Human T cells recovered from human/Balb radiation chimeras are hypersensitive to human immunodeficiency virus type 1 infection.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated by virus-encoded regulatory proteins, as well as by a variety of cