bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous development of protective anti-T cell receptor autoimmunity targeted against a natural EAE-regulatory idiotope located within the 39-59 region of the TCR-V beta 8.2 chain.
Authors: Hashim G A , Offner H , Wang R Y , Shukla K , Carvalho E , Morrison W J , Vandenbark A A
Published: December 2022

The development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats is mediated by V beta 8.2+ T cells specific for myelin basic protein. One consequence of this biased expression of V beta 8.2 is the spontaneous development of regulatory T cells and antibodies against residues 39-59 of the V beta 8.2 sequence. Moreover, a synthetic V beta 8.2-39-59 peptide could induce protection against and speed recovery from EAE. T cells and antibodies specific for V beta 8.2-39-59 could transfer protection from EAE. Recently, we reported that the protective T cell epitope is subsumed within the V beta 8-44-54 sequence. We now report that protection induced by V beta 8-44-54 lasted at least 102 days and produced "split tolerance," enhancing anti-myelin basic protein antibody titers but reducing anti-myelin basic protein T cell frequency. The shorter V beta 8-44-54 peptide induced a distinct set of antibodies that did not cross-react with the longer V beta 8.2-39-59 peptide, although both specificities could stain V beta 8.2+ T cells and were equally protective against EAE. However, the V beta 8.2-39-59 peptide, but not the V beta 8-44-54 peptide, would appear to represent the natural idiotope: antibodies to V beta 8.2-39-59 that develop spontaneously during EAE could be boosted to higher titers only by the V beta 8.2-39-59, but not by other TCR peptides from the V beta 8.2 sequence, including V beta 8-44-54 that contains the functional T cell epitope. These results suggest that natural processing of the TCR V beta-chain favors the formation of a peptide that resembles the V beta 8.2-39-59 sequence. The B cell epitope present on the V beta 8-44-54 sequence was evident only in the absence of residues 39-43 and 55-59, suggesting that the two peptides possess distinct conformations. However, the V beta 8-44-54 B cell epitope is most likely expressed on the V beta 8.2+ T cells, either as a low affinity determinant on the intact TCR alpha/beta heterodimer or as a cryptic epitope bound in the cleft of surface MHC molecules.
Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides.
Authors: Geluk A , Van Meijgaarden K E , Janson A A , Drijfhout J W , Meloen R H , De Vries R R , Ottenhoff T H
Published: December 2022

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.
Characterization of gamma delta T lymphocytes at the maternal-fetal interface.
Authors: Heyborne K D , Cranfill R L , Carding S R , Born W K , O'Brien R L
Published: December 2022

A specific subset of gamma delta T lymphocytes bearing a V gamma 6V delta 1-encoded TCR is known to populate the normal nonpregnant murine uterine and vaginal epithelium. However, gamma delta T lymphocytes residing at the maternal-fetal interface during pregnancy have not yet been investigated. Using mAb and cytofluorographic analysis, we analyzed gamma delta TCR-bearing lymphocytes obtained from placental/decidual tissues of allogeneic (C57B1/10 X BALB/c and BALB/c X C57B1/10) pregnancies. Gestations were analyzed at several time points during the second half of pregnancy, with lymphocytes from both maternal spleen and nonpregnant C57B1/10 uteri analyzed as controls. We found that all gamma delta T lymphocytes at the maternal-fetal interface are maternally derived. Relative to the total T lymphocyte population, the percentage of gamma delta TCR-bearing T lymphocytes at the maternal-fetal interface is enriched three- to four-fold compared with maternal spleen, and twofold compared with nonpregnant uteri. Although one-third of gamma delta T lymphocytes from pregnant animals express a cell-surface marker associated with activation (IL-2R), gamma delta cells from uteri of nonpregnant mice fail to express IL-2R. In terms of absolute numbers, we estimate that reproductive-tract gamma delta T cells are increased nearly 100-fold in pregnant animals compared with nonpregnant animals. To characterize the TCR-gamma delta repertoire in the placenta/decidua, we generated 21 TCR-gamma delta-bearing hybridomas from lymphocytes in this tissue. Analysis of these hybridomas revealed at least six distinct gamma delta receptor types expressed at the maternal-fetal interface, with V gamma 6V delta 1 encoded TCR representing the predominant population. As specific resident constituents of the reproductive tract, gamma delta T lymphocytes may be involved in regulating a variety of physiologic and pathophysiologic events in reproductive biology.
C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.
Authors: Imai T , Fukudome K , Takagi S , Nagira M , Furuse M , Fukuhara N , Nishimura M , Hinuma Y , Yoshie O
Published: December 2022

