bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of granulocyte-macrophage colony-stimulating factor on rat alveolar macrophage anticryptococcal activity in vitro.
Authors: Chen G H , Curtis J L , Mody C H , Christensen P J , Armstrong L R , Toews G B
Published: December 2022

Cryptococcus neoformans, a pathogenic fungus usually acquired by inhalation, causes the most common lethal mycosis in AIDS. The resident lung phagocytes, alveolar macrophages (AM phi), inhibit growth of C. neoformans poorly unless activated by cytokines such as IFN-gamma. In this study, we examined the effect of rat AM phi of the potent hematopoietic and M phi-activating cytokine, granulocyte-macrophage CSF (GM-CSF), alone and in combination with other cytokines. Rat AM phi monolayers were preincubated with 0.1 to 1000 U/ml GM-CSF without or with other recombinant cytokines, and then were incubated with viable C. neoformans (strain H99/C3D). Growth inhibition was assessed by counting cryptococcal CFU at 24 and 48 h of coculture; AM phi proliferation was assessed by measuring both uptake of [3H]TdR and AM phi numbers. AM phi preincubated with GM-CSF for 5 days (but not for shorter periods) inhibited growth of C. neoformans. Anticryptococcal activity required direct contact of AM phi with C. neoformans, but once induced by preincubation, did not require continued exposure to GM-CSF. Induction of anticryptococcal activity by GM-CSF was dose dependent (maximal induction at 250 U/ml), and was due to both increased ingestion and killing. GM-CSF induced AM phi proliferation, but anticryptococcal activity was not due totally to increases in AM phi numbers, indicating AM phi activation by GM-CSF. GM-CSF-induced AM phi proliferation was increased by IL-6, unchanged by IL-8, and abolished by LPS or IFN-gamma. However, IL-6 did not increase GM-CSF-induced anticryptococal activity. The combination of GM-CSF and IFN-gamma showed rapid and sustained anticryptococcal activity, unlike either cytokine alone. Our in vitro data suggest that the combination of GM-CSF and IFN-gamma may have beneficial effects on host defense against C. neoformans in vivo.
Differences between human eosinophils and neutrophils in the function and expression of sialic acid-containing counterligands for E-selectin.
Authors: Bochner B S , Sterbinsky S A , Bickel C A , Werfel S , Wein M , Newman W
Published: December 2022

Both neutrophils and eosinophils have been shown to bind to the inducible endothelial cell adhesion molecule E-selectin. For neutrophils, one of the reported ligands for E-selectin is the sialylated Lewis X Ag (sLe(x)). To analyze the counterligands on eosinophils for E-selectin, adhesion assays were performed in which purified leukocytes were allowed to adhere to a soluble recombinant form of the molecule immobilized on plastic plates. Eosinophils, like neutrophils, bound to immobilized E-selectin, but significantly more neutrophils than eosinophils adhered in this assay. Consistent with the greater ability of neutrophils to bind E-selectin was the observation by flow cytometry that neutrophils expressed significant levels of sLe(x) and a sialylated dimeric form of the Le(x) Ag (sialyl-dimeric Le(x), or sialyl-stage-specific embryonic Ag-1, recognized by mAb FH6), whereas the expression of these epitopes on eosinophils was extremely low or undetectable. Expression was similar on eosinophils from allergic and nonallergic donors, and was not altered on eosinophils after induction of L-selectin shedding in vitro by treatment with platelet-activating factor. For both eosinophils and neutrophils, treatment with sialidase was associated with the complete elimination of sLe(x) and sialyl-dimeric Le(x) surface expression, and abolished leukocyte adhesion to E-selectin. Another glycosidase, endo-beta-galactosidase, which specifically cleaves the beta 1-4 galactose linkage to N-acetyl-glucosamine when it exists in an extended chain form such as that found in sialyl-dimeric Le(x), significantly inhibited eosinophil and neutrophil adhesion and expression of sialyl-dimeric Le(x). Such treatment also reduced sLe(x) expression on eosinophils, while having little effect on total neutrophil sLe(x) expression. For both eosinophils and neutrophils the sialylated ligand did not appear to be a glycoprotein because pretreatment of leukocytes with several proteases had no effect on adhesion to E-selectin or on expression of sLe(x) and sialyl-dimeric Le(x). These data suggest that eosinophils, like neutrophils, use sialylated, protease-resistant structures to bind to E-selectin, although the eosinophil expresses much lower levels of these structures on its surface. A major proportion of the sLe(x)-containing E-selectin ligand on the surface of eosinophils appears to be in the form of sialyl-dimeric Le(x), whereas this represents a minor proportion on the surface of neutrophils. Based on results using endo-beta-galactosidase, it appears that these cells may rely disproportionately upon the cell surface sialyl-dimeric Le(x) to bind to E-selectin.(ABSTRACT TRUNCATED AT 400 WORDS)
Fc gamma R-mediated endocytosis and expression of cell surface Fc gamma RIIb1 and Fc gamma RIIb2 by mouse bone marrow culture-derived progenitor mast cells.
Authors: Lobell R B , Austen K F , Katz H R
Published: December 2022

Mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC) bind IgG immune complexes through Fc receptors for IgG (Fc gamma R) but express minimal Fc gamma RIII on their surfaces. BMMC do not degranulate appreciably when their Fc gamma R are perturbed with the rat anti-mouse Fc gamma RII/III mAb 2.4G2 and F(ab')2 mouse anti-rat IgG (MAR). In contrast, after their Fc gamma R were cross-linked with mAb 2.4G2 and Na125I-labeled MAR at 37 degrees C, BMMC rapidly internalized the complex. To identify the Fc gamma R species expressed on the surface of BMMC and therefore implicated in the endocytic response, two rabbit antipeptide antisera were raised, one against a sequence common to the cytoplasmic regions of Fc gamma RIIb1 and Fc gamma RIIb2 and the other to a unique cytoplasmic region of Fc gamma RIIb1. When Fc gamma R were immunoprecipitated with mAb 2.4G2 from detergent extracts of BMMC, digested with N-glycosidase F, subjected to SDS-PAGE, and immunoblotted with the Fc gamma RIIb1- and Fc gamma RIIb1/b2-specific antibodies, BMMC were found to express Fc gamma RIIb1 and Fc gamma RIIb2. Selective immunoprecipitation of plasma membrane-localized Fc gamma RIIb1 and Fc gamma RIIb2 from [3H]leucine-labeled BMMC showed that their ratio at the cell surface was similar to their initial biosynthetic ratio. Thus, in contrast to mature serosal mast cells that degranulate on binding of IgG complexes, immature mast cells, of which BMMC are a prototype, may have a role in the clearance of complexes without concomitant release of proinflammatory mediators.
Bacterial endotoxin regulation of cytokine receptors on murine bone marrow cells: in vivo and in vitro study.
Authors: Shieh J H , Peterson R H , Moore M A
Published: December 2022

Bacterial endotoxin (LPS) modulation of CSF-1, granulocyte-macrophage (GM)-CSF, G-CSF, IL-1, TNF, and Kit Ligand receptors on murine bone marrow cells (BMC) in vivo and in vitro was investigated. In vivo LPS reduced the binding of hrIL-1 (> 90%), mrTNF (> 62%), mrGM-CSF (> 42%) and hrG-CSF (> 91%) to BMC within 2 h, but elevated IL-1 binding (> 8.4-fold) between 8 to 48 h. In vitro, LPS decreased G-CSF and IL-1 binding after 8 h yet increased TNF binding in a dose-dependent manner, suggesting that in vivo, the LPS up-regulation of IL-1 binding to BMC was indirect. Because in vivo LPS elevated the levels of TNF, IL-6, GM-CSF, and glucocorticoids, we further examined GM-CSF and TNF modulation of cytokine receptors on BMC in vivo. In vivo, TNF decreased the binding of TNF (> 88%), G-CSF (> 89%), and IL-1 (> 73%) within 30 min, but increased IL-1 binding (> 4.8-fold) after 10 h. In contrast, in vitro TNF decreased IL-1 binding after 8 h, implying that in vivo TNF up-regulation of Il-1 binding to BMC was also due to an indirect mechanism. However, GM-CSF increased IL-1 binding to BMC both in vivo and in vitro after 8 h. Further studies showed that in vitro GM-CSF and dexamethasone synergistically increased IL-1 binding to BMC in a time- and dose-dependent manner. This synergistic modulation depended on synthesis of protein and mRNA, and was due to an increase in receptor number rather than an increase in receptor affinity. Because in vivo, LPS and LPS-induced cytokines (IL-1 and TNF) elicited the secretion of glucocorticoid and CSF activities, our results revealed a mechanism for LPS up-modulation of IL-1R on BMC in vivo.
Soluble Fc gamma RIIIa is present in plasma and is derived from natural killer cells.
Authors: de Haas M , Kleijer M , Minchinton R M , Roos D , von dem Borne A E
Published: December 2022

Fc gamma RIII (CD16), a receptor for complexed IgG, is encoded by two very homologous genes: Fc gamma RIIIA and Fc gamma RIIIB. NK cells and macrophages express Fc gamma RIIIa, whereas only neutrophils constitutively express Fc gamma RIIIb. In a previous study we found that soluble (s)Fc gamma RIII in plasma seemed to originate only from neutrophils. However, CD16 mAb, directed against different epitopes of Fc gamma RIII, precipitated a glycoprotein from plasma of homozygous Fc gamma RIIIB gene-deficient donors. This glycoprotein migrated in a similar way as did released Fc gamma RIIIa derived from NK cells, whereas Fc gamma RIIIa released by cultured monocytes migrated differently and appeared to be more heavily glycosylated on SDS-PAGE. After deglycosylation, the M(r) of the plasma sFc gamma RIIIa was similar to that of released Fc gamma RIIIa. Moreover, V8-protease maps were identical. Therefore, we conclude that sFc gamma RIIIa is also present in plasma and is derived from NK cells. Because sFc gamma RIII levels are hardly detectable in the plasma of most homozygous Fc gamma RIIIB gene-deficient donors, we suspect that the sFc gamma RIIIa level is negligible compared with the level of sFc gamma RIIIb in plasma of healthy donors. Two patients with an NK cell lymphocytosis had a high plasma level of sFc gamma RIIIaNK. Furthermore, high levels of sFc gamma RIIIaNK were found in plasma of two patients with rheumatoid arthritis. Thus, the level of sFc gamma RIIIaNK might reflect either an increase in circulating NK cells or an enhanced release of Fc gamma RIIIaNK in certain diseases. This study shows that an assay that discriminates between sFc gamma RIIIa and sFc gamma RIIIb is necessary for the interpretation of sFc gamma RIII levels in patients.
Thymic epithelial cell lines that mediate positive selection can also induce thymocyte clonal deletion.
Authors: Hugo P , Kappler J W , Godfrey D I , Marrack P C
Published: December 2022

