traffic is discussed. TCA4 is also a chemoattractant of cultured mesangial cells. Neutralizing anti-TCA4 mAb was used to demonstrate the specificity of TCA4-mediated cell migration. Finally, competitive binding studies with a SV40-transformed mouse mesangial cell line demonstrated that other murine beta-chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and thymus-derived chemotactic agent 3) do not compete for TCA4 binding.
Priming with IL-4 and IL-13 during HIV-1 infection restores in vitro IL-12 production by mononuclear cells of HIV-infected patients.
Authors: Marshall J D , Robertson S E , Trinchieri G , Chehimi J
Published: December 2022

The production of proinflammatory cytokines can be regulated by several factors that exert activating or inhibitory effects. IL-4, IL-10, IL-13, TGF-beta, and PGE2 have demonstrated a very wide range of potent macrophage-deactivating activities and, specifically, down-regulation of the production of many proinflammatory monokines. IL-12 plays a key role during immune response by providing a link between natural resistance and adaptive immunity. We and others have previously shown an impairment in IL-12 production by PBMC from HIV-1-infected individuals in response to various stimuli, but defining the mechanism responsible remains elusive. In this study, we observed that pretreatment of PBMC from patients with IL-4 or IL-13 for 24 h primes the cells for enhanced production of IL-12 in response to Staphylococcus aureus, and almost completely restores their deficient IL-12 production when compared with healthy controls. Although this priming effect was completely abrogated by IL-10 and PGE2, IL-10 was produced equivalently by untreated and IL-4- or IL-13-pretreated PBMC from both patients and controls. Additionally, indomethacin, which shuts off PGE2 synthesis, and cAMP-blocking reagents failed to restore or enhance IL-12 production. The priming effect of IL-4 and IL-13 is at the transcription level for both p40 and p35 genes. This complete restoration of IL-12 production by Th2-associated cytokines was unexpected in light of the mutually antagonistic roles of IL-12 and IL-4 in promoting Th1 or Th2 immune responses.
Telomerase is up-regulated in human germinal center B cells in vivo and can be re-expressed in memory B cells activated in vitro.
Authors: Hu B T , Lee S C , Marin E , Ryan D H , Insel R A
Published: December 2022

The extensive proliferation that B lymphocytes undergo in germinal centers could compromise generation of long term B cell memory if there occurs shortening of the telomeres of germinal center B cells with cell division. Telomere length, which is thought to act as a "mitotic clock" for somatic cells that dictates cellular senescence, can be preserved by the enzyme telomerase. Human tonsil germinal center B cells consistently expressed 100- to 1000-fold higher levels of telomerase activity than naive or memory B cells, which had no or very low detectable activity, as analyzed by the telomere repeat amplification protocol assay. In vitro stimulation of human memory B cells through surface Ig or CD40 was capable of up-regulating telomerase. The findings suggest that longevity of B cell memory is maintained, despite multiple cell divisions in the generation of a memory B cell, by up-regulation of telomerase in germinal center and activated memory B cells.
Correction of defective expression in MHC class II deficiency (bare lymphocyte syndrome) cells by retroviral transduction of CIITA.
Authors: Bradley M B , Fernandez J M , Ungers G , Diaz-Barrientos T , Steimle V , Mach B , O'Reilly R , Lee J S
Published: December 2022

