bluuebunny/crossref_metadata_2025 huggingface dataset:
T cells from tumor-immune mice nonspecifically expanded in vitro with anti-CD3 plus IL-2 retain specific function in vitro and can eradicate disseminated leukemia in vivo.
Authors: Crossland K D , Lee V K , Chen W , Riddell S R , Greenberg P D , Cheever M A
Published: December 2022

The therapeutic efficacy of adoptive immunotherapy of cancer has been shown to positively correlate with the dose of tumor-immune T cells transferred. Therefore, the success of this therapy is critically dependent on the ability to procure large numbers of functionally active T cells. Previous studies in animal models have shown that the limited therapeutic efficacy of a small number of immune T cells can be greatly enhanced by expansion of T cells in vitro to greater numbers before transfer in vivo. Optimal regimens for T cell expansion in vitro have generally employed the use of intermittent stimulation of the TCR with specific Ag followed by exogenous IL-2. The use of IL-2 alone does not provide for requisite episodic up-regulation of IL-2R. Stimulation of the invariant CD3 portion of the TCR/CD3 complex with antibody to CD3 (anti-CD3) represents an alternative method of up-regulating IL-2R and has been used to nonspecifically induce the growth of Ag-specific T cell lines and clones long-term in vitro with maintenance of function and specificity. The current study examined whether resting T cell populations containing small numbers of memory tumor-specific T cells could be rendered more effective in tumor therapy by nonspecific expansion in vitro with anti-CD3 plus IL-2. Spleens from C57BL/6 mice previously immunized to FBL-3, a syngeneic virus-induced leukemia, were nonspecifically stimulated with anti-CD3 plus IL-2. The resultant T cells were expanded in number, were nonlytic to FBL-3 but retained the ability to become lytic upon specific stimulation by FBL-3, and were effective in specific tumor therapy. The Ag-specific anti-tumor immune function declined on a per cell basis after each cycle of anti-CD3-induced T cell expansion. However, the approach resulted in a substantial increase in total T cell number and an overall net increase in the function of the effector T cell population. Thus, stimulation of tumor-immune T cell populations with anti-CD3 plus IL-2 represents a nonspecific method for expanding the number of specific effector T cells for cancer therapy.
Subcellular localization of perforin and serine esterase in lymphokine-activated killer cells and cytotoxic T cells by immunogold labeling.
Authors: Ojcius D M , Zheng L M , Sphicas E C , Zychlinsky A , Young J D
Published: December 2022

CTL, NK cells, and lymphokine-activated killer (LAK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by forming large pores on the plasma membrane of the target cell. Other proteins besides perforin are found in the cytoplasmic granules of effector lymphocytes, and these include a family of serine esterases. Ultrastructural immunogold labeling studies with antibodies against perforin and a serine esterase (MTSP-1, also known as granzyme A and SE-1) show that all the granules of LAK cells and a CTL cell line contain perforin and serine esterase. For both LAK cells and CTL, perforin has been located mostly in the fine granular matrix of the granules, whereas gold particles corresponding to serine esterase have been found in both the matrix and the cap regions of the granules. Results from double immunogold labeling indicate that perforin and serine esterase colocalize to the same granules.
Mutations affecting antigen processing impair class II-restricted allorecognition.
Authors: Cotner T , Mellins E , Johnson A H , Pious D
Published: December 2022

Both exogenously derived and endogenously derived Ag generally require processing for their optimal binding and presentation by class I and class II major histocompatibility proteins. It is not known whether steps involved in Ag processing also affect the recognition of alloreactive T cells. We have recently described B cell mutants which have general defects in the processing and presentation of a variety of exogenous Ag to class II restricted T cells. In this report we have studied the ability of these processing mutants to stimulate a set of anti-DR3-specific alloreactive T cells clones. These processing/presentation mutants express normal MHC class II molecules, both in terms of primary sequence and cell surface abundance, but they appear unable to generate effective peptide-MHC complexes. When tested for their ability to stimulate MHC class II alloreactive T cell clones, only one of four T cell clones was stimulated by these mutants; the other three alloreactive T cell clones were not stimulated by either of two different mutants. Both of these mutants express normal levels of the accessory molecules, LFA-3 and ICAM-1. The inability of these mutants to stimulate three of four alloreactive clones indicates that the capacity to be recognized by many alloreactive T cells is linked to the Ag processing capacity of a stimulator cell.
Lectin-mediated induction of IL-4-producing CD4+ T cells.
Authors: Röcken M , Müller K M , Saurat J H , Hauser C
Published: December 2022

