bluuebunny/crossref_metadata_2025 huggingface dataset:
Local suppression of IFN-gamma in hepatic granulomas correlates with tissue-specific replication of Leishmania chagasi.
Authors: Wilson M E , Sandor M , Blum A M , Young B M , Metwali A , Elliott D , Lynch R G , Weinstock J V
Published: January 2023

BALB/c mice are susceptible to infection with the visceralizing species of Leishmania, Leishmania chagasi. The parasite load initially rises in the liver and spontaneously subsides, whereas parasite multiplication begins later and remains lower in the spleen. To investigate whether this organ-specific multiplication of L. chagasi correlates with localized immune responses, we compared cytokine production by splenic vs hepatic immune cells. Livers from infected mice contained granulomas harboring intracellular L. chagasi amastigotes, whereas few amastigotes were present in the spleen. FACS analysis granuloma cells showed granuloma lymphocytes expressed a memory/effector phenotype. Granuloma cells cultured in vitro produced IL-10 and IL-6 but no detectable IFN-gamma, IL-4 or IL-5. In contrast, splenocytes from the same animals secreted IFN-gamma, IL-4, IL-6, and IL-10. T cells were depleted from granuloma cells by immune lysis, and the results indicated that IL-10 and IL-6 were derived at least in part from a non-T cell compartment. Paradoxically, FACS-purified Thy-1+ granuloma lymphocytes were able to produce IFN-gamma in the absence of other granuloma cells, suggesting IFN-gamma production might usually be inhibited by a granuloma-associated non-T cell element. Coculture of splenocytes with either granuloma cells or supernatants from granuloma cultures inhibited the usual splenocyte production of IFN-gamma and IL-4 but not IL-10. Thus, there may be a unique granuloma-associated suppressive factor accounting for the absence of IFN-gamma in hepatic granuloma cultures. It may be the absence of IFN-gamma in the liver and presence in the spleen that allows or inhibits parasite survival, respectively, in these different locations.
IL-7 up-regulates HIV-1 replication in naturally infected peripheral blood mononuclear cells.
Authors: Smithgall M D , Wong J G , Critchett K E , Haffar O K
Published: January 2023

We have investigated the effect of exogenous IL-7 on replication of HIV-1 in PBMCs isolated from asymptomatic, chronically infected donors. We show that IL-7 induced virus replication and increased proviral DNA levels in CD8- PBMC cultures. IL-7 also increased the levels of doubly spliced HIV-1 tat RNA in these cultures. In comparison, IL-2 induced lower levels of virus production than IL-7, but had a more pronounced effect on cell proliferation. The IL-7-mediated increase in virus replication was not inhibited by neutralizing mAbs to IL-1 beta, IL-2, IL-6, or TNF-alpha, and was only partially dependent on ligation of the T cell accessory molecule CD28. CD8+ cells inhibited the increase in viral replication following IL-7 stimulation, but did not prevent virus replication following ligation of CD3 in the presence of IL-7. The data shows that IL-7 regulates HIV-1 replication in naturally infected PBMCs.
H-2Dp transgene alters natural killer cell specificity at the target and effector cell levels. Comparison with an H-2Dd transgene.
Authors: Sentman C L , Olsson-Alheim M Y , Lendahl U , Kärre K
Published: January 2023

The expression of MHC class I molecules is an important determinate of natural killer (NK) cell specificity. The missing self hypothesis proposes that NK cells express receptors for self-MHC class I molecules so that target cells that share MHC class I alleles with the NK cells are not killed by those NK cells. However, some effector cells fail to kill some allogeneic target cells suggesting that shared motifs between different MHC class I alleles can interact with the effector cell class I receptors and prevent lysis. We have used transgenic mice to critically assess whether different MHC class I alleles can exert common influences on NK cell specificity at the host/effector and target cell levels. The specificity of NK cells have been compared between C57BL/6 (H-2b) mice and B6DP (H-2b, H-2Dp) and D8 (H-2b, H-2Dd) transgenic mice. The data indicate that H-2Dp and H-2Dd confer similar protection and specific lysis, such that NK cells from either of the H-2Dp or H-2Dd transgenic mice kill nontransgenic target cells yet they do not kill either of the transgenic target cells. The expression of an H-2Dp transgene also provides protection for C57BL/6 lymphoblasts from allogeneic BALB/c (H-2d) NK cells. Furthermore, H-2Dp and H-2Dd transgenic target cells are lysed to a similar extent by H-2k effector cells. These data suggest that H-2Dp and H-2Dd may be able to inhibit the same NK cell population. This may occur through a shared motif recognized by the same receptor, or different motifs recognized by different, but co-expressed receptors.
IL-1 receptor antagonist (IL-1ra) expression, function, and cytokine-mediated regulation during mycobacterial and schistosomal antigen-elicited granuloma formation.
Authors: Ruth J H , Bienkowski M , Warmington K S , Lincoln P M , Kunkel S L , Chensue S W
Published: January 2023