Negative selection of potentially autoreactive thymocytes occurs mainly in the thymus and is thought to be induced primarily by interaction with bone marrow-derived cells. However, some studies have also reported a role for radioresistant thymic cells, which are probably epithelial in origin, in the deletion of thymocytes reacting to endogenous superantigens. We have previously demonstrated that thymic epithelial cell lines could induce thymocyte-positive selection in vivo. In this study, we assessed the potential of these cells to delete thymocytes reacting to the staphylococcal enterotoxin A or B superantigens in vitro. In the presence of staphylococcal enterotoxin A or B we found that all thymic epithelial cell lines used in this study were capable of activating T cell hybrids or deleting CD4+CD8+ thymocytes expressing an appropriate TCR. The extent of superantigen-mediated thymocyte deletion mediated by thymic epithelial cell lines was comparable to that mediated by a thymic macrophage cell line. Similar results were obtained with three phenotypically distinct thymic cell lines, suggesting that the ability to induce thymocyte deletion might be a general feature of various subsets of thymic epithelium. The observations provided in this study, combined with our previous demonstration that the same thymic epithelial cell lines can participate in positive selection, suggest that a given stromal cell population might be capable of taking part both in positive and negative selection of thymocytes.
Reduction of CTL antipeptide response mediated by CD8+ cells whose class I MHC can bind the peptide.
Authors: Sambhara S R , Miller R G
Published: December 2022

Primary CTL responses can be generated in vitro against defined peptides in association with class I MHC molecules. We show here that if cells obtained from a 5-day MLR are also included in the cultures, the response is greatly reduced if the added cells both carry CD8 and can bind the peptide. Our interpretation is that the added MLR cells are acting as deletional APC or veto cells. Peptide-specific CTL precursors recognize the peptide on the class I MHC of the CD8+ MLR cells and then receive a negative signal via CD8 on these cells. In support of this, when MLR cells carrying the Lyt-2.1 allele of CD8 were used to down-regulate the response of Ly-2.2+ responder cells, inclusion of anti-Ly-2.1 mAb in the cultures partially reversed the response reduction. Similar signaling may occur in vivo. When mice were injected i.v. with syngeneic lymphoid cells incubated with a peptide which they could bind, the response against that peptide was specifically reduced in a subsequent in vitro assay.
MHC-encoded proteasome subunits LMP2 and LMP7 are not required for efficient antigen presentation.
Authors: Yewdell J , Lapham C , Bacik I , Spies T , Bennink J
Published: December 2022

LMP2 and LMP7 are proteins encoded by MHC genes that are tightly linked to the genes encoding TAP, the transporter that conveys peptides from the cytosol to the endoplasmic reticulum for assembly with MHC class I molecules. LMP2 and LMP7 are subunits of a subset of proteasomes, large molecular assemblies with multi-proteolytic activities believed to degrade damaged and unwanted cellular proteins. Like TAP and class I molecules themselves, expression of LMP genes is enhanced after exposure of cells to IFN-gamma. These findings implicate LMP2 and LMP7 in the cytosolic production of antigenic peptides. Doubts have been cast, however, on the role of LMP2 and LMP7 in Ag processing, because cells lacking these proteins possess class I molecules that contain peptides quantitatively and qualitatively indistinguishable from the peptides bound to class I molecules derived from normal cells. In this paper we show that cells lacking LMP2 and LMP7 present seven TAP-dependent determinants derived from viral proteins. For two determinants, the kinetics of presentation are shown to be similar for LMP-expressing and -nonexpressing cells. We also demonstrate biochemically that peptide is not limiting in the assembly of class I molecules in LMP-nonexpressing cells. These findings provide additional evidence that LMP2 and LMP7 are not required for efficient Ag presentation, and suggest that these proteins have either a more specialized role in the production of class I-associated peptides, or are not involved in the processing of proteins for association with class I molecules.
Stimulation of tumor-draining lymph node cells with superantigenic staphylococcal toxins leads to the generation of tumor-specific effector T cells.
Authors: Shu S , Krinock R A , Matsumura T , Sussman J J , Fox B A , Chang A E , Terman D S
Published: December 2022

In animal studies, lymph nodes (LN) draining progressive tumors contain immunologically sensitized but functionally deficient T cells. These preeffector cells can differentiate into mature effector cells on stimulation in vitro with anti-CD3 and IL-2. However, anti-CD3 react with all T cells and the activated cell population expressed a broad but normal distribution of V beta phenotypes. In this study, we examined the feasibility of using bacterial superantigens to stimulate tumor-draining LN cells. Because of their TCR V beta restriction, superantigen activation may afford a means to identify T cell subsets that are important in the antitumor immune response. Stimulation of draining LN cells with staphylococcal enterotoxins A (SEA) or B (SEB) followed by culture in IL-2 resulted in selective activation and expansion of V beta 3 and V beta 11 or V beta 3 and V beta 8 T cells, respectively. However, in adoptive immunotherapy, SEB- but not SEA-activated cells mediated the regression of established pulmonary metastases. To define the relative antitumor effects of V beta 3 and V beta 8 T cells, SEB-activated cells were depleted of either V beta 3 or V beta 8 T cells with mAb and magnetic beads. The antitumor effects were demonstratably diminished after V beta 8 cell depletion but enhanced after V beta 3 cell depletion. Using antigenically distinct MCA 205 and 207 sarcomas, tumor regression mediated by the activated cells was found to be immunologically specific for the tumor that stimulated the draining LN. Furthermore, the SEB-activated cells were virtually all T cells consisting of approximately equal proportions of CD4+ and CD8+ cells and the collaboration of the two T cell subsets was required for in vivo antitumor effects. However, the helper function of CD4+ cells could be facilitated by the administration of exogenous IL-2. Despite their in vivo antitumor reactivity, SEB-activated cells did not exhibit tumor cytotoxicity in the 4-h 51Cr release assay. However, they secreted IFN-gamma on specific stimulation with tumor cells. Taken together, these results provide for the first time clear evidence of the functional significance of superantigen interactions with immunologically committed T cells and suggest a preferential V beta use that might be associated with the T cell immune response to progressively growing tumors.
Metalloprotease and serine protease are involved in cleavage of CD43, CD44, and CD16 from stimulated human granulocytes. Induction of cleavage of L-selectin via CD16.
Authors: Bazil V , Strominger J L
Published: December 2022

