bluuebunny/crossref_metadata_2025 huggingface dataset:
Human V gamma 9-V delta 2 T cell receptor-gamma delta lymphocytes show specificity to Daudi Burkitt's lymphoma cells.
Authors: Sturm E , Braakman E , Fisch P , Vreugdenhil R J , Sondel P , Bolhuis R L
Published: January 2023

Peripheral blood TCR-gamma delta cells with different functional V gamma or V delta gene rearrangements represent two nonoverlapping subsets. The major subset uses the V gamma 9 and the V delta 2 gene segments and the minor subset the V delta 1 gene segments in its functional TCR rearrangement. Upon in vitro activation, these TCR-gamma delta lymphocytes display MHC-unrestricted lytic activity, against a wide variety of tumor cells of distinct histologic origin. Here we show that fresh TCR-gamma delta lymphocytes that express a V gamma 9-V delta 2 encoded TCR display a specific proliferative response to Daudi, Burkitt's lymphoma cells. Moreover, cloned V gamma 9-V delta 2 lymphocytes show the capacity to lyse Daudi cells, whereas none of the cloned V gamma 1 TCR-gamma delta lymphocytes shows such specificity. Nucleotide diversity at the V-D-J junction of the TCR-V delta 2 gene did not contribute to this Daudi cell specificity. Comparison of the MHC-unrestricted cytolytic capacities of the V gamma 9-V delta 2 and the V delta 1 clones using a panel of distinct types of tumor target cells showed that on average, the level of MHC unrestricted lysis of V gamma 9-V delta 2 clones against these tumor cells exceeded that of V delta 1 clones. However, in contrast to all these tumor cell lines, only the Daudi cells showed such an absolute distinction in susceptibility to lysis by V gamma 9-V delta 2 and V delta 1 clones. V gamma 9-V delta 2 clones that were generated with a stimulator cell other than Daudi did not lyse their stimulator cells but nevertheless showed specific cytolysis of Daudi cells. The specific proliferation to and cytolysis of Daudi cells of the entire V gamma 9-V delta 2 subpopulation of TCR-gamma delta lymphocytes is reminiscent of a superantigen response.
Differential expression of homing-associated adhesion molecules by T cell subsets in man.
Authors: Picker L J , Terstappen L W , Rott L S , Streeter P R , Stein H , Butcher E C
Published: January 2023

The ability of lymphocyte populations to recognize and bind high endothelial venules during homing into lymphoid tissues and sites of chronic inflammation is critically dependent on their expression of certain homing-associated adhesion molecules known as homing receptors (HR). In animal models, certain lymphocyte populations, particularly subsets of memory or previously activated lymphocytes, demonstrate tissue-selective homing behavior, and it has been hypothesized that differential expression of HR accounts for this selective migration. In this study, we analyzed expression of human HR--the Dreg 56/Leu 8-defined peripheral lymph node (PLN) HR (also known as LECAM-1), H-CAM (CD44), and alpha 4-integrins (CD49d; VLA-4)--among subsets of thymocytes and peripheral blood T cells to identify populations with differential homing potential. In the thymus, these three HR classes are differentially regulated relative to phenotypically defined maturational stages, but are all expressed on the mature, surface CD3high subset. In the peripheral blood, virgin T cells (LFA-3/CD58low) show uniform high expression of the PLN HR, uniform relatively low expression of H-CAM and alpha 4-integrin, and lack markers of tissue association--the mucosal and cutaneous lymphocyte associated Ag (MLA and CLA Ags) defined by mAb Ber ACT8 and HECA-452, respectively. In contrast, circulating memory T cells (LFA-3/CD58high) are bimodal with respect to PLN HR expression, show uniform high expression of H-CAM and alpha 4-integrin, and contain essentially all the CLA and MLA Ag-bearing T cells. The circulating skin-associated T cell subset (CLA Ag+; 10 to 15% of total T cells) is predominantly PLN HR+, and shows high levels of both the alpha 4- and beta 1-integrin chains. The distinct mucosa-associated T cell subset (MLA Ag+; 1 to 3% of peripheral blood T cells) is predominantly PLN HR-; and is alpha 4high, but beta 1low. These findings indicate the independent regulation of homing-associated adhesion molecules among populations of memory/previously activated T cells, and suggest that the expression patterns of these molecules contribute to, or perhaps determine, the tissue distribution of these subsets.
Human thymic epithelial cells produce IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor.
Authors: Le P T , Lazorick S , Whichard L P , Yang Y C , Clark S C , Haynes B F , Singer K H
Published: January 2023

The development of conditions for culturing normal human thymic epithelial (TE) cells free from contaminating stromal cells has allowed us to characterize a number of cytokines produced by TE cells. Using cDNA probes for human IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor (LIF), we identified mRNA for these cytokines by RNA blot analysis of total RNA preparations derived from TE cells. We demonstrated that TE cells produced IL-6 transcripts and that TE cell culture supernatants contained IL-6 biologic activity, as determined by the ability to support proliferation of the T1165 plasmacytoma line. The 1.0-kilobase (kb) transcript of granulocyte-monocyte-CSF was also detected in TE cell-derived total RNA. TE cell culture supernatants contained LIF activity, as determined by proliferation of the murine cell line DA-1a, and a 4.0-kb LIF transcript was detected in TE cell-derived total RNA preparations. The 4.0-kb LIF transcript from TE cell-derived total RNA corresponded in size to the LIF transcripts in PMA-activated T lymphocytes. Thus, using biologic assays and RNA blot analysis, we demonstrated that cultured normal human TE cells produced both immunoregulatory cytokines and cytokines that drive various differentiation stages of human hematopoiesis. Our findings support the hypothesis that TE cells may play a role in providing cytokines that are important for the proliferation and differentiation of hematopoietic precursor cells that migrate to the thymus during fetal and postnatal human thymic development.
Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers.
Authors: Lee J M , Watts T H
Published: January 2023

