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D F Lake

Publications and source records attributed to D F Lake.

At least 19 recordsLinked to original sources
PubMed2001

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes.

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.

Antigen Presentation↗
PubMed2001

Dendritic cells pulsed with Coccidioides immitis lysate induce antigen-specific naive T cell activation.

Coccidioidomycosis, an infection endemic to the southwestern United States, is caused by the fungus Coccidioides immitis. Coccidioidal infection is overcome by the development of cell-mediated immunity. This study evaluated the role of dendritic cells (DCs) in the initiation of coccidioidal immunity in nonimmune individuals. It was demonstrated that DCs pulsed with the coccidioidal antigen preparation, toluene spherule lysate (TSL), induce DC maturation, autologous lymphocyte proliferation, and antigen-specific lymphocyte responses from nonimmune donors. Furthermore, TSL-primed lymphocytes secreted interferon-gamma after restimulation with TSL or antigen 2/proline-rich antigen, a subcomponent of TSL, but they did not do so when restimulated with ovalbumin or unpulsed DCs. The results demonstrate that DCs generated from individuals not exposed to C. immitis can specifically prime lymphocytes for coccidioidal antigens and that the response generated by the lymphocytes is characteristic of a cellular immune response.

Antigens, Fungal↗
PubMed1999

Construction and binding analysis of recombinant single-chain TCR derived from tumor-infiltrating lymphocytes and a cytotoxic T lymphocyte clone directed against MAGE-1.

The TCR is responsible for the specificity of cytotoxic T lymphocytes (CTL) by recognizing peptides presented in the context of MHC. By producing recombinant soluble TCR, it is possible to study this interaction at the molecular level. We generated single-chain TCR (scTCR) from tumor infiltrating lymphocytes (TIL) and one CTL clone directed against melanoma-associated antigen (MAGE)-1. Sixty-eight day anti-MAGE-1 TIL and one cloned anti-MAGE-1 CTL were analyzed by PCR for their Valpha and Vbeta gene usage. The TIL population showed a restriction in Valpha and Vbeta usage with only Valpha4 and Valpha9 and Vbeta2 and Vbeta7 expressed. The anti-MAGE-1 CTL clone demonstrated absolute restriction with only Valpha12 and Vbeta1 expressed. DNA sequence analysis was performed on all V regions. For the TIL, each possible Valpha-Vbeta combination (i.e. Valpha4-Vbeta2, Valpha9-Vbeta2, Valpha4-Vbeta7 and Valpha9-Vbeta7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria. From the anti-MAGE-1 TIL, Valpha4-Vbeta2 scTCR demonstrated binding activity to HLA-A1(+) cells pulsed with MAGE-1 peptide. Results obtained from screening a panel of our scTCR constructs on HLA-A1(+) cells pulsed with MAGE-1 peptide or irrelevant peptide demonstrated that Vbeta2 plays a significant role in binding to the MAGE-1 peptide. Amino acid alignment analysis showed that each Vbeta sequence is distinctly different from the others. These findings demonstrate that soluble TCR in single-chain format have binding activity. Furthermore, the results indicate that in TCR, like antibodies, one chain may contribute a dominant portion of the binding activity.

Amino Acid Sequence↗
PubMed1997

Analysis of autoantibodies to T-cell receptors among HIV-infected individuals: epitope analysis and time course.

Individuals seropositive for human immunodeficiency virus type 1 (HIV) express elevated levels of autoantibodies (AAbs) directed against recombinant T-cell receptors (TCRs) and synthetic peptide epitopes duplicating beta chain markers. We performed longitudinal studies of anti-TCR AAbs in HIV-1-infected individuals, making comparisons with uninfected sera and sera from other individuals infected with a nonviral agent. We determined levels of autoantibodies by titration using enzyme-linked immunosorbent assay (ELISA) and developed a means for characterizing "autoantibody CDR recognition spectrotypes" for individual sera. Antibody levels against certain defined synthetic epitopes were substantially elevated in HIV-infected subjects relative to reactivities by control groups. Individual sera showed relatively high AAb levels to a subset of CDR1 peptide epitopes. Two patients who subsequently developed AIDS showed particular reactivity to Vbeta2.1, 8.1, 10.1, and 22.1 epitopes. Our results show that production AAbs to TCR Vbeta epitopes is a general consequence of HIV infection. The response is individual but shows some restriction and shifts in AAb subpopulations often occur with time.

Adult↗
PubMed1997

Physical and epitope analysis of a recombinant human T-cell receptor V alpha/V beta construct support the similarity to immunoglobulin.