Cultured murine CD4+ T cells have been shown to differentiate into IL-2 or IL-4-producing subsets. The factors responsible for the development of CD4+ T cells which produce IL-2 but not IL-4 and cells capable of producing IL-4 but not IL-2 are unknown. Here we describe a system that allows the controlled induction of IL-2- or IL-4-producing T cells after one single round of activation. Freshly isolated CD8-depleted T cells were activated with various polyclonal T cell activators for 48 h, washed, and then expanded under different conditions. IL-2 and IL-4 production were induced by restimulation of T cells and were measured with CTLL cells that respond to both cytokines and mAb to IL-2 and IL-4. T cells produced mainly IL-2 and small amounts of IL-4 when restimulated after expansion culture for 12 days with rIL-2 alone. However, after expansion for 12 days in the presence of rIL-2 plus Con A, we observed a 30- to 100-fold up-regulation of IL-4 activity and a 100-fold down-regulation of IL-2 when assessed by responses of CTLL cells incubated with the supernatant of restimulated T cells and by responses of CTLL cells cocultured with restimulated cells. An increase of IL-4 and decrease of IL-2 was also observed when the results were based on the cell numbers at the beginning of the expansion culture. The induction of IL-4 and the down-regulation of IL-2 1) were not reproduced with alpha-methyl-mannoside-treated supernatant of Con A-stimulated spleen cells, 2) were not dependent on the presence of large numbers of APC, 3) did not result from differential consumption of lymphokines after restimulation, 4) were not due to a difference in the time course of IL-2 or IL-4 release in either T cell population, and 5) were obtained regardless of the agents used to activate or to restimulate the T cells. Because Con A remained detectable on the T cell surface and because expansion of activated T cells with IL-2 plus Con A for several days was necessary, our results indicate that mainly IL-4-producing CD4+ T cells can be induced by prolonged engagement of T cell surface molecules.
Prednisone inhibits the appearance of inflammatory mediators and the influx of eosinophils and basophils associated with the cutaneous late-phase response to allergen.
Authors: Charlesworth E N , Kagey-Sobotka A , Schleimer R P , Norman P S , Lichtenstein L M
Published: December 2022

To better define the effect of systemic glucocorticoids on the cutaneous early and late phase response (LPR), nine atopic subjects were examined in a double-blind cross-over study using skin chambers fixed over denuded skin blisters. A challenge was carried out by placing allergen in the chamber for 60 min in subjects who received either a 3-day pretreatment with 60 mg/day of prednisone or placebo. Skin chamber cell counts and inflammatory mediators (histamine, PGD2, and leukotriene C4 (LTC4)) were measured at hourly intervals for 12 h. Prednisone pretreatment did not alter the immediate skin erythema or release of histamine but ablated the late secondary erythema and rise in histamine. The median histamine values during h 10, 11, and 12 in the placebo and prednisone pretreatment visits were 3.73 and 0.22 ng/ml, respectively (p less than or equal to 0.02). Prednisone did not alter PGD2 production; however, LTC4 production was suppressed during the LPR. The cumulative median LTC4 values during h 7, 8, and 9 were 5.6-fold (p less than or equal to 0.05) more after placebo than after prednisone pretreatment. Prednisone altered cellular traffic more dramatically than it did inflammatory mediators. The influx of eosinophils, which peaked during the 9th and 10th h in placebo-treated patients, was completely blocked by prednisone (p less than or equal to 0.02) for every h from 6 through 12. The influx of basophils, which started during the 9th h and peaked during the 12th h in placebo-treated patients, was suppressed at all time points (p less than or equal to 0.02) in prednisone-treated patients. There was no significant alteration in neutrophil transit into the skin chambers induced by prednisone. We suggest that the selective blockade of eosinophil and basophil influx by prednisone and the associated decrease in inflammatory mediators may contribute to the blockade of the clinical expression of the cutaneous LPR.
Characterization of a membrane antigen of Leishmania amazonensis that stimulates human immune responses.
Authors: Burns J M , Scott J M , Carvalho E M , Russo D M , March C J , Van Ness K P , Reed S G
Published: December 2022

To investigate human immune responses to defined leishmania Ag we have begun to characterize biochemically and immunologically, an abundant 42-kDa surface Ag of Leishmania amazonensis, a causative agent of human leishmaniasis. We have shown that this Ag, La gp42, is expressed on the surface of L. amazonensis promastigotes, being anchored to the membrane by a glycosyl-phosphatidylinositol moiety. As demonstrated by lectin blotting studies, La gp42 is glycosylated, binding both Con A and wheat germ agglutinin. Immunologically, La gp42 is strongly recognized by sera from patients with different forms of leishmaniasis as well as by patients with Chagas' disease. In addition, we show that purified La gp42 stimulates the proliferation of human T lymphocytes obtained from several leishmaniasis patients. Finally, the N-terminal sequence of La gp42 was obtained and a serologically cross-reactive 42-kDa protein with a homologous sequence was identified in Leishmania major.
Tumor necrosis factor-independent IL-6 production during murine listeriosis.
Authors: Havell E A , Sehgal P B
Published: December 2022

We report that TNF, IL-6, and IFN-alpha/beta are produced by mice during either sublethal or lethal Listeria monocytogenes infections. The quantities of these cytokines in infected spleens increase and decrease in concordance with bacterial numbers in these organs. While all of these cytokines were present in Listeria-infected spleens, only IL-6 and IFN-alpha/beta were found in the peripheral circulation. Inasmuch as TNF has been reported to be responsible for the production of IL-6 in vivo following the inoculation of a lethal dose of the Gram-negative bacterium, Escherichia coli (Fong et al., 1989. J. Exp. Med. 170: 1627), experiments were undertaken to determine whether IL-6 production elicited by the Gram-positive bacterium, L. monocytogenes, was also TNF-dependent. It was found that the passive immunization of mice with neutralizing antibodies specific for TNF shortly before i.v. injection of a lethal or sublethal Listeria inoculum resulted in the complete neutralization of endogenously produced TNF, and in the progressive multiplication of bacteria in infected organs. It was also found that the anti-TNF IgG treatment resulted in a progressive increase in the amounts of Listeria-induced IL-6 present in spleen and blood, until the death of the host. These findings indicate that Listeria-induced IL-6 production in mice occurs primarily through a TNF-independent pathway, and correlates directly with the severity of the infection.
Isolation and expression of cDNA encoding the murine homologues of CD1.
Authors: Balk S P , Bleicher P A , Terhorst C
Published: December 2022

The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.
Targeting human cytotoxic T lymphocytes to kill heterologous epidermal growth factor receptor-bearing tumor cells. Tumor-infiltrating lymphocyte/hormone receptor/recombinant antibody.
Authors: Gillies S D , Wesolowski J S , Lo K M
Published: December 2022

A genetically engineered conjugate between an anti-CD3 antibody and epidermal growth factor (EGF) was tested for its ability to mediate the lysis of receptor-bearing cells by human CTL. This construct was made by fusing an EGF coding sequence to the 3' end of the human gamma-1 H chain gene sequence and expressing the modified gene in transfected cells together with the V regions of a mouse antibody specific for the human T cell marker, CD3. The resulting conjugate was able to compete with EGF for its receptor and, at extremely low concentrations, was able to mediate the lysis of receptor-bearing tumor targets by a tumor-infiltrating lymphocytes line or by a CTL line established from peripheral blood. The construction of such conjugates by genetic engineering represents a general approach to the direct expression of highly specific hetero-bifunctional reagents without the necessity of further in vitro manipulations.
Murine T cell responses to melittin and its analogs.
Authors: Fehlner P F , Berg R H , Tam J P , King T P
Published: December 2022

The 26-residue peptide melittin from bee venom elicits high IgG1 and IgE responses in selected strains of mice. The antibody responses were shown previously to be specific mainly for the region of residue 20-26. The T cell epitope of melittin in H-2d-restricted mice is now found to be primarily in residue 11-19, corresponding to an alpha-helical amphiphilic segment of the molecule. Melittin-specific T cell lines have varying responses to different structural analogs of the melittin T cell epitope, and the results indicate that the antigenicity of T cell epitope peptides depend more on their primary structure than on their secondary structure. Melittin-specific T cell clones are found to be CD4+ and secrete IL-4, and are restricted to presentation on I-Ad or I-Ed. The I-Ad- or I-Ed-restricted clones differ in their responses to different analogs of melittin.
Distinct pathways of CD4 and CD8 cells induce rapid target DNA fragmentation.
Authors: Ju S T
Published: December 2022

When activated with either Con A, a CD3-specific mAb, or Ag-pulsed B lymphoma (LK35.2) cells, CD4 (Th1) clones quickly induce DNA fragmentation in target cells followed by release of 51Cr-labeled intracellular materials. Both activated CD4 clones and CD8 (CTL) cells fragment target DNA into electrophoretically identical "ladder" pattern made of approximately 200 bp. The effect of various metabolic inhibitors on the ability of CD4 and CD8 cells to induce target DNA fragmentation was studied. Little effect was observed with the DNA synthesis inhibitor, mitomycin C. The RNA synthesis inhibitor, actinomycin D, and the protein synthesis inhibitor, cycloheximide, strongly inhibited the ability of CD4 cells, but not CD8 cells, to induce target DNA fragmentation. In contrast, target DNA fragmentation by CD8 cells, but not by CD4 cells, was inhibited by cholera toxin. Although cyclosporin A inhibited CD4 cells to fragment target DNA during the early phase (90 min) of E:T interaction, this inhibition was not sustained in the later phase (210 min) of the assay. Zinc ions inhibited the ability of both CD4 and CD8 cells to fragment target DNA. Treatment of effectors and targets with these inhibitors, followed by washings, demonstrated that the action of these inhibitors on effector cells alone is sufficient to inhibit target DNA fragmentation. The strong correlation among these parameters of DNA fragmentation and Cr-release assays supports the hypothesis of programed cell death. Although distinct cytolytic pathways are used by CD4 and CD8 cells to kill targets, both pathways deliver a signal that activates endonuclease(s), fragments target DNA, causes Cr-release, and lyses target cells. Taken together with our previous studies, the present findings demonstrate that activated cytolytic CD4 clones do not use perforin, serine proteases, and TNF as mediators for resistant target DNA fragmentation.
Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.
Authors: Shearman C W , Kanzy E J , Lawrie D K , Li Y W , Thammana P , Moore G P , Kurrle R
Published: December 2022

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.
Expression of VHIII-associated cross-reactive idiotype on human B lymphocytes. Association with staphylococcal protein A binding and Staphylococcus aureus Cowan I stimulation.
Authors: Shokri F , Mageed R A , Maziak B R , Jefferis R
Published: December 2022

It has been demonstrated that staphylococcal protein A (SPA) has an "alternative" binding site with specificity for human Ig H chain V region of the VHIII subgroup. Because the major mitogenic component of Staphylococcus aureus Cowan I (SAC) is SPA, it is possible that SAC stimulates a subpopulation of B cells expressing Ig of the VHIII H chain subgroup. In the present study, we have investigated further the relationship between SPA binding and the expression of VHI- or VHIII-associated cross-reactive idiotype (CRI) on the surface of tonsillar B lymphocytes enriched for the expression or nonexpression of the CRI, and we examined the Ig secreted by cell lines established from these populations of B cells by EBV transformation. The VHIII CRI (D12)-enriched population yielded 21 cell lines, with 67% of them secreting SPA-reactive Ig; in contrast, only 6% (1 of 16) of VHI CRI-expressing lines secreted SPA-reactive Ig. The CRI-negative B cell population yielded 54 cell lines, of which 20% secreted SPA-reactive Ig, as might be anticipated because a majority of VHIII Ig+ B cells will be CRI-. SAC stimulation of CRI+ and CRI- populations showed preferential stimulation of the D12 population. These data support the proposal that SAC stimulation of human B cells is mediated through binding of SPA by its alternative binding site to IgV regions of the VHIII subgroup.
The role of polymorphic amino acids of the MHC molecule in the selection of the T cell repertoire.
Authors: Bhayani H R , Hedrick S M
Published: December 2022

Allelic variants of MHC molecules expressed on cells of the thymus affect the selection and the specificity of the T cell repertoire. The selection is based on either the direct recognition by the TCR of the MHC molecules, or the recognition of a complex determinant formed by self-peptides bound to MHC molecules. In an analysis of the T cell repertoire in bone marrow chimeras that express allelic forms of MHC class II molecules in the thymus epithelium, we find that amino acid substitutions that are predicted to affect peptide binding influence the selection of the T cell repertoire during thymic selection.
Phenotype, ontogeny, and repertoire of CD4-CD8- T cell receptor alpha beta + thymocytes. Variable influence of self-antigens on T cell receptor V beta usage.
Authors: Takahama Y , Kosugi A , Singer A
Published: December 2022

We have characterized CD4-CD8- double negative (DN) thymocytes that express TCR-alpha beta and represent a minor thymocyte subpopulation expressing a markedly skewed TCR repertoire. We found that DN TCR-alpha beta + thymocytes resemble mature T cells in that they (a) are phenotypically CD2hiCD5hiQa2+HSA-, (b) appear late in ontogeny, and (c) are susceptible to cyclosporin A-induced maturation arrest. In addition, we found that DNA sequences 5' to the CD8 alpha gene were demethylated relative to their germline state, suggesting that DN TCR-alpha beta + thymocytes are derived from cells that had at one time expressed their CD8 alpha gene locus. Because DN TCR-alpha beta + thymocytes are known to express an unusual TCR repertoire with significant overexpression of V beta 8, we were interested in examining the possible role played by self-Ag in shaping their TCR repertoire. It has been suggested that DN TCR-alpha beta + thymocytes are derived from potentially self-reactive thymocytes that have escaped clonal deletion by down-regulating their surface expression of CD4 and/or CD8 determinants. However, apparently inconsistent with such an hypothesis, we found that the frequency of DN thymocytes expressing various anti-self TCR (V beta 6, V beta 8.1, V beta 11, V beta 17a) were not increased in strains expressing their putative self-Ag, but instead were either unaffected or significantly reduced in those strains. With regard to V beta 8 expression among DN TCR-alpha beta + thymocytes, V beta 8 overexpression in DN TCR-alpha beta + thymocytes appeared to be independent of, and superimposed on, the developmental appearance of the basic DN thymocyte repertoire. Even though V beta 8 overexpression appeared to be generated by a mechanism distinct from that generating the rest of the DN TCR-alpha beta + thymocyte repertoire, we found that super-Ag against which V beta 8 TCR react introduced into the neonatal differentiation environment also significantly reduced, rather than increased, the frequency of DN TCR-alpha beta + V beta 8+ thymocytes. Thus, the present study is consistent with DN TCR-alpha beta + thymocytes being mature cells derived from CD8+ precursors, and documents that their TCR repertoire can be influenced, at least negatively, by either self-Ag or Ag introduced into the neonatal differentiation environment. However, we found no evidence to support the hypothesis that DN TCR-alpha beta + thymocytes are enriched in cells expressing TCR reactive against self-Ag.
Influenza A-specific, HLA-A2.1-restricted cytotoxic T lymphocytes from HLA-A2.1 transgenic mice recognize fragments of the M1 protein.
Authors: Engelhard V H , Lacy E , Ridge J P
Published: December 2022

Previous studies have indicated that in transgenic mice expressing human class I MHC molecules, it is difficult to demonstrate a significant CTL response to a viral Ag in the context of the transgenic molecule. In this paper, a procedure is reported for the isolation of influenza-specific murine CTL restricted by the human class I molecule HLA-A2.1. The principal specificity of such CTL is for a fragment of the influenza M1 protein that has been previously shown to be immunodominant for human HLA-A2.1-restricted CTL. CTL of this specificity were also established through the use of peptide-pulsed rather than virus-infected stimulators. The dependence of murine CTL recognition upon peptide length and HLA-A2 structure was established to be similar to that previously reported for human CTL. However, the fine specificity of CTL maintained on virus-infected stimulators was somewhat different from that of CTL maintained with M1 peptide. This suggests that differences in surface density or peptide structure between peptide-pulsed and virus-infected stimulators may result in the outgrowth of T cells with different receptor structures. The immunodominance of the M1 peptide determinant in both mice and humans suggests that species-specific differences in TCR structure, Ag-processing systems, and self-tolerance are of less importance than limitations on the ability of antigenic peptides to bind to appropriate class I molecules. These results thus establish the utility of the transgenic system for the identification of human class I MHC-restricted T cell epitopes.
Truncation analysis of several DR binding epitopes.
Authors: O'Sullivan D , Sidney J , Del Guercio M F , Colón S M , Sette A
Published: December 2022

Peptide regions crucial for binding to four different DR alleles (DR1, DR2, DR5, and DR52a) have been localized in five unrelated DR binding peptides (dynorphin 1-13, sperm whale myoglobin 132-153, influenza hemagglutinin 307-319, pigeon cytochrome c 88-104, and tetanus toxoid 830-843) by testing panels of truncated analogs for DR binding. It was found that in most cases, different DR alleles recognize almost identical, albeit distinct, core regions, suggesting that different DR alleles may recognize similar structures on their peptide ligands. Furthermore, it was found that these core regions, notwithstanding their derivation from unrelated sequences, share a common structural pattern. When the sequences of several other unrelated determinants were scrutinized, the structural motif identified was present in some, but absent in other good DR binders, suggesting that good DR binding capacity of peptide molecules may be compatible with more than one single sequence pattern.
Genetic control of the immune response in mice to Leishmania mexicana surface protease.
Authors: López J A , Reins H A , Etges R J , Button L L , McMaster W R , Overath P , Klein J
Published: December 2022

Congenic mouse strains were tested in the lymphocyte proliferation assay for their response to the purified surface protease of Leishmania mexicana (gp63). The data obtained allow us to distinguish three different patterns of response, influenced both by H-2 (class II) and non-H-2 genes. Mice of the C57BL/10 (B10) background carrying H-2 haplotypes b,q, and r were found to be high responders; those carrying H-2 haplotypes d, j, v, and z were low responders; and those with H-2a, H-2f, H-2k, H-2p, and H-2u haplotypes were intermediate responders. Studies with H-2 recombinant strains indicated that the high responsiveness on the B10 background was determined by the Ab allele and the low responsiveness influenced by the Ad allele. Other genes besides H-2 appear to have a role in the immune response as shown by the fact that some strains with BALB, DBA, or C3H background differed in their pattern of responsiveness from B10 background strains carrying the corresponding H-2 haplotypes. By using recombinant protein, the influence of the leishmanial surface lipophosphoglycan that might co-purify with gp63, on the MHC restriction of the response to gp63 was excluded. The immune response to gp63 did not correlate with susceptibility of mouse strains to cutaneous infection with L. mexicana promastigotes.
V gamma 3 T cell receptor rearrangement and expression in the adult thymus.
Authors: Aguilar L K , Belmont J W
Published: December 2022

Rearrangement and expression of the V gamma 3-J gamma 1 TCR has been found in murine dendritic epidermal cells (DEC) and fetal thymus. By using the polymerase chain reaction technique, V gamma 3-J gamma 1 rearrangements and RNA expression were detected in the murine adult thymus. Individual genomic and cDNA junctions were cloned and sequenced. In genomic DNA, 55% (16/29) of V gamma 3-J gamma 1 junctional sequences had N regions ranging in length from 1 to 12 nucleotides resulting in considerable junctional diversity. Only 5% (2/42) of cDNA sequences had N regions. The canonical DEC sequence represented 36% (15/42) of the cDNA sequences. Thus, fetal-type V gamma 3-J gamma 1 rearrangements lacking N regions were preferentially expressed in adult thymocytes, some of which may be DEC precursors. The developmental stages in which V gamma 3-J gamma 1 rearrangements are generated were studied by using polymerase chain reaction to detect circular rearrangement products. Active V gamma 3-J gamma 1 rearrangement was detected in thymuses from fetal, newborn, and 2-wk-old mice but not in 5-wk or 8-wk-old (adult) mice. V gamma 2, one of the most common V gamma rearrangements in the adult, was found to be actively rearranging to J gamma 1 in the adult thymus. However, V gamma 2-V gamma 3 replacement rearrangement was not found. These results support the hypotheses that adult thymocytes with rearranged V gamma 3-J gamma 1 are persistent from earlier developmental stages and represent a separate lineage from those with V gamma 2-J gamma 1 rearrangements.
The W element is a positive regulator of HLA-DRA transcription in various DR+ cell types.
Authors: Cogswell J P , Austin J , Ting J P
Published: December 2022