C33 Ag was originally identified by mAb inhibitory to syncytium formation induced by human T cell leukemia virus type 1. The Ag was shown to be a highly heterogeneous glycoprotein consisting of a 28-kDa protein and N-linked oligosaccharides ranging from 10 to 50 kDa. In the present study, cDNA clones were isolated from a human T cell cDNA expression library in Escherichia coli by using mAb C33. The identity of cDNA was verified by immunostaining and immunoprecipitation of transfected NIH3T3 cells with mAb. The cDNA contained an open reading frame of a 267-amino acid sequence which was a type III integral membrane protein of 29.6 kDa with four putative transmembrane domains and three putative N-glycosylation sites. The C33 gene was found to belong to a newly defined family of genes for membrane proteins, such as CD9, CD37, CD53, CD63, and TAPA-1, and was identical to R2, a cDNA recently isolated because of its strong up-regulation after T cell activation. Availability of mAb for C33 Ag enabled us to define its distribution in human leukocytes. C33 Ag was expressed in CD4+ T cells, CD19+ B cells, CD14+ monocytes, and CD16+ granulocytes. Its expression was low in CD8+ T cells and mostly negative in CD16+ NK cells. PHA stimulation enhanced the expression of C33 Ag in CD4+ T cells by about 5-fold and in CD8+ T cells by about 20-fold. PHA stimulation also induced the dramatic size changes in the N-linked sugars previously shown to accompany human T cell leukemia virus type 1-induced transformation of CD4+ T cells.
Long term expression of IL-4 in vivo using retroviral-mediated gene transfer.
Authors: Chambers C A , Kang J , Hozumi N
Published: December 2022

Th cell subsets regulate immune responses by cell-cell interaction and secretion of cytokines. IL-4 is one of the cytokines secreted by Th cells important for cellular and humoral, particularly IgG1 and IgE responses. To study the role of IL-4 in T cell development and regulation of immune responses in vivo, low IgE-responder C57BL/6 mice were reconstituted with bone marrow cells that had been infected with recombinant retrovirus expressing a high level of IL-4. The reconstituted mice expressed retroviral IL-4 transcripts (9/10) even 8 mo postreconstitution. Physiologically significant levels of IL-4 were detected in the majority of the provirus-positive animals tested (5/8). Ectopic expression of exogenous IL-4 in hematopoietic cells had dramatic effects on T cell development resulting in changes in CD4:CD8 ratios. Moreover, the levels of serum IgG1 and, with antigenic stimulation, IgE were also increased. These results demonstrate that the retrovirus gene transfer system can be used to study the effects of ectopic cytokine gene expression in vivo on Ig isotype regulation and T helper cell subset differentiation.
The HLA-E gene encodes two differentially regulated transcripts and a cell surface protein.
Authors: Ulbrecht M , Honka T , Person S , Johnson J P , Weiss E H
Published: December 2022

An HLA-E-specific oligonucleotide probe was used to study the expression of HLA-E. This probe detects two HLA-E transcripts, 1.8 and 2.7 kb in size, which are present in varying ratios in all tissues and cell lines investigated. We demonstrate that alternative poly(A) site usage accounts for the differential regulation of the two HLA-E mRNA species. Sequence analysis of three cDNA clones, representing the two transcripts of HLA-E, and of an HLA-E gene encoded by cosmid cd3.14, revealed identity of gene and cDNA in the 3' untranslated region. S1 nuclease protection assays confirmed that the two HLA-E transcripts are not alternative splicing products. Introduction of cd3.14, together with human beta 2 m into the murine myeloma cell line P3X63-Ag8.653, resulted in a cell surface expression of an HLA-class I heavy chain detectable by indirect immunofluorescence whereas transfection into the human beta 2m expressing mouse L cell line, J27 was negative with regard to cell surface expression. Cell surface labeling of transfectants and immunoprecipitation with a monomorphic HLA class I-specific antibody or an antibody against human beta 2m confirmed the presence of an HLA-E H chain on the cell surface. These results indicate that the HLA-E gene codes for a class I H chain that can be expressed on the cell surface.
Regulation of IL-2 receptor subunit genes in human monocytes. Differential effects of IL-2 and IFN-gamma.
Authors: Espinoza-Delgado I , Longo D L , Gusella G L , Varesio L
Published: December 2022

We investigated the effects of IFN-gamma and IL-2 on IL-2R alpha and beta mRNA expression in human monocytes. Low basal expression of IL-2R beta mRNA was detected in fresh monocytes. Stimulation of monocytes with IL-2 induced a significant increase of IL-2R beta mRNA, but did not induce IL-2R alpha mRNA. In contrast, stimulation of monocytes with IFN-gamma-induced IL-2R alpha mRNA, but did not modify IL-2R beta mRNA. Five U/ml of IFN-gamma induced IL-2R alpha mRNA and 2.2 nM of IL-2 induced IL-2R beta mRNA, both within 3 h. Nuclear run-on experiments demonstrated that the induction of IL-2R alpha mRNA by IFN-gamma is controlled, at least in part, at the transcriptional level. In contrast, the enhancement of IL-2R beta mRNA by IL-2 is controlled at a posttranscriptional level and is associated with an increase in the half-life of IL-2R beta mRNA. The results of studies on the cytotoxic activity and on the expression of c-fms mRNA of monocytes activated by the combination of IFN-gamma and IL-2 show that pretreatment with IFN-gamma renders monocytes more sensitive to activation by IL-2. These results demonstrate that the IL-2R alpha and IL-2R beta subunits are induced by different lymphokines through distinct mechanisms and that both receptor subunits can influence the response of monocytes to IL-2.
Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity.
Authors: Ehlers S , Mielke M E , Blankenstein T , Hahn H
Published: December 2022