Retrovirus-mediated gene transfer was used to restore expression to MHC class II-negative patient cells from complementation group A(II) of MHC class II immunodeficiency or bare lymphocyte syndrome (BLS). The cells of these patients do not transcribe MHC class II genes due to a defect in the trans-acting factor, CIITA. We constructed a vector, pGAG/Ii-CIITA, with the MHC class II-associated invariant chain promoter driving CIITA expression. Cocultivation with the virus producer line was consistently shown to be the optimal method for infection of all cell types. The induction of MHC class II expression after virus infection was rapid, and high levels of expression were achieved in cell lines within 1 wk of infection. In addition, expression was easily detectable even in peripheral blood cells of a BLS patient within a few days. Cell lines maintained in vitro for several months remained positive, and the proportion of cells with surface expression of DR was correlated with the number of integrated proviruses. Moreover, transduced B lymphoblastoid cell lines readily established tumors in CB17-scid/scid mice, and the MHC class II-positive cells demonstrated a clear competitive advantage in vivo. Ultimately, we hope to use this transduction system to restore normal immune function to a BLS patient for which no other therapeutic option currently exists.
Distinct methylation states of the CD8 beta gene in peripheral T cells and intraepithelial lymphocytes.
Authors: Hamerman J A , Page S T , Pullen A M
Published: December 2022

The CD8 coreceptor is expressed on both immature and mature T cells as either an alphabeta heterodimer or an alpha alpha homodimer. Thymocytes and peripheral T cells express CD8 alphabeta, whereas TCR alphabeta+ intraepithelial lymphocytes (IEL) express CD8 alpha alpha or CD8 alphabeta, and the majority of TCR gammadelta+ IEL bear CD8 alpha alpha. The presence of CD8 beta enhances the signaling and adhesion properties of the CD8 alphabeta coreceptor and is necessary for efficient T cell development in the thymus, but is not required for the extrathymic maturation of CD8 alpha alpha+ IEL. To address whether CD8 alpha alpha+ IEL express CD8 beta during their development, we examined the methylation state of cytosines in the CD8 beta gene 5' regulatory region to identify those for which the methylation state inversely correlates with expression of the CD8 beta protein. We identified four such cytosines that were demethylated in CD8 beta-expressing thymocytes and T cells. Interestingly, these cytosines were also demethylated in CD4+ lymph node T cells that had transiently expressed CD8 beta during their development. The methylation state of these cytosines was examined in DNA purified from TCR alphabeta+ CD8 alpha alpha+ and TCR alphabeta+ CD8 alphabeta+ IEL, as well as from TCR gammadelta+ CD8 alpha alpha+ and CD3- CD8 alpha alpha+ IEL. The methylation pattern for TCR alphabeta+ CD8 alpha alpha+ IEL DNA was distinct from that seen for DNA from CD4+ lymph node cells, suggesting that TCR alphabeta+ CD8 alpha alpha+ IEL have not previously expressed CD8 beta. Analysis of DNA from CD3- CD8 alpha alpha+ IEL indicated that the unique methylation pattern of the CD8 beta gene in TCR alphabeta+ CD8 alpha alpha+ IEL DNA was not due to transcription of the CD8 alpha gene or the influence of the gut microenvironment.
Identification of a STAT6 domain required for IL-4-induced activation of transcription.
Authors: Lu B , Reichel M , Fisher D A , Smith J F , Rothman P
Published: December 2022

Tyrosine phosphorylation of STAT6 in response to IL-4 results in the formation of STAT6 homodimers that bind specific DNA elements. Although binding sites for STAT6 have been shown to be important for the function of several IL-4-inducible promoters, the role of STAT6 in this activation has not been defined. To determine whether STAT6 is a transcriptional activator, different portions of the carboxyl terminus of STAT6 were fused to the yeast Gal4 protein DNA binding domain. Analysis of these chimeric Gal4-STAT6 proteins demonstrates that a 140-amino-acid proline-rich region of the carboxyl terminus of STAT6 contains a region that activates transcription. Truncation mutants of STAT6 that lack this domain cannot activate transcription and are capable of repressing transcription stimulated by a wild-type STAT6 protein. Strikingly, the ability of IL-4 to induce transcription from the Ig germline epsilon promoter is suppressed by overexpression of a carboxyl-terminal deletion mutant of STAT6. These studies demonstrate that the carboxyl terminus of STAT6 contains an activating domain required for the induction of genes by IL-4.
Effect of mitogenic stimulation and DNA methylation on human T cell DNA methyltransferase expression and activity.
Authors: Yang J , Deng C , Hemati N , Hanash S M , Richardson B C
Published: December 2022