The W and P elements are cis-acting transcriptional regulators of the human class II MHC gene HLA-DRA necessary for maximal promoter activity in DR+ B cells. Proteins that bind specifically to W may mediate promoter activity from a DNA segment containing W and P (W/P). In this report, we demonstrate that a -143 to -123 bp region that lacks P is sufficient to mediate the W elements function and to bind the W proteins W-B1 and W-B2 in the Raji B-lymphoblastoid cell line. In contrast to previous reports, we find that W/P is not a B cell-specific element; rather that its promoter activity parallels active transcription of the endogenous DRA gene. In addition, we show that tissue-restricted regulation by W/P is not correlated with the ubiquitous (W-B1) and lymphoid-specific (W-B2) distribution of the W binding proteins. We suggest that mechanisms in addition to binding to W may account for W/P-dependent transcriptional activation in DR+ cells.
An autosomal recessive gene that delays expression of lupus in BXSB mice.
Authors: Kofler R , McConahey P J , Duchosal M A , Balderas R S , Theofilopoulos A N , Dixon F J
Published: December 2022

We report the generation and serologic, cellular, histologic, and genetic characteristics of a BXSB/MpJScr substrain, termed BXSB/MpJScr-ll/ll, that has lost early-life male lupus disease. Classic genetic analysis suggested that delayed disease expression results from the action of a single autosomal recessive gene. This putative gene, referred to as ll (long-lived), causes a significant delay in expression of autoimmune serology (total serum IgG and anti-nuclear antibodies levels), monocytosis, and of immune complex-mediated histopathologic changes such as glomerulonephritis, arteritis, and myocardial infarction. Presumably as a consequence of the delayed immunopathology male BXSB/MpJScr-ll/ll mice live three to four times longer than regular BXSB/MpJScr. This strain might be useful for analysis of single genes responsible for severe autoimmune disease expression.
Effect of cyclosporin A on lymphopoiesis. III. Augmentation of the generation of natural killer cells in bone marrow transplanted mice treated with cyclosporin A.
Authors: Kosugi A , Shearer G M
Published: December 2022

The effects of cyclosporin A (CsA) on the generation of NK cells were studied using syngeneic bone marrow transplanted mice subsequently treated with CsA (BMT/CsA mice). In contrast to a severe reduction in T cells that was reported previously, these mice exhibited a marked enhancement of splenic NK activity. The enhanced NK activity was mediated by NK1.1+, Thy-1- cells as assessed by antibody plus complement treatment, and was concomitant with an absolute increase in the numbers of NK1.1+ cells as assessed by flow cytometry. Because the depletion of host-derived, mature NK cells by injection of anti-asialo GM1 antibody before bone marrow reconstitution did not affect the enhancement of NK activity, CsA appeared to augment the generation of NK cells from bone marrow precursors. To investigate a possible relationship between the enhancement of NK activity and the maturational arrest of T cells in the thymus induced by CsA, mice were thymectomized, followed by irradiation, bone marrow reconstitution, and CsA treatment. These mice exhibited as strong enhancement of splenic NK activity as BMT/CsA mice, suggesting that the CsA-induced effect on NK cells is distinct from its effect on T cell development in the thymus. Taken together, these results are the first demonstration of the positive effect of CsA on NK cell generation and may be of importance in clinical bone marrow transplantation.
An analysis of synthetic peptide restriction by HLA-DR alleles in T cells from human subjects, naturally sensitized by Streptococcus mutans.
Authors: Childerstone A , Altmann D , Haron J A , Wilkinson D , Trowsdale J , Lehner T
Published: December 2022

T cells from most human subjects show significant in vitro proliferative responses to a 185-kDa surface Ag from Streptococcus mutans as well as to synthetic peptides derived from the sequence of a Mr 3800 streptococcal Ag. T cells from subjects expressing each of the alleles from DR1 to DR7 responded to synthetic peptides of 17 or 21 amino acid residues. Furthermore, inhibition studies with mAb to HLA class I and class II Ag showed that the DR Ag was a restriction molecule for the proliferative responses. Mouse L cells transfected with DR1, DR2, DR4, DR5, and DR7 were used to confirm the permissive nature of the responses. An analysis of the fine specificity of the responses showed that the minimum peptides capable of stimulating T cells from subjects with different DR types varied by one or two residues. For DR2 and DR3 the shortest peptide was residues 6-15, an additional serine (residue 5) was required for DR1 and DR7 and an aspartic acid (residue 4) for DR4, DR5, and DR6. Successful oral-mucosal bacterial colonisation in humans, by a largely commensal Streptococcus, might be associated with the permissive nature of the HLA-DR restriction of the response to a major streptococcal cell surface peptide. The peptide recognised in association with the HLA-DR molecule may induce an immune response that prevents central entry of the organism from the peripheral mucosal site.
Enhancement of lymphokine-activated T killer cell tumor necrosis factor receptor mRNA transcription, tumor necrosis factor receptor membrane expression, and tumor necrosis factor/lymphotoxin release by IL-1 beta, IL-4, and IL-6 in vitro.
Authors: Dett C A , Gatanaga M , Ininns E K , Cappuccini F , Yamamoto R S , Granger G A , Gatanaga T
Published: December 2022

