Monoclonal antibody against human T cell leukemia virus p19 defines a human thymic epithelial antigen acquired during ontogeny.

Using monoclonal antibody 12/1-2 against a 19,000-dalton human T cell leukemia virus (HTLV) protein (anti-p19), previously demonstrated to be reactive with HTLV-infected human cells, but not in numerous other uninfected cells, we found a reactive antigen to be expressed on the neuroendocrine component of human thymic epithelial cells but not on any other normal epithelial or neuroendocrine human tissues. Moreover, this reactive antigen is acquired on neuroendocrine thymic epithelium during thymic ontogeny--first appearing on fetal thymic epithelial cells between 8 and 15 wk gestation. While only a portion of thymic epithelial cells in the subcapsular cortical region of 15- and 24-wk fetal thymuses contained anti-p19+ epithelial cells, the entire subcapsular cortical region of newborn thymus epithelium was anti-p19+. By age 3 yr, normal subjects' entire subcapsular cortical and medullary thymic epithelium was anti-p19+. Using antibody against HTLV core protein, p24, and c-DNA probes for HTLV DNA, neither HTLV-specific p24 protein nor proviral DNA could be demonstrated in anti-p19+ thymic epithelial tissue. However, thymic epithelial extracts, disrupted HTLV extracts, as well as purified HTLV p19 antigen all inhibited the binding of anti-p19 antibody to thymic epithelium. Thus, anti-p19 may recognize a determinant on an HTLV-encoded 19,000-dalton structural protein that is shared by human thymic epithelium. Alternatively, anti-p19 defines a host encoded protein that is selectively expressed by normal thymic epithelium, and is induced to be expressed in HTLV-infected malignant T cells.

Monoclonal antibodies against human T cell leukemia-lymphoma virus (HTLV) p24 internal core protein. Use as diagnostic probes and cellular localization of HTLV.

Four monoclonal antibodies, human T cell leukemia-lymphoma virus (HTLV) 6, 7, 8, and 9, which react with the 24,000 dalton internal core protein of HTLVI, have been developed. These monoclonal antibodies reacted with only HTLV-infected cells and not with a broad spectrum of normal, neoplastic, mitogen-stimulated, or virus-infected cells and tissues. HTLV 6, 7, 8, and 9 identified at least two different antigenic determinants on HTLV p24 that were also recognized by antibodies present in HTLV+ patient sera. Monoclonal antibodies HTLV 6, 7, 8, and 9 reacted in indirect immunofluorescence assays with HTLV p24 localized at the cell surface of 5-d cultures of HTLV-infected T cells and, as well, reacted with T cells infected with HTLVII, a new type of HTLV isolated from a patient (MO) with a T cell variant of hairy cell leukemia. Thus, HTLV 6, 7, 8, and 9 should prove to be useful diagnostic reagents in the identification of HTLV- and HTLVII-infected T cells.

Conversion of an immunogenic human immunodeficiency virus (HIV) envelope synthetic peptide to a tolerogen in chimpanzees by the fusogenic domain of HIV gp41 envelope protein.

The fusogenic (F) domain of human immunodeficiency virus (HIV) gp41 envelope (env) protein has sequence similarities to many virus and mediates the fusion of HIV-infected cells. During a survey of the immunogenicity of HIV env peptides in chimpanzees, we have observed that HIV peptide immunogenicity was dramatically altered by the NH2-terminal synthesis of the gp41 F domain to an otherwise immunogenic peptide. We compared two hybrid peptide types comprised of T helper (Th) and B cell epitopes of HIV gp120 env protein for their immunogenicity in chimpanzees. The Th-B epitope hybrid peptides contained the HIV gp120 Th cell determinant, T1 (amino acids [aa] 428-440)-synthesized NH2 terminal to gp120 V3 loop peptides, which contain B cell epitopes that induce anti-HIV-neutralizing antibodies (SP10IIIB [aa 303-321] and SP10IIIB [A] [aa 303-327]). The F-Th-B peptide contained the HIV gp41 F domain of HIVIIIB gp41 (aa 519-530)-synthesized NH2 terminal to the Th-B peptide. Whereas Th-B peptides were potent immunogens for chimpanzee antibody and T cell-proliferative responses, the F-Th-B peptide induced lower anti-HIV gp120 T and B cell responses. Moreover, immunization of chimpanzees with F-Th-B peptide but not Th-B peptides induced a significant decrease in peripheral blood T lymphocytes (mean decrease during immunization, 52%; p < 0.02). Chimpanzees previously immunized with F-Th-B peptide did not respond well to immunization with Th-B peptide with T or B cell responses to HIV peptides, demonstrating that the F-Th-B peptide induced immune hyporesponsiveness to Th and B HIV gp120 env determinants. These observations raise the hypothesis that the HIV gp41 env F domain may be a biologically active immunoregulatory peptide in vivo, and by an as yet uncharacterized mechanism, promotes primate immune system hyporesponsiveness to otherwise immunogenic peptides.

Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS.

Peripheral blood lymphocytes from patients with the acquired immunodeficiency syndrome (AIDS) or with signs or symptoms that frequently precede AIDS (pre-AIDS) were grown in vitro with added T-cell growth factor and assayed for the expression and release of human T-lymphotropic retroviruses (HTLV). Retroviruses belonging to the HTLV family and collectively designated HTLV-III were isolated from a total of 48 subjects including 18 of 21 patients wih pre-AIDS, three of four clinically normal mothers of juveniles with AIDS, 26 of 72 adult and juvenile patients with AIDS, and from one of 22 normal male homosexual subjects. No HTLV-III was detected in or isolated from 115 normal heterosexual subjects. The number of HTLV-III isolates reported here underestimates the true prevalence of the virus since many specimens were received in unsatisfactory condition. Other data show that serum samples from a high proportion of AIDS patients contain antibodies to HTLV-III. That these new isolates are members of the HTLV family but differ from the previous isolates known as HTLV-I and HTLV-II is indicated by their morphological, biological, and immunological characteristics. These results and those reported elsewhere in this issue suggest that HTLV-III may be the primary cause of AIDS.

Identification and physicochemical characterization of a tumor-associated antigen from canine transmissible venereal sarcoma.

A tumor antigen associated wtih canine transmissible venereal sarcoma (CTVS) has been identified and partially characterized. The antigen was demonstrated in 3-M KCI and saline extracts of washed CTVS cells. Rabbit anti-CTVS antisera absorbed wih glutaraldehyde cross-linked normal dog serum and pooled homogenates of canine spleen, thymus, lymph node, and liver were used to identify the antigen. The specificity of the rabbit anti-CTVS antiserum for the CTVS antigen was established by use of immunodiffusion, two-dimensional electrophoresis, and the enzyme-linked immunosorbent assay (ELISA). Extracted CTVS antigen was fractionated by Sephadex G-200 chromatography, and the antigen factions were identified with the use of absorbed rabbit anti-CTVS antiserum and the ELISA technique. Antigen activity was detected in samples with estimated molecular weights greater than 70,000 daltons. Antigen fractions reacted with absorbed anit-CTVS antiserum to form a single precipitation band in immunodiffusion studies. Extraction of CTVS antigen with 3 M KCl and saline in the presence of a protease inhibitor did not significantly alter the antigen activity or molecular weight of the CVTS antigen. Cytoplasmic and nucleolar fluorescence were observed in an indirect immunofluorescence test with the use of acetone-fixed CTVS cells and absorbed anti-CTVS antiserum. The CTVs antigen activity was greatly reduced by trypsin digestion, by incubation for 1 hour at 65 degrees C, and by exposure to pH 2.8, 4.0, and 11.0 for 1 hour each.

Safety and immunogenicity of an HLA-based HIV envelope polyvalent synthetic peptide immunogen. DATRI 010 Study Group. Division of AIDS Treatment Research Initiative.