The genetic organization and protein structure of T-cell receptors (TCR) and immunoglobulins (Ig) are remarkably similar. Through recombinant, physical, and peptide-based immunological studies we demonstrated that rabbit antisera generated against a recombinant single-chain TCR (scTCR) react with defined peptide epitopes of their constituent TCR alpha and beta chains. These antisera cross-react with the lambda light-chain Mcg as well as with peptides duplicating its covalent structure. Conversely, rabbit antisera generated to human lambda light chains cross-reacted with the recombinant scTCR. Rabbit anti-lambda antibodies purified on an scTCR affinity column bound to T-cell lines and to T and B lymphocytes from peripheral blood. Circular dichroism analysis demonstrated plots characteristic of beta-sheets for both Mcg and recombinant scTCR. Antisera directed against TCR alpha-chain synthetic peptides reacted with scTCR, Mcg lambda light-chain protein, synthetic peptides from regions of sequence homology in beta-chains, and Mcg. Based upon this homology and the serological cross-reactions which reflect conformational determinants, we suggest that the V alpha/V beta antigen-binding domain of this particular monoclonal scTCR construct is substantially similar to the conformational structure of lambda light chains.

Amino Acid Sequence↗
PubMed1996

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Advance Directives↗
PubMed1996

Autoantibodies to T-cell receptor beta chains in human heart transplantation: epitope and spectrotype analyses and kinetics of response.

Autoantibodies against T-cell receptors have been found in two alloimmunization situations in humans: renal transplantation and pregnancy. We carried out longitudinal studies of human heart transplant recipients monitoring their autoantibody production to a recombinant single chain T-cell receptor V alpha/V beta construct, a set of nested, overlapping peptides duplicating the complete covalent structure of an individual T-cell receptor beta chain and a set of peptides duplicating the first complementarity determining segments of 24 distinct human V beta gene products in order to define the time course, epitope specificity and recognition heterogeneity of the response. Autoantibodies against intact and peptide-defined V beta and C beta determinants were generated following human heart allotransplantation. The responses generally show an increase following transplantation that subsequently decreases with time, a result which is consistent with a single immunization. However, some patients showed elevated responses as long as 12 months following the transplant. Autoantibody anti-CDR1 spectrotype analyses detected individual differences among patients, but 5 of 8 patients characterized in detail showed elevated IgG binding to CDR1 peptide epitopes of V beta 6.1, 21.1 and 22.1 gene products. Autoantibodies to CDR1 epitopes of V beta 7.1 and 8.1 were high pretransplant and remained high, although the relative increases with respect to the pretransplant values were not as impressive as those for the above CDR1 epitopes and others usually present in low quantity, e.g. anti-V beta 2.1, 3.1 and 24.1. Although there was great disparity between the MHC haplotypes of donors and recipients, and individual differences among patients, the degree of restriction in the autoantibody response was surprising and suggests a common step in recognition and regulation of the response to allografts.

Amino Acid Sequence↗
PubMed1995

A human monoclonal antimelanoma single-chain Fv antibody derived from tumor-infiltrating lymphocytes.

With the development of recombinant DNA technology, it has become feasible to clone, construct, and express fully human immunoglobulin molecules. Here we report a novel methodology to make human antitumor single-chain Fv (scFv) antibodies from tumor-infiltrating B lymphocytes. We isolated and expanded tumor-infiltrating B lymphocytes from melanomas in the presence of Epstein-Barr virus. The transformed B cells secreting tumor-specific antibodies were identified and cloned by limiting dilution. From one B cell clone with specific melanoma reactivity, we captured the immunoglobulin variable region genes VH and Vk by PCR, sequenced the genes, and linked them together by PCR assembly with the use of a (Gly4Ser)3 linker. The scFv gene was then cloned into the pET21d vector and expressed. The obtained scFv protein with a M(r), of 29,000 was purified and biotinylated for further characterization. The scFv demonstrated specific tumor reactivity to 21 of 24 different melanoma cell lines and not to 14 nonmelanoma tumor cell lines, such as breast, ovarian, and colon cancer cells lines; normal human melanocytes as well as normal human leukocytes. These results were obtained in (a) a tumor cell ELISA, (b) fixed cell immunofluorescence, and (c) live cell flow cytometry. The immunoprecipitation results indicated that a protein antigen of M(r) 45,000 was recognized by the scFv. Since we reported previously that about 70% of human tumors of different histological types contain tumor-infiltrating B lymphocytes producing specific antitumor antibodies, this approach offers a rapid, effective method by combining in vitro B-cell expansion and PCR gene cloning to elucidate the repertoire of the human antitumor immune response and to make human monoclonal antitumor antibody molecules.

Amino Acid Sequence↗
PubMed1995

Autoantibodies against peptide-defined epitopes of T-cell receptors in retrovirally infected humans and mice.