Co-culture with IL-2 can induce human T lymphocytes to proliferate and become nongenetically restricted, lymphokine-activated killer (LAK) cells in vitro. Our studies were conducted with long term cultured, human T-LAK cells from peripheral blood, which are 95 to 99% CD3+. We found that proliferating 7 to 10-day human T-LAK cells express TNFR, by using a 125I-TNF binding assay. Additional treatment of these cells with the cytokines IL-1 beta, IL-4, or IL-6 rapidly up-regulated 55-kDa TNFR mRNA transcription and doubled TNFR membrane expression. Further studies revealed that these cytokines also increased the release of TNF and lymphotoxin (LT). Antibody neutralization studies indicated that IL-1 induces release of both TNF and LT; however, IL-4 and IL-6 induce primarily LT release. These results further support the concept that these cytokines are involved in the regulation of TNF/LT release, TNFR synthesis, and TNFR membrane expression. It is apparent that cytokines and their membrane receptors are involved in the autocrine/paracrine control of T cell proliferation, differentiation, and expression of functional activity after IL-2 stimulation in vitro.
A novel role of growth hormone and insulin-like growth factor-I. Priming neutrophils for superoxide anion secretion.
Authors: Fu Y K , Arkins S , Wang B S , Kelley K W
Published: December 2022

Growth hormone (GH) and the GH-dependent growth promoting peptide, insulin-like growth factor-I (IGF-I), are both potent signals for priming human and porcine polymorphonuclear neutrophils (PMN) to secrete superoxide anion (O2-). PMA, opsonized-zymosan, or FMLP could all be used as triggering stimuli to demonstrate priming by GH or IGF-I. As positive controls, IFN-gamma and LPS also primed both human and porcine PMN for enhanced O2- release. However, only the LPS-mediated enhancement was inhibited by polymyxin B, which demonstrates that the priming induced by GH, IGF-I, or IFN-gamma was not caused by LPS contamination. Furthermore, a specific antibody to GH abrogated priming induced by this molecule. In contrast to IGF-I, the closely related molecule insulin was unable to prime PMN even at pharmacologic levels. Insulin, at pharmacologic levels, antagonized the priming mediated by IGF-I but had no effect on GH priming. A mAb directed against the human IGF-I receptor blocked the enhanced secretion of O2- by human PMN that was caused by IGF-I, but not GH, indicating that neutrophil priming induced by GH was not mediated by inducing extracellular release of IGF-I. However, priming PMN by both GH and IGF-I required de novo protein synthesis, because cycloheximide completely abrogated enhanced O2- secretion that was caused by these growth factors. These data show that a classic pituitary hormone (GH), as well as its widely recognized growth promoting peptide (IGF-I), are involved in regulating an important functional activity of both porcine and human PMN. Inasmuch as GH and IGF-I have recently been demonstrated to be synthesized by leukocytes, these data support the possibility that both of these proteins could act in a paracrine fashion as cytokines to prime PMN for an enhanced respiratory burst.
Activation of phospholipase D in a rat mast (RBL 2H3) cell line. A possible unifying mechanism for IgE-dependent degranulation and arachidonic acid metabolite release.
Authors: Lin P Y , Wiggan G A , Gilfillan A M
Published: December 2022

RBL 2H3 cells (a model of mast cell function) were sensitized with anti-TNP IgE (0.5 micrograms/ml) and triggered to secrete both histamine and arachidonic acid (AA) metabolites by the addition of TNP-OVA (0 to 100 ng/ml). After a 3-min delay, the release of both groups of mediators proceeded in a parallel manner. In cells labeled with [14C]-AA, TNP-OVA produced a rapid increase in phosphatidic acid (PA), and subsequently, 1,2-diacylglycerol (DAG) and intracellular AA levels. Concurrently, there was a decrease in [14C]-AA labeled phosphatidylcholine. The release of labeled AA from phosphatidylcholine in response to TNP-OVA was paralleled by a liberation of free choline but no evidence of liberation of phosphorylcholine. When ethanol (0.05 to 2% v/v) was included in the culture medium, phosphatidylethanol was synthesized at the expense of PA and DAG, with a resulting inhibition of secretion. D,1 propranolol, an inhibitor of PA phosphohydrolase, inhibited the IgE-dependent production of [14C]-DAG, and [14C]-free fatty acid but not [14C]-PA. The IgE-dependent release of both histamine and AA metabolites was completely inhibited by pretreatment with propranolol. Taken together, the above results suggest that phospholipase D is activated upon cross-bridging of IgE receptors on the surface of RBL 2H3 cells and that this may be a pivotal step in the signal transduction cascade leading to the release of both presynthesized and de novo synthesized mediators.
Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1.
Authors: Luscinskas F W , Cybulsky M I , Kiely J M , Peckins C S , Davis V M , Gimbrone M A
Published: December 2022

