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J A Bluestone

Publications and source records attributed to J A Bluestone.

At least 181 records · Page 10Linked to original sources
PubMed1991

Anti-CD3 monoclonal antibody therapy. An approach toward optimization by in vitro analysis of new anti-CD3 antibodies.

Several problems remain associated with anti-CD3 monoclonal antibody therapy, including first-dose reactions, recurrent rejection, and the host humoral response to the xenogeneic mAb. One approach toward optimization of anti-CD3 mAb therapy involves the use of anti-CD3 mAbs of defined idiotype, isotype, and epitope specificity, so that the desired T cell activation and suppression properties are obtained and neutralization by antiidiotypic antibody is avoided. The purpose of the present study was to define the contribution of mAb isotype and epitope specificity in determining T cell activation and suppression potencies and to evaluate the idiotypic relationships among ten anti-CD3 mAbs and one anti-TCR alpha beta mAb selected for this study. Epitope mapping by flow cytofluorometry indicated that one mAb, OKT3D, possesses an epitope specificity distinct from that of other anti-CD3 mAbs. Analysis of early T cell activation, proliferation, and lymphokine production (TNF-alpha, gamma-IFN, and GM-CSF) indicated that mAb isotype exerted a profound effect on activation potency (IgG2a much greater than IgG1 greater than IgG2b), whereas epitope specificity exerted a minor effect. CTL inhibition studies demonstrated an epitope effect with highest inhibition potencies observed with OKT3D IgG1 and OKT3D IgG2b mAbs. Idiotypic analysis of nine mAbs indicated that all anti-CD3 mAbs except OKT3E possess idiotypes distinct from that of OKT3. Thus, two anti-CD3 mAbs, OKT3D IgG1 and OKT3D IgG2b, possess high immune suppression potency, low activation potency, and idiotypes distinct from OKT3. In conclusion, selection of anti-CD3 mAbs of defined idiotype and appropriate T cell activation and suppression properties may provide a means for mitigating problems associated with OKT3 therapy.

Antibodies, Anti-Idiotypic↗
PubMed1991

Specificity of gamma delta receptor bearing T cells.

T cells expressing the gamma delta receptor heterodimer can recognize a broad array of different antigens, including classical and non-classical major histocompatibility complex (MHC) proteins, MHC-like CD1 antigens, and bacterial antigens such as the mycobacterial heat shock proteins and staphylococcal enterotoxins. Reactivity to self antigens including autologous stress proteins implicates TCR gamma delta T cells in autoimmune disease. It is as yet unclear whether the responses of gamma delta T cells specific for soluble proteins are restricted by conventional or non-classical MHC molecules. Correlations of TCR gamma delta usage with specificity suggest that, like TCR alpha beta, sequences encoded within both the V regions and the V(D)J junctions are important in determining receptor specificity.

Animals↗
PubMed1990

Extrathymic selection of TCR gamma delta + T cells by class II major histocompatibility complex molecules.

The influence of MHC antigens on TCR gamma delta usage in CD8+ intraepithelial lymphocytes (IELs) was examined using a pan-reactive and V delta 4 region-specific MAb. While an average of 30% of IELs from the majority of mice of various MHC haplotypes were V delta 4+, a 2-fold or greater percentage of IELs from H-2k mice were V delta 4+. Analysis of IELs from F1 mice indicated that the increase in TCRs using V delta 4 was likely to be the result of positive selection. The V delta 4 usage patterns of IELs from recombinant inbred strains and from mice recombinant within H-2 revealed that the increase in V delta 4 usage mapped to H-2 and required I-E expression. Moreover, selection of TCRs using V delta 4 occurred in chimeric mice in the absence of a thymus. The results demonstrate an extrathymic selective mechanism for gamma delta TCRs of CD8+ IELs and suggest that these cells may exhibit MHC class II-restricted antigen recognition.

Animals↗
PubMed1990

Treatment with monoclonal anti-CD3 antibody protects against lethal Sendai virus infection by induction of natural killer cells.

C57BL/6 mice are protected from a lethal pneumonia caused by Sendai virus when treated with low doses of mAb directed to the CD3 Ag. The protective mechanism is not due to an accelerated Sendai virus-specific Th cell, CTL, or antibody response but to a strong NK cell response via the in vivo induction of lymphokines. Antibodies directed against the NK1.1 and asialo GM1 marker totally reversed the protective effect of anti-CD3 treatment. In vivo treatment with rIL-2 also induced NK activity and induced antiviral protection. Treatment with anti-CD3 protects when given in a narrow time window (1 day before until 1 day after Sendai virus inoculation), indicating that NK activity is protective in the early phase of virus infection.