Granulomas (GR) mediated predominantly by Th1/type 1 (IFN-gamma) and Th2/type 2 (IL-4, IL-5, IL-10) cytokines were induced by i.v. injection of sensitized CBA/J mice with carbohydrate beads coated with Mycobacterium tuberculosis or Schistosoma mansoni egg Ags, respectively. GR macrophages (Mphi) from types 1 and 2 GR both produced IL-1ra, but the former showed accelerated IL-1ra-producing capacity, releasing two- to threefold greater amounts on day 4 than those of type 2 GR, as measured by sandwich ELISA. In vivo depletion of IL-1ra exacerbated GR size and augmented regional cytokine production in both types of responses. To determine the critical cytokines mediating IL-Ira expression, oil-elicited peritoneal Mphi were exposed to graded doses (0.1 to 10 ng/ml) of cytokines (IL-1beta, IL-2, IL-4, IL-10, IL-12, IFN-gamma, and TNF-alpha) for 24 h, then stimulated with opsonized zymosan. Of the cytokines tested, IFN-gamma and TNF-alpha were the best costimuli for IL-1ra production in the presence of zymosan, whereas IL-1beta, IL-10, and IL-12 were not active. In vivo depletion of IL-4, IL-10, IL-12, IFN-gamma, or TNF-alpha with 5 mg of cytokine-specific neutralizing rabbit IgG revealed that IFN-gamma and TNF-alpha were required for maximal IL-1ra production by Mphi. Furthermore, the delayed IL-1ra production by type 2 GR Mphi could be related to later TNF-alpha production. Our findings indicate that IL-1ra is a common regulatory product of inflammatory Mphi and is particularly promoted by type 1 cytokines, IFN-gamma, and TNF-alpha.
Neutrophil lactoferrin release induced by IgA immune complexes can be mediated either by Fc alpha receptors or by complement receptors through different pathways.
Authors: Zhang W , Lachmann P J
Published: January 2023

Our previous results showed that neutrophil secondary granule release, indicated by release of lactoferrin, was a slow process when induced by IgA immune complexes (IC) formed in heat-inactivated serum, but became very fast if IgA IC were formed in normal human serum. This phenomenon did not apply to the IC of other Ab isotypes. In this paper, we demonstrate that the fast lactoferrin release is caused by complement, mainly due to the deposition of C3b and iC3b on IgA IC. Either CR1 or CR3 can mediate the response and both receptors have to be blocked to prevent it. Complement also influences FcalphaR-mediated lactoferrin release, in that this is enhanced by the anaphylatoxin peptides, C5a and C5a(desArg). Divalent cations are required for FcalphaR and CR3- but not for CR1-mediated lactoferrin release. Genistein, a protein tyrosine kinase inhibitor, totally inhibits FcalphaR-mediated response, but has little effect on CR1-mediated response. Therefore, it is clear that different pathways of intracellular signaling are utilized. In addition, stimulation through FcalphaR promotes the receptor up-regulation, which is abolished by the presence of EDTA or genistein.
Ultraviolet B radiation sensitizes a murine epidermal dendritic cell line (XS52) to undergo apoptosis upon antigen presentation to T cells.
Authors: Kitajima T , Ariizumi K , Bergstresser P R , Takashima A
Published: January 2023

Ultraviolet B irradiation of skin leads to immunologic tolerance, rather than immunity against newly introduced Ag, by altering the function of Langerhans cells, skin-specific members of the dendritic cell (DC) family. Using the murine epidermal-derived DC line, XS52, which retains important features of resident Langerhans cells, we have tested the hypothesis that UV radiation delivers a signal leading to apoptosis. XS52 cells, when exposed to modest fluences (25-100 J/m2) of radiation, underwent apoptosis during a subsequent 6-h incubation with LPS or upon 6-h coculture with the keyhole limpet hemocyanin-specific Th1 clone HDK-1 in the presence of Ag. Specifically, XS52 cells treated in this way exhibited diminished cell viability, DNA laddering, and condensed staining of DNA. By contrast, none of these changes was induced by radiation alone, LPS alone, or coculture with T cells and Ag. Likewise, neither UV radiation plus T cells nor radiation plus Ag were sufficient to induce apoptosis, indicating that both T cells and Ag are required to induce apoptosis in the UV-sensitized cells. XS52 cells remained fully susceptible to T cell-mediated apoptosis even 16 h after irradiation, indicating the persistence of the sensitized state. These observations establish a model in which UV radiation induces a first event in which DC become sensitive to a second, apoptotic signal that is delivered by Ag-specific interaction with T cells or by LPS. We suggest that DC undergoing apoptosis deliver unusual activation signals to T cells during Ag presentation, signals that lead to cellular unresponsiveness rather than to effective immunity.
Defective B cell development in Snell dwarf (dw/dw) mice can be corrected by thyroxine treatment.
Authors: Montecino-Rodriguez E , Clark R , Johnson A , Collins L , Dorshkind K
Published: January 2023

Snell dwarf (dw/dw) mice are deficient in anterior pituitary hormones due to a mutation in the gene encoding the Pit-1 transcription factor. Bone marrow B cell development is also suppressed in the mice, providing circumstantial evidence that one or more anterior pituitary-derived products, or factors induced by them, are required for normal B lymphopoiesis. However, concluding that this is the case is dependent on showing that hormonal treatment of dwarf mice reverses their B cell defects. dw/dw mice were treated with growth hormone (GH), insulin-like growth factor-I (IGF-I), or thyroxine in an attempt to restore bone marrow B lymphopoiesis. GH and IGF-I increased the number of B lineage cells in the bone marrow and spleen but did not restore the frequency of bone marrow pre-B cells to normal. However, bone marrow cellularity in thyroxine-treated dw/dw mice was comparable to that in control animals, and both the frequency and absolute number of B lineage cells had increased to normal or even above normal. Taken together, these data indicate that endocrine factors, especially those regulated by the hypothalamic-pituitary-thyroid axis, are potent B lymphopoietic factors.
Peptide sequence requirements for the recognition of HLA-B*2705 by specific natural killer cells.
Authors: Peruzzi M , Parker K C , Long E O , Malnati M S
Published: January 2023