rIL-1 beta treatment of cultured human endothelial cells (HEC) promotes polymorphonuclear leukocyte (PMN) adhesion and transmigration. Using in vitro quantitative monolayer adhesion and videomicroscopic transmigration assays, we have examined the contributions of endothelial-leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and the leukocyte adhesion complex, CD11/CD18, to these processes. Maximal enhancement of PMN adhesion and transmigration were observed after 4 h of rIL-1 beta treatment, when surface expression of ELAM-1 had peaked and ICAM-1 was modestly increased. Blocking mAb directed to either ELAM-1 or ICAM-1 inhibited greater than 90% of the up-regulated PMN transmigration. Blocking mAb directed to either CD11a/CD18 (LFA-1, a ICAM-1 counter-receptor), CD11b/CD18 (Mo-1), or CD18 (common beta 2-integrin) also blocked greater than 90% of PMN transmigration. At later time points (24 or 48 h), ELAM-1 surface expression was markedly decreased, whereas ICAM-1 expression was increased over the 4-h level; PMN adhesion remained elevated (approximately 50 to 60% of 4 h level), but transmigration returned to levels seen with unactivated HEC. These data indicate that PMN interaction with at least two distinct HEC adhesion molecules is necessary for transendothelial migration and suggests that PMN adhesion and transmigration, although interrelated, are mechanistically distinct processes.
Platelet-activating factor induces phospholipid turnover, calcium flux, arachidonic acid liberation, eicosanoid generation, and oncogene expression in a human B cell line.
Authors: Schulam P G , Kuruvilla A , Putcha G , Mangus L , Franklin-Johnson J , Shearer W T
Published: December 2022

Platelet-activating factor is a potent mediator of the inflammatory response. Studies of the actions of platelet-activating factor have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of platelet-activating factor on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that platelet-activating factor significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, lyso-platelet-activating factor, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that platelet-activating factor from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas lyso-platelet-activating factor was again ineffective. We further demonstrate the impact of platelet-activating factor binding to B cells by measuring platelet-activating factor induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, platelet-activating factor was capable of inducing transcription of the nuclear proto-oncogenes c-fos and c-jun. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates platelet-activating factor induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, platelet-activating factor is shown here to have a direct and profound effect on a pure B cell line.
Inducible nuclear expression of NF-kappa B in primary B cells stimulated through the surface Ig receptor.
Authors: Liu J L , Chiles T C , Sen R J , Rothstein T L
Published: December 2022

Constitutive expression of NF-kappa B has been associated with developmental maturity in B cells on the basis of studies using continuously growing cell lines and plasmacytomas; however, little is known about the behavior of NF-kappa B in primary, mature B cells. In the present work, the regulation of NF-kappa B expression was studied by analyzing subcellular fractions of adult murine splenic B cells with the electrophoretic mobility shift assay using a kappa B-containing oligonucleotide. Although nuclear extracts from resting B cells contained kappa B-binding activity, additional kappa B-binding activity was present in cytosolic fractions in a form that became apparent after treatment with detergent. Competition analysis indicated that the DNA binding activity detected by electrophoretic gel mobility shift assay was specific for the kappa B motif, and UV photo-cross-linking showed the molecular size of kappa B-binding protein to be similar to that of the DNA binding subunit of NF-kappa B. Nuclear expression of kappa B-binding activity was markedly induced by treatment of B cells with phorbol ester or with LPS. Most notably, kappa B-binding activity was induced after surface IgR cross-linking, and the mechanism of this induction involved PKC. Further, anti-Ig-induced activity was superinduced in the presence of cycloheximide. These results indicate that nuclear NF-kappa B is rapidly induced as a result of B cell stimulation, and further suggest that NF-kappa B may play a specific role in mature B cells after ligand binding to surface Ig distinct from its postulated developmental role as a stage-specific factor involved in kappa-enhancer function.
Cytotoxic T cell clones isolated from ovarian tumor-infiltrating lymphocytes recognize multiple antigenic epitopes on autologous tumor cells.
Authors: Ioannides C G , Freedman R S , Platsoucas C D , Rashed S , Kim Y P
Published: December 2022

CTL clones were developed from tumor infiltrating lymphocytes (TIL) from the ascites of a patient with ovarian carcinoma by coculture of TIL with autologous tumor cells and subsequent cloning in the presence of autologous tumor cells. These CTL clones expressed preferential cytolytic activity against autologous tumor cells but not against allogeneic ovarian tumor cells and the NK-sensitive cell line K562. The cytolytic activity of these CTL against autologous tumors was inhibited by anti-TCR (WT31 mAb), anti-HLA class I, and anti-CD3 mAb but not by the NK function antibody Leu 11b. Cloning of the autologous tumor cells in vitro revealed that the CTL clones of the ovarian TIL expressed differential abilities to lyse autologous tumor cell clones. The specificity analysis of these autologous tumor specific CTL suggested that they recognize several antigenic determinants present on the ovarian tumor cells. Our results indicate the presence of at least three antigenic epitopes on the tumor cells (designated OVA-1A, OVA-1B, and OVA-1C), one of which (OVA-1C) is unstable. These determinants are present either simultaneously or separately, and six types of ovarian clones can be distinguished on the basis of their expression. These results indicate that CTL of the TIL detect intratumor antigenic heterogeneity. The novel heterogeneity identified within the ovarian tumor cells in this report may be of significance for understanding cellular immunity in ovarian cancer and developing adoptive specific immunotherapeutic approaches in ovarian cancer.