Animals↗
PubMed1990

T cell receptor delta-gene expression and diversity in the mouse spleen.

Thirteen T cell hybridomas and two cell lines expressing gamma delta TCR were generated from B10 and B6 CD3+CD4-CD8- splenic T cells. At least three different types of gamma-chains (V gamma 2-C gamma 1, C gamma 2, and C gamma 4) were shown to be expressed in these cells. Analysis of V delta gene segment usage in these hybridomas was performed by Northern blot hybridizations using probes specific for the V delta 1 through V delta 6 gene families and electrophoretic analysis of polymerase chain reaction amplified DNA obtained using V region-specific oligonucleotide primers for the V delta gene families as well as for V alpha 10, V alpha 4, and V alpha 11. These analyses revealed that V delta 5 gene segments are used by a high percentage of splenic gamma delta CD4-CD8- T cells (53% of the cells in our panel). Other V delta segments expressed by our panel of cells were V delta 2, V delta 4, V delta 6, and V alpha 10, indicating that the V delta repertoire expressed in the spleen is similar, but possibly not identical to the adult thymus repertoire. Sequence analysis of the V-D-J joinings of the delta-chain messages revealed substantial diversity, indicating that the delta-chain repertoire expressed in peripheral lymphoid organs uses a significant portion of the potential diversity predicted for these chains. These results demonstrate that, in spite of the low numbers of gamma delta T cells in peripheral lymphoid organs, the diversity of their TCR is extensive enough to play an important role in the immune response.

Amino Acid Sequence↗
PubMed1990

Structural basis of Kbm8 alloreactivity. Amino acid substitutions on the beta-pleated floor of the antigen recognition site.

We have analyzed the functional significance of the four amino acid differences between the parental H-2Kb and mutant H-2Kbm8 glycoproteins. Six bm8 variants including single substitutions at residues 22, 23, 24, and 30 as well as paired substitutions at residues 23, 30 and 22, 24 were generated and transfected into L cells. Surface expression of these H-2Kb variants was analyzed using monoclonal antibodies which bind to well-defined H-2Kb epitopes. No alterations introduced into the conformational structure of H-2Kb by the amino acid substitutions were detected. The effect of these substitutions on CTL recognition was initially analyzed using the following bulk CTL: either H-2Kb anti-H-2Kbm8, H-2Kbm8 anti-H-2Kb, or third party anti-H-2Kb. The alloreactivity between H-2Kb and H-2Kbm8 is dominated by the amino acid substitution at residue 24 (Glu----Ser). The complete bm8 phenotype, however, also requires the additional substitution at residue 22 (Tyr----Phe). The H-2Kbm8 anti-Kb bulk CTL reacted with both variant H-2Kbm8 molecules containing single substitutions at amino acid positions 22 or 24 but not the variant molecule containing both substitutions. Further analysis using three individual H-2Kbm8 anti-Kb CTL clones indicated the complexity of the self Kbm8 phenotype. Clone 8B1.20 did not react to changes in residues 22 or 24. The 8B1.32 clone reacted with the change at residue 22 but not with the change at residue 24, although the 8B1.54 clone reacted with the change at residue 24 but not with the change at residue 22. The changes in residues 23 (Met----Ile) and/or 30 (Asp----Asn) did not impact significantly on the alloantigenic properties of Kbm8 as determined by both the bulk and cloned CTL populations. According to the three-dimensional class I structure the substitution at amino acid 24 is inaccessible to the TCR. The location of this substitution within the Ag recognition site implies that altered peptide binding, and not a disruption of MHC residues that interact with the TCR, is responsible for the alloreactivity between H-2Kb and H-2Kbm8.

Amino Acid Sequence↗
PubMed1990

Expression of a T cell receptor gamma-chain (V gamma 1.1J gamma 4C gamma 4) transgene in mice influences T cell receptor ontogeny and thymic architecture during development.