The anamnestic response to infection with Listeria monocytogenes is characterized by the rapid elimination of normally lethal doses of bacteria and accelerated granuloma formation. These phenomena are mediated by listeria-specific memory T cells within the first 24 h after reinfection. In order to elucidate the mechanisms operative during this decisive phase of infection, we conducted a comprehensive kinetic and quantitative analysis of cytokine gene expression in the livers of naive and immune mice. Organs were removed at 30 min, and 1, 2, 6, and 24 h after primary and secondary infections, and PCR3-assisted messenger RNA (mRNA) amplification was performed on matched samples using primers specific for IL-1 beta, IL-6, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, IL-10, IL-4, IL-2, IL-3 and I1-2Rp55. The cytokine pattern characteristic of secondarily infected animals differed qualitatively by the expression of mRNA for IL-2, IL-2Rp55, IL-3, and IL-4, demonstrating the accumulation and activation of specific T cells in the livers as early as 1 to 2 h after reinfection. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection almost completely abrogated the differentiated cytokine profile typical of the anamnestic response. Using competitive PCR for semiquantitative determination of mRNA levels, the amount of IL-1 beta and IL-6 mRNA was found to be very similar during primary and secondary infection, whereas TNF-alpha mRNA was found to be increased by approximately 10-fold 2 h and IFN-gamma mRNA by approximately 50 to 100-fold 6 h after reinfection when compared with a primary challenge. Combined in vivo depletion of both CD4+ and CD8+ T cells before reinfection resulted in a substantial (approximately 10-fold) decrease in IFN-gamma mRNA expression. To correlate these findings with cytokine secretion, spleen cells from naive and immune as well as normal and CD4+ and CD8+ cell depleted mice infected 6 h previously were cultured for 48 h, and supernatants were analyzed for the amount of the above mentioned cytokines. Semiquantitative PCR-assisted mRNA amplification is demonstrated to be a superior tool in dissociating the mediators of innate resistance from those operative in protective immunity and granuloma formation.
Studies in Specific Hypersensitiveness
Authors: Coca A. F. , Milford E. L.
Published: December 2022

House Dust Extract The use of house dust extract has been limited by the weak concentration of the specific excitant in the fluid extract. Even when the extracting fluid was used in a quantity just sufficient to wet the dust, the extracts were active in the intradermal test in a dilution of at most 1:10, and they were never capable of causing a diagnostic cutaneous reaction with the scratch method.

We have endeavored to concentrate the extract after dialysis against buffered saline solution by evaporation with the use of an electric fan, without heating, and by evaporation in vacuo at 37°C., with the aid of a current of nitrogen gas. Neither of these procedures accomplished a concentration of the activity of the extract. No such concentrated preparation was found capable of causing a diagnostic cutaneous reaction with the scratch technic.
Anaphylaxis
Authors: Elmore Mary E.
Published: December 2022

This work is a continuation of a study of the antigenic properties of these organisms previously reported (1). The forms used were Euglena gracilis, Stichococcus sp., Chlamydomonas sp., and two species of Chlorella. In spite of the low agglutinin and precipitin titers obtained, experiments were carried out to determine whether it might be possible to sensitize guinea pigs to these organisms, either actively with a protein extract, or passively with the serum of the treated rabbits.

The Dale test was carried out with 33 virgin guinea pigs. Table 1 summarizes the results with certain of these selected to report upon here.

In the case of active sensitization to Euglena gracilis clearly positive reactions were obtained with the majority of living horns. Immune rabbit serum with a sedimentation titer as previously described did not passively sensitize. With the other antigens ten to twelve daily injections were necessary for sensitization.
Anaphylaxis
Authors: Sherwood Noble P. , Downs C. M.
Published: December 2022

Summary and Conclusions

Passive sensitization of turtles was accomplished by injecting them with some of the high titered immune rooster serum used to passively sensitize chick embryos. The reaction was specific and similar to that in the chick embryo. It consisted in a marked slowing of the heart, increase in distole and a decrease in amplitude and a sinking of the heart to a lower level. There was an apparent rise in tone probably due to the latter phenomenon. In 2 turtles that showed positive results for passive sensitization, the hearts survived eighteen hours with regular rhythms. Sensitivity had not returned during this time. Reversed anaphylaxis was not demonstrated in 7 turtles and a doubtful positive was obtained in 1. The incubation period for the latter was two hours. The shortest incubation period for passive sensitization was four hours while the average was twenty-four to forty-eight hours. High titered immune serum from rabbits failed to passively sensitize in a short series of 16 turtles.
Parenteral Denaturization of Foreign Proteins
Authors: Manwaring W. H. , Marino H. D. , Azevedo J. L.
Published: December 2022

Titrations by means of rabbit precipitin indicate that horse proteins injected intravenously into normal dogs are retained apparently quantitatively in the circulation for at least ten days. Nevertheless, transfusions from these dogs into partially exsanguinated normal recipients show that these proteins are so altered by the seventh day as almost completely to lose their specific sensitizing power.

It is conceivable that this parenteral denaturization is an initial stage in specific antibody formation.
Bacteriophage and Phagocytosis
Authors: Smith George H.
Published: December 2022

Of the many phenomena which appear to be initiated or, at least, intensified through the action of bacteriophage that of bacterial dissolution has received by far the greatest amount of attention. This is but natural since the process of bacterial dissolution through this agent is by far the most spectacular manifestation of its activity. It does not necessarily follow, from either the biological point of view or from the standpoint of immunology, that this manifestation of bacteriophage is the most significant.