OBJECTIVE: To evaluate the safety and immunogenicity of a polyvalent (PV) HIV envelope synthetic peptide immunogen, C4-V3. The immunogen comprised four peptides containing T-helper epitopes from the fourth constant region (C4) of gp120 of HIV-1MN, and T-helper, cytotoxic T-lymphocyte HLA-B7-restricted, and B-cell neutralizing epitopes from the gp120 third variable region (V3) of four clade B HIV-1 isolates, HIV-1MN, HIV-1RF, HIV-1EV91, and HIV-1Can0A. DESIGN: A pilot, Phase I controlled trial [Division of AIDS Treatment Research Initiative (DATRI) 010] conducted at a single center. METHODS: Ten HIV-infected, HLA-B7-positive patients with CD4 cells > 500 x 10(6)/l were enrolled. Eight patients received the C4-V3 PV immunogen emulsified in incomplete Freund's adjuvant in five intramuscular injections over 24 weeks, and two controls received incomplete Freund's adjuvant alone. All subjects were followed for 52 weeks. RESULTS: Four out of eight C4-V3 PV recipients generated at least fourfold rise in serum antibody titers to at least three immunogen peptides in contrast to none of the control subjects. Four out of eight C4-V3 PV recipients and none of the controls had an at least fourfold rise in neutralizing antibodies to either HIV-1MN, HIV-1RF, or HIV-1(4489-5) laboratory-adapted HIV isolates. 3H-Thymidine incorporation assays of peripheral blood mononuclear cells increased at least fivefold over the baseline stimulation index to at least one of the immunogen peptides in two consecutive post-immunization timepoints in five out of eight C4-V3 PV recipients versus none of the controls. CD4 cell counts and plasma HIV RNA levels did not change in patients who received either C4-V3 PV or adjuvant alone. Adverse events consisted primarily of grade 1 injection site reactions in six subjects (four C4-V3 recipients, two controls). CONCLUSIONS: C4-V3 PV synthetic peptides demonstrated both immunogenicity and safety in HIV-infected patients.

Mother-to-child transmission of human T-cell lymphotropic virus type I (HTLV-I) in Jamaica: association with antibodies to envelope glycoprotein (gp46) epitopes.

To study mother-to-child transmission of HTLV-I in Jamaica, we screened antenatal patients in Kingston, Jamaica, from 1983 to 1985. Of 2,329 women, 81 (3.5%) were HTLV-I seropositive. Two to three years later, 36 seropositive mothers were recontacted, and blood was drawn from them and their children. All sera were tested for HTLV-I antibodies, and mother's sera were additionally tested for HTLV-I whole-virus antibody titer, syncytium-inhibition neutralizing antibody titer, and titers to six synthetic peptides from the HTLV-I envelope glycoprotein gp46. Seventeen of 74 (23%) [95% confidence interval (CI) 15-34%] children were seropositive. HTLV-I transmission was associated with breast-feeding duration > 6 months [relative risk (RR) 3.2; CI 0.4-22.1], maternal age > 30 years (RR 2.8; CI 1.0-7.8), and higher maternal whole-virus antibody titer (RR 3.3; CI 1.3-8.5). After controlling for higher whole-virus antibody titer, transmission remained associated with higher titer of neutralizing antibody and higher titer of antibody to the peptide sp4a1, corresponding to amino acids 196-209 of the gp46 envelope glycoprotein. We conclude that mother-to-child transmission of HTLV-I in Jamaica is associated with longer duration of breast-feeding, older age, and higher HTLV-I antibody titer, in particular to a certain immunogenic portion of the gp46 envelope glycoprotein.

Human T-cell lymphotropic virus I and adult T-cell leukemia: report of a cluster in North Carolina.

PURPOSE: Human adult T-cell leukemia-lymphoma is a malignant, proliferative disease of CD4+ lymphocytes associated with infection with human T-cell lymphotropic virus type I (HTLV-I). Following the presentation of a patient who was infected with the virus, we undertook a study of his family members and sexual contacts to see if a cluster of infected persons could be identified. CASE REPORT: A black heterosexual North Carolina native with a history of drug abuse presented with jaundice, and pancytopenia subsequently developed. He then became hypercalcemic and leukemic, with high numbers of circulating, morphologically abnormal CD4+ lymphocytes. RESULTS: As determined by radioimmunoassay and immunoblot analyses, the serum of the index case contained antibodies against core proteins (p19 and p24) of HTLV-I. When cultured in vitro with interleukin-2, the lymphocytes expressed HTLV-I specific core proteins. The virus recovered from these T cells was transmitted to cord blood T cells, which became immortalized for continuous growth in vitro, expressed HTLV-I p19 protein, and displayed characteristic C-type particles by electron microscopy. Studies of family members and sexual contacts, all of whom were black, heterosexual central North Carolina natives, revealed five of 28 whose serum had anti-HTLV-I antibodies as determined by radioimmunoassay and immunoblot. Neither the patient nor the seropositive family/contacts had antibodies against human immunodeficiency virus proteins. Four of the six people with HTLV-I infection had no history of intravenous drug abuse. Three of the five seropositive family/contacts had circulating, morphologically abnormal lymphocytes suggestive of "preleukemic" or "smoldering" human adult T-cell leukemia-lymphoma.