In this report, we have characterized T cell ontogeny in mice transgenic for the V gamma 1.1J gamma 4C gamma 4 (TcR gamma 4) TCR chain gene by analyzing the T cell surface phenotype and microarchitecture of the transgenic lymphoid organs during development. The first wave of V gamma 3J gamma 1C gamma 1 TCR+ thymocytes that appear in the thymus at day 14 of gestation were virtually absent in the TCR gamma 4 transgenic mice. However, comparable levels of total gamma delta+ thymocytes were present in the TCR gamma 4 transgenic mice, suggesting that these thymocytes expressed a transgene receptor. In addition, the appearance of significant numbers of TCR alpha beta+ T cells occurred earlier (day 17 of fetal development) in the transgenic thymus. After birth, thymuses from TCR gamma 4 transgenic mice were characterized by a consistent increase in the number of TCR gamma delta+ thymocytes and a transient increase (between 2 and 4 wk of age) in the absolute number of TCR+ thymocytes (2- to 4-fold) compared with thymuses from normal mice. Immunohistologic analysis of the thymus, spleen, and lymph nodes from 2-day and 3-wk-old transgenic mice showed significant differences to controls only in regions harboring mature, functional T cell subsets which may be the result of bidirectional signaling between lymphocyte subsets (influenced by transgene expression) and stromal elements. The results demonstrate that striking differences, leading to the earlier appearance of mature T cells, occurred in both the fetal and neonatal thymus and periphery of mice that expressed a TCR gamma 4 transgene. These findings are consistent with the hypothesis that expression of the TCR gamma 4 chain gene, early in ontogeny, has a positive influence on the development of the T cell compartment.

Animals↗
PubMed1990

Mechanism of tumor rejection in anti-CD3 monoclonal antibody-treated mice.

The present study was undertaken to determine the mechanism of tumor rejection in mice treated with low dose anti-CD3 mAb. It was found that treated mice developed nonrestricted antitumor cytolytic spleen cells of the Thy-1+, asialo GM-1+, CD4-, CD8- phenotype. Although these cells might play a role in immunopotentiating some immune responses, in vivo depletion studies using anti-asialo GM-1 mAb demonstrated that these cells were not involved in the rejection of the progressor tumor, 1591-PRO4L, by anti-CD3 mAb-treated mice. Mice treated with anti-CD3 did develop lasting tumor specific immunity as demonstrated by their ability to reject PRO4L on tumor rechallenge while being unable to reject an unrelated UV-induced tumor. The specificity of this memory implicated T cells in the response to PRO4L in anti-CD3-treated mice. Using in vivo T cell subset depletion of anti-CD3-treated animals, it was shown that both CD4+ and CD8+ T cells are required for anti-CD3-induced tumor rejection. The CD4+ cells provide helper function and are only required in the early rejection period, whereas CD8+ cells are required throughout the immune response. In fact, examination of rejecting tumors from treated animals revealed the presence of tumor-specific CD8+ cytolytic T cells capable of cytolysis immediately after removal from the rejecting PRO4L tumor. Thus, in vivo treatment with anti-CD3 mAb likely results in the pan-stimulation of the entire T cell population, which enhances the generation of specific CD8+ T cells, which then eliminate the tumor.

Animals↗
PubMed1990

Self-reactive gamma delta T cells are eliminated in the thymus.

The genes encoding a gamma delta T-cell receptor specific for a major histocompatibility complex class I molecule encoded by the TIa locus have been inserted into the mouse germ line. In mice that do not express the TIa-encoded determinant, transgenic gamma delta T cells are a functional component of the CD4-CD8- 'double-negative' T cells in the thymus and peripheral lymphoid organs. In mice that express the TIa-encoded determinant, there are no transgenic gamma delta T cells in peripheral lymphoid organs, and there are no thymocytes expressing normal levels of the transgenic gamma delta T-cell receptor.

Animals↗
PubMed1990

Cloning of an interleukin-4 inducible gene from cytotoxic T lymphocytes and its identification as a lipase.

Interleukin-4 (IL-4) has been demonstrated to be an important lymphokine for the generation of cytotoxic T lymphocytes (CTLs). Here we describe an IL-4 inducible gene specifically expressed in CTLs. By sequence homology, this gene is likely to be the mouse homolog of pancreatic lipase. Oocyte translation of in vitro transcribed mRNA results in the expression of a protein with lipase activity, and Northern analysis of various tissues and a large panel of hematopoietic cell types demonstrates that this gene is expressed only in the pancreas and CTLs. Lysates of CTLs grown in IL-4, but not in IL-2, exhibit lipase activity. Furthermore, Northern analysis of CTLs grown in the presence of IL-4 for as little as 5 days demonstrates a marked induction of lipase mRNA, which correlates with enhanced cytolysis by these cells. These results suggest that this lipase may have an important role in CTL effector function.