CD43, CD44, CD16, and L-selectin have been previously shown to be enzymatically cleaved from stimulated leukocytes. However, little is known about the enzymes involved in these processes. Here, metalloprotease(s) inhibitable by 1,10-phenanthroline together with serine protease(s) inhibitable by N alpha-p-tosyl-L-lysine chloromethyl ketone and 3,4-dichloroisocoumarin are shown to be involved in the cleavage of CD43, CD44, and CD16 but not in the cleavage of L-selectin on granulocytes. In addition, mAbs that recognize these individual receptors and induce their specific cleavage did not initiate cleavage of the others. In one case only, L-selectin, cleavage was also triggered by mAbs interacting with CD16 (the low affinity Fc gamma R). Thus, this mechanism represents a novel pathway of L-selectin cleavage induction.
The induction of persistence of I-A expression by macrophages from Bcgr mice occurs via a protein kinase C-dependent pathway.
Authors: Brown D , Faris M , Hilburger M , Zwilling B S
Published: December 2022

We have described conditions by which MHC class II (I-A) glycoproteins can be induced to be differentially expressed after treatment of macrophages with rIFN-gamma. Treatment of macrophages from BCG-resistant mice with 1 U of rIFN-gamma induced transient I-A expression that decayed in the presence of cycloheximide. Subsequent treatment of these macrophages with 100 U of rIFN-gamma induced the persistence of I-A that was not affected by cycloheximide. The aim of this investigation was to define, by pharmacologic intervention, the second signals that resulted in the induction of persistence of I-A. Treatment of the macrophages that transiently expressed I-A with PMA resulted in the induction of persistence. When we compared the effect of different protein kinase C (PKC) inhibitors with the induction of persistence by rIFN-gamma, we found that H-7 blocked the induction of persistence only when added before or at the same time as the addition of a high dose of rIFN-gamma. In contrast, the addition of staurosporine to macrophages as late as 2 h after treatment with high doses of rIFN-gamma inhibited the induction of I-A persistence. The addition of a high dose of rIFN-gamma to macrophages previously treated with a low dose of rIFN-gamma resulted in the synergistic activation of PKC. The effect of H-7 and of staurosporine on the activation of PKC activity coincided with the effect of these inhibitors on the induction of persistent I-A expression. Tyrosine kinase inhibitors genistein and herbimycin did not affect the induction of I-A persistence nor of PKC activation. Antibody to the IFN-gamma receptor inhibited PKC activation. Finally, the addition of the high dose of rIFN-gamma to macrophages from BALB/c.Bcgs mice, previously treated with the low dose of rIFN-gamma, failed to activate high levels of PKC activity attained after similar treatment of macrophages from BALB/c.Bcgr mice. One effect of the Bcg gene may be to regulate the activation of PKC activity.
Premature escape of double-positive thymocytes to the periphery of young mice. Possible role in autoimmunity.
Authors: Bonomo A , Kehn P J , Shevach E M
Published: December 2022

Thymectomy of 3-day-old mice induces organ-specific autoimmune disease. To define a relationship between the development of T cells early in the neonatal period and autoimmunity, we studied the thymus and peripheral lymphoid tissues of 3-day-old mice. Lymph nodes, but not spleens, of 3- to 4-day-old mice contained a significant number of thymus-derived CD4+CD8+ cells that are phenotypically similar to CD4+CD8+ thymocytes in their level of expression of CD3 and HSA. It is likely that the prematurely exported cells are the progenitors of autoreactive T cells because the lymph nodes from 3- to 4-day-old male, but not female, mice which express a transgenic TCR specific for the H-Y Ag contained a large number of CD4+CD8+Tg+ as well as CD4-CD8+Tg+ T cells. Thus, the neonatal thymus is capable of exporting immature T cells that in the absence of a thymus may differentiate into autoimmune effector cells.
CD40 preferentially costimulates activation of CD4+ T lymphocytes.
Authors: Cayabyab M , Phillips J H , Lanier L L
Published: December 2022

CD40 is a membrane differentiation antigen constitutively expressed on B cells that induces B cell growth and Ig synthesis after ligation with anti-CD40 mAb or with the recently identified CD40 ligand (CD40L). CD40L is rapidly induced on T cells after activation with anti-CD3 mAb or mitogens. While CD40-CD40L interactions are clearly beneficial to B cells, we speculated that a reciprocal costimulation of T cells might also occur. We have used genetic transfection to demonstrate that interactions between human small, resting T cells and CD40+ murine transfectants substantially augmented anti-CD3 induced T cell proliferation and resulted in the generation of CTL. T cell proliferation costimulated by CD40 was IL-2 dependent. The ability of CD40+ transfectants to costimulate T cell proliferation was specific in that VCAM-1+, CD54+, CD72+, CD56+, CD31+, and fas+ transfectants in the same host cells were inactive. CD4+ T cells preferentially responded to CD40 costimulation, whereas CD8+ T cells were substantially less reactive. By contrast, costimulation with B7 transfectants induced equivalent proliferation in the CD4+ and CD8+ T cell subsets. In addition, adult naive and memory T cells, as well as cord blood T cells, were responsive to CD40. These findings suggest that the CD40-CD40L costimulation pathway may allow for selective expansion of CD4+ T cells after interaction with CD40-bearing APC. The relatively restricted expression of CD40 on APC, as well as on medullary and cortical thymic epithelium, indicates a possible role for this interaction in T cell differentiation and activation.
Adjuvant-free in vivo targeting. Antigen delivery by alpha 2-macroglobulin enhances antibody formation.
Authors: Chu C T , Oury T D , Enghild J J , Pizzo S V
Published: December 2022