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.
Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation.
Authors: Thompson R A , Ballas Z K
Published: January 2023

Guanine ribonucleosides, substituted at the C8 position with either a bromine or a thiol group, have recently been shown to regulate several immunologic responses. We have previously shown that 8-mercaptoguanosine (8MG) can replace the requirement for cytokines in the generation of MHC-restricted CTL. In this paper, we examined the ability of 8MG to induce MHC-nonrestricted killer cells. We found that 8MG did not induce significant lytic activity from normal resting lymphocytes. However, 8MG was able to synergize with minimal amounts of IL-2 in inducing lytic activity similar to lymphokine-activated killers (LAK) in that both NK-sensitive and NK-resistant tumor cells were killed. Both the precursors and effectors of 8MG-LAK activity were similar to NK cells and were CD4- CD8- asialo-GM1+ NK1.1+. Similar to IL-2-induced LAK, 8MG-LAK were B220+. 8MG appeared to "stage" these precursor lymphocytes to become more responsive to IL-2 because optimal induction of 8MG-LAK required preincubation with 8MG before the addition of IL-2. This "staging" appeared to be due to the release of a "second signal" since it was readily inhibited by cyclosporine A. Anti-IFN-alpha beta was as efficient as cyclosporine A in inhibiting 8MG-LAK generation, whereas anti-IFN-gamma and anti-IL-1 did not exhibit significant inhibition. These findings suggest that 8MG can be of possible utility as an IL-2-sparing agent in LAK generation from NK cells.
Long-term CD4+ memory T cells from the spleen lack MEL-14, the lymph node homing receptor.
Authors: Bradley L M , Atkins G G , Swain S L
Published: January 2023

We have characterized the surface phenotype and function of long-lived, Ag-specific memory CD4+ T cells generated in vivo by immunization with keyhole limpet hemocyanin (KLH). CD4+ T cells from the spleens of mice primed more than 2 mo previously with KLH, produced high levels of IL-2 and IL-3, and low levels of IL-4 and IFN-gamma in response to in vitro restimulation with specific Ag. The KLH-primed T cells mediated carrier-specific helper activity for the antibody production by NIP-primed B cells in secondary in vitro responses to NIP-KLH. Subsets of CD4+ T cells from KLH-primed mice were isolated on the basis of surface CD45RB (23G2) by magnetic separation and were examined for functional capacity in several assays of Ag-specific recall. Virtually all of the secretion of IL-2, IL-3, IL-4, and IFN-gamma in response to restimulation with Ag in vitro was associated with, and considerably enriched in, the CD45RB- subset of CD4+ T cells. Similarly, carrier-specific helper function and Ag-specific proliferation in vitro were also confined to the CD45RB-, CD4+ subset of T cells, confirming the previous association of this surface phenotype with memory Th cell activity. We also examined expression of the lymphocyte homing receptor, MEL-14 (gp90MEL), which is required for lymphocyte extravasation to peripheral lymph nodes and is present in high levels on naive T cells. MEL-14 positive and negative subsets of CD4+ T cells from long term KLH-primed mice were evaluated for Ag-specific memory function in terms of lymphokine production, Ag-induced proliferation, and helper activity. Each of these functions was associated exclusively with the MEL-14- subset of CD4+ T cells, which exhibited responses comparable to the CD45RB- subset. These data indicate that memory Th cell function in the spleen is contained within the MEL-14-, CD45RB- subset of CD4+ T cells and suggest that memory helper cells may have different patterns of recirculation from naive T cells.
Cytokine-stimulated human mesothelial cells produce chemotactic activity for neutrophils including NAP-1/IL-8.
Authors: Goodman R B , Wood R G , Martin T R , Hanson-Painton O , Kinasewitz G T
Published: January 2023

To test the hypothesis that mesothelial cells play a role in regulating inflammatory responses within the pleural space, we examined neutrophil chemotactic activity released by cytokine-stimulated mesothelial cells. Human mesothelial cells were isolated from patients with transudative pleural effusions and cultured. The purity of the cell population was assessed by morphologic, immunocytochemical, and biochemical characteristics. Confluent fourth passage mesothelial cell plates were exposed to varying concentrations of the recombinant human cytokines IL-1 alpha, TNF-alpha, or IFN-gamma, or Escherichia coli endotoxin (LPS). Polymorphonuclear neutrophil (PMN) chemotactic activity in the conditioned media was measured in microchemotaxis chambers. Although none of the cytokines demonstrated inherent chemotactic activity, each stimulated mesothelial cells to produce PMN chemotactic activity in a dose-dependent manner. TNF-alpha stimulated the release of the greatest quantity, whereas stimulation with IFN-gamma and IL-1 alpha resulted in the release of lesser but still significant quantities of PMN chemotactic activity. By contrast, LPS did not increase the basal level of chemotactic activity produced by the cells. The cytokine-induced chemotactic activity was proteinaceous, required de novo synthesis, and had a predominant m.w. of 10,000. Significant quantities of immunoreactive neutrophil-activating peptide-1 (NAP-1)/IL-8 were detected in mesothelial cell supernatants after stimulation with each of the cytokines. The neutrophil chemotactic activity of supernatants from mesothelial cells stimulated with either IL-1 alpha or IFN-gamma was completely neutralized with rabbit anti-human NAP-1/IL-8 polyclonal antiserum. The same antiserum neutralized the majority, but not all, of the neutrophil chemotactic activity in supernatants from TNF-stimulated mesothelial cells. Stimulated mesothelial cells also expressed an inducible mRNA transcript that hybridized with a specific oligonucleotide probe for human NAP-1/IL-8. These observations provide a mechanism whereby mesothelial cells could respond to inflammatory stimuli in the underlying lung and regulate inflammatory responses within the pleural space.
IFN-gamma-induced L-arginine-dependent toxoplasmastatic activity in murine peritoneal macrophages is mediated by endogenous tumor necrosis factor-alpha.
Authors: Langermans J A , Van der Hulst M E , Nibbering P H , Hiemstra P S , Fransen L , Van Furth R
Published: January 2023