NK clones that were inhibited by target cell expression of HLA-B*2705 displayed peptide-specific recognition of HLA-B*2705. To evaluate the specificity of this recognition, synthetic versions of 14 endogenous ligands of HLA-B*2705 were tested for their ability to provide protection from NK-mediated lysis by binding to surface HLA-B*2705 molecules on RMA-S cells deficient in the transporter for Ag presentation. Several unrelated peptides inhibited lysis by the same NK clones. Despite similar capacities to stabilize HLA-B*2705 molecules on RMA-S cells, the 14 peptides differed widely in their abilities to provide protection. Single amino acid substitutions in both a protective and a nonprotective peptide revealed the importance of residues 7 and 8 in the peptide for recognition by NK clones, thus localizing the peptide influence to a polymorphic region of the alpha-helix of HLA class I molecules known to control discrimination among allelic variants of HLA-B and HLA-C by NK cells.
Differential activation of the ERK, JNK, and p38 mitogen-activated protein kinases by CD40 and the B cell antigen receptor.
Authors: Sutherland C L , Heath A W , Pelech S L , Young P R , Gold M R
Published: January 2023

B cell antigen receptor (BCR)-induced apoptosis in the WEHI-231 B lymphoma cell line can be prevented by engaging CD40. We have used this cell line to investigate the role of mitogen-activated protein (MAP) kinases in integrating BCR and CD40 signaling. Each of the three types of MAP kinases, the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and p38, phosphorylates a distinct set of transcription factors. Thus, activating different combinations of MAP kinases could lead to distinct biological responses. We found that BCR engagement in WEHI-231 cells caused a 15- to 20-fold activation of ERK2 and a 2- to 3-fold stimulation of ERK1. CD40 did not activate either of these kinases, nor did it affect BCR-induced ERK activation. In contrast, CD40 engagement caused a 50- to 70-fold increase in JNK activity. BCR cross-linking caused a modest (4- to 8-fold) increase in JNK activity by itself and also potentiated CD40-induced JNK activation. Finally, CD40 caused strong activation of the p38 kinase as well as MAPKAP kinase-2, a downstream target of p38. BCR engagement caused only weak activation of the p38 pathway. In summary, the BCR strongly activates ERK2 and weakly activates ERK1, JNK, and p38, while CD40 markedly stimulates the JNK and p38 kinases. Thus, activation of only ERK2 correlates with apoptosis in WEHI-231 cells, whereas full activation of all three MAP kinase pathways correlates with cell survival. The role of MAP kinases in regulating these responses remains to be tested.
Follicular dendritic cell-derived antigen and accessory activity in initiation of memory IgG responses in vitro.
Authors: Wu J , Qin D , Burton G F , Szakal A K , Tew J G
Published: January 2023

Follicular dendritic cells (FDC) release Ag in developing germinal centers in the form of immune complex-coated bodies (iccosomes). Iccosomes are endocytosed by B cells, and the B cells process and present the FDC-derived Ag to T cells. By 3 days after Ag challenge, Ab-forming cells producing IgG specific for the iccosomal Ag emerge from the developing germinal centers and home to the bone marrow, where most Ab in a secondary response are produced. In addition to providing Ag, FDC exhibit potent accessory activities that promote B cell proliferation. These observations prompted the hypothesis that both FDC-derived Ag and FDC-derived Ag-independent secondary signal(s) are essential for optimal secondary Ab responses. To test this hypothesis, methods were developed to separate Ag-bearing iccosomes from intact FDC, and we then examined the ability of isolated iccosomes to elicit secondary Ab responses in vitro in the presence and absence of intact FDC. In the absence of FDC, iccosomes bearing the appropriate Ag elicited only minimal levels of specific IgG. Proliferation studies revealed that iccosomes lacked the FDC accessory activity necessary to augment B cell proliferation. When a source of FDC lacking the relevant Ag but exhibiting accessory activity was added to the iccosomal/Ag/lymphocyte mixture, dramatic increases in IgG specific for the iccosomal Ag were obtained (increases were from low ng/ml to microg/ml levels of specific IgG). The results suggest the concept that Ag on FDC or on iccosomes signals B cells through B cell Ag receptor, the iccosome provides these B cells with Ag necessary to process and elicit T cell help, and a secondary signal(s) necessary to optimize the memory response is delivered to B cells by FDC in an Ag-nonspecific fashion.
Defect of thymocyte emigration in a T cell deficiency strain (CTS) of the mouse.
Authors: Yagi H , Matsumoto M , Nakamura M , Makino S , Suzuki R , Harada M , Itoh T
Published: January 2023