The proteinase "inhibitor" alpha 2-macroglobulin (alpha 2M) is able to entrap and form covalent linkages with diverse proteins during a transient proteinase-activated state. These complexes are rapidly endocytosed after binding to receptors present on macrophages and other cells. We have previously shown that compared to free hen egg lysozyme (HEL), alpha 2M-complexed HEL undergoes enhanced macrophage uptake, processing, and presentation to T hybridoma clones in vitro. Inasmuch as it is not clear whether T hybridoma responses accurately reflect primary immune responses in vivo, we studied antibody production in rabbits using two Ag complexed with either human alpha 2M (H alpha 2M) or a homologous protein purified from rabbit plasma, alpha 1-macroglobulin (R alpha 1M). Pathogen-free NZW rabbits received s.c. injections with adjuvant-free preparations of free HEL or porcine pancreatic elastase (PPE), H alpha 2M-HEL-PPE complexes, R alpha 1M-HEL-PPE complexes, or mixtures of the uncomplexed proteins. Complexing the Ag to alpha 2M resulted in 10 to 500-fold higher IgG titers compared to uncomplexed controls. Injection of Ag complexed to either H alpha 2M or R alpha 1M resulted in levels of anti-HEL IgG comparable to those elicited by emulsification in CFA. Inasmuch as inflammatory proteinases such as neutrophil elastase can initiate covalent complex formation with alpha 2M, we propose that "proteinase-activated" alpha 2M may mediate receptor-enhanced Ag uptake by macrophages, resulting in augmented Ag processing and antibody production.
Structural requirements for peptides that stimulate a subset of gamma delta T cells.
Authors: Fu Y X , Vollmer M , Kalataradi H , Heyborne K , Reardon C , Miles C , O'Brien R , Born W
Published: December 2022

Hybridomas representing the V gamma 1-positive subset of murine gamma delta T cells secrete lymphokines in response to synthetic peptides representing a short segment of the mycobacterial 60-kDa heat shock protein (HSP-60). Here we show the TCR dependency of this response by transfection of productively rearranged TCR genes derived from an HSP-60 reactive gamma delta T cell hybridoma. We also have defined structural requirements for the stimulatory peptide. The smallest HSP-60 peptide capable of stimulating these hybridomas is seven amino acids long, representing positions 181-187, and having the sequence FGLQLEL. Amino acid-substituted derivatives of this peptide, and another containing the same core, p180-190, revealed amino acids essential for stimulatory activity. Phenylalanine in position 181 and leucine in position 183 seem to be required for stimulation of all HSP-60 reactive cells, whereas others are only required by some. Clonal differences in the responses to these peptides provide indirect evidence for cognate TCR-peptide interactions. The smallest stimulatory peptide, p181-187, represents an area not well conserved among HSP-60 molecules of other species, and stimulates a mycobacteria-specific response unlike the earlier observed cross-reactive responses of the same hybridomas with longer HSP-60 peptides derived from mycobacteria and other species (our manuscript in preparation). We propose that the TCR-dependent multiclonal gamma delta T cell response to HSP-60 peptides and derivatives, which in some ways resembles superantigen responses and in other ways resembles responses to conventional Ag, may be a separate, third type of Ag response by T cells.
Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity.
Authors: Liang S , Mozdzanowska K , Palladino G , Gerhard W
Published: December 2022

Immunity that cross-reacts between influenza type A viruses of distinct subtypes is called hetero(sub)typic (Het-I). We have studied Het-I by challenging PR8-immune mice with the heterosubtypic virus X31. Het-I did not prevent infection by X31 but, at its height, strongly aided in recovery. The nature of the effector mechanisms involved was investigated by simultaneous challenge with X31 and an immunologically unrelated influenza type B virus and by depleting individual lymphocyte subsets in PR8-immune mice before challenge. The study showed the following: 1) The effector mechanisms were intimately associated with immune recognition events. 2) In the nose, depletion of CD8+ or CD4+ T cells led to partial reduction of Het-I, and simultaneous depletion of both T cell subsets abrogated Het-I almost completely. This T cell-mediated immunity was short lived and had disappeared 4 to 5 mo after induction. 3) In trachea and lung, depletion of CD8+ T cells led to a partial reduction of Het-I, whereas depletion of CD4+ T cells was without significant effect. The CD8-mediated component appeared short lived, whereas the residual immunity (in CD4/8-depleted mice) was long lived and persisted past 7 mos after induction. 4) Depletion of NK cells did not significantly reduce the strength of Het-I in either nose or lung. In conclusion, the study shows that Het-I in this system is mediated by a complex combination of immune mechanisms that differ, in part, between upper and lower respiratory tract.
A peptide antigen antagonist prevents the differentiation of T cell receptor transgenic thymocytes.
Authors: Spain L M , Jorgensen J L , Davis M M , Berg L J
Published: December 2022

The developmental fate of an immature T cell is determined in the thymus. Depending on the specificity of its TCR, a thymocyte receives signals to either die or differentiate. We have used fetal thymic organ cultures derived from TCR transgenic mice to examine the role of MHC/peptide ligands in T cell selection. Single amino acid substituted peptide analogues of the Ag recognized by the transgenic TCR were examined for their ability to enhance or interfere with positive selection. We have identified a nonstimulatory peptide analogue that interferes with the differentiation of transgenic CD4+8+ thymocytes into CD4+8- cells. We also show that this peptide, substituted in a TCR contact residue, is a competitive antagonist for activation of the T cell hybridoma expressing the same TCR. These observations demonstrate a novel mechanism for tolerance induction in the thymus.
Natural antibody and complement-mediated antigen processing and presentation by B lymphocytes.
Authors: Thornton B P , Vĕtvicka V , Ross G D
Published: December 2022

