Phenotypic characterization and ontogeny of mesodermal-derived and endocrine epithelial components of the human thymic microenvironment.

Using murine monoclonal antibodies TE-4 and TE-7 raised against human thymic stroma, we identified two distinct and mutually exclusive thymic microenvironment components: the thymic endocrine epithelium (TE-4+) and mesodermal-derived fibrous stroma (TE-7+). TE-4-reactive epithelium did not react with antibody TE-7, contained thymosin alpha 1 and keratin, and expressed other known markers of thymic endocrine epithelium (A2B5 and p19). Moreover, TE-4+ thymic epithelial cells strongly expressed class I (HLA-A, -B and -C) and class II (Ia-like) major histocompatibility complex (MHC) antigens. In contrast, TE-7+ thymic fibrous stroma did not react with antibody TE-4, did not contain thymosin alpha 1 nor keratin, and did not express the thymic endocrine epithelium markers A2B5 and p19. TE-7+ thymic stromal cells weakly expressed class I and did not express class II MHC antigens. Both TE-4+ and TE-7+ thymic microenvironment compartments were identifiable in thymus from 7 wk gestation through adult life. At 7 wk fetal gestation, TE-7+ stroma surrounded a cylindrical TE-4+, A2B5+ thymic epithelial rudiment. Between 10 and 15 wk fetal gestation, TE-7+ thymic stroma surrounded early thymic lobules. By 15 wk fetal gestation, antibody TE-4 defined subcapsular cortical and medullary zones of endocrine thymic epithelium, while antibody TE-7 bound to interlobular fibrous septae, vessels, and thymic fibrous capsule. While otherwise specific for endocrine thymic epithelium, antibody TE-4 reacted with the basal layer of squamous epithelium in skin, tonsil, conjunctiva, and upper esophagus.

Resistance of CD7-deficient mice to lipopolysaccharide-induced shock syndromes.

CD7 is an immunoglobulin superfamily molecule involved in T and natural killer (NK) cell activation and cytokine production. CD7-deficient animals develop normally but have antigen-specific defects in interferon (IFN)-gamma production and CD8(+) CTL generation. To determine the in vivo role of CD7 in systems dependent on IFN-gamma, the response of CD7-deficient mice to lipopolysaccharide (LPS)-induced shock syndromes was studied. In the high-dose LPS-induced shock model, 67% of CD7-deficient mice survived LPS injection, whereas 19% of control C57BL/6 mice survived LPS challenge (P < 0.001). CD7-deficient or C57BL/6 control mice were next injected with low-dose LPS (1 microgram plus 8 mg D-galactosamine [D-gal] per mouse) and monitored for survival. All CD7-deficient mice were alive 72 h after injection of LPS compared with 20% of C57BL/6 control mice (P < 0.001). After injection of LPS and D-gal, CD7-deficient mice had decreased serum IFN-gamma and tumor necrosis factor (TNF)-alpha levels compared with control C57BL/6 mice (P < 0.001). Steady-state mRNA levels for IFN-gamma and TNF-alpha in liver tissue were also significantly decreased in CD7-deficient mice compared with controls (P < 0.05). In contrast, CD7-deficient animals had normal liver interleukin (IL)-12, IL-18, and interleukin 1 converting enzyme (ICE) mRNA levels, and CD7-deficient splenocytes had normal IFN-gamma responses when stimulated with IL-12 and IL-18 in vitro. NK1.1(+)/ CD3(+) T cells are known to be key effector cells in the pathogenesis of toxic shock. Phenotypic analysis of liver mononuclear cells revealed that CD7-deficient mice had fewer numbers of liver NK1.1(+)/CD3(+) T cells (1.5 +/- 0.3 x 10(5)) versus C57BL/6 control mice (3.7 +/- 0.8 x 10(5); P < 0.05), whereas numbers of liver NK1.1(+)/CD3(-) NK cells were not different from controls. Thus, targeted disruption of CD7 leads to a selective deficiency of liver NK1.1(+)/ CD3(+) T cells, and is associated with resistance to LPS shock. These data suggest that CD7 is a key molecule in the inflammatory response leading to LPS-induced shock.

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.

Cytotoxic antibodies to cloned rat islet cells in serum of patients with diabetes mellitus.

We have found complement-dependent cytotoxic antibodies in the serum of 8 of 24 patients with insulin-dependent diabetes mellitus using a 51Cr cytotoxicity assay with monolayers of cloned rat islet cells (clones RINm 5F and RINm 14B). In contrast, complement-dependent cytotoxicity with 51Cr release greater than 24% was found with sera from 34 controls or from 5 patients with polyglandular failure without diabetes, and was present in only 1 serum our of 12 from patients with insulin-independent diabetes mellitus. The prevalence of antibodies appears to decrease with duration of insulin-dependent diabetes, and in one patient studied, cytotoxic antibodies were present at the time of diagnosis of diabetes. Cytotoxicity is independent of insulin synthesis, as evidenced by the linear correlation of cytotoxicity of sera for the insulin-producing clone RINm 5F and the somatostatin-producing clone RINm 14B. The present study identifies nonspecies-specific cytotoxic antibodies in the serum of patients with diabetes mellitus, and the assay used should facilitate studies of humoral immunity in the pathogenesis of diabetes mellitus.

Production of monoclonal antibodies reacting with rat islet cell membrane antigens.

The present communication describes the generation of twelve lymphocyte hybrid cell lines whose antibodies react with the rat islet cell line RIN, and the initial characterization of the tissue specificity and functional properties of these antibodies. Since antibodies recognizing islet cell differentiation antigens were sought, these permanent hybrid cell lines were developed from cultures whose antibodies, by radioimmunoassay, bound minimally or not at all to rat red blood cells (cell line A3C1 is an exception). Antibodies from five of the cell lines by radioimmunoassay react significantly with cultured rat fibroblasts in addition to their reaction with RIN cells. Antibody F41-5D6 by indirect immunofluorescence reacted with sections of the RISL transplantable islet cell tumor and specifically with islet cells in sections of rat pancreas. Four antibodies (F41-1G3, 5B5, 6C5, and 5A5), by indirect immunofluorescence, reacted with sections of the RISL transplantable islet cell tumor but not with sections of normal pancreas. Seven of the antibodies were cytotoxic to cultured RIN cells and seven antibodies share the useful property of reacting with protein A. This study demonstrates the feasibility of producing monoclonal antibodies to islet cell differentiation antigens and describes several antibodies which should be useful reagents in studies of the physiology and pathophysiology of the islet cell plasma membrane.

Removal of fibroblasts from human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages.

A complement fixing IgM monoclonal antibody (1B10) that reacts with surface membrane molecules of human fibroblasts, tissue macrophages, and peripheral monocytes was produced. In Western blot analysis of detergent extracts of cultured human foreskin fibroblasts, antibody 1B10 detected protein bands of Mr 43,000 and 72-80,000. We used the 1B10 antibody with complement to eliminate most 1B10 positive nonepithelial cells from thymic epithelial (TE) cell cultures, thereby allowing us to grow highly enriched populations of human TE cells.

Transfer plate radioassay using adsorbed anti-insulin antibody to detect insulin secreted by islet cell cultures.

A solid-phase radioimmunoassay for detection of insulin synthesized by islet cell clones is described. This assay employs anti-insulin antibody adsorbed onto fenestrated polyvinyl chloride 96-well plates ('transfer plates'). The calibrated aperture in the bottom of each transfer plate well permits fluid to enter the wells when transfer plates are lowered into microculture wells containing insulin. With this assay it is possible to rapidly screen hundreds of islet cell cultures for insulin production. We have used this assay to facilitate cloning of the RIN rat insulinoma cell line. The assay readily detects insulin synthesis by RIN cells and [125I]insulin is not displayed by culture medium from cells which do not produce insulin. The transfer plate format should be applicable to semiautomate other radioimmunoassays.