The rôle of the bacteriophage in inciting or in accelerating microbic dissociation may, perhaps, be a matter of far greater biological import than is actual bacterial dissolution, and from the standpoint of immunology it may very well be that, under certain circumstances at least, the influence of bacteriophage upon phagocytosis may be far more significant than is the direct action of bacteriophage upon the bacterial cell.
The Immunological Significance of the Tuberculous Tissue
Authors: Dienes L.
Published: December 2022

The introduction of antigens directly into the tuberculous lesions incites more antibody production than the subcutaneous and intravenous treatment of the tuberculous animal. Under appropriate conditions only the injection of the antigens in the tuberculous lesions is effective.

It is very probable that the antibodies are formed in the tuberculous tissue and that the formation of the pathological tissue is the cause of the increased antibody production after tuberculous infection.
The Effect of Chloroform on the Immunizing Action of Vaccine Virus
Authors: Duran-Reynals F.
Published: December 2022

Summary and Conclusions

When vaccine virus was so far inactivated by chloroform that no eruption resulted from the intradermal injection of very large doses, no immunity was induced in the animals.

When the chloroform virus produced any lesion whatever, the rabbits subsequently proved immune when tested by a second injection of untreated virus.

These experiments support the general idea that inactivated vaccine virus is incapable of giving rise to immunity.
Some Causes of Variation in the Laboratory Interpretation of Complement Fixation by Different Methods
Authors: Wilson M. A. , Forbes Mary V. , Nedley Rose M. , Honig Florence
Published: December 2022

The observations reported in this paper have been emphasized by their recurrence in several studies of the complement fixation test in which we had the coöperation of other laboratories.

In 1922 we made a study of the Kolmer (1) (2) standard method for complement fixation test as an aid to the diagnosis of syphilis, in coöperation with the laboratories of New York University and of New York Hospital. We had difficulty in drawing conclusions from those comparison tests because of the different criteria for interpretation of the two methods employed by the coöperating laboratories.

In 1925 we participated in the work of the American Public Health Association Committee on Standardization of the Wassermann Test. Here, again, we had to take into consideration various criteria for interpretation before an accurate comparison could be made of the tests by the different laboratories.
Functional consequences of engagement of the T cell receptor by low affinity ligands.
Authors: Alexander J , Snoke K , Ruppert J , Sidney J , Wall M , Southwood S , Oseroff C , Arrhenius T , Gaeta F C , Colón S M
Published: December 2022

The mechanisms involved in TCR antagonism by Ag analog/MHC have been analyzed. A detailed structure-activity relationship study indicated that modification of any of the major T cell contact residues of the peptide molecule can yield a powerful antagonist. It was also shown that as the analog structure increased in similarity to the Ag, the capacity to antagonize Ag-TCR interaction increased up to the point that the analogs themselves became antigenic. These data strongly suggest an affinity-related mechanism whereby a certain affinity is required for signaling through the TCR, and that below this level there can be sufficient affinity to engage the receptor such that triggering does not occur and antagonism can be detected. Taking advantage of this information, antagonist peptides active down to the 10 nM range were engineered. Thus, this approach demonstrates for the first time a rational approach to designing effective, selective low m.w. compounds with high potential in treatment of allergies and autoimmune diseases.
T cell antigen receptor-eta subunit. Low levels of expression and limited cross-species conservation.
Authors: Jensen J P , Cenciarelli C , Hou D , Rellahan B L , Dean M , Weissman A M
Published: December 2022

The eta-subunit of the TCR is derived from an alternative splice product of the TCR-zeta gene. The eta-subunit has been extensively characterized in murine T cells, where up to 10% of TCR bear zeta-eta heterodimers rather than zeta-homodimers. In contrast to the significant levels of eta found in murine T cells, in human PBL an eta-like region is expressed spliced to upstream exons of the zeta-gene at no more than 0.25% the level of zeta-mRNA. Analysis of genomic DNA from five additional mammalian species demonstrated that eta-like sequences are highly conserved at a nucleic acid level. The protein sequences encoded in the eta-regions from various species ranged from 28 to 92 amino acids in length, with the first seven deduced amino acids of eta-being common to all species. Within three to five amino acids of this region, all species have five consecutive charged amino acids. Beyond this point, due to translation in different reading frames, there is no significant amino acid homology. The findings of low level of expression of eta-RNA, limited cross-species conservation on a protein level, and the lack of an established functional role for this alternative splice product raise questions as to the potential roles that this subunit may play in TCR function.
Effector functions of a mouse IgG that lacks the entire CH1 domain. C1q binding and complement fixation in the absence of antigen.
Authors: Mizutani R , Igarashi T , Tanaka T , Shimada I , Arata Y
Published: December 2022