Normal immune responses to primary protein Ags (those not seen previously by the immune system) have been shown to require C3 and the C3 receptor CR2 (CD21). This investigation tested the hypothesis that natural Abs to the primary protein Ag keyhole limpet hemocyanin (KLH) exist in normal serum and will form immune complexes (IC) that activate C and generate bound iC3b/C3dg, thereby promoting B cell CR2-dependent Ag processing. Both IgM and IgG anti-KLH were detectable in sera from 11 normal donors. IC generated with fresh serum bore iC3b and bound to CR1 (CD35), CR2, and CR3 (CD11b/CD18). Further treatment of IC with serum containing rCR1 formed IC-bearing C3dg that bound only to CR2. When ICs were mixed with B lymphoblastoid cell clones, CR2-dependent processing of the KLH occurred that was dependent on bound C3dg and CR2. Processing occurred regardless of whether the B cells bore KLH-specific surface Ig, although the efficiency of processing was greater with KLH-specific B cells. Both KLH-specific and nonspecific B cell clones presented KLH to KLH-specific T cells. The binding of KLH IC by normal B lymphocytes induced expression of the B7/BB1 Ag (CD80), the required co-stimulatory ligand for T cell CD28. Blocking experiments indicated that although bound C3 and CR2 were required to mediate IC binding to B cells, induction of CD80 expression required the secondary ligation of IC-associated IgG to B cell FcRII (CD32). These data support the hypothesis that responses to primary protein Ags involve IgG natural Abs and C3 that mediate Ag processing and presentation via B cell CR2 and FcRII.
Coordinate regulation of multiple human lymphokine genes by Oct-1 and potentially novel 45 and 43 kDa polypeptides.
Authors: Kaushansky K , Shoemaker S G , O'Rork C A , McCarty J M
Published: December 2022

The hematologic response to inflammation is critically dependent on the release of a family of glycoproteins termed CSFs. Central to this process is the coordinated T lymphocyte production of three of these proteins, granulocyte macrophage-CSF (GM-CSF), IL-3, and IL-5. In the course of our studies of the transcriptional regulation of IL-3 expression we noted that the same or similar proteins that bound to the downstream-activating region of the human IL-3 gene were also captured using DNA probes derived from sequences present in the GM-CSF and IL-5 genes. In this study, DNA-protein cross-linking techniques and reporter gene analyses were used to define the nuclear proteins that mediate transcriptional enhancement of these genes at one cis-acting site. These studies establish that Oct-1, a member of the POU family of transcription factors, binds to regions of the GM-CSF, IL-3, and IL-5 promoters and contributes to basal T cell gene transcription. In addition, although the c-jun proto-oncogene product was detected in stimulated human T cells, two additional T cell-specific proteins (45 and 43 kDa) bind to the GM-CSF, IL-3, and IL-5 promoters and are functionally and immunologically distinct from c-jun or OAP40 (jun-D). These proteins contribute to transcriptional enhancement of the IL-3 and GM-CSF genes in stimulated T cells. Together, these data suggest that the coordinate regulation of several lymphokine genes is based on responses to shared proteins. These studies further our understanding of the networks of transcriptionally active proteins responsible for the host response to inflammatory stimuli.
Expression cloning and characterization of a human IL-10 receptor.
Authors: Liu Y , Wei S H , Ho A S , de Waal Malefyt R , Moore K W
Published: December 2022

cDNA clones encoding a human IL-10R (hIL-10R) from a Burkitt lymphoma cell line, BJAB, express a 90 to 110 kDa polypeptide in COS7 cells that binds hIL-10 specifically. The predicted amino acid sequence of hIL-10R is 60% identical and 73% similar to mouse IL-10R (mIL-10R). rIL-10R expressed in an IL-3-dependent mouse pro-B cell line (Ba/F3) binds hIL-10 with high affinity (200 to 250 pM), and the transfected cells exhibit a proliferative response to hIL-10. Mouse IL-10 does not bind to hIL-10R, and hIL-10R-expressing Ba/F3 cells do not respond to the mouse cytokine, observations consistent with the known species specificity of IL-10. Expression of hIL-10R mRNA seems to be restricted mainly to human hemopoietic cells and cell lines. In a number of human T cell clones, expression of hIL-10R mRNA is down-regulated after activation of the cells with anti-CD3 Ab and phorbol ester. The hIL-10R gene is on human chromosome 11. Like mIL-10R, hIL-10R is structurally related to IFNR. Because IL-10 inhibits macrophage activation by IFN-gamma, this relationship suggests possible shared receptor or signal transduction pathway components.
Administration of anti-granulocyte mAb RB6-8C5 impairs the resistance of mice to Listeria monocytogenes infection.
Authors: Czuprynski C J , Brown J F , Maroushek N , Wagner R D , Steinberg H
Published: December 2022