HIV type 1 V3 region primer-induced antibody suppression is overcome by administration of C4-V3 peptides as a polyvalent immunogen.

The extreme variability of HIV-1 immunogenic regions has hampered attempts to design immunogens capable of inducing broadly reactive neutralizing anti-HIV antibody responses. We have begun to study the immune responses generated to a polyvalent mixture of HIV envelope gp120 synthetic peptides, and to determine the ability of each component of a polyvalent immunogen to prime and boost immune responses to each immunogen component. A major concern regarding the use of a polyvalent mixture of HIV-1 immunogens is that the phenomenon of "original antigenic sin," or HIV-1 primer-induced suppression of antibody responses to a subsequent boost by a second HIV-1 variant, may occur and prevent effective anti-HIV immune responses. Using a prototypic four-valent HIV peptide envelope immunogen in BALB/c mice, we observed two types of primer-induced antibody suppression: "original antigenic sin" with primer-induced suppression of antibody responses to only the boosting immunogen, and a second, novel form of primer-induced antibody suppression, with inhibition of antibody responses not only to the priming immunogen but also to all other immunogens in the polyvalent immunogen mixture as well. Importantly, either reversing the sequence of administration of the immunogens or administration of all four components as a polyvalent mixture completely overcame both forms of HIV-1 primer-induced antibody suppression.

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.

Human erythrocyte antigens. III. Characterization of a panel of murine monoclonal antibodies that react with human erythrocyte and erythroid precursor membranes.

Human erythrocyte membrane proteins express antigens which serve as markers for erythroid differentiation as well as targets for human blood group alloantibodies. We have produced and characterized a new panel of five monoclonal antibodies to erythrocyte membrane proteins. Three monoclonal antibodies (E3, E4, E5) were specific for erythrocyte glycophorins. One antibody (E3) identified the sialoglycoprotein alpha and beta homologous regions proximal to the plasma membrane, a second antibody (E4) was specific for sialoglycoprotein alpha, while a third (E5) was a sialoglycoprotein-beta-specific antibody. Two antibodies (E6 and TE10) to the 65,000-dalton chymotrypsin cleavage product of band 3 were also produced. These antibodies constitute a new panel of probes for investigation of normal erythroid differentiation, erythroleukemia, and the expression of normal and anomalous blood group antigens.

The human thymic microenvironment: cortical thymic epithelium is an antigenically distinct region of the thymic microenvironment.

The thymic microenvironment is a complex tissue essential for normal T cell maturation. Prothymocytes in the subcapsular cortical (SCC) region of the thymus undergo cell division and migrate to the inner cortex. The majority of cortical thymocytes cease dividing and die, but a minority are exported to the periphery. We have previously shown thymic hormones in SCC and medullary thymic epithelium and have identified a monoclonal antibody (TE-4) that defines human endocrine thymic epithelium. However, no marker that selectively defines cortical thymic epithelium has been available. In this study, we have produced two monoclonal antibodies, TE-3A and TE-3B, raised against human thymic stroma that bind to an intracellular antigen in cortical but not medullary thymic epithelium. In double immunofluorescence assays in which we used anti-keratin, anti-thymosin alpha 1, and anti-endocrine thymic epithelium antibodies (TE-4, A2B5), TE-3+ SCC epithelium was TE-4+ and contained keratin and thymosin. alpha 1. In contrast, TE-3+ inner cortical epithelium was TE-4/A2B5 nonreactive and did not contain thymosin alpha 1. An ontogeny study of seven fetal and five neonatal thymuses demonstrated that expression of the TE-3 antigen was acquired at 10 wk fetal gestation. Using TE-3 antibody, we observed sequential stages of separation of cortical and medullary epithelium from 12 to 20 wk fetal gestation. In dysplastic (severe combined immunodeficiency disease) thymuses, strands of TE-3+ nonendocrine cells encircled nests of TE-4+ endocrine epithelium. Thus, human cortical thymic epithelium is antigenically distinct from endocrine medullary epithelium. Antibodies against the TE-3 antigen define an intracellular molecule that may reflect a specialized function of cortical thymic epithelium.