C1q binding and complement fixation were examined of a short-chain IgG2a antibody that lacks the entire CH1 domain. This short-chain antibody has been reported to have a low level of constitutive complement-activating activity in the absence of Ag. Two-dimensional SDS/PAGE and cation-exchange chromatography have demonstrated that two types of IgG2a proteins are secreted by the short-chain IgG2a antibody producing cell line. It has been shown that 1) the difference between these two types of the IgG2a proteins is whether the two L chains are linked by a disulfide bridge or not, and 2) C1q-binding and complement-activating activities are expressed only when the inter L chain disulfide bridge does not exist. A molecular model is presented for the active form of the short-chain IgG2a antibody.
Multiple cis-acting elements are required for proper transcription of the mouse V delta 1 T cell receptor promoter.
Authors: Punturieri A , Shirakata Y , Bovolenta C , Kikuchi G , Coligan J E
Published: December 2022

To gain insight into the developmentally regulated expression of the mouse TCR V delta-gene segments, we have investigated the role of the 5' promoter region of the V delta 1-gene. Transient transfection assays showed that a construct encompassing 267 nucleotides upstream from the mapped transcriptional start site was capable of driving promoter activity when transfected into V delta 1+ T cells. The inclusion of an additional 459-bp 5' segment to this construct did not affect promoter activity. However, a deletion of 222 5' nucleotides from the same construct dramatically decreased promoter activity. In vivo genomic footprinting localized several protein-DNA interactions to the stretch of DNA shown to have transcriptional activity. A computer analysis revealed that the segments of DNA participating in these protein-DNA interactions were identical to the previously described cyclic AMP response element (CRE), E box, and leukemia virus E26 cis-acting elements. Transient transfection assays performed with -267 bp constructs containing mutations at each of the localized cis-acting elements revealed that the CRE, E box, and Ets elements work together in driving promoter activity and that the CRE and Ets elements are the most important for driving transcription. Gel mobility shift analyses showed that each of these cis-acting elements is capable of binding specific nuclear factors present in V delta 1-expressing cells. These data indicate that multiple transcription factors acting in concert are responsible for V delta 1 gene expression.
Relationship of C5a receptor modulation to the functional responsiveness of human polymorphonuclear leukocytes to C5a.
Authors: Van Epps D E , Simpson S J , Johnson R
Published: December 2022

The relationship of C5a receptor expression on human polymorphonuclear leukocytes (PMN) to the functional response of these cells to C5a was studied using flow cytometry. C5a receptor expression was determined with a fluorescein conjugate of C5a and oxidative burst activity was monitored by conversion of dichlorofluorescein to dichlorofluorescein (DCF) as a measure of H2O2 production. These studies showed that after incubation of PMN with increasing concentrations of C5a, and allowing for internalization of bound ligand, more than 40% of the cell surface C5a receptors were internalized before the DCF response to optimal concentrations of C5a was decreased below the levels for untreated control cells. Although C5a responsiveness was lost after preincubation with 10(-8) M C5a, cells remained responsive to formyl peptide. In other studies, cells were preincubated with unlabeled C5a under conditions that provided for internalization of nearly all C5a receptors. PMN were then cultured for up to 90 min and monitored for C5a receptor reexpression and return of cell function. In these studies, the DCF response of PMN to C5a returned to 100% much earlier than the cells regained full expression of C5a receptors. The DCF response to formyl peptide remained intact throughout the period of C5a receptor reexpression. These studies showed that once > 40% of the original population of C5a receptors are reexpressed on the PMN, that these cells regain 100% of their functional responsiveness to C5a in the DCF assay. Evaluation of the affinity and number of C5a receptors using 125I-labeled C5a after receptor reexpression showed that maximal receptor reexpression was approximately 73% of that obtained with control cells and the Kd of reexpressed receptors was 0.60 vs 0.94 nM for control cells. These studies demonstrate that only a portion of the total C5a receptors expressed on PMN are essential to stimulate a 100% functional response in PMN and that the reexpressed receptors are capable of transducing a signal that activates the oxidative burst in these cells.
Nonmitogenic anti-CD3F(ab')2 fragments inhibit lethal murine graft-versus-host disease induced across the major histocompatibility barrier.
Authors: Blazar B R , Taylor P A , Snover D C , Bluestone J A , Vallera D A
Published: December 2022

We have investigated the in vivo administration of nonmitogenic anti-CD3F(ab')2 fragments for the prevention of lethal graft-vs-host disease (GVHD) in irradiated recipients of fully allogeneic bone marrow cells plus splenocyte (BMS) inocula. Recipients of anti-CD3F(ab')2 fragments administered for 1 mo post-bone marrow transplantation (BMT) had 100% survival without clinical or histopathological evidence of GVHD. Controls given saline injections succumbed by 39 days post-BMT. Similar results were obtained in groups of recipient mice given BMS in which T cells were depleted by in vitro anti-Thy-1.2 plus C' treatment. Further studies were undertaken to define mechanistic differences in the two approaches. Using Ly-5 congenic sources of donor bone marrow and spleen, we determined that anti-CD3F(ab')2 fragments induced TCR modulation and T cell depletion. Mature splenic-derived CD4+ cells were depleted to a greater extent than CD8+ cells. Early post-BMT, recipients receiving injections with control saline had the highest number of CD4+ and CD8+ cells (which may cause GVHD) followed by recipients of anti-CD3F(ab')2 fragments, with the fewest CD8+ cells observed in the anti-Thy-1.2 + C' treated group. CD3+CD4-CD8- cells (which may suppress GVHD generation) were present in higher numbers early post-BMT in recipients given anti-CD3F(ab')2 fragments as compared to recipients given anti-Thy-1.2 + C'-treated BMS. In long term survivors, a mononuclear T cell containing infiltrate without evidence of destruction was observed in sites of GVHD (lung and liver), consistent with a "Quilty" effect, which was not observed in either of the other two groups. Although survivors were tolerant of donor skin grafts and rejected third party grafts, recipients given anti-CD3F(ab')2 fragments but not anti-Thy-1.2 + C'-treated BMS had vigorous anti-host proliferative responses. These results demonstrate that although in vitro anti-Thy-1.2 + C' treatment of BMS (which is highly depletionary) and in vivo administration of anti-CD3F(ab')2 fragments (which is modulatory and less depletionary) are both effective strategies for GVHD, the cellular events involved in achieving GVHD prevention are indeed different.
Ig CDR3-like region of the CD4 molecule is involved in HIV-induced syncytia formation but not in viral entry.
Authors: Corbeau P , Benkirane M , Weil R , David C , Emiliani S , Olive D , Mawas C , Serre A , Devaux C
Published: December 2022