Intranasal immunization is superior to vaginal, gastric, or rectal immunization for the induction of systemic and mucosal anti-HIV antibody responses.

Vaginal anti-HIV antibody responses may be beneficial, and possibly required, for vaccine-induced protection against HIV infection acquired through receptive vaginal intercourse. We have previously determined that intranasal immunization with a hybrid HIV peptide and cholera toxin induced vaginal anti-HIV IgA responses in BALB/c and C57BL/6 mice. To determine if vaginal, gastric, or rectal boosting would enhance the induction of vaginal anti-HIV IgA responses over those observed with intranasal immunization only, C57BL/6 mice were intranasally immunized with the hybrid HIV peptide T1SP10MN(A) and cholera toxin (days 0 and 14) and boosted via the vaginal, gastric, or rectal route (days 7 and 28). Four intranasal immunizations was superior to all other immunizations evaluated for the induction of plasma anti-peptide IgG, vaginal anti-peptide IgG and IgA, and peptide-specific delayed-type hypersensitivity. In addition, intranasal priming with gastric boosting was associated with greatly elevated total serum IgE concentrations whereas intranasal immunization only was associated with only a modest increase in total serum IgE. These results suggest that intranasal immunization is a viable route of immunization for the induction of systemic and mucosal anti-HIV immune responses.

Purification of envelope glycoproteins of human T cell lymphotropic virus type I (HTLV-I) by affinity chromatography.

The external envelope glycoprotein (gp46) and transmembrane glycoprotein (gp21) of human T-cell lymphotropic virus type I (HTLV-I) were isolated from lysates of HTLV-I-infected HUT-102 cells by affinity chromatography. Fifty ml aliquots of packed HUT-102 cells were extracted with 1% Triton X-100, and lysates were treated sequentially with an affinity column containing IgG from an HTLV-I+ human subject followed by chromatography of the bound fraction over a lentil lectin column. The identity of the purified envelope proteins was confirmed with a human monoclonal antibody (0.5 alpha) to gp46 and with rabbit antisera raised to a synthetic peptide from the C-terminus of gp21. Affinity-purified envelope glycoproteins were bound to microtiter wells and used in radioimmunoassay to detect murine and human anti-envelope antibodies to gp46 and gp21 molecules.

Detection of immune complexes in sera of dogs with canine transmissible venereal sarcoma (CTVS) by a conglutinin-binding assay.

The canine transmissible venereal sarcoma (CTVS) is capable of extended growth in an allogeneic host. Since immune complexes can enhance allograft survival in other animal models, we used an enzyme-linked conglutinin-binding assay to determine the presence and amount of circulating immune complexes in dogs with CTVS. With the conglutinin-binding assay, 23 of 64 dogs (36 per cent) bearing CTVS had concentration of immune complexes 3 standard deviations greater than those detected in normal canine serum. When dogs with circulating immune complexes were separated into 3 groups based on whether the size of CTVS had increased (progressor), decreased (regressor) or remained the same (steady-state) during the week before collection of serum, no significant difference was found in the amounts of immune complexes in sera from progressor dogs. In 3 regressor dogs that received a second transplant of CTVS, the mean concentration of circulating immune complex was significantly greater than the mean for progressor dogs. In progressor dogs, amounts of immune complexes decreased with increasing tumour volume. Following sucrose density gradient ultracentrifugation analysis of sera from a normal and progressor dog, 27S complexes containing both IgG and IgM were detected in serum from a progressor dog. Thus, it appears that the conglutinin-binding assay is a useful and sensitive method for detecting immune complexes in canine serum.

Developmental regulation of lymphocyte-specific protein 1 (LSP1) expression in thymus during human T-cell maturation.