Amino Acid Sequence↗
PubMed1990

Polymorphisms and diversity of T-cell receptor-gamma proteins expressed in mouse spleen.

Bulk populations of T-cell receptor (Tcr) gamma delta-expressing splenocytes from different inbred strains of mice were examined for the diversity of Tcr gamma delta proteins. Immunoprecipitations with anti-C gamma 1/2, anti-C gamma 4, and anti-V gamma 1 sera demonstrated that splenocytes from B10.BR, C57BL/6, and C57L strains of mice expressed the same array of Tcr gamma proteins, namely V gamma 1-C gamma 2, V gamma 1-C gamma 4, and V gamma 2-C gamma 1, although the Tcr gamma delta heterodimers observed for each of these strains were biochemically distinct. Examination of bulk splenic Tcr gamma delta heterodimers from several other inbred strains of mice demonstrated that each of the strains could be categorized into one of three basic phenotypes. For several reasons, the differences observed between the strains appeared to be solely dependent on polymorphisms of the Tcrg loci. First, F1 mice co-expressed both parental Tcr gamma delta phenotypes. Second, the distinguishing polymorphism between mice of phenotype 1 and phenotypes 2 or 3 was due to the presence of an N-linked glycosylation site within the Tcrg-Cl gene segment, previously described for BALB.B and C57BL/6 Tcrg-Cl genes. Finally, the V gamma 1-C gamma 4 polymorphism between mice of phenotype 3 and phenotypes 1 or 2 was due to differences in core protein size. Furthermore, the three defined Tcr gamma chains were expressed independently of the major histocompatibility complex (MHC) haplotype. Although no striking qualitative differences in Tcr gamma delta heterodimers were observed between strains (including those with autoimmune disorders), a quantitative difference in the relative amount of C gamma 4-encoded proteins was observed on Tcr gamma delta splenocytes from both newborn euthymic and adult athymic mice when compared to adult Tcr gamma delta splenocytes from euthymic mice. These results demonstrate that genetic polymorphisms exist among different mouse strains and suggest that selective developmental pressures may govern Tcr gamma delta expression.

Animals↗
PubMed1990

Freshly isolated Thy-1+ dendritic epidermal cells express T cell receptor gamma delta-CD3.

Within murine epidermis exists a population of Thy-1+ DEC which express membrane Thy-1 antigen, but lack CD8 or CD4 antigen. We examined freshly obtained non-cultured Thy-1+ DEC both by immunofluorescence and by biochemical techniques to identify the protein products of the T cell receptor (TCR) and the associated CD3 complex on these cells. Virtually all of the Thy-1+ DEC are brightly positive in CD3 expression with immunofluorescence using the monoclonal antibody 145-2C11. By immunoprecipitation, using this same antibody and polyclonal anti-TCR-gamma antibody, the only TCR heterodimer detected on the freshly isolated Thy-1+ DEC is the gamma delta heterodimer. These findings suggest that in the phenotype and TCR expression, Thy-1+ DEC are analogous to CD8-, CD4- early fetal thymocytes.

Amino Acid Sequence↗
PubMed1990

Anti-CD3 F(ab')2 fragments are immunosuppressive in vivo without evoking either the strong humoral response or morbidity associated with whole mAb.

The use of anti-CD3 monoclonal antibodies to treat organ allograft rejection has been complicated by (1) the morbidity associated with the initial dose, (2) the humoral response of the patients against the mAbs, and (3) the generalized immunosuppression induced by the mAbs. We investigated the potential of F(ab')2 fragments of the anti-murine-CD3 mAb, 145-2C11, to avoid these complications in the murine model. Both whole mAb and F(ab')2 fragments induced T cell depletion. However, injection of F(ab')2 fragments of anti-CD3 mAb did not cause T cell activation, and did not induce the morbidity and mortality observed following injection of whole mAb. The humoral response against the F(ab')2 fragments was significantly reduced compared with the response against the whole mAb. Furthermore, repeated administration of F(ab')2 fragments of anti-CD3 mAb resulted in prolongation of allogeneic skin graft survival superior to that seen following treatment with a single dose of whole mAb. Finally, while T cells from mice treated with whole mAb displayed profound suppression of in vitro CTL generation, T cells from mice treated with F(ab')2 fragments had significant in vitro CTL function. These results suggest that the use of F(ab')2 fragments of anti-CD3 mAb may offer significant advantages over whole mAb for the induction and maintenance of immunosuppression.

Animals↗