The HIV1 envelope glycoprotein gp120 binding site has been previously mapped by genetic studies to the CDR2-like region of the first domain of the CD4 molecule. mAb reactive with epitopes linked to this region (e.g., OKT4A) inhibit both HIV entry into CD4-positive cells and syncytia formation. A second area of this domain, the CDR3-like region, has been shown to be involved in gp120-CD4 interactions, but its role remained so far unclear. We show here that a mAb specific for the CDR3-like region of the CD4 receptor, 13B8-2, actually blocks soluble gp120 binding to CD4, inhibits HIV-induced cell-cell fusion, and prevents viral production by infected cells. However, this mAb fails to inhibit the binding of viral particles to cell-surface CD4 and their entry into CD4-positive cells. These results strongly suggest i) that soluble gp120 and virion-anchored gp120 bind CD4 in distinct manners, ii) that gp120-CD4 interactions required for viral entry and syncytia formation are different, and iii) that mAb binding to the CDR3-like region of the first domain of CD4 affects a post-entry step of the HIV replicative cycle.
Simultaneous presence, in one serum, of four monoclonal antibodies that might correspond to different steps in a clonal evolution from polyreactive to monoreactive antibodies.
Authors: Houdayer M , Bouvet J P , Wolff A , Magnac C , Guillemot J C , Borche L , Dighiero G
Published: December 2022

Three monoclonal IgG of different subclasses (IgG1, IgG2, and IgG4) and one IgA1 were isolated from the serum of patient Per suffering from an immunocytic sarcoma. All four monoclonal Ig shared the same N-terminal sequence of their H chains (VH3). Furthermore, their kappa-chains exhibited identical isoelectric charges and N-terminal sequences (VK2) and expressed the same private idiotope. A strong antitubulin activity was found in IgA1Per and in two of the three monoclonal IgGPer. The specificity was restricted to tubulin for IgA1Per and IgG4Per, whereas IgG1Per also displayed significant polyreactive bindings and IgG2Per failed to react with any of the Ag tested. The monoreactive IgG4Per, as well as the polyreactive IgG1Per, bound a large peptide in the central part of both alpha and beta subunits of tubulin (amino acid position 100 to 300). In contrast, the monoreactive IgA1Per bound to a rarely detected epitope close to residue 310 of these subunits. The tubulin epitope recognized by polyreactive IgG1Per was similar to that of germ-line-encoded polyreactive antibodies. It is hypothesized that IgG4Per- and IgA1Per-producing cells derive from the IgG1Per polyreactive clone after somatic events leading to the production of monoreactive antibodies.
Cytokine expression by germinal center cells.
Authors: Butch A W , Chung G H , Hoffmann J W , Nahm M H
Published: December 2022

Germinal centers (GC) primarily consist of B cells along with a small number of T cells (5 to 10%) and follicular dendritic cells (FDC) (< or = 1%). Although extensive Ag-driven B cell proliferation and maturation occurs in GC, very little is known about the role of cytokines in the development of GC B cells. Therefore, to identify cytokines present in the GC microenvironment that may influence B cell development, we systematically examined cytokine gene expression by GC cells. GC T cells (CD57+/CD4+), GC B cells (CD77+), and FDC (HJ2+) were isolated from human tonsils by cell sorting using a flow cytometer. Freshly isolated GC cells were examined for mRNA expression for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-alpha, and IFN-gamma using reverse transcription polymerase chain reaction. Freshly isolated GC T cells consistently expressed IL-4 mRNA (11 of 12 tonsils), whereas CD57- Th cells (mostly non-GC Th cells) were often negative for IL-4 mRNA. When the other nine cytokine mRNA were studied, freshly isolated CD57+ Th cells occasionally expressed mRNA for IL-10, TNF-alpha, and IFN-gamma. CD57- Th cells were occasionally positive for IL-1 beta, IL-10, IFN-gamma, and TNF-alpha, and negative for IL-2 and IL-6. Freshly isolated GC B cells as well as FDC failed to express detectable quantities of mRNA for all 10 cytokines that were studied. Thus, IL-4 is the only cytokine out of 10 that is consistently expressed in GC and may be important for the development of B cells in GC. After stimulation of CD57+ Th cells with PWM, production of IL-4 mRNA was dramatically reduced, whereas CD57- Th cell production of IL-4 was greatly augmented. This finding indicates that GC T cells may differ from other Th cells in cytokine gene expression and that results of cytokine production obtained after in vitro stimulation do not always reflect in vivo results.
Analysis of IL-2, IL-4, and IFN-gamma-producing cells in situ during immune responses to protein antigens.
Authors: Bogen S A , Fogelman I , Abbas A K
Published: December 2022