As bone marrow progenitors migrate to the thymus, differentiate into immunologically competent T cells, then leave the thymus and home to the peripheral lymphoid organs, each migration or homing is an essentially critical process for T cell differentiation. Homing of thymocytes into peripheral lymphoid organs has been extensively analyzed. On the contrary, little is known about the mechanism whereby mature thymocytes emigrate from the thymus to peripheral lymphoid organs. In this work, we show that the mutant T cell deficiency strain (CTS) of mouse, previously described as a peripheral T cell deficiency strain, has a novel defect of mature thymocyte emigration process. Flow-cytometric analysis demonstrated a marked decrease in T lymphocytes in peripheral lymphoid organs and an accumulation of mature thymocytes in the thymus. Histologic study revealed the high concentration of thymocytes within the giant perivascular space at thymic corticomedullary junction. These accumulating cells closely resemble normal peripheral T lymphocytes in terms of cell surface phenotype and responsiveness to mitogens or allogeneic cells. They were found to express the Mel-14 Ag, the homing receptor to lymph nodes, and had a high capability of homing to peripheral lymphoid organs when explanted thymocytes were injected i.v. The intrathymic labeling methods detected hardly any emigrating CTS thymocytes from the thymus. These results indicate that emigration defect lies in the thymus of CTS mice, and suggest that this mutation is due to abnormal releasing mechanisms occurring between the thymus and the bloodstream. Complete delineation of the precise mechanisms of this defect, however, requires further studies.
Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules.
Authors: Sidney J , Southwood S , del Guercio M F , Grey H M , Chesnut R W , Kubo R T , Sette A
Published: January 2023

The HLA-B7-like binding supertype includes several different HLA-B molecules. Herein, the primary and secondary anchor specificities of the five most common HLA-B7-like molecules (B*0702, B*3501, B51, B*5301, and B*5401) were defined by the use of molecular binding assays, analogue peptides, and large sets of peptides corresponding to naturally occurring sequences. All five B7-like molecules analyzed preferentially bound 9-mers, with a stringent requirement for proline in position 2, while a variety of hydrophobic or aromatic residues were well tolerated at the C-terminal anchor position. Although most peptides bound in an allele-specific fashion, approximately 20% of the binders identified were degenerate and bound at least three of the five B7-like molecules analyzed with affinities of 500 nM or less. It was also noted that, in general, peptides that bind with high affinity to any given one B7-like molecule were also most frequently capable of degenerate binding. Prominent roles for secondary anchors in positions 1 and 3 were observed for most B7-like molecules, and secondary anchor motifs were utilized to derive an HLA-B7-like supermotif. The validity of this B7-like supermotif was tested by a blind prediction set. Finally, the B7-like supermotif was utilized to derive a general strategy for rationally engineering peptide analogues of naturally occurring sequences with greatly increased binding affinity and degeneracy. Such engineered supermotif binding peptides may be of significant utility in the development of peptide-based vaccines against chronic viral diseases and cancer.
DNA vaccination primes MHC class I-restricted, simian virus 40 large tumor antigen-specific CTL in H-2d mice that reject syngeneic tumors.
Authors: Schirmbeck R , Böhm W , Reimann J
Published: January 2023

We investigated the stimulation of a MHC class I-restricted response of CTL to the SV40 large tumor Ag (T-Ag) by different vaccination strategies in H-2d and H-2b mice. Immunization with plasmid DNA or exogenous T-Ag, or infection with SV40, primed CTL to T-Ag in H-2b mice; these three different types of Ag delivery primed T-Ag-specific CTL populations with similar epitope/restriction specificities. In H-2d mice, i.m. immunization with plasmid DNA, but neither immunization with exogenous protein Ag nor SV40 infection, primed CTL to T-Ag. T-Ag-specific H-2d CTL were primed by DNA-based immunization in vivo, expressed the CD4-CD8+ phenotype, and were L(d)-restricted. In H-2d (DBA/2) mice, T-Ag-specific immune responses primed by plasmid DNA injection, but not those primed by exogenous T-Ag or SV40 infection, mediated CD8+ CTL-dependent rejection of T-Ag-expressing P815/T tumor grafts. The data indicate that immunization by plasmid DNA injection is an efficient strategy to induce class I-restricted CTL responses against oncogene-encoded Ags of low immunogenicity that mediate tumor rejection.
Molecular bases of C7 deficiency: three different defects.
Authors: Fernie B A , Orren A , Sheehan G , Schlesinger M , Hobart M J
Published: January 2023

The molecular basis of C7 deficiency has been investigated in two Irish families and a number of Israeli families of Moroccan Sephardic Jewish origin. Exon PCR and sequencing revealed a heterozygous point mutation at the 3' splice acceptor site of intron 1 in one Irish family. In the other Irish family, exons 7 and 8 failed to amplify and they were shown to be deleted. Marker haplotype studies of the C6 and C7 gene region and Southern blots show that the Irish family with the splice defect also segregate for the deletion, which is not easily detected in heterozygotes. The Israeli C7-deficient cases all share a C7 haplotype and are homozygous for a mis-sense mutation in exon 9. However, one individual is heterozygous for markers at adjacent C6 loci, showing that there has been an intergenic recombination and suggesting that the deficiency mutation is of appreciable antiquity.
Dexamethasone potently enhances phorbol ester-induced IL-1beta gene expression and nuclear factor NF-kappaB activation.
Authors: Wang Y , Zhang J J , Dai W , Lei K Y , Pike J W
Published: January 2023