Mice injected i.p. with RB6-8C5 mAb experienced a profound depletion of neutrophils in the bloodstream and spleen and significant impairment of their resistance to experimental infection with Listeria monocytogenes. Control mice survived i.v. inoculation with 5 x 10(4) L. monocytogenes; whereas, most RB6-8C5 mAb-treated mice inoculated i.v. with as few as 10 L. monocytogenes died within 6 days. RB6-8C5 mAb treatment was particularly deleterious when given within the first 24 h after i.v. inoculation with L. monocytogenes; however, some adverse effect was observed even when administration was delayed until 3 or 5 days after bacterial inoculation. Histopathologic examination of the livers of RB6-8C5 mAb-treated mice revealed necrotic foci that were characterized by few inflammatory cells and massive numbers of Gram-positive bacteria within hepatocytes. Additional evidence that the effects of RB6-8C5 mAb administration were chiefly due to neutrophil depletion include: 1) the effects of RB6-8C5 mAb treatment occurred more rapidly than what is generally seen in mice treated with anti-T cell mAbs, 2) similar results were observed with normal and scid mice, 3) RB6-8C5 mAb administration did not diminish delayed-type hypersensitivity nor the ability of spleen cells from immunized mice to transfer resistance, and 4) natural killer cell activity was unaffected by RB6-8C5 mAb administration. The results of this study provide additional evidence in support of the importance of neutrophils in the early stage of innate resistance to murine listeriosis.
A role for nonspecific (cyclosporin A) or specific (monoclonal antibodies to ICAM-1, LFA-1, and IL-10) immunomodulation in the prolongation of skin allografts after antigen-specific pretransplant immunization or transfusion.
Authors: Gorczynski R M , Wojcik D
Published: December 2022

C3H/HEJ mice given pretransplant immunization via the portal vein with irradiated B10.BR spleen cells or non-irradiated B10.BR peripheral blood leukocytes showed delayed rejection of B10.BR but not BALB.K skin grafts. No increased graft survival was seen if pretransplant immunization was given by the lateral tail vein. Addition of a single treatment with cyclosporin A after pretransplant immunization via the portal vein led to indefinite graft survival in over 70% of recipients. As reported earlier, increased graft survival in vivo was correlated with preferential production of IL-4 (from Th2 type cells) rather than IL-2 from lymphocytes stimulated in vitro. When mice were given pretransplant transfusion via the lateral tail vein and a combination of anti-LFA-1 and anti-ICAM-1 mAbs, increased graft survival was again seen. In addition, in vivo injection of anti-IL-10 mAbs (but not anti-IFN-gamma or anti-IL-4 mAbs) abolished the delayed rejection seen when mice received pretransplant immunization via the portal vein. In all cases, delayed graft rejection in vivo was correlated with preferential activation of Th2-type cells, as assessed by lymphokine production from cells harvested from treated mice and activated in vitro.
Human cutaneous allergic late-phase response is inhibited by soluble IL-1 receptor.
Authors: Mullarkey M F , Leiferman K M , Peters M S , Caro I , Roux E R , Hanna R K , Rubin A S , Jacobs C A
Published: December 2022

The late-phase allergic reaction (LPR) occurs 4 to 8 h after allergen exposure and probably causes the symptoms of chronic allergic disease. To determine the effects of soluble IL-1 receptor on the cutaneous LPR, we performed a prospective, randomized, double-blind, placebo-controlled study on 15 allergic subjects. Intradermal injections of allergen were placed on subjects' forearms, followed by immediate subcutaneous injections at the same site of either 1, 10, 25, 50, or 100 micrograms of rhu IL-1R to three subjects in each dosage group. Placebo was given to matched allergen-injected sites on the contralateral arm. Erythema, induration, and itching were recorded for each site. Sites were biopsied at 8 h for immunohistologic evaluations. Rhu IL-1R significantly reduced the clinical reaction at all concentrations. At 1 and 10 micrograms, measurements of LPR were significantly less (p < 0.05) than at placebo sites at several time points from 2 to 8 h. At higher concentrations, LPR was suppressed at rhu IL-1R and placebo sites, suggesting a systemic effect of rhu IL-1R. Histologic evaluation and indirect immunofluorescence for eosinophil granule major basic protein, neutrophil elastase, and mast cell tryptase showed no statistical differences between rhu IL-1R and placebo sites or among doses. IL-1 plays an important role in the generation of allergic LPR. While microgram quantities of rhu IL-1R inhibited the clinical signs and symptoms of LPR, its effects on the allergic inflammatory infiltrate are yet to be defined. In this short term trial, rhu IL-1R was neither immunogenic nor toxic.
Production of interleukin-1 receptor antagonist, inflammatory chemotactic proteins, and prostaglandin E by rheumatoid and osteoarthritic synoviocytes--regulation by IFN-gamma and IL-4.
Authors: Seitz M , Loetscher P , Dewald B , Towbin H , Ceska M , Baggiolini M
Published: December 2022

This study analyzes the effects of the T cell cytokines IL-4 and IFN-gamma on the spontaneous and stimulated production of IL-8, MCP-1, IL-1 receptor antagonist (IL-1ra), and PGE by synoviocytes from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells from both sources constitutively released IL-8 and MCP-1, but no IL-1ra or PGE. Stimulation with IL-1 beta or TNF-alpha massively increased chemokine production and induced the generation of PGE and low amounts of IL-1ra. The constitutive or cytokine-stimulated release of IL-8 was inhibited by IFN-gamma, but not by IL-4. The constitutive or IL-1 beta-stimulated release of MCP-1, by contrast, was markedly enhanced by IL-4 and IFN-gamma. Both cytokines, however, had only borderline effects on the release stimulated by TNF-alpha. The yield of IL-1ra was strongly enhanced by IFN-gamma in all cases, whereas the effect of IL-4 was pronounced only in IL-1 beta-stimulated OA synoviocytes. IL-4, on the other hand, markedly decreased the release of PGE, which was less susceptible to IFN-gamma. The observed effects on chemokines, IL-1ra expression, and PGE release by synoviocytes suggest that IFN-gamma and IL-4 are important regulatory elements in the inflamed synovium and may exert anti-inflammatory effects.
Evidence for a direct physical interaction of membrane IgM, IgD, and IgG with the B29 gene product.
Authors: Lankester A C , van Schijndel G M , Frommé J , Cordell J L , van Lier R A , van Noesel C J
Published: December 2022