Immunohistochemistry has been used to define the patterns and kinetics of IL-2, IL-4, and IFN-gamma production at the sites of Ag exposure and in the lymphoid tissues of immunized mice, and to examine the anatomic relationships between cytokine-producing T cells and various APC or Ag-stimulated B cells. The earliest detectable cytokine response to administration of a protein Ag in adjuvant was the appearance of IFN-gamma-producing NK cells at the site of immunization by day 3. T lymphocytes producing IL-2, IL-4, and IFN-gamma were initially detected in draining lymph nodes and spleen within 7 days after immunization, and IL-2-producing cells were present at the immunization site several weeks later. Thus, T cell activation is initiated within lymphoid tissues, and these cells migrate back to depots of Ag. The IFN-gamma produced by NK cells early after immunization may regulate the phenotype of the subsequent Ag-specific T cell response. Using a hapten to which the antibody response is oligoclonal and dominated by a single idiotype, Ag-stimulated (idiotype-producing) B cells could also be detected by immunohistochemistry. These B cells were present in the same areas of lymphoid tissues as cytokine-producing T lymphocytes. Two-color staining showed that idiotype-producing B cells were in close proximity to both IL-2- and IL-4-producing T cells, suggesting that T cells producing either of these cytokines could provide helper function for the B cells. Finally, after subcutaneous immunization with adjuvant, IL-2+ T cells were found adjacent to F4/80+ macrophages, suggesting that macrophages function as important APC in this response.
A mutant antigen-presenting cell defective in antigen presentation expresses class II MHC molecules with an altered conformation.
Authors: Dang L H , Lien L L , Benacerraf B , Rock K L
Published: December 2022

Ag presentation by APC to class II MHC-restricted T cells involves a sequence of events: 1) intracellular processing of protein Ag into immunogenic peptides, 2) specific binding of peptides to class II MHC molecules, and then 3) transport of the MHC-peptide complexes to the plasma membrane. The critical event in the activation of T cells by APC is the recognition of MHC-associated antigenic determinants by the TCR/CD3 complex. In this report we describe the isolation and characterization of a mutant APC with a defect in an intracellular process that results in its inability to form MHC-peptide complexes for recognition by T cells. The mutant APC cannot present many different protein Ag with both I-A and I-E molecules but is able to present processing-independent peptides. The functional defect in the mutant APC is not caused by either a decrease in expression or a structural mutation in class II MHC molecules. Further, there is no mutation in the invariant chain (li) and it displays a normal kinetics of association and dissociation from the class II MHC molecules during biosynthesis. Although the mutation is not in the genes encoding for the class II MHC molecules or li, the mutant APC expresses class II MHC molecules with distinct serological epitopes suggestive of an altered conformation. Pulse-chase experiments suggest that a conformational difference between I-Ad molecules of wild-type and mutant cells occurs after the class II molecules exit from the endoplasmic reticulum but while they are still associated with li. The mutant cell produces few compact (SDS-resistant) class II heterodimers. This mutant APC provides a tool for studying the cell biology of Ag processing and presentation.
Phosphatidylserine enhances the ability of epidermal Langerhans cells to induce contact hypersensitivity.
Authors: Girolomoni G , Pastore S , Zacchi V , Cavani A , Marconi A , Giannetti A
Published: December 2022

Phosphatidylserine (PS) modulates several immune functions in vitro, including T cell activation, antibody and cytokine production, and macrophage growth. In the present work we studied the effects of PS on the induction of contact hypersensitivity (CH) in mice. BALB/c mice painted with PS (9.4-75 mg/kg) and with a sensitizing dose of DNFB or oxazolone on the same skin site exhibited a dose-dependent augmentation of CH reactions to either DNFB (> 60%) or oxazolone (> 35%), respectively. Bovine brain PS-enriched phospholipid mixture, lyso-PS, and dipalmitoyl-PS also induced similar enhanced CH responses, whereas phosphatidylglycerols had no effect. Increased CH was observed only when PS was applied from 2 days before to 12 h after DNFB. Immunization of naive syngeneic mice with skin grafts that were treated with PS and DNFB also led to enhanced (> 50%) CH responses. In addition, immunization by iv injection of epidermal cell suspensions enriched for Langerhans cells (LC) or of purified LC that were treated with PS (1-100 microM, 30 min, 37 degrees C), and then modified in vitro with DNBS (1 mg/ml, 30 min, 37 degrees C) led to increased (> 30-75%) CH responses in recipient syngeneic animals. Finally, adoptive transfer of DNFB-immune lymph node cells obtained from mice that were treated with PS induced augmented CH responses in recipient animals. The results suggest that PS is capable of up-regulating the induction of CH in mice by stimulating the APC function of epidermal LC.
Functional CD4 T cell subset interplay in an intact immune system.
Authors: Murray J S , Madri J , Pasqualini T , Bottomly K
Published: December 2022