The synthetic glucocorticoid dexamethasone, an immunosuppressive and anti-inflammatory agent, was investigated for its effect on PMA-mediated expression of the inflammatory cytokine IL-1beta in the human monocytic leukemic cell line THP-1. PMA alone induced the production of low levels of IL-1beta in THP-1 cells, whereas dexamethasone alone had no effect. However, dexamethasone potently enhanced PMA-mediated IL-1beta production. Using a selective and potent inhibitor of protein kinase C, we found that synergistic interaction between PMA and dexamethasone requires protein kinase C activation. PMA has been known to activate nuclear factor NF-kappaB in THP-1 cells. Using an oligonucleotide probe corresponding to an NF-kappaB DNA-binding motif of the IL-1beta gene promoter in gel electrophoresis mobility shift assays, we demonstrated that PMA-induced NF-kappaB activation was greatly potentiated by dexamethasone. Our results indicate that glucocorticoids can be positive regulators of inflammatory cytokine gene expression during monocytic cell differentiation.
Melatonin enhances IL-2, IL-6, and IFN-gamma production by human circulating CD4+ cells: a possible nuclear receptor-mediated mechanism involving T helper type 1 lymphocytes and monocytes.
Authors: Garcia-Mauriño S , Gonzalez-Haba M G , Calvo J R , Rafii-El-Idrissi M , Sanchez-Margalet V , Goberna R , Guerrero J M
Published: January 2023

This paper shows that melatonin is able to activate human Th1 lymphocytes by increasing the production of IL-2 and IFN-gamma in vitro. Th2 cells appear not to be affected by melatonin, since IL-4, which is mostly produced by Th2 cells, is not modified by the hormone. Melatonin also enhances IL-6 production by PBMCs. The activation by melatonin of IL-6 production is apparently related to the presence of monocytes, rather than to Th2 cells, in the cell preparation, since PBMCs depleted of monocytes (CD14+ cells) were not activated. Activation of PBMCs by melatonin was dependent on the dose and, measured by cytokine production, was observed only when cells were either not activated or only slightly activated by low concentrations of PHA, or when cell activation was achieved by incubating the cells with previously irradiated cells. Using a different approach to identify what type of cells among the PBMC subsets was activated by melatonin, the expression of CD69, a marker of cell activation, was studied. Melatonin increased the percentage of cells expressing the CD69 Ag in CD4+ but not in CD8+ cells. We have also achieved enhanced production of IL-2 and IL-6 using CGP 52608, a specific ligand of the putative nuclear melatonin receptor RZR/ROR, raising the possibility of direct effects of melatonin on gene regulation in both Th1 cells and monocytes. The results suggest that melatonin may be involved in the regulation of human immune functions by modulating the activity of Th1 cells and monocytes via nuclear receptor-mediated transcriptional control.
Targeting of natural killer cells to mammary carcinoma via naturally occurring tumor cell-bound iC3b and beta-glucan-primed CR3 (CD11b/CD18).
Authors: Vetvicka V , Thornton B P , Wieman T J , Ross G D
Published: January 2023

Previous reports have suggested that malignant cells frequently generate a humoral immune response that is ineffective in tumor destruction. Despite coating tumors with IgM and IgG that activate the C system via the classical pathway, normal membrane regulators of C (e.g., membrane cofactor protein and CD59) prevent cytotoxicity. Moreover, C3 deposition on tumors does not result in cytotoxic recognition by phagocytes or NK cells bearing C3 receptors capable of mediating destruction of C3-opsonized bacteria or yeast. The current investigation showed that freshly excised mammary tumors bore IgM, IgG, and C3 detectable by flow cytometry. Normal sera contained natural IgM and IgG Abs reactive with breast tumor cell lines, and IgG Ab titers were increased in patients with breast cancer. Breast tumor cell lines incubated in normal serum from AB+ individuals activated the classical, but not the alternative, pathway of C and became coated with C3. Despite exhibiting membrane-bound C3, serum-opsonized breast tumor cell lines were not killed by CR3 (CD11b/CD18)-bearing NK cells. Priming of NK cell CR3 with small soluble yeast beta-glucan polysaccharides enabled CR3-dependent killing of these same C3-bearing tumor cell lines. Tests of mammary carcinoma cells from freshly excised tumors demonstrated that they also bore sufficient amounts of opsonic C3 for cytotoxic recognition by NK cells bearing polysaccharide-primed CR3, whereas they were largely resistant to NK cells bearing unprimed CR3. This study demonstrates the potential utility of using naturally occurring opsonic C3 on tumor cells for specific immunotherapeutic targeting by NK cells and phagocytes bearing polysaccharide-primed CR3.
Compromised allograft rejection response in transgenic mice expressing antisense sequences to retinoic acid receptor beta2.
Authors: Chen H , Bérard J , Luo H , Landers M , Vinet B , Bradley W E , Wu J
Published: January 2023

Transgenic mice expressing antisense sequences to retinoic acid receptor beta2 (AS-RARbeta2 mice) were generated. The transgene was expressed in T cells, macrophages, and non-T spleen cells. These AS-RARbeta2 mice had four- to sevenfold higher endogenous RARbeta2 messages in their macrophages, comparing with their nontransgenic littermates, but the RARbeta protein level was significantly lower in these cells. This suggests that the antisense message interferes with RARbeta2 translation, and there is a feedback control of the RARbeta2 mRNA level in the macrophages. The endogenous RARbeta2 message was not detectable in T and B cells of either the transgenic or nontransgenic mice. These mice appeared to be healthy upon visual inspection. When transplanted with allogenic cardiac grafts heterotopically, the mice showed significantly compromised rejection response. These mice had a decrease of the macrophage population in spleen, using the nontransgenic littermates as references. The generation of alloantigen-specific T cell cytotoxicity was decreased in these AS-RARbeta2 mice. These results suggest that macrophage development and T cell function are affected by antisense expression of RARbeta2, and that anomaly in these cells might be contributing factors to the compromised immune response observed in the AS-RARbeta2 mice.
Type 1 versus type 2 cytokine release by Vbeta T cell subpopulations determines in vivo antitumor reactivity: IL-10 mediates a suppressive role.
Authors: Aruga A , Aruga E , Tanigawa K , Bishop D K , Sondak V K , Chang A E
Published: January 2023