Autoantibodies directed against peptide-defined epitopes of T-cell receptors occur in the serum of healthy humans with the levels and isotypic expression dependent upon physiological changes (aging, pregnancy) or upon the development of autoimmune disease. We carried out investigations of autoantibodies against Tcr peptide-defined epitopes in retroviral infections of humans (HIV-1) and mice (LP-BM5 strain of murine leukemia virus) to determine whether infection with these agents disrupted the regulation of the production of these antibodies. Retroviral infection in humans resulted in increased levels of autoantibody production against putative immunoregulatory regions of the Tcr beta chain (V beta CDR1 and Fr3), a result reflecting a disruption of regulation. In addition, antigenic mimicry was observed with a cross-reaction shared between the common portion of the V3 neutralizing loop of the HIV-1 gp120 molecule and the joining segment of T-cell receptors (J beta). Infection of mice with the defective retrovirus resulted in the induction of antibodies directed particularly against V beta CDR1 peptide-defined determinants. Analysis of the virally induced response to a set of 8 CDR1 peptide epitopes indicated a selectivity to the process. It was possible to partially reverse aberrant cytokine changes correlated with the onset of murine MAIDS by administration of T-cell receptor peptides in saline. These results show that retroviral infection in humans and mice has a profound dysfunctional effect on the regulation of autoantibodies to T-cell receptors. The function of these autoantibodies in the immunopathogenesis of acquired immunodeficiency remains to be determined.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Characterization of autoantibodies directed against T cell receptors.

Recently it has been observed that administration of intravenous immunoglobulin (IVIG) can have profound effects on a wide variety of diseases related to the dysregulation of the immune system. The mechanisms which explain these activities are poorly understood. Human IVIG and various Cohn plasma fractions contain autoantibodies directed against T cell receptors (Tcr). Previous studies have shown that IVIG contains autoantibodies against T cell receptor peptides. In order to further our understanding of autoantibody specificities, a single chain Tcr (scTcr) was constructed by recombinant DNA techniques from the variable alpha and variable beta chains of the Jurkat cell line. Anti-Tcr autoantibodies were isolated from IVIG and Cohn fractions I + III using a scTcr affinity column. This scTcr affinity purified material reacted with the surfaces of T cells at 10 micrograms/ml whereas non-purified IVIG did not. Sera from patients with rheumatoid arthritis (RA) as well as serum from patients with systemic lupus erythematosus (SLE) reacted with the scTcr at levels above that of normals.

Animals↗
PubMed1994

Evolutionary conservation and molecular cloning of the recombinase activating gene 1.

A 700-bp fragment of the recombinase activating gene 1 (RAG-1) was cloned from several evolutionarily distant (sandbar shark, paddlefish, goldfish, axolotl and pig) species using PCR. The nucleotide and deduced amino acid sequences revealed a highly conserved region that has remained essentially unaltered during 400 million years of evolution; e.g., shark and human sequences were 75% identical at the nucleic acid level and 87% as protein. The RAG-1 mRNA levels in the shark were analyzed using semi-quantitative PCR to reveal expression patterns contrary to normal mammalian expression. These results establish that the genetic mechanisms for Ig gene rearrangement are present in all extant gnathanstomes.

Amino Acid Sequence↗
PubMed1994

Autoantibodies to the alpha/beta T-cell receptors in human immunodeficiency virus infection: dysregulation and mimicry.

Autoimmune reactivity is a consequence of infection with human immunodeficiency virus (HIV). We studied serological cross-reactions of purified pooled IgG from sera of HIV-infected individuals by using nested sets of synthetic overlapping peptides duplicating the covalent structures of T-cell receptors (TCRs) and immunoglobulin light chains and report that two processes of autoantibody production occur. (i) IgG autoantibodies to putative regulatory variable domain CDR1 and FR3 epitopes (where CDR is complementarity-determining region and FR is framework region) are present in pooled IgG from HIV-infected individuals at levels 10-fold greater than that in pooled IgG from healthy humans. (ii) Anti-TCR autoimmunization involves antigenic mimicry between a conserved peptide stretch of the major neutralizing V3 loop determinant of HIV-1 gp120 and the conserved FR4 segment of the TCR V beta. Affinity-purified antibodies to the synthetic V3 loop peptide bound to a recombinant single-chain TCR and to a synthetic TCR joining segment peptide containing the FR4 sequence. Conversely, affinity-purified autoantibodies from pooled IgG from HIV-infected individuals to the TCR peptide bound the V3 loop peptide and a single-chain TCR. Inhibition studies indicated that the cross-reactive immunizing antigen was the V3 loop. These results bear upon the impact of HIV infection on immune regulation and on the selection of peptides for vaccine development.

Amino Acid Sequence↗
PubMed1994

Construction and serological characterization of a recombinant human single chain T cell receptor.