The human thymic microenvironment. Phenotypic characterization of Hassall's bodies with the use of monoclonal antibodies.

The human thymic microenvironment is important in promotion of T cell maturation, particularly during early stages of thymic ontogeny. Hassall's bodies (HB) are epithelial swirls in the human thymic medulla that are thought to be derived from endocrine medullary thymic epithelium. To study the ontogeny and function of various components of the human thymic microenvironment, we have produced four monoclonal antibodies (TE-8, TE-15, TE-16, and TE-19) that selectively reacted in thymus with HB. Antibodies TE-8 and TE-16 reacted with the cells forming the outer rim of the HB swirl. Antibody TE-19 reacted with the entire cellular portion of HB and with epithelial cells immediately surrounding HB. Granular foci in the cellular swirls of greater than 90% of HB reacted with antibody TE-15. During thymic ontogeny, the antigens defined by antibodies TE-8, TE-15, TE-16, and TE-19 were first detected in fetal thymus on HB beginning at 16 wk gestation, the age when HB morphologically appear in the thymus. Aberrant expression of the antigens corresponding to antibodies TE-8, TE-15, TE-16, and TE-19 was observed on thymic tissue from individuals with severe cellular immunodeficiency disease. In human skin, antibodies TE-8, TE-16, and TE-19 reacted with the stratum granulosum; antibody TE-15 reacted with the stratum corneum. Thus, with the use of antibodies TE-8, TE-15, TE-16, and TE-19, we have identified HB as antigenically distinct regions of endocrine thymic epithelium. Furthermore, we have shown that these anti-HB reagents also selectively react with epidermal keratinocytes in the terminal stages of keratinocyte maturation.

C-terminal region of human T cell lymphotropic virus type I (HTLVI) p19 core protein is immunogenic in humans and contains an HTLVI-specific epitope.

To study the human host response to viral structural proteins during HTLV type I infection, five synthetic peptides matching the N-terminal and C-terminal regions of HTLVI p19 core protein were used to identify antigenic sites on p19 that were immunogenic in man. In radioimmunoassay and immunoprecipitation experiments, antibodies in 16 of 18 HTLVI+ patient sera reacted with a synthetic peptide matching the C-terminal 11-amino acid sequence of p19, whereas only two sera contained antibodies that reacted with other N- or C-terminal region p19 synthetic peptides. Polyclonal rabbit antisera to N- and C-terminal peptides reacted with a native viral protein of 19,000 daltons and with gag-encoded precursors of p19. Six monoclonal antibodies against native viral p19 were screened for reactivity to the five synthetic peptides. One of six antibodies (13B12) reacted with the C-terminal synthetic peptide of p19. Antibody 13B12 did not react with HTLVII or HTLVIII proteins or with HTLVIII-infected cells, nor did it cross-react with a wide variety of HTLV-uninfected normal host tissues. Thus, the C-terminus of p19 contains an antigen that is highly immunogenic in most HTLVI-infected patients and is HTLVI specific.

AD2, a human molecule involved in the interaction of T cells with epidermal keratinocytes and thymic epithelial cells.

Interactions between T cells and epithelial cells of the thymus are essential for normal T cell development, and interactions between T cells and skin epidermal keratinocytes occur in the context of inflammatory skin diseases and cutaneous T cell malignancies. On the basis of observations that the T cell ALL cell line, HSB, bound to IFN-gamma activated epidermal keratinocytes (41 +/- 5% of EK with three or more HSB cells bound), whereas the CTCL cell line H9 bound poorly (8 +/- 3%), we have raised mAb 13H12 that identified a 36 kDa molecule, termed AD2, that was highly expressed on HSB but not on H9 T cells. mAb 13H12 inhibited the binding of HSB T cells to IFN-gamma-treated epidermal keratinocytes (43 +/- 5% inhibition, p < 0.01), inhibited the binding of peripheral blood T cells to IFN-gamma-treated EK (62 +/- 3% inhibition, p < 0.001), and inhibited the binding of IFN-gamma-treated human thymic epithelial cells to thymocytes (39 +/- 3% inhibition, p < 0.01). Although AD2 was expressed at a low level on all T cells, AD2 was highly expressed on the CD3-CD4-CD8-, CD3-CD4low+ CD8-, and CD3-CD4+ CD8+ subsets of immature thymocytes in the thymic subcapsular and inner cortex. Taken together, these data suggest a role for the AD2 molecule in interactions of T cells with epithelial cells of skin and thymus.