We have previously reported that CD8+ tumor-draining lymph node (TDLN) cells activated with anti-CD3 and IL-2-mediated tumor regression in adoptive immunotherapy. In this study, we examined the TCR Vbeta repertoire usage of TDLN cells with respect to cytokine release profiles and therapeutic efficacy in vivo. The majority of the whole population of TDLN cells after activation with anti-CD3 were composed of Vbeta3+, -5+, -7+, -8+, and -11+ cells. Enrichment of Vbeta subsets of TDLN cells by in vitro activation with anti-Vbeta mAb revealed Vbeta8+ cells released high amounts of IFN-gamma and granulocyte/macrophage-CSF (GM-CSF) with minimal amounts of IL-10 in response to tumor and mediated tumor regression in vivo. In contrast, enriched populations of Vbeta5+, Vbeta7+, and Vbeta11+ cells released low amounts of IFN-gamma and GM-CSF with high levels of IL-10 and had no in vivo antitumor reactivity. In vitro depletion of specific Vbeta subsets from the whole TDLN pool confirmed that the profile of cytokines released correlated with in vivo antitumor function. Therapeutic efficacy mediated by TDLN cells required the release of IFN-gamma and GM-CSF since in vivo neutralization of both cytokines inhibited tumor regression. The administration of anti-IL-10 mAb abrogated the suppressed antitumor response manifested by adoptively transferred TDLN cells, which elaborated increased levels of IL-10. Our study documents that type 1 cytokine release (i.e., IFN-gamma and GM-CSF) promotes in vivo tumor Ag recognition, in contrast to type 2 release (i.e., IL-10), which suppresses this interaction, and discriminates the functional activity of Vbeta subpopulations of effector cells.
Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells.
Authors: Jaleco A C , Blom B , Res P , Weijer K , Lanier L L , Phillips J H , Spits H
Published: January 2023

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.
Transgenic expression of a novel thymic epithelial cell antigen stimulates abberant development of thymocytes.
Authors: Takeuchi T , Kuro-o M , Miyazawa H , Ohtsuki Y , Yamamoto H
Published: January 2023

We previously reported a novel thymic stromal cell Ag, HS9, as a potent molecule participating in intrathymic T cell development. HS9 Ag is expressed on thymic stromal cells especially in the cortex but not on thymocytes. In the present study, we isolated and characterized a novel cDNA, N14, encoding HS9 Ag. Sequencing analysis of N14 cDNA has revealed it to be a novel one without any significant homology to previously reported functional molecules. COS7 cells transfected with expression vectors harboring N14 cDNA became reactive with HS9-specific mAb. Northern blot analysis and in situ hybridization revealed that several tissues that are positive for HS9 mAb expressed N14 mRNA. To examine the role of this molecule in T cell development, transgenic mice were generated. In situ hybridization and immunohistochemical study showed that the transgene was significantly overexpressed on both cortical and medullar thymic stromal cells but not on thymocytes. Flow cytometric analyses showed that the percentages of mature CD4- CD8+ or CD4+ CD8- thymocytes in transgenic mice were approximately twice and triple, respectively, those in control littermates. Moreover, substantial CD4+ CD8+ thymocytes appeared to have high levels of TCR compared with peripheral T cells. Histologic examination revealed that transgenic mice had thin cortex and relatively developed medulla. These data indicate the critical role of the N14 gene in T cell development.
Normal junctional diversification of immune receptors in p53-deficient mice.
Authors: Wang C , Bogue M A , Roth D B , Meek K
Published: January 2023

One of the strategies that the immune system utilizes to generate Ab and TCR diversity is programmed imprecision of coding joint formation. This is accomplished by both nucleotide loss and random nucleotide addition (N segments) to the termini of immune receptor coding segments before resolution. Although it has been known for more than a decade that terminal deoxynucleotidyl transferase is the enzyme responsible for N segment addition, the enzymes responsible for nucleotide loss have not been identified. Recently, the p53 tumor suppressor protein was shown to have an intrinsic exonuclease activity; we reasoned that p53 as an exonuclease might be responsible for coding end processing during V(D)J recombination. Thus, we examined nucleotide loss from Ig and TCR-beta coding joints in mice lacking p53. We find no significant difference in the degree of nucleotide loss in coding joints isolated from these animals as compared with littermate controls. Thus, we conclude that p53 does not play a role in removal of nucleotides from coding termini during V(D)J recombination. Additionally, recent evidence has surfaced suggesting that p53 may play an important checkpoint role in early thymocyte differentiation. More specifically, it has been suggested that p53 is required to prevent thymocytes from maturing to the double-positive stage in the absence of a functionally rearranged TCR-beta allele. Our data suggest that TCR-beta selection is not affected in p53-deficient, V(D)J rearrangement-proficient mice.
Interferons up-regulate STAT1, STAT2, and IRF family transcription factor gene expression in human peripheral blood mononuclear cells and macrophages.
Authors: Lehtonen A , Matikainen S , Julkunen I
Published: January 2023