DNA methylation, a mechanism modifying gene expression, is mediated in part by the enzyme DNA methyltransferase. Reduced levels of T cell DNA methyltransferase have been observed in lupus-like diseases, and increased levels have been reported in malignancies. Little is known concerning the regulation of human DNA methyltransferase. In this report we demonstrate that mitogenic T cell stimulation causes an increase in DNA methyltransferase mRNA and enzyme activity. We also show that pharmacologic inhibition of T cell DNA methylation causes an increase in the rate of DNA methyltransferase mRNA transcription and a corresponding increase in mRNA levels and enzyme activity. This suggests that DNA methyltransferase is itself regulated in part by DNA methylation status, possibly representing a feedback mechanism. DNA methylation inhibition also resulted in an increase in Ha-ras and c-jun mRNA levels, overexpression of which increases DNA methyltransferase in murine systems. These results thus identify two mechanisms regulating levels of human T cell DNA methyltransferase and raise the possibility that abnormalities in either could contribute to disorders associated with altered DNA methylation.
Virtually identical enhancers containing a segment of homology to murine 3'IgH-E(hs1,2) lie downstream of human Ig C alpha 1 and C alpha 2 genes.
Authors: Chen C , Birshtein B K
Published: December 2022

We have isolated sequences downstream of human Ig C alpha 1 and C alpha 2 genes and have identified two enhancers in these regions. One enhancer is located approximately 9 kb downstream of C alpha 1, and the second enhancer is located approximately 11 kb downstream of C alpha 2. These approximately 1.6-kb enhancers are virtually identical to each other except for varying numbers of a approximately 53-bp motif. The C alpha 2-associated enhancer contains four copies of this motif in tandem, whereas the C alpha 1-associated enhancer has only a single copy. Within the human enhancers is a 177-bp segment that is homologous to a 191-bp segment of one of four enhancers from the 3' regulatory region of murine (and rat) DNA, namely 3'IgH-E(hs1,2). Like the murine and rat enhancers, both human enhancers are flanked by inverted repeats; furthermore, the human enhancers generally appear to be inverted with respect to each other. The evolutionarily conserved region of homology has substantial core enhancer activity. Contained within this region are the single octamer and one copy of the approximately 53-bp motif, both of which contribute to the activity of the full-length enhancer. A comparison of the DNA sequences and the results of transient transfection assays imply that the human C alpha-associated enhancers may be regulated (in part) differently than the murine enhancer 3'IgH-E(hs1,2).
Differential induction of apoptosis by virulent Mycobacterium tuberculosis in resistant and susceptible murine macrophages: role of nitric oxide and mycobacterial products.
Authors: Rojas M , Barrera L F , Puzo G , Garcia L F
Published: December 2022

Resistance and susceptibility of macrophages to mycobacteria are under the control of the Bcg/Nramp1 gene, which also controls the NO- production in response to macrophage activators. There is recent evidence indicating that mycobacteria induces apoptosis in infected macrophages. Using murine macrophage lines, congenic at the Bcg/Nramp1 gene, this report shows that B10R are more prone than B10S macrophages to undergo apoptosis after exposure to live virulent Mycobacterium tuberculosis H37Rv (Mtb) or PPD, as determined by cell viability, DNA fragmentation, hypoploidy, and the terminal deoxynucleotide transferase dUTP-biotin nick-end labeling assay. Induction of apoptosis correlated with NO- production. Aminoguanidine and anti-TNF-alpha inhibited NO- production and apoptosis. B10R and B10S macrophages were equally affected by sodium nitroprusside, a donor of NO-, but its effect, mainly in B10R cells, was enhanced by the presence of Mtb. Nonvirulent mycobacteria induced lower levels of NO- and did not cause cell death. Killed Mtb, mannose-capped lipoarabinomannan (ManLAM), and LPS rescued macrophages from apoptosis albeit induce NO-. These findings suggest the existence of opposite pathways: metabolically active mycobacteria promotes apoptosis whereas their structural components inhibit it. Apoptosis may be a critical mechanism by which Nramp1 gene controls the macrophage infection with virulent mycobacteria.
The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection.
Authors: Livingston B D , Crimi C , Grey H , Ishioka G , Chisari F V , Fikes J , Grey H , Chesnut R W , Sette A
Published: December 2022