Immunodominant sites of human T cell lymphotropic virus type 1 envelope protein for murine helper T cells.

HTLV-I (human T cell lymphotropic virus type 1) is the retrovirus causally related to adult T cell leukemia/lymphoma and is also associated with a neurological disorder, tropical spastic paraparesis, or HTLV-I-associated myelopathy. The development of these two different diseases among HTLV-I-infected individuals may depend in part on differences in their T cell immunity associated with a difference of HLA phenotype. Peptides corresponding to 17 sites in the HTLV-I envelope protein were tested for their antigenicity for lymph node cells from B10.BR, B10.D2, B10.A(5R), and B10.HTT congenic mice, representing four independent MHC haplotypes, immunized with the native envelope protein. Ten of the 17 tested sites were predicted to be amphipathic alpha-helical sites and all of them were found to be antigenic for at least one of the four MHC congenic strains of mice. Three of the 17 sites were amphipathic 3(10)-helical sites and four sites were predicted to be non-helical sites: none of the 3(10)-helical sites were antigenic and only one of four non-predicted sites was found to be immunodominant. Furthermore, three potent immunodominant peptides, V1E1 (342-363), V1E8/SP4a (191-209), and V1E10 (141-156) were also shown to be immunogenic; i.e., these peptides could be used to immunize mice to elicit proliferative responses of lymph node cells to the native HTLV-I envelope protein. Furthermore, these three peptides were able to prime animals for an enhanced antibody response to the native protein. Because this priming followed the same Ir gene control as the proliferative response, it probably reflects the ability of these peptides to prime helper T cells. The localization of immunodominant sites in HTLV-I envelope protein in mice may be useful for finding antigenic and immunogenic sites in humans, for developing a peptide vaccine for the virus, and possibly for aiding in prognosis for the development of different disease manifestations of HTLV-I infection.

Mapping of immunogenic regions of human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins with env-encoded synthetic peptides and a monoclonal antibody to gp46.

Antigenic sites on human T cell leukemia virus type I (HTLV-I) gp46 and gp21 envelope glycoproteins that are immunogenic in man were studied with envelope gene (env)-encoded synthetic peptides and a mAb to HTLV-I gp46 envelope glycoprotein. Antibodies in 78% of sera from HTLV-I seropositive subjects reacted with synthetic peptide 4A (amino acids 190 to 209) from a central region of HTLV-I gp46. Human anti-HTLV-I antibodies also bound to synthetic peptides 6 (29% of sera) and 7 (18% of sera) from a C-terminal region of gp46 (amino acids 296 to 312) and an N-terminal region of gp21 (amino acids 374 to 392), respectively. mAb 1C11 raised to affinity-purified HTLV-I gp46 reacted with gp46 external envelope glycoprotein and gp63 envelope precursor in immunoblot assay and also bound to the surface of HTLV-I+ cells lines HUT-102 and MT-2. Antibody 1C11 did not react with HTLV-II or HIV-infected cells or with a broad panel of normal human tissues or cell lines. In competitive RIA, anti-gp46 antibody 1C11 was inhibited from binding to gp46 either by antibodies from HTLV-I seropositive subjects or by HTLV-I env-encoded synthetic peptide 4A, indicating that 1C11 bound to or near a site on gp46 within amino acids 190 to 209 also recognized by antibodies from HTLV-I-seropositive individuals. When tested in syncytium inhibition assay, mAb 1C11 did not neutralize the infectivity of HTLV-I. Thus, HTLV-I infection in man is associated with a major antibody response to a region of gp46 within amino acids 190 to 209 that is on the surface of virus-infected cells.