The lymphocyte specific protein 1 (LSP1) phosphoprotein is an F-actin binding molecule restricted to cells of hematopoietic origin in mice and humans. LSP1 is localized to the internal surface of the plasma membrane, the cytoplasm, and NP-40-insoluble actin filaments and is thought to mediate cytoskeleton-driven responses in activated leukocytes that involve receptor capping, cell-cell interactions and cell motility. Here, we generated two monoclonal antibodies (MAbs), 5E3 and 14G8, that are specific for human LSP1 to define the expression of LSP1 throughout human T-cell development. Both MAbs reacted with a 52-kDa protein in BW5147 cells transfected with human LSP1 cDNA in pcDNA3, but not in cells transfected with cDNA in an antisense orientation, indicating the specificity of 5E3 and 14G8 for human LSP1. In developing T cells, LSP1 was expressed on human fetal liver CD7+ NK and T-cell precursors, the CD7+, CD3-, CD4-, CD8- human stem cell line DU-528, and on CD4-, CD8- double-negative (DN) thymocytes. Immunohistochemistry and three-color flow cytometry analysis of fetal or postnatal thymocytes revealed that LSP1 was increasingly expressed during intrathymic human T-cell maturation. While immature CD4+CD8+ double-positive (DP) thymocytes expressed low to undetectable levels of LSP1, mature CD4+CD8- and CD4-CD8+ single-positive (SP) thymocytes expressed high levels of LSP1. Thus, LSP1 is developmentally regulated during T-cell maturation within the human thymus and may play a functional role in the motility of DN and SP thymocytes.

Tumor size, leukocyte adherence inhibition and serum levels of tumor antigen in dogs with the canine transmissible venereal sarcoma.

Tumor antigen (TA) associated with the canine transmissible venereal sarcoma (CTVS) was detected in the sera of dogs bearing the tumor. Rabbit antisera specific for tumor antigen and 3 M KCl extracts of CTVS cells were used in both a competitive enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA to quantify levels of circulating TA. In a study of 29 dogs bearing the transplanted CTVS, levels of circulating TA correlated positively with tumor volume. In a longitudinal study of four dogs receiving a transplant of 10(8) viable CTVS cells, circulating CTVS antigen was detected transiently 2 days after transplantation, while persistent levels of TA associated with increasing tumor volume were demonstrable 19-34 days after transplantation. In three of four tumor-bearing dogs, levels of serum TA correlated inversely with values obtained with peripheral blood leukocytes in the leukocyte adherence inhibition (LAI) assay; elevated levels of circulating TA found in dogs with large (greater than 7 cm3) tumors were associated with decreased LAI reactivity of peripheral blood leukocytes. TA could not be detected in sera 48-72 h after surgical removal of CTVS whereas LAI reactivity of peripheral blood leukocytes to CTVS antigen rebounded 1-3 weeks following tumor excision. Results of this study support the use of the competitive ELISA and LAI techniques in assessing levels of circulating tumor antigen, tumor burden and tumor-specific immunity.

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.

Effect of bismuth salts on systemic and mucosal immune responses to orally administered cholera toxin.

While the antimicrobial and antisecretory effects of bismuth salts are well documented, little is known regarding their effects on immune responses to enterotoxins such as that of V. cholerae or to orally administered vaccine antigens. To evaluate the effects of Pepto Bismol (PB) on the induction of systemic and mucosal immune responses to cholera toxin (CT), C57BL/6 mice were orally administered 10 micrograms CT and PB, or mice were pretreated with PB 30 min prior to CT administration. When co-administered with CT, PB attenuated serum IgG1, IgG2a, IgG2b and IgG3 anti-CT responses in a dose-dependent manner and also reduced levels of circulating anti-CT IgA and total serum IgE. Similarly, anti-CT intestinal IgA responses were also decreased. However, when administered 30 min prior to CT, PB had little to no effect on serum or intestinal anti-CT immunoglobulin responses. Administration of bismuth subsalicylate (BSS), the active component of PB, or sodium salicylate did not reduce immune responses to CT, suggesting that the combination of BSS plus other constituents contained within PB contributed to the decreased immune response to CT. Moreover, bismuth subgallate alone inhibited antibody responses to CT. Our data are consistent with the hypothesis that, when administered orally with CT, PB and bismuth subgallate create a physical barrier to antigen uptake.