A single chain T cell receptor (scTcr) was constructed from the complete V alpha and V beta regions of Jurkat T-cell receptor alpha/beta chain genes using molecular cloning techniques. The recombinant scTcr reacted with a panel of rabbit antisera generated against synthetic 16-mer peptides duplicating the amino acid sequence of Jurkat V alpha and beta chains but not with antisera directed against peptides from the constant domain. Autoantibodies present in sera from systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients bound the scTcr in ELISA assays. The recombinant scTcr described here should prove to be a useful reagent with which to study T-cell receptor activity in serological and functional assays.

Autoantibodies↗
PubMed1994

Molecular cloning, expression and mutagenesis of an anti-insulin single chain Fv (scFv).

The immunoglobulin variable region genes of a murine anti-insulin IgG-producing hybridoma were rescued and cloned into a bacterial expression vector. The variable regions of the gamma heavy chain and the kappa light chain were expressed independently and together as a single chain antibody (scFv). The variable heavy chain alone demonstrated the ability to bind to insulin. The kappa light chain did not show any binding activity towards insulin. The scFv was constructed by PCR assembly using a (Gly4Ser)3 linker between the carboxyl end of the variable heavy chain and the amino terminus of the kappa light chain. The scFv bound insulin at an IC50 of 3.5 x 10(-8) M whereas the parent antibody bound insulin at 1.0 x 10(-8) M. Mutagenesis of the variable heavy chain complementarity determining regions (CDR) indicated that CDR1 and CDR3 were important for binding to insulin. Position 99 in CDR3 of the heavy chain was found to be a critical position for the ability of the scFv to bind to insulin.

Amino Acid Sequence↗
PubMed1992

Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro.

OBJECTIVE: The purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1. DESIGN: Based upon previous studies involving the generation of HuMAb that neutralize other enveloped viruses, we thought it feasible to generate HuMAb that might neutralize HIV-1. METHODS: A HuMAb was generated by fusing splenic B-cells from an HIV-positive patient with a mouse myeloma cell line. Flow cytometry was used to determine surface reactivity of the HuMAb on HIV-infected and non-infected cells. Radioimmunoprecipitation was employed to elucidate the antigen recognized by the HuMAb. A cell survival assay was used to determine the ability of the HuMAb to neutralize divergent isolates of HIV-1 in the presence or absence of complement. A gp120-CD4 inhibition enzyme-linked immunosorbent assay (ELISA) was developed in order to initiate studies to determine the mechanism of neutralization by the HuMAb. RESULTS: An anti-HIV HuMAb was generated that neutralized two HIV-1 isolates (IIIB and MN) without complement and which neutralized one divergent isolate (RF) and one clinical isolate in the presence of complement. This HuMAb, designated S1-1, was found, by flow cytometric analysis, to react with the surface of HIV-1-infected but not with uninfected cells. Radioimmunoprecipitation analysis demonstrated that S1-1 binds to native HIV gp120, but not dithiothreitol (DTT)-treated gp120. In addition, HuMAb S1-1 did not bind to denatured HIV antigens in Western blot analysis. HuMAb S1-1 effectively inhibited the binding of gp120 to soluble CD4 in ELISA. CONCLUSIONS: These results suggest that the epitope recognized by S1-1 is conformational and conserved among diverse HIV-1 isolates and may represent an uncharacterized HIV neutralizing domain within or close to the CD4 binding domain on gp120. HuMAb S1-1 might have a role to play in vaccine development or passive immunotherapy.

Antibodies, Monoclonal↗
PubMed1980

Cross-linking of cytochrome oxidase subunits with difluorodinitrobenzene.

Cytochrome c oxidase was treated with 1,5-difluoro-2,4-dinitrobenzene at molar ratios (DFDNB:oxidase) varying from 5 to 625. At the lowest ratio, there was virtually no effect of the probe on oxidase activity or on migration of oxidase subunits on sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis. At ratios of 25 and greater, there was loss of oxidase activity and a change of the pattern of subunit migration on sodium dodecyl sulfate electrophoresis. (i) Activity loss was probably a result of severely perturbing the cytochrome c binding site since oxidase activity with a low molecular weight reductant (N,N,N',N'-tetramethylphenylenediamine) was unaltered. Also unaltered were the oxidized, reduced, and carbon monoxide binding spectra of the treated oxidase. (ii) The staining pattern on sodium dodecyl sulfate electrophoresis showed that subunits III and VI disappeared from their normal positions on the gel. A new band of higher molecular weight accompanied their loss from the gel indicating that the two subunits were being cross-linked. Subunits III and VI are thus shown to have two reactive groups within 4.8 A (1 A = 0.1 nm) of one another. This proximity has not been detected with other probes that react with the same groups.

Animals↗