Activated murine peritoneal macrophages inhibit the intracellular proliferation of Toxoplasma gondii and produce a number of cytokines, such as TNF-alpha and IL-1. Both TNF-alpha and IL-1 have been reported to be involved in the immune response against various microorganisms, but the mechanisms responsible for these effects are not known. In the present study it was investigated whether endogenously produced TNF-alpha and IL-1 are involved in the activation of peritoneal macrophages by rIFN-gamma leading to toxoplasmastatic activity and the production of reactive nitrogen intermediates. The rIFN-gamma-induced toxoplasmastatic activity was inhibited by neutralizing antibodies against mouse TNF-alpha in a dose-dependent and time-dependent way, but neutralizing antibodies against mouse IL-1 alpha and IL-1 beta did not affect this activity. Involvement of TNF-alpha in the induction of toxoplasmastatic activity was confirmed by our finding that rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma inhibited the intracellular proliferation of T. gondii. No synergistic activity of rIL-1 and rIFN-gamma on the inhibition of T. gondii proliferation was found. Both rTNF-alpha and rIL-1 alpha alone inhibited the intracellular proliferation of T. gondii only slightly. Because it has been reported recently that activated macrophages produce reactive nitrogen intermediates that are essential in the induction of toxoplasmastatic activity, we investigated whether these intermediates are involved in the TNF-dependent induction of toxoplasmastatic activity. Neutralizing antibodies against mouse TNF-alpha inhibited also the release of NO2- by rIFN-gamma-activated macrophages almost completely. Macrophages incubated with rTNF-alpha in combination with a nonactivating concentration of rIFN-gamma released substantial amounts of NO2-, but rTNF-alpha and rIL-1 alpha alone, and the combination of rIL-1 alpha and a nonactivating concentration of rIFN-gamma induced only little NO2(-)-release by macrophages. To assess whether reactive nitrogen intermediates act directly or indirectly on the intracellular proliferation of T. gondii, macrophages were incubated with the L-arginine analog NG-monomethyl-L-arginine or the NADPH-inhibitor diphenylene iodonium, both inhibitors of the generation of reactive nitrogen intermediates. Good correlation was found between toxoplasmastatic activity and the release of NO2- during the 24-h activation period before infection of the macrophages with T. gondii, but no correlation was found between toxoplasmastatic activity and the release of NO2- during infection of the macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
Identification of a novel gene expressed in activated natural killer cells and T cells.
Authors: Dahl C A , Schall R P , He H L , Cairns J S
Published: January 2023

We have isolated a cDNA clone from a human activated NK cell-derived cDNA library that identifies a transcript (NK4) that is selectively expressed in lymphocytes. The expression of this transcript is increased after activation of T cells by mitogens or activation of NK cells by IL-2 (lymphokine-activated killer cells). The transcript levels demonstrated by Northern blot analysis increase by 12 h after activation, remain high for at least 48 h, and require protein synthesis for expression. Southern blot analysis of B lymphoblastoid lines derived from 18 unrelated individuals reveal variable banding patterns suggestive of polymorphism within the NK4 gene. No homology was found between the sequence of the coding region of this transcript and any sequences in the GenBank data base. Sequence homology to the U1 small nuclear RNA was found within the 3' untranslated region immediately upstream of the site of polyadenylation, suggesting a possible role for U1 in the polyadenylation process. Sequence analysis indicates the transcript would encode a protein having a mass of 27 kDa. The presence of a signal sequence and lack of a transmembrane region suggests that the protein is secreted. In addition, the protein contains an RGD sequence that may be involved in cellular adhesion. This transcript appears to encode a novel product common to the activation pathways of both NK cells and T cells.
Influence of MHC genes on spontaneous recovery from Friend retrovirus-induced leukemia.
Authors: Miyazawa M , Nishio J , Wehrly K , Chesebro B
Published: January 2023