Immunization with human collagen IV has been shown to lead to the selective activation of Th-1 and Th-2-like cells in vivo depending on the I-A genotype of the mice. Mice expressing I-As generate Th-1 cells, and mice expressing I-Ab generate Th-2 cells after immunization. We examined the response of (bxs)F1 hybrid mice to determine the types of CD4 T cell activated. We found that Th-1-like responses dominated, in that CD4 T cells from human collagen IV-primed mice displayed good proliferative responses and little ability to induce antibody formation, and secreted IFN-gamma and not IL-5. Most of the Th-1-like activity of F1 hybrid T cells was I-As restricted. Inhibition of Th-1 cell priming using anti-I-As antibody administered with the priming Ag in vivo revealed Th-2-like activity, in that the CD4 T cells now secreted IL-5 and not IFN-gamma and induced antibody formation. Additional studies indicated that anti-IFN-gamma treatment in vivo also revealed Th-2 cells, suggesting that I-As-restricted CD4 T cells producing or controlling the production of IFN-gamma suppress Th-2 priming.
Regulated expression of a receptor for hyaluronan-mediated motility on human thymocytes and T cells.
Authors: Pilarski L M , Miszta H , Turley E A
Published: December 2022

A receptor for hyaluronan-mediated motility (RHAMM) has been shown to promote cell locomotion. Among human T lineage lymphocytes, RHAMM is expressed only on a subset of thymocytes, being absent on mature peripheral T cells from blood, spleen, and lymph node. Among thymocytes, RHAMM is selectively expressed on a subset of CD3+ CD45RA+R0+ cells, and functions in motility as shown by the ability of anti-RHAMM to reduce the speed of thymocyte locomotion from 11 microns/minute to 3 microns/min. Although freshly isolated multi-negative (MN) thymocytes (CD3-4-8-19-) lack RHAMM, its expression is induced on day 3 of culture in a variety of conditions that support differentiation, as assessed by acquisition of CD3. When MN thymocytes are cultured on plates coated with fibronectin, expression of RHAMM is prolonged, but on uncoated surfaces, its expression is transient and lost by day 7 of culture with PHA or IL-2. Culture of MN thymocytes on thymic epithelial layers, with or without IL-2, resulted in a lack of RHAMM expression. Because in the absence of epithelial cells, RHAMM is expressed, the effect appears to be one of inhibition. Although expression of RHAMM by MN thymocytes cultured with IL-2 on uncoated surfaces is transient, addition of cyclosporin A resulted in prolonged expression. These observations are consistent with the view that cyclosporin A inactivates a RHAMM-directed inhibitory mechanism. Mature peripheral blood T cells transiently express RHAMM upon culture with PHA, PMA, or IL-2. T cells that expressed RHAMM after culture with PMA alone lacked RHAMM when stimulated by mitogenic CD2 antibodies with or without CD28 antibody, indicating inhibition of RHAMM expression. Thus expression of RHAMM is regulated by a RHAMM-directed inhibitory mechanism induced by stimulation through CD2/CD28. A similar mechanism may operate in thymocyte/epithelial cell cultures. These results suggest the inhibition of RHAMM during early, presumably sessile, thymic progenitor development, followed by its induction during developmental stages when locomotion is required. The apparently strong negative regulatory control over RHAMM expression by microenvironmental factors and by known thymic and T cell signaling molecules supports this view.
Role of TNF-alpha in CD8+ cytotoxic T lymphocyte-mediated lysis.
Authors: Ratner A , Clark W R
Published: December 2022

The possibility that lymphokines such as TNF-alpha produced by CD8+ CTL are responsible for acute (short term) target cell damage induced by CTL has been debated for many years. However, the slow kinetics of TNF-induced target cell death stands in sharp contrast to the rapid target cell lysis mediated by CTL. We find that cloned CD8+ CTL activated through their TCR secrete TNF-alpha. On the other hand, our cloned CTL also have a membrane form of TNF-alpha, and they kill TNF-alpha-sensitive target cells not recognized through the TCR in a slow (18-h) lytic reaction using this surface-associated TNF-alpha. There is no secreted TNF-alpha release during this interaction. Cyclosporin A and protein synthesis inhibitors block TNF-alpha secretion, but have no effect on slow lysis mediated by the CTL. On the other hand, TNF-alpha-resistant variants are greatly resistant to slow lysis, and antibodies to TNF-alpha strongly inhibit this slow lysis. Thus, although secreted TNF-alpha does not seem to be the mechanism behind slow lysis, some form of TNF-alpha, most likely the membrane-associated form, must be involved. Not only does surface TNF-alpha appear to be biologically active in these CTL, but its expression is enhanced severalfold upon activation of the CTL through the TCR. This may be important in vivo, where surface TNF-alpha could preserve the localized nature of cytolysis and endow a CTL with an additional, albeit slower, mechanism of cell lysis. Finally, we find that although activated CTL clearly use the membrane form of TNF-alpha in slow lysis, they appear not to use TNF-alpha, in any form, during acute lysis, even under conditions in which degranulation and perforin assembly are blocked.