We have previously described the development of a lipopeptide-based vaccine, Theradigm-HBV, capable of inducing CTL responses in humans. This vaccine incorporates the HLA-A2.1-restricted CTL epitope hepatitis B core Ag 18-27, linked to the universal helper T lymphocyte (HTL) epitope tetanus toxoid (TT) 830-843. Herein, we report the results of a phase I trial designed to examine the effects of Theradigm-HBV dose and regimen on the magnitude and duration of the memory CTL response. A total of four injections of up to 5 mg/dose were found to be a safe and effective means of generating substantial memory CTL responses. Precursor frequency analysis demonstrated CTL responses of similar magnitude to those previously observed in patients who successfully cleared hepatitis B virus infection and to influenza-specific memory CTL responses induced by natural exposure to influenza virus. Theradigm-HBV induced CTL responses that persisted for more than 9 months after the last injection. HTL responses were associated with significant CTL responses, and sustained HTL activity was necessary for development of persistent memory CTL activity. These results represent the first demonstration of the importance of HTL activity for development of long-lived memory CTL responses in humans. In conclusion, our results show that lipopeptides safely induce specific CTL activity in humans of such magnitude and persistence as to be of potential therapeutic significance.
Differential expression of cyclic nucleotide phosphodiesterase 3 and 4 activities in human T cell clones specific for myelin basic protein.
Authors: Ekholm D , Hemmer B , Gao G , Vergelli M , Martin R , Manganiello V
Published: December 2022

Little is known concerning the relative distribution and function of the different cyclic nucleotide phosphodiesterases (PDEs) in lymphocytes. Recent reports, however, have indicated that specific PDE4 inhibitors were effective in treatment of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. The therapeutic effect of PDE4 inhibitors is thought to be related to inhibition of autoreactive CD4+ T cells specific for myelin basic protein (MBP) or other myelin proteins. Human autoreactive CD4+ T lymphocyte clones (TCC), specific for the immunodominant MBP epitope (amino acids 83-99), contain PDE3 and PDE4, two PDEs that exhibit a high affinity for cAMP. Amplification of TCC mRNA by reverse transcription-PCR indicated that TCC PDE3 mRNA was of the PDE3B, not PDE3A, subtype. Different TCC contained different proportions of PDE3 and PDE4, and their activities increased during Ag (MBP) stimulation. Specific PDE3 (cilostamide) and PDE4 (rolipram) inhibitors suppressed [3H]thymidine incorporation in TCC. Since it is believed that many autoimmune diseases are at least partially mediated by autoreactive CD4+ T cells, these observations may have important implications not only for the treatment of multiple sclerosis but also for other autoimmune diseases.
Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice.
Authors: Toyabe S , Seki S , Iiai T , Takeda K , Shirai K , Watanabe H , Hiraide H , Uchiyama M , Abo T
Published: December 2022

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.
Conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts.
Authors: Vély F , Vivier E
Published: December 2022

Immunoreceptor tyrosine-based inhibition motifs (ITIMs) consist of a 6-amino acid stretch (ILV)xYxx(LV). ITIMs are expressed in the intracytoplasmic domain of transmembrane receptors, which extinguish cell activation induced by receptors bearing immunoreceptor tyrosine-based activation motifs. The recent identification of new members of the ITIM-bearing receptor family (such as the signal-regulatory proteins) extends the ITIM concept to the negative regulation of growth factor- and oncogene-induced activation in nonhemopoietic cells. In addition, it appears that a feature of the family of ITIM-bearing receptors is the existence of noninhibitory/activatory counterparts devoid of intracytoplasmic ITIMs and characterized by the presence of a charged amino acid residue in their transmembrane domain.
Induction of thymocyte deletion by purified single peptide/major histocompatibility complex ligands.
Authors: Lesage S , Charron J , Winslow G , Hugo P
Published: December 2022

We report a novel in vitro approach that allows study of the consequences of TCR ligation on thymocytes in the absence of thymic stromal cells. Hence, thymocytes were incubated either in the presence of recombinant antigenic peptide/MHC complexes, which represent ligands of physiologic affinities, or with anti-TCR mAb, a ligand of supraphysiologic affinity. Whereas TCR cross-linking with mAb led to thymocyte deletion, incubation with peptide/MHC ligands did not trigger cellular apoptosis. However, the addition of a costimulatory signal (provided by anti-CD28 mAb) allowed the induction of apoptosis following TCR binding to peptide/MHC ligands, and it increased the levels of cell death obtained through mAb-mediated TCR cross-linking. Requirement for accessory signals seen with TCR stimulation by peptide/MHC complexes argues in favor of qualitative differences between TCR engagement by ligands of either physiologic or supraphysiologic affinity.
Memory, but not naive, peripheral blood B lymphocytes differentiate into Ig-secreting cells after CD40 ligation and costimulation with IL-4 and the differentiation factors IL-2, IL-10, and IL-3.
Authors: Kindler V , Zubler R H
Published: December 2022

The ligation of CD40 on B lymphocytes by CD40 ligand, transiently expressed on activated Th cells, provides a key activation signal required for the germinal center B cell response. In vitro, human B cell activation has been investigated extensively by coculturing tonsillar B cells with CD32-transfected fibroblasts coated with anti-CD40 Abs, in the presence of cytokines (the CD40 system). When tonsillar IgD+ B cells are cultured in the CD40 system with IL-4, cells proliferate and switch to IgG, but they display a block of differentiation illustrated by the persistence of IgD expression on cycling B cells. In this study, we analyzed the responses of peripheral blood B lymphocyte fractions, which may contain fewer in vivo activated cells than those from tonsils. While the differentiation block was confirmed with peripheral naive B cells cultured in the CD40 system with IL-4, it was also observed with the combination of IL-2, IL-10, and IL-3 alone or together with IL-4 (persistence of >90% IgD+ cells, including 24-60% IgD+, IgG+ cells, and <6% IgD+, IgA+ cells after 8 days). IgD+, IgG-, and IgA- (naive) B cells secreted 70-fold less Ig than IgG+, IgA+ (memory) B cells in response to anti-CD40 plus IL-2, IL-10, and IL-3. IgG-, IgA- B cells, or IgD-, IgM+, which should include IgM+ memory cells, strongly secreted IgM, but no IgG. In conclusion, only memory B cells secreted Ig; like memory T cells, their activation requirements to differentiate into effector cells seem less stringent than those of the naive cells.
Positive and negative selection of functional mouse CD4 cells by porcine MHC in pig thymus grafts.
Authors: Zhao Y , Sergio J J , Swenson K , Arn J S , Sachs D H , Sykes M
Published: December 2022