Genes influencing the rate of spontaneous recovery from erythroleukemia induced by a low dose of Friend virus complex were located in the right and left portions of the mouse MHC. The right side gene was most likely the previously described Rfv-1 in the H-2D region. Using the B6.C-H-2bm12 mutant mice, the left side gene was mapped to the A beta class II locus. The A beta b was a resistant allele and A beta k and A beta bm12 were susceptible alleles. Genes at this class II locus controlled the responsiveness of Th cells to envelope glycoprotein of Friend murine leukemia helper virus and affected the class switching of virus-neutralizing antibodies from IgM to IgG in FV-infected mice.
Human antibody response in human peripheral blood leukocyte/severe combined immunodeficient chimeric model is dependent on B and T cell costimulation via CD40/CD40 ligand.
Authors: Chen F A , Williams S S , Fanslow W C , Bankert R B
Published: January 2023

We have used a human/severe combined immunodeficient (SCID) mouse chimeric model (in which human PBLs are engrafted into SCID mice) to investigate in vivo the role of the CD40/CD40 ligand (CD40L) interaction in the generation of humoral immunity by engrafted human cells. It is established in this work that the thymic dependent, multiclonal, humoral immune response of human lymphocytes to mouse erythrocyte protein Ags is inhibited significantly when CD40/CD40L ligation is prevented in vivo. Suppression of the response was observed with administration of neutralizing Abs specific for either CD40 or CD40L, or with the soluble fusion protein human CD40-Fc. Human anti-mouse erythrocyte Abs and the total human Ig levels in the sera of the SCID mice were suppressed for up to 10 wk when the neutralizing agents were administered at the time of human leukocyte engraftment, i.e., coincident with Ag stimulation, and subsequently at 2 and 4 days after Ag stimulation. These results are the first to demonstrate the ability of CD40/CD40L blocking agents to inhibit the human Ag-specific humoral immune response in vivo, and they sustain the notion that these blocking agents may be utilized clinically to eliminate immune responses associated with autoimmunity or allergy. In addition to confirming the importance of the CD40/CD40L interaction of T and B cells in vivo, it is established in this work that the mouse erythrocyte-specific humoral immune response of the lymphocytes in the human PBL/SCID mouse model is a bona fide T cell-dependent response, and that this is a viable model with which to study human lymphocyte activation in vivo. These results also suggest that the costimulatory activation of human B and T cells is not dependent on the microenvironment of the germinal centers.
Differences in the binding of blocking anti-CD11b monoclonal antibodies to the A-domain of CD11b.
Authors: Violette S M , Rusche J R , Purdy S R , Boyd J G , Cos J , Silver S
Published: January 2023

CD11b/CD18 (Mac-1) is a leukocyte integrin that plays a critical role in neutrophil adhesion and the initiation of acute inflammatory responses. Several Mac-1 blocking mAbs bind to the A-domain of CD11b, a approximately 200 amino acid region in the N-terminal portion of the protein that is involved in ligand binding and Mac-1 functional activity. We examined several CD11b blocking mAbs for different patterns of binding to A-domain. We used human/murine chimeric CD11b expression constructs and deletions of the A-domain to examine binding. We describe the binding characteristics of mAbs 60.1, LM2/1, LPM19C, M170, 44, and 904. All of these mAbs, except for 60.1, bind to the C-terminal half of the human A-domain (CD11b181-316). mAb 60.1 was unique in that it required regions of the N- and C-terminal ends of the A-domain for binding. mAbs 60.1, LPM19C, 904, and 44 all required the A-domain to be intact for binding. This suggests that these CD11b mAbs recognize a conformational epitope. LM2/1 was capable of binding to a fragment of the A-domain, CD11b285-300. Inasmuch as this system has been used to define different mAb binding sites, it may be used to analyze specific ligand binding sites in the A-domain of CD11b.
Sequences of V kappa L subgroup light chains in Fanconi's syndrome. Light chain V region gene usage restriction and peculiarities in myeloma-associated Fanconi's syndrome.
Authors: Rocca A , Khamlichi A A , Touchard G , Mougenot B , Ronco P , Denoroy L , Deret S , Preud'homme J L , Aucouturier P , Cogné M
Published: January 2023

Certain monoclonal Ig light chains (LC) are responsible for marked disturbances of proximal tubule cell functions (Fanconi's syndrome, FS). In patients with FS, intracellular crystal-like inclusions containing LC determinants are commonly found in plasma cells, macrophages, and renal tubular cells. In an attempt at understanding the pathogenesis of myeloma-associated FS, we recently determined the first complete primary sequence of a kappa-LC (CHEB) responsible for the disease. We now report on the primary structure of three other kappa-LC of the V kappa l variability subgroup associated with FS (TRE, TRO, and DEL). After PCR amplification, cDNA encoding these LC were sequenced. CHEB, TRE, and TRO LC genes were found to be highly homologous to the same germline gene O2/O12. These patients had numerous intracellular crystals, whereas the fourth patient, DEL, had no detectable crystals. The LC from the latter patient was homologous to another germline gene, O8/O18. Comparison of these LC sequences to previously reported LC of the V kappa l subgroup allowed identification of a number of unusual amino acid substitutions in the V region that had rarely or never been previously described at the corresponding positions. Some of these unusual substitutions affect highly conserved amino acids located either in an external loop (residue 30) or in inner (residues 48 and 55) and outer (positions 63 and 72) beta-sheets that may be important for the structure and binding properties of the kappa-chains. These and several other substitutions, some of them shared with amyloidogenic kappa-LC, could induce conformational alterations and represent a determinant pathogenic factor.
Induction of antigen-specific T and B cell immunity in colon carcinoma patients by anti-idiotypic antibody.
Authors: Somasundaram R , Zaloudik J , Jacob L , Benden A , Sperlagh M , Hart E , Marks G , Kane M , Mastrangelo M , Herlyn D
Published: January 2023

Polyclonal goat anti-idiotypic Abs directed against anti-human gastrointestinal carcinoma mAb GA733 were administered to 13 colon cancer patients who had their primary tumor and lymph node metastases removed before immunotherapy. Patients received four s.c. doses (0.5 to 8 mg each) of alum-precipitated anti-idiotypic Ab. Seven of the 13 patients produced anti-anti-Ids that bound specifically to the GA733 epitope on tumor cells and shared idiotopes with mAb GA733. In four of the seven responding patients, anti-Id therapy specifically modulated T cell responses. In two patients who did not demonstrate GA733 Ag/anti-Id-reactive T cells before therapy, anti-Id administration induced CD4+, MHC class II-dependent T cells that specifically proliferated in culture in response to stimulation with either anti-Id or GA733 Ag. In two other patients who did demonstrate Ag/anti-Id-reactive T cells before therapy, anti-Id administration transiently induced lymphocytes that suppressed the proliferative responses of cultured pretherapy lymphocytes to stimulation with anti-Id or GA733 Ag. Nine of the 13 treated patients showed no evidence of disease after 39 to 86 mo of observation. Five of these patients developed Ag-specific Ab3 and one had, in addition, a T cell response.
Cytokine and chemokine expression in tumors of mice receiving systemic therapy with IL-12.
Authors: Tannenbaum C S , Wicker N , Armstrong D , Tubbs R , Finke J , Bukowski R M , Hamilton T A
Published: January 2023

The cellular and molecular mechanisms of IL-12-mediated anti-tumor activity have been examined. BALB/c mice bearing established s.c. RENCA or CT26 tumors that were treated daily with IL-12 showed essentially complete tumor regression while tumors in untreated animals grew progressively. Examination of inflammatory gene expression in tumor tissue from treated vs untreated mice revealed the selective expression of IFN-gamma and the IFN-gamma-inducible CXC chemokine IP-10. Immunohistologic analysis demonstrated that tumors from treated mice were heavily infiltrated with CD8+ T cells and Mac-1+ mononuclear cells. Tumor regression in IL-12-treated mice was associated with expression of the lytic effector molecules perforin and granzyme B. These findings support the hypothesis that the anti-tumor function of IL-12 treatment depends upon the induced expression of IFN-gamma by T cells and/or NK cells, the amplification of the immune response mediated by IFN-gamma-induced expression of chemoattractant cytokines, and the IL-12-dependent potentiation of the cytolytic effector function of recruited CD8+ T cells.
Adjuvant-free hsp70 fusion protein system elicits humoral and cellular immune responses to HIV-1 p24.
Authors: Suzue K , Young R A
Published: January 2023

Heat shock proteins are major targets of the immune response to bacterial and parasitic pathogens. Mycobacterium tuberculosis hsp70 is an especially powerful Ag containing multiple B and T cell epitopes. We investigated whether M. tuberculosis hsp70 can be used as an adjuvant-free carrier to stimulate the humoral and cellular immune response to an accompanying protein. A recombinant hsp70 protein expression vector was developed that permits the production of any protein fused to the amino terminus of mycobacterial hsp70. We found that a recombinant HIV p24-hsp70 fusion protein produced with this vector elicited both humoral and cellular immune responses against p24 in mice when administered in saline in the absence of adjuvant. Covalent linkage of hsp70 to p24 was essential to elicit immune responses to p24 under these conditions. The anti-p24 IgG1 Abs induced in p24-hsp70-immunized mice persisted at high levels for more than 1 yr after immunization. These results demonstrate that the antigenic properties of M. tuberculosis hsp70 can be exploited to enhance the humoral and cellular immune response to an attached protein.
Uncoupling IL-2 production from apoptosis and TNF production by changing the signal through the TCR.
Authors: Glickstein L , Macphail S , Stutman O
Published: January 2023

T cells may discriminate between stimuli in a variety of ways, including the presence of cytokines or other costimulatory signals, the type of Ag (peptide, superantigen, or allorecognition), or the magnitude of the signal through the TCR. We have used anti-CD3 stimulation of T hybridomas to examine signals generated through the TCR in the absence of exogenous APCs. Soluble whole anti-CD3, but not F(ab')2 anti-CD3, was able to stimulate the T hybridomas to produce IL-2. Plastic-bound anti-CD3, in contrast, stimulated TNF production, G1 arrest, and apoptosis by the T hybridoma. Engagement of the CD4 coreceptor on these cells had no effect on the overall pattern of signaling observed. Although TNF production was correlated with apoptosis, anti-TNF treatment did not prevent cell death or G1 arrest. The response of the T hybridoma to both forms of anti-CD3 included significant IL-2 production even at the lowest dose tested. However, soluble anti-CD3 at the highest dose tested elicited only minor apoptosis, while plastic-bound anti-CD3 elicited significant apoptosis even at the lowest dose. The difference in response was not evident at the level of phosphotyrosine proteins two min after cross-linking of the TCR.
Retrovirus-mediated transfer of viral IL-10 gene prolongs murine cardiac allograft survival.
Authors: Qin L , Chavin K D , Ding Y , Tahara H , Favaro J P , Woodward J E , Suzuki T , Robbins P D , Lotze M T , Bromberg J S
Published: January 2023

A murine heterotopic, nonvascularized cardiac allograft model was used to examine the effects of the immunosuppressive cytokine, viral IL-10 (vIL-10), delivered by gene transfer on graft rejection. Retroviral-mediated gene transfer and expression of vIL-10 significantly prolonged allograft survival, without conventional systemic immunosuppression, from 12.1 +/- 0.8 days to 39.4 +/- 2.5 days (p < 0.0001). The effect was specific, dose dependent, and restricted to the site of transplantation. PCR analysis demonstrated specific expression of the transferred gene within the allograft. Analysis of the cellular infiltrate in the allografts showed a reduction in T cells and alloantigen-specific cytotoxic T cells and IL-2 producing helper T cells. Thus, the transient local expression of a gene encoding an immunosuppressive protein within a graft can generate local immunosuppression, making gene therapy a viable approach for facilitating transplantation.
Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor.
Authors: Brodbeck W G , Liu D , Sperry J , Mold C , Medof M E
Published: January 2023

Decay-accelerating factor (DAF) is a cell-associated C regulatory protein that protects host cells from autologous C attack. It functions intrinsically in host cell surface membranes to rapidly dissociate autologous classical and alternative pathway C3 convertases whenever these amplifying enzymes assemble on host cell surfaces. It is composed of four contiguous approximately 70 amino acid long regions termed short consensus repeats (SCRs) that share homology with similar units in other C3 convertase regulatory proteins. It is attached to the cell surface membrane by a glycoinositol phospholipid (GPI) anchor that is added posttranslationally. In this study, we prepared rGPI-anchored DAF proteins devoid of individual SCRs. We then incorporated the GPI-anchored products into sheep erythrocyte (Esh) hemolytic intermediates and examined their abilities to intrinsically regulate classical or alternative pathway activation. We found that classical pathway C3 convertase regulatory function resides within SCR-2 and SCR-3, while alternative pathway C3 convertase regulatory function resides within SCR-2, -3, and -4. Functional comparisons of the variant DAF proteins in fluid phase C3 activation assays established that the differences reflect domain-specific interactions rather than changes in the spatial arrangement of SCRs above the cell surface. In accordance with these findings, we found that variant DAF molecules containing SCR-1, -2, and -3, but not SCR-4, function to selectively inhibit classical pathway activation.
Di- and trinucleotide target preferences of somatic mutagenesis in normal and autoreactive B cells.
Authors: Smith D S , Creadon G , Jena P K , Portanova J P , Kotzin B L , Wysocki L J
Published: January 2023

During Ag-driven development of memory B cells, Ab V genes are modified by somatic mutagenesis. Although V gene somatic mutations have important biologic consequences in both physiologic and autoimmune Ab responses, little is known about the mechanism of mutation, or whether it operates normally in autoreactive B cells. To approach these issues, we analyzed somatic mutations in Ab genes for evidence of sequence-specific target preferences. Our analysis was confined to noncoding segments of V genes so that the intrinsic characteristics of the somatic mutation process could be reliably dissociated from the indirect but substantial influences of cellular selection. We consistently observed that some dinucleotides, GC and TA in particular, mutated at frequencies that were higher than expected based on their frequency of occurrence. Most of the dinucleotide mutation preferences could not be extrapolated directly from mononucleotide mutation preferences. Specific trinucleotides, including AGC, TAC, and their inverse repeats (GCT, GTA), also mutated more frequently than expected. These and other mutation characteristics were virtually indistinguishable in V genes of normal and autoreactive B cells. An analysis of mutations in published flanking sequences confirmed the target preferences, as did an examination of reported "hot spots" within coding V sequences. The shared preferences in coding and noncoding regions of V genes suggests that somatic mutations are generated de novo. Collectively, our findings indicate that the somatic mutation process exhibits sequence-specific preferences, consistent with an untemplated mechanism, and appears to operate similarly in normal and autoreactive B cells.
Mucosal IFN-gamma gene transfer inhibits pulmonary allergic responses in mice.
Authors: Li X M , Chopra R K , Chou T Y , Schofield B H , Wills-Karp M , Huang S K
Published: January 2023

Th2 cytokines are associated with the airway inflammation and hyperreactivity in allergic asthma and are potential targets for developing novel therapies. The efficacy of a mucosal cytokine-gene transfer approach was examined in a mouse model for allergic asthma. We showed first that mucosal IFN-gamma gene transfer results in a significant expression of IFN-gamma in the pulmonary epithelium. Significantly, this approach inhibits both Ag- and Th2-cell-induced pulmonary eosinophilia and airway hyperreactivity. These findings suggest that mucosal IFN-gamma gene transfer is effective in modulating pulmonary allergic responses and provides a basis for developing a novel therapeutic approach.
Residues critical for H-L disulfide bond formation in human IgA1 and IgA2.
Authors: Chintalacharuvu K R , Morrison S L
Published: January 2023