Characterization of the surface topography and putative tertiary structure of the human CD7 molecule.

The CD7 gp40 molecule is a member of the Ig gene superfamily and is expressed on T cell precursors before their entry into the thymus during fetal development. N-terminal amino acids 1-107 of CD7 are highly homologous to Ig kappa-L chains whereas the carboxyl-terminal region of the extracellular domain of CD7 is proline-rich and has been postulated to form a stalk from which the Ig domain projects. To define potential functional regions of CD7, we have studied the surface topography of the CD7 Ag by synthesizing peptides corresponding to linear sequences within the CD7 extracellular domains, by raising polyclonal anti-CD7 rabbit sera against these peptides, and by computer analysis of the primary CD7 amino acid sequence. Polyclonal anti-CD7 sera were studied using indirect immunofluorescence, RIA, radioimmunoprecipitation, and Western blot assays. Computer analysis was performed comparing the CD7 sequence with all other known protein sequences. We found that three CD7 epitopes defined by peptides CD7-1A (AA 1-38), CD7-4 (AA 48-74), and CD7-7 (AA 129-146) were available for binding antibody on the surface of the CD7 molecule. Using computer analysis, we transposed the amino acid sequence of the CD7 Ig kappa-like N-terminal domain of CD7 onto the spatial coordinates of REI, a previously reported Ig kappa-molecule highly homologous (48%) to the CD7 N-terminal Ig-like region. Based on computer analysis of this putative CD7 three-dimensional structure, both the CD7-1A and CD7-4 regions protruded from the surface of the N-terminal domain of the CD7 molecule. Finally, comparison of the CD7 transmembrane sequence with CD4 and HIV transmembrane sequences and with respiratory syncytial virus fusion sequences demonstrated similar sequence motifs among these molecules.

Monoclonal antibodies reactive with human T cell lymphotropic virusI (HTLVI) p19 internal core protein: cross-reactivity with normal tissues and differential reactivity with HTLV types I and II.

Three monoclonal antibodies to human T cell lymphotropic virus type I (HTLVI) p19 internal core protein, designated as alpha HTLV-2, 3, and 4, have been developed. In indirect immunofluorescence (IF) assays, these antibodies reacted with acetone-fixed cytocentrifuge preparations of culture HTLVI-infected peripheral blood leukocyte (PBL) from a patient (SD) with Japanese adult T cell leukemia and with infected HUT-102 T cells but not with cultured normal PBL. Anti-p19 antibodies alpha HTLV-2, 3, and 4 all reacted with the same HTLVI p19 identified both by antibodies in HTLVI+ patient sera and by antisera raised against two synthetic peptides encoded by the p19 gag region of HTLVI. Partial proteolytic cleavage of p19 immunoprecipitates obtained with antibodies alpha HTLV-2, 3, and 4 produced a 17,000-dalton cleavage product, in agreement with the size of the fragment predicted from the nucleic acid sequence of the HTLVI p19 gag region. Antibodies alpha HTLV-3 and 4 reacted with HTLVI but not HTLVII proteins and were useful diagnostic probes in identifying HTLVI- but not HTLVII-infected lymphoid cells in immunofluorescence assays. In addition to reacting with HTLVI p19, antibodies 2 and 4 also cross-reacted with a wide variety of HTLV-uninfected normal and neoplastic cells and tissues. In addition, HTLVI+ patient sera contained antibodies that competed for binding to the antigenic site on p19 recognized by antibody 4. Thus, anti-p19 monoclonal antibodies alpha HTLV-2 and 4 reacted with a 19,000-dalton viral-encoded protein of HTLVI and cross-reacted with normal host tissues, while anti-p19 antibody alpha HTLV-3 was specific for HTLVI p19 core protein.