Mucosal immunity to HIV-1: systemic and vaginal antibody responses after intranasal immunization with the HIV-1 C4/V3 peptide T1SP10 MN(A).

To optimize mucosal immune responses to the HIV-1 peptide vaccine candidate T1SP10 MN(A), we intranasally immunized BALB/c and C57BL/6 mice with C4/V3 HIV-1 peptide together with the mucosal adjuvant cholera toxin (CT). Four doses over a 4-wk period resulted in peak serum anti-peptide IgG titers of > 1:160,000 in BALB/c mice and > 1:520,000 in C57BL/6 mice, and significant levels (>1:30,000) persisted in both strains of mice for longer than 6 mo. Furthermore, intranasal immunization with peptide and CT induced serum IgG reactivity to HIV-1 gp120 and HIV-1(MN) neutralizing responses. The primary anti-peptide IgG subclass was IgG1, suggesting a predominant Th2-type response. In addition to elevated serum anti-peptide A responses, intranasal immunization with T1SP10 MN(A) and CT induced both vaginal anti-peptide IgG and IgA responses, which persisted for 91 days in both strains of mice. Vaginal anti-HIV IgA was frequently associated with secretory component, suggesting transepithelial transport of IgA into vaginal secretions. Cervical lymph nodes contained the highest relative concentration of anti-T1SP10 MN(A) IgG-producing cells, while the spleen was the next major site of anti-T1SP10 MN(A) IgG-producing cells. Ag-specific proliferative responses were also detected in cervical lymph node and spleen cell populations after intranasal immunization with T1SP10 MN(A) and CT. In addition, intranasal immunization with T1SP10 MN(A) and CT was able to induce anti-HIV cell-mediated immunity in vivo as indicated by the induction of delayed-type hypersensitivity. Therefore, intranasal immunization with hybrid HIV peptides provides a noninvasive route of immunization that induces both long-lived systemic and mucosal Ab responses as well as cell-mediated immunity to HIV.

Characterization of an antigen shared by human thymic epithelium and human T cell leukemia virus p19 Gag protein.

The molecular basis for cross-reactive antibody binding to human T cell leukemia virus type I (HTLV-I) p19 core protein and human thymic epithelium has been defined with two monoclonal antibodies (mAbs), 12/1-2 and 13B12, raised to HTLV-I p19. The mAb 12/1-2 has previously been shown to react with HTLV-I p19, HTLV-II p22, and antigens of normal human thymic epithelium, placenta, and foreskin, whereas mAb 13B12 binds only to the carboxyl terminus of HTLV-I p19. In the present study, mAb 12/1-2 bound to a subset of Triton X-100-insoluble intermediate filaments in human thymic epithelium also recognized by antikeratin antibodies AE1 and AE3. The mAb 12/1-2 also reacted in Western blot assays with proteins of 54, 46, and 40 kDa present in extracts of human thymic epithelium and with hexameric peptides containing overlapping sequences of HTLV-I p19 with the amino acids IPP (amino acids 117-119). In contrast, the HTLV-I-specific mAb 13B12 did not bind to human thymic epithelium and reacted with a single hexameric peptide containing the carboxy-terminal HTLV-I p19 sequence IPPPYV (amino acids 117-122). Binding of mAb 12/1-2 to thymic epithelium could be inhibited by adsorption with peptide SP-79 containing a C-terminal sequence (amino acids 112-125) of p19. The crossreactive IPP site is within a region of p19 that has been previously shown to be highly immunogenic in HTLV-I-infected individuals and that is also encoded by genes or mRNA of human cytokeratin 17, keratin 4, epidermal cytokeratin 2, and 50-kDa type I epidermal keratin. Thus, our studies define the sequence of a cross-reactive antigen on HTLV-I p19 that is also associated with keratin intermediate filaments from human thymic epithelium and other normal human tissues and that could serve as a focus of an autoimmune response during HTLV-I infection.

Human erythrocyte antigens: II. The In(Lu) gene regulates expression of an antigen on an 80-kilodalton protein of human erythrocytes.

We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.