There are two subclasses of human IgA, IgA1 and IgA2. IgA2 exists as two known allotypes, IgA2 m(1) and IgA2 m(2) with a recently reported novel IgA2 (IgA2(n)) possibly representing a third allotype. The variants of human IgA differ in their H and L chain disulfide-bonding pattern; in IgA1, IgA2(n), and IgA2 m(2), a disulfide bond connects a cysteine residue in CH1 of the H chain with the L chains while human IgA2 m(1) has been reported to lack a covalent bond between the H and L chains. Here we have used site-directed mutagenesis to demonstrate that Cys133 is essential for the formation of the H-L disulfide bond in IgA1. However, IgA2 m(2) and the IgA2(n) but not IgA2 m(1) form an H-L disulfide in the absence of Cys133. IgA2 m(1) differs from IgA2 m(2) and the IgA2(n) at two positions in CH1; IgA2 m(1) has Pro212 and Pro221 whereas IgA2 m(2) and the IgA2(n) have Ser212 and Arg221. Our studies demonstrate that it is the presence of Pro221 in IgA2 m(1) that interferes with the H-L disulfide in the absence of Cys133. Contrary to what has been previously reported, protein purified from culture supernatants of IgA2 m(1) show some HL, H2L2, and H4L4J, suggesting that IgA2 m(1) can exist either as a form lacking H-L disulfide bonds or as a form with H-L disulfides.
HMG box-activating factors 1 and 2, two HMG box transcription factors that bind the human Ig heavy chain enhancer.
Authors: Stevens S , Ordentlich P , Sen R , Kadesch T
Published: January 2023

We present the isolation of two cDNAs that encode distinct, yet related, proteins that bind the HE2 region of the human Ig heavy chain (IgH) enhancer. Designated HMG box-activating factors (HAF) 1 and 2, the two proteins are new members of the HMG box family of DNA binding proteins. Both are potent transcription activators when expressed 1) as GAL4 fusions targeted to promoters containing GAL4 operators, or 2) as intact proteins targeted to minimal promoters containing binding sites derived from the IgH enhancer. HAF-1 and HAF-2 mRNAs are apparently expressed in both B cells and non-B cells. However, activity generated by the isolated HE2 region in B cells is dependent on both an intact HAF-1/HAF-2 binding site and at least one additional site that has been reported previously to bind a B cell-restricted protein. Our results suggest a collaborative role for either or both HAF-1 and HAF-2 in establishing the B cell activity of the human IgH enhancer.
Induction of antigen-specific tolerance for the treatment of ongoing, relapsing autoimmune encephalomyelitis: a comparison between oral and peripheral tolerance.
Authors: Kennedy K J , Smith W S , Miller S D , Karpus W J
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune demyelinating disease of the central nervous system that serves as an animal model for multiple sclerosis. Various forms of Ag-specific tolerance have been used prophylactically to prevent development of acute EAE. Here we compare the induction of Ag-specific tolerance using two regimens, proteolipid protein 139-151 (PLP139-151) peptide-coupled splenocytes and oral administration of PLP139-151, for efficacy in the reduction of established, chronic clinical EAE. PLP139-151-coupled splenocytes and not oral administration of PLP139-151 was able to down-regulate established EAE, including subsequent relapses. PLP139-151 peptide-coupled splenocytes were effective at reducing Ag-specific T cell proliferation and IL-2 and IFN-gamma production, while concomitantly increasing IL-4 production. Oral administration of PLP139-151 did not reduce IL-2 or IFN-gamma production and appeared to increase Ag-specific T cell proliferation. Neither multiple high nor low doses of PLP139-151 were effective at decreasing ongoing clinical EAE or PLP139-151-specific IL-2 and IFN-gamma production. These results suggest that PLP139-151 peptide-induced tolerance is an efficacious treatment for ongoing, R-EAE when the peptide is coupled to chemically fixed splenocytes and not when given orally.
Role of adhesion molecules of the selectin-carbohydrate families in antibody-dependent cell-mediated cytoxicity to schistosome targets.
Authors: Trottein F , Nutten S , Papin J P , Leportier C , Poulain-Godefroy O , Capron A , Capron M
Published: January 2023

The role of adhesion molecules in antibody-dependent cell-mediated cytotoxicity (ADCC) of macrophages toward the extracellular parasite Schistosoma mansoni was investigated by using 1) a panel of blocking mAbs directed against adhesion molecules and 2) different soluble ligands as candidate inhibitors of ADCC. The results show that the beta2-integrin Mac-1 (CD11b/CD18), L-selectin (CD62-L), and the carbohydrate determinant sialyl Lewis(x) (sLe(x); sCD15) are required for macrophage effector function toward schistosomula targets. On the other hand, the parasite counter-receptors involved in ADCC were found to share common motifs with the mammalian selectin-carbohydrate families. One family of parasite receptor(s) involved in ADCC carries the Lewis(x) (Le(x); CD15) carbohydrate structure, whereas a second family of receptor(s) appears to display selectin-like properties with affinity for the sLe(x) tetrasaccharide. Immunostaining experiments confirmed that schistosomula express on their surface hostlike molecules recognized by anti-Le(x) (CD15) and by anti-human E-selectin (CD62-E) mAbs. The double receptor-ligand interaction between macrophages and parasite targets provides new insights into the biologic roles of selectins and Le(x)-related structures in immunity against helminthic parasites.
Activation and migration of CD8 T cells in the intestinal mucosa.
Authors: Kim S K , Reed D S , Heath W R , Carbone F , Lefrançois L
Published: January 2023

The activation of CD8 T cells was studied using transgenic mice expressing an MHC class I-restricted OVA-specific TCR (OT-I). Immunization of OT-I mice resulted in activation of lymph node T cells and up-regulation of expression of B220, CD11a, and CD44 and caused a preferential loss of mucosal-type T cells from the periphery. Immunization induced dramatic changes in intestinal lymphocytes, including the appearance of a CD11a high population that resembled peripheral T cells and induced a primary cytolytic response. Activation of adoptively transferred OT-I cells induced migration of CD8 T cells into the lamina propria and intraepithelial lymphocyte compartments. The results suggest that Ag presentation in the intestinal epithelium occurs, and that CD8 cells activated in the periphery can readily enter the mucosa during an ongoing immune response.
The orientation of a T cell receptor to its MHC class II:peptide ligands.
Authors: Hong S C , Sant'Angelo D B , Dittel B N , Medzhitov R , Yoon S T , Waterbury P G , Janeway C A
Published: January 2023