Specific tolerance to discordant xenogeneic donors can be achieved by grafting of fetal pig thymic and liver tissue (FP THY/LIV) to T cell and NK cell-depleted, thymectomized (ATX) mice. Mouse CD4+ T cells develop in FP THY/LIV grafts, and demonstrate remarkably normal immune function, including host-restricted responses to keyhole limpet hemocyanin. We have therefore studied the role of host MHC class II in the development of mouse T cells in FP THY/LIV grafts by comparing their development in ATX MHC class II-deficient (IIKO) and wild-type (H-2b) mice. Mouse CD4+ T cells repopulated T/NK cell-depleted, ATX IIKO mice after grafting with FP THY/LIV, indicating that pig MHC can positively select mouse CD4 cells. Expression of TCR, MHC class I, Qa-2, heat-stable Ag, and CD45RB among double-positive and CD4 single-positive (SP) graft thymocytes in wild-type recipients was similar to that in normal mouse thymi, whereas CD4 SP thymocytes in grafts of IIKO mice showed increased Qa-2 and decreased heat-stable Ag expression, suggesting an increased level of maturity. Double-positive cells in grafts of IIKO mice also expressed higher than normal levels of Qa-2. Deletion within the grafts of Vbeta3+, Vbeta5.1/5.2+, and Vbeta11+ but not Vbeta6+, Vbeta7+, or Vbeta8.1/8.2+ mouse CD4 SP thymocytes in ATX IIKO mice demonstrated that swine leukocyte Ag participates in negative selection of the T cell repertoire. Therefore, porcine MHC mediates positive and negative selection of mouse thymocytes, but host class II MHC molecules also regulate thymocyte maturation in xenogeneic thymic grafts.
Human autoreactive and foreign antigen-specific T cells resist apoptosis induced by soluble recombinant CD95 ligand.
Authors: Zipp F , Martin R , Lichtenfels R , Roth W , Dichgans J , Krammer P H , Weller M
Published: December 2022

Mature T cells are susceptible to activation-induced cell death in the periphery. Activation-induced cell death is thought to involve CD95/CD95 ligand interactions in vivo. Here we report that stimulated, CD45RO+ human T cell lines specific for myelin basic protein or tetanus toxoid from multiple sclerosis patients and healthy individuals resist apoptosis induced by soluble recombinant CD95 ligand in vitro. In contrast, the same CD95 ligand effectively kills Jurkat T lymphoma and human malignant glioma cells. The resistance of the T cell lines is not due to a lack of CD95 expression at the cell surface and is not overcome by coexposure to CD95 ligand and inhibitors of RNA or protein synthesis. The expression level of BCL-2 is lower in Jurkat than in Ag-specific T cells. After exposure to soluble CD95 ligand, Jurkat T cells, but not Ag-specific T cells, exhibit loss of BCL-2 and BCL-X expression whereas BAX expression is not affected. Surprisingly, Ag-specific T cells are rather sensitive to CD95 ligand expressed at the cell surface of N2A neuroblastoma cells. Accessory molecules expressed by the CD95 ligand-expressing effector cell are dispensable for apoptosis since the T cells are equally sensitive to agonistic APO-1 Ab. Further studies are required to determine whether resistance to soluble CD95 ligand-mediated apoptosis is a possible escape mechanism for T cells from peripheral deletion that may have relevance for autoimmune disorders.
Amplified follicular immune complex deposition in mice lacking the Fc receptor gamma-chain does not alter maturation of the B cell response.
Authors: Vora K A , Ravetch J V , Manser T
Published: December 2022

The role of Ag-Ab complexes (or immune complexes; ICs) in the regulation of the maturation of the B cell immune response was investigated in mice perturbed in the deposition and retention of such complexes. Loss of surface expression of Fc gammaRI and Fc gammaRIII due to targeted disruption of the common FcR gamma-chain gene results in dramatically increased deposition of ICs on follicular dendritic cells (FDCs) in germinal centers (GCs), attributed to altered clearance of circulating ICs. Despite these changes in the trapping of ICs by FDCs, serum Ab production, V gene hypermutation, isotype class switching and Ab affinity maturation are overtly unaltered. Thus, substantially augmenting B cell cognate Ag density on FDCs does not alter the outcome of the maturation of the B cell response. The significance of this finding in terms of the currently accepted model for the generation of B cell memory is discussed.
CD4 T cell anergy in murine AIDS: costimulation via CD28 and the addition of IL-12 are not sufficient to rescue anergic CD4 T cells.
Authors: Andrews C , Swain S L , Muralidhar G
Published: December 2022