IFN signaling is mediated by binding of IFNs to their receptors and subsequent activation of Janus tyrosine kinase (JAK)-STAT signaling pathway. Stimulation of cells with IFN-alpha leads to the assembly of IFN-stimulated gene factor 3 transcription factor complex formed by STAT1, STAT2, and p48 protein. IFN-gamma signaling is mediated by homodimeric STAT1 protein. Although these signaling molecules are expressed constitutively, there is also evidence of transcriptional regulation by IFNs. We have characterized the expression of STAT and IFN regulatory factor (IRF) family transcription factors in primary human blood mononuclear cells and macrophages in response to IFN-alpha and IFN-gamma stimulation. We show that IFN-alpha and IFN-gamma rapidly and efficiently enhanced STAT1, STAT2, p48, and IRF-1 gene expression. IFN-gamma induced IRF-1 gene expression more strongly than IFN-alpha. Stimulation experiments in the presence of protein synthesis inhibitor, cycloheximide, suggested that these genes were activated directly by IFNs. IRF-2 gene was apparently only weakly responsive to IFNs in these cells. When macrophages were pretreated with low doses of IFN-gamma and then stimulated with IFN-alpha, clearly enhanced formation of specific transcription factor complexes was detected. This suggests that higher intracellular levels of STAT1, STAT2, and p48 protein may result in enhanced signal transduction for cytokines utilizing these transcription factors.
Recombinant human granulocyte-macrophage colony-stimulating factor (CSF), but not recombinant human granulocyte CSF, down-regulates the recombinant human stem cell factor-dependent differentiation of human fetal liver-derived mast cells.
Authors: Du Z , Li Y , Xia H , Irani A M , Schwartz L B
Published: January 2023

The effects of recombinant human granulocyte CSF (rhG-CSF) and recombinant human granulocyte-macrophage CSF (rhGM-CSF) on the recombinant human stem cell factor (rhSCF)-dependent development of human mast cells from fetal liver progenitors were examined. Mast cells were identified by immunohistochemical staining for tryptase and by flow cytometric analysis of surface Kit expression. Only rhGM-CSF affected mast cell development. When rhGM-CSF (1, 10, or 100 ng/ml) and rhSCF (50 ng/ml) were added to cell cultures from day 0, both the percentage and absolute numbers of mast cells were diminished after 4 wk compared with cultures exposed to rhSCF alone. Half of the maximal response was achieved at a dose of rhGM-CSF between 0.1 and 1 ng/ml. The Kit+ cells developing in the presence of rhGM-CSF and rhSCF exhibited an intensity of surface Kit expression comparable to that of cells exposed to rhSCF alone. Also, if the initial exposure to rhGM-CSF was delayed for 1 to 3 wk, attenuation of mast cell development waned. These findings are consistent with uncommitted progenitor cells being diverted to nonmast cell lineages by rhGM-CSF, while cells committed to a mast cell lineage, albeit immature, appear to be resistant to the lineage directives of rhGM-CSF. Exposure of fetal liver cells to rhGM-CSF for 1 to 3 days before addition of rhSCF further diminishes the number of mast cells that develop compared with the simultaneous addition of these growth factors on day 0. Whether administration of rhGM-CSF to humans before or together with rhSCF diminishes the mast cell hyperplasia that occurs with rhSCF alone remains to be determined.
Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes.
Authors: Tanabe S , Heesen M , Yoshizawa I , Berman M A , Luo Y , Bleul C C , Springer T A , Okuda K , Gerard N , Dorf M E
Published: January 2023

The mRNA for the seven-transmembrane-spanning G protein-coupled receptor fusin/CXCR-4 is expressed in primary mouse astrocyte cultures and the transformed mouse microglial cell line, N9. Cell surface expression of fusin in these cells was confirmed by staining with a polyclonal anti-fusin Ab. The functional capacity of this chemokine receptor was examined by evaluating the calcium responses following stimulation of glial cells with the CXC-chemokine, stromal-derived cell factor-1alpha (SDF-1alpha). Both astrocytes and microglial cells mobilized calcium following stimulation with chemically synthesized SDF-1alpha. SDF-1alpha- and carbachol-mediated calcium responses of astrocytes were partially inhibited by treatment with pertussis toxin (PTx), suggesting receptor coupling to a combination of G alpha(i) and other G proteins. In contrast, the calcium responses of microglial cells to SDF-1alpha were completely PTx sensitive, while responses to carbachol stimulation were PTx resistant. The ability of SDF-1alpha to induce glial cell migration was also examined. Synthetic SDF-1alpha was a potent chemoattractant for mouse microglial cells at ligand concentrations of 10 to 500 ng/ml; peak responses were noted at 100 ng/ml. In contrast, astrocytes did not migrate toward a gradient of SDF-1alpha. The failure of SDF-1alpha to induce astrocyte migration was specific, as another chemokine, macrophage inflammatory protein-1alpha, triggered astrocyte chemotaxis.
CD7-mediated regulation of integrin adhesiveness on human T cells involves tyrosine phosphorylation-dependent activation of phosphatidylinositol 3-kinase.
Authors: Chan A S , Mobley J L , Fields G B , Shimizu Y
Published: January 2023

The functional activity of integrin receptors on T cells is dynamically regulated so that T cells can alternate rapidly between adhesive and nonadhesive states. The CD7 Ag is one of several molecules on T cells that can transduce intracellular signals that rapidly up-regulate integrin-mediated adhesion. We demonstrate in this report that the signaling pathway that CD7 utilizes to regulate integrin activity involves the lipid kinase phosphatidylinositol 3-kinase (PI 3-K). CD7 stimulation of both Jurkat T cells and resting human peripheral blood CD4+ T cells results in rapid association and activation of PI 3-K with CD7. Phosphopeptide competition assays demonstrate that the association of CD7 with PI 3-K is dependent on tyrosine phosphorylation of the SH2 binding motif Tyr-Glu-Asp-Met (YEDM) in the CD7 cytoplasmic domain. A role for PI 3-K in the regulation of integrin function by CD7 is demonstrated by: 1) the ability of two structurally distinct PI 3-K inhibitors, wortmannin and LY294002, to inhibit CD7-mediated increases in beta1 integrin function of human T cells; and 2) inhibition of CD7-mediated activation of beta3 integrin function in human T cells by expression of a dominant negative form of the p85 subunit of PI 3-K. These results demonstrate that the CD7 Ag on human T cells is coupled to PI 3-K and that this association is relevant to CD7-mediated signaling events, specifically CD7-induced increases in integrin adhesiveness. Furthermore, these studies provide important new evidence implicating PI 3-K in the regulation of integrin adhesiveness by multiple cell surface signaling receptors.
CD4+ T cells from IRF-1-deficient mice exhibit altered patterns of cytokine expression and cell subset homeostasis.
Authors: McElligott D L , Phillips J A , Stillman C A , Koch R J , Mosier D E , Hobbs M V
Published: January 2023

Interferon regulatory factor-1 (IRF-1) is a member of a family of transcription factors that regulate an array of genes involved in cell growth, differentiation, and death. Analysis of cytokine expression by stimulated CD4+ cells from IRF-1(-/-) and IRF-1(+/+) mice revealed that IRF-1 deficiency resulted in an elevated production of Th2-related cytokines and a compensatory decrease in the expression of naive cell- and Th1-related cytokines. The altered cytokine profiles of IRF-1(-/-) cells could be explained, in part, by a shift in the representation of subsets of CD4+ cells; IRF-1(-/-) mice exhibited a decreased percentage of naive cells (a major source of IL-2) but increased numbers of memory or effector cells (the source of Th2-related cytokines). We analyzed purified, phenotypically matched memory/effector cells from IRF-1(-/-) and IRF-1(+/+) mice and found that the increased Th2:Th1 cytokine ratio was still evident in the IRF-1(-/-) group, thus suggesting that IRF-1 is involved in the polarization of the cytokine repertoire in CD4+ cells. Our data indicate that IRF-1 plays an important role in the maintenance of CD4+ cell subset homeostasis and in the expression of cytokines by naive and memory/effector cells.
Cloning and analysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry.
Authors: Moriuchi M , Moriuchi H , Turner W , Fauci A S
Published: January 2023

The chemokine receptor CXCR4 (also designated fusin and LESTR) is a cofactor for fusion and entry of T cell-tropic strains of HIV-1. CXCR4 is expressed in various cell types; however, the mechanisms involved in the regulation of its expression remain unknown. To delineate these mechanisms, approximately 1.2 kb of DNA from the immediate 5' upstream region of CXCR4 gene was cloned, sequenced, and characterized. Transient expression assays using CXCR4 promoter/luciferase gene reporter constructs revealed that stimulation with PMA plus ionomycin up-regulates the CXCR4 promoter activity in the A3.01 CD4+ T cell line and PBL and that a DNA fragment from -93 to +59 relative to the transcription start site contributes markedly to the basal and induced activity. This fragment contains a consensus TATA box, two potential GC boxes, and a potential nuclear respiratory factor (NRF)-1 binding site, which were confirmed by gel mobility shift assays and footprinting analysis. Mutagenesis studies revealed that a NRF-1 site is especially important for the basal and induced activity of the CXCR4 promoter. Transient expression assays further revealed that stimulation of PBL with either IL-2 or Abs to CD3 and CD28 enhances the CXCR4 promoter activity. Inducibility of the CXCR4 promoter activity by T cell stimulation suggests that overexpression of CXCR4 may be one of the mechanisms whereby immune activation and/or perturbation of the cytokine network up-regulate HIV expression and replication and thus contribute to the progression of HIV disease.
TNF-alpha and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells.
Authors: Barks J L , McQuillan J J , Iademarco M F
Published: January 2023

Vascular cell adhesion molecule-1 (VCAM-1) is expressed on the endothelium and vascular smooth muscle in atherosclerosis, where it is thought to recruit alpha4 integrin-positive leukocytes, which play a role in disease progression. In this study, we show an increase of VCAM-1 expression on vascular smooth muscle cells (VSMC) results in increased adhesion of alpha4 integrin-positive lymphocytes. Additionally, we examine the regulation of VCAM-1 expression by cytokines in cultured VSMC. Previously in endothelial cells, we have demonstrated that TNF-alpha increases transcription of the VCAM-1 gene, whereas IL-4 acts to increase VCAM-1 mRNA stability. The combination of a cytokine that increases transcription with a cytokine that stabilizes mRNA results in a synergistic increase in VCAM-1 expression. In this study, we show that the combination of TNF-alpha with IL-4 also resulted in a synergistic increase in VCAM-1 expression on VSMC; however, the mechanism of cytokine activation differed. In contrast to endothelial cells, IL-4 stimulated VCAM-1 gene transcription in the VSMC, but there was little effect of TNF-alpha alone. Additionally, the synergy between TNF-alpha and IL-4 appears to result, at least in part, from a cooperative transcriptional mechanism.