The B cell Ag-receptor complex is composed of membrane immunoglobulin (mIg) and the mb-1/B29 heterodimer. In order to obtain insight into the architecture of the B cell receptor complex, we have looked for conditions that disrupt all disulfide bridges in the complex without affecting the noncovalent interaction between the mIg heavy chain and one or both members of the associated heterodimer. We show that in the presence of the reducing agent beta-mercaptoethanol the m mu, m delta, and m gamma heavy chains remain selectively associated with the B29 members. Our findings implied that if isotype-related differences exist between the mIg-associated dimers, they may reside in B29 and not, as initially suggested, in mb-1. However, sequence analyses of B29 gene transcripts from B cells expressing mIgM, mIgD, or mIgG only revealed no differences in their nucleotide composition. Thus, in spite of their close physical interaction with mIg heavy chain classes, which are significantly distinct in the C-terminal regions, no isotype-specific forms of B29 seem to exist.
IL-2 reverses human T cell unresponsiveness induced by thymic epithelium in SCID-hu mice.
Authors: Schols D , Vandekerckhove B , Jones D , Roncarolo M G
Published: December 2022

In this study we investigated the mechanism responsible for the unresponsiveness of thymus-reactive T cells obtained from severe combined immunodeficient (SCID) mice constructed with human fetal liver (FL) stem cells from donor A and an allogeneic human fetal thymus (FT) from donor B (A/B SCID-hu mice). These A/B SCID-hu mice have a human thymus containing B cells, macrophages, and dendritic cells from FL donor A but thymic epithelial cells from FT donor B. The repertoire of human T cells developing in this chimeric thymus is depleted of T cells specific for the HLA Ags of the FL donor, whereas T cells reactive against the HLA Ags expressed by the FT donor are still present. However, these thymocytes failed to proliferate and expressed low levels of the activation markers CD25, CD71, and HLA-DR after stimulation with the EBV-LCL of the FT donor in primary MLRs. This unresponsiveness could be completely reversed by IL-2. Restoration of T cell responsiveness was Ag specific and a unique property of IL-2. The T cells produced very low levels of IL-2 when stimulated with the HLA Ags of FT donor B, whereas they secreted normal levels of IL-2 after activation by third party alloantigens. Low IL-2 production was also observed at the clonal level. CD4+ T cell clones from A/B SCID-hu mice, specific for the HLA Ags of B, produced significantly less IL-2 and granulocyte macrophage-CSF than control CD4+ T cell clones. However, these T cell clones synthesized normal levels of IL-2 and granulocyte macrophage-CSF after stimulation with combinations of PMA/Calo or PMA/anti-CD3 mAb. Thus, T cells that differentiate in a chimeric thymus containing allogeneic host thymic epithelium are rendered tolerant to the HLA Ags expressed by the thymic epithelial cells. This tolerance results in the presence of T cells that do not proliferate properly after Ag-specific stimulation. This lack of proliferation is primarily related to their inability to produce sufficient levels of IL-2 and can be restored by exogenous IL-2.
Effect of antibody valency on interaction with cell-surface expressed HIV-1 and viral neutralization.
Authors: Cavacini L A , Emes C L , Power J , Duval M , Posner M R
Published: December 2022

F(ab) and F(ab')2 fragments of the human mAb, F105, were compared to intact IgG1 for binding to the CD4 binding site of HIV-1/gp120 on the surface of infected cells and viral neutralization. F105 IgG1 and F(ab')2 bound to IIIB, MN, and RF infected cells and neutralized these strains in an identical fashion, whereas strain-specific differences were observed in F(ab) activity. Although F105 F(ab) bound with equivalent affinity to IIIB-infected cells, there was a 4- to 10-fold decrease in the neutralization of IIIB by monovalent F(ab) compared to the bivalent molecules. F105 F(ab) demonstrated both diminished binding and neutralization of the MN strain and failed to bind or neutralize the RF strain. When cooperativity of V3 loop antibody (V3ab) with F105 IgG and fragments was examined, the binding of F105 IgG and F(ab')2 to IIIB-, MN-, or RF-infected cells was modestly enhanced by V3ab; viral neutralization was substantially enhanced by the combination of V3ab and F105 IgG and F(ab')2. The combination of F105 F(ab) with V3ab also resulted in significant cooperative neutralization of IIIB and MN, but the lack of F105 F(ab) binding and neutralization of RF was unaltered by V3ab. These results suggest that bivalent interaction may be important in binding and neutralization of virus, and support the notion that this interaction may depend on conformational changes in oligomeric gp120 on intact virions and cell surface rather than on affinity or steric effects.
Expression of the NK-TR gene is required for NK-like activity in human T cells.
Authors: Chambers C A , Gallinger S , Anderson S K , Giardina S , Ortaldo J R , Hozumi N , Roder J
Published: December 2022

NK cells lyse target cells without previous immune sensitization. A small subset of T cells also exhibits NK-like activity, which is distinct from TCR-mediated, MHC-restricted, and MHC-unrestricted cytotoxicity. We recently cloned a gene, NK-TR, which is postulated to be part of the NK target-recognition/triggering complex. To determine whether the NK-TR gene product is requisite for NK-like killing, stable antisense transfectants were generated by using a human T cell clone with NK-like activity. Two distinct antisense regions of the sequence were used to generate the transfectants alpha NK-TR and alpha Cyclo. Transfectants lost the ability to lyse NK-sensitive targets but did not lose lectin-mediated cytotoxic activity. This effect was not seen with the control vector transfectant cell line. The loss of NK-like activity by the antisense transfectant alpha NK-TR correlated with the specific decrease in endogenous NK-TR mRNA and protein. These results demonstrate the requirement for the NK-TR protein for NK-like killing. Moreover, the results have important implications for examining developmental relationship between T and NK cells and the possible roles for T cells with NK-like activity in vivo.