Murine acquired immunodeficiency syndrome (MAIDS) is a fatal disease induced by a mixture of retroviruses known as BM5. It is characterized by splenomegaly, lymphadenopathy, hypergammaglobulinemia, loss of T and B cell function, and development of B cell lymphomas. As the disease progresses, by wk 8 of infection, CD4 T cell response to Ags and mitogens is severely curtailed and the CD4 T cell population becomes anergic. We examined responses of anergic CD4 T cells upon addition of a costimulatory signal (anti-CD28) and a cytokine (IL-12), which might help to restore the function of cells. We report that proliferation and cytokine production were restored in the early stages of infection by the strategies we tested, but not at later stages when anergy was well established. We also examined the effect of the same treatments on anergy of CD4 T cells from thymectomized, BM5-infected mice to determine whether the rescue seen was due to cells freshly derived from the thymus. We report that proliferation and cytokine production decreased in thymectomized mice even at wk 4 of infection, indicating that cells that are freshly derived from thymus are the ones responding to treatment. This study indicates that once anergy has been established in MAIDS, it cannot be reversed by providing costimulation via CD28 and IL-12. Anergy of CD4 T cells in MAIDS appears to be different from that seen in other systems, both in underlying cause and in the ability of the cells to revert to a normal state.
Fas ligand expression is restricted to nonlymphoid thymic components in situ.
Authors: French L E , Wilson A , Hahne M , Viard I , Tschopp J , MacDonald H R
Published: December 2022

The cell surface receptor Fas (Apo-1/CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in activation-induced death of mature T cells and in killing mediated by cytolytic T cells. The role of the Fas pathway in apoptosis associated with thymic selection events is, however, controversial. Although Fas and FasL are known to be expressed in the thymus, the nature and in vivo localization of FasL-expressing cells have not been determined. Using recently developed anti-FasL Abs in combination with in situ hybridization on tissue sections, we show in this work that FasL-expressing cells are present in the thymus, particularly within the medulla. FasL mRNA was detected readily in thymic stromal cell extracts, but not in isolated thymocytes. Moreover, immunohistochemical analysis of serial tissue sections stained with Abs against FasL in conjunction with epithelial and dendritic cell markers indicated that both thymic epithelial and dendritic cells express FasL in situ. The coexistence of FasL-expressing stromal cells and Fas-expressing thymocytes may have important implications for the role of the Fas pathway in apoptosis associated with thymic selection events.
The Lck SH2 phosphotyrosine binding site is critical for efficient TCR-induced processive tyrosine phosphorylation of the zeta-chain and IL-2 production.
Authors: Lewis L A , Chung C D , Chen J , Parnes J R , Moran M , Patel V P , Miceli M C
Published: December 2022

The lymphocyte-specific tyrosine kinase Lck is essential for TCR-mediated signal transduction. This is in part due to its enzymatic activity as a tyrosine kinase responsible for TCR-induced tyrosine phosphorylation of zeta and CD3 receptor subunits. In addition to its catalytic domain, the Lck protein contains SH3 and SH2 domains capable of associating with other signaling molecules. It has been proposed that phosphotyrosine binding by the Lck SH2 domain may enhance substrate tyrosine phosphorylation by facilitating the processive phosphorylation of multiple sites within the TCR complex. Alternatively or additionally, it may function in adapter activity for facilitating required protein-protein interactions. Previous experiments demonstrate that overexpression of a constitutively activated form of Lck (F505) in the BI-141 T cell hybridoma leads to the Lck kinase activity-dependent enhancement of TCR-mediated signals. Here we demonstrate that mutation of amino acids important for SH2 phosphotyrosine binding significantly compromises the ability of F505 to enhance TCR-mediated protein tyrosine phosphorylation and Ag-induced IL-2 production in BI-141. Examination of the effects of TCR-regulated phosphorylation of the Lck substrate zeta provides in vivo evidence for a role for the Lck SH2 domain in the processive phosphorylation of a multiply phosphorylated substrate.