The TCR found on CD4 T cells recognizes peptides bound to self MHC class II molecules as well as non-self MHC class II molecules. We have used the receptor on a cloned T cell line called D10.G4.1 (D10) to perform a structure-function analysis of this interaction. The D10 T cell clone recognizes not only a peptide from conalbumin (CA-wt) bound to syngeneic I-Ak against which it was raised, but also the allogeneic MHC molecules I-A(b,v,p,q,d). In the present study, we show that residue 30 in complementarity-determining region 1 (CDR1) of the TCR alpha-chain interacts with the I-A alpha-chain at hvr2 (residues 52, 53, and 55). We also show that residue 51 in CDR2 of the TCR alpha-chain interacts with the peptide at peptide residue 2. Finally, we show that residue 29 in CDR1 of the TCR beta-chain affects recognition of the glutamic acid at residue 66 in the I-A beta-chain. These data suggest an orientation of TCR relative to its peptide:MHC class II ligands. We argue that this orientation will be shared by all CD4 TCRs, and that it is only subtly different from the common orientation proposed for receptors binding to MHC class I.
Syk-independent tyrosine phosphorylation and association of the protein tyrosine phosphatases SHP-1 and SHP-2 with the high affinity IgE receptor.
Authors: Kimura T , Zhang J , Sagawa K , Sakaguchi K , Appella E , Siraganian R P
Published: January 2023

Aggregation of the high affinity IgE receptor (Fc epsilonRI), a member of the immune receptor family, results in the activation of protein tyrosine kinases and downstream signaling pathways. The two cytoplasmic Src homology 2 domain-containing protein tyrosine phosphatases, SHP-1 (also called SH-PTP1, PTP1C or HCP) and SHP-2 (also known as SH-PTP2, PTP1D, PTP2C, or Syp), are expressed in the RBL-2H3 rat mast cell line. Here we report that aggregation of Fc epsilonRI induced the tyrosine phosphorylation of both SHP-1 and SHP-2. This phosphorylation was independent of the presence of the protein tyrosine kinase Syk. Both SHP-1 and SHP-2 associated with Fc epsilonRI. Whereas SHP-1 was constitutively associated with the receptor, SHP-2 coprecipitated with Fc epsilonRI only after receptor aggregation. Fusion proteins containing either the full-length or the Src homology 2 domains of SHP-2 directly bound to the tyrosine-phosphorylated beta, but not the gamma, subunit of Fc epsilonRI. In the reciprocal experiments, synthetic phosphorylated peptides based on the immunoreceptor tyrosine-based activation motif of the beta, but not the gamma, subunit precipitated SHP-2. In contrast, neither fusion proteins nor synthetic peptides detected interaction between SHP-1 and Fc epsilonRI. In vitro, both SHP-1 and SHP-2 dephosphorylated tyrosine-phosphorylated beta and gamma subunits of Fc epsilonRI. Therefore, SHP-1 and SHP-2 associate with Fc epsilonRI by different mechanisms and can regulate the extent of the tyrosine phosphorylation of the receptor subunits. Thus, unlike other immune cells in which inhibitory molecules are recruited by accessory proteins, Fc epsilonRI bind molecules that both activate and inhibit signal transduction.
Intracytoplasmic delivery of listeriolysin O by a vaccinal strain of Bacillus anthracis induces CD8-mediated protection against Listeria monocytogenes.
Authors: Sirard J C , Fayolle C , de Chastellier C , Mock M , Leclerc C , Berche P
Published: January 2023

The facultative intracellular pathogen Listeria monocytogenes secretes a 58-kDa hemolysin, listeriolysin O (LLO), that allows bacteria to access the cytoplasm and to multiply inside infected cells. LLO is also a protective Ag required for the development of specific immunity. We studied the capacity of a new bacterial vector, derived from an attenuated strain of Bacillus anthracis, to deliver in vivo LLO and to induce protection against L. monocytogenes infection. The hly gene encoding LLO was fused to a B. anthracis regulatory region induced in vivo and was integrated into a resident plasmid of this bacterium. This recombinant strain secreted a functional LLO in vitro and inside phagosomes of bone marrow macrophages. This LLO production enabled the conversion of the extracellular replicating B. anthracis into an intracytoplasmic bacterium. LLO+ B. anthracis thus mimicked the intracellular behavior of L. monocytogenes in macrophages. Specific protection of mice against lethal doses of L. monocytogenes was induced by immunization with LLO+ B. anthracis. The immunity was mediated by CD8+ T lymphocytes and was associated with the activation of LLO-specific MHC class I-restricted CD8+ CTL, able to recognize the immunodominant H-2d-restricted epitope 91-99 of LLO. This study, therefore, suggests that intracytoplasmic delivery of LLO by B. anthracis is sufficient to induce a MHC class I-restricted CD8-mediated protection against L. monocytogenes. The LLO+ B. anthracis recombinant strain represents a potential vector for delivering foreign Ags involved in CD8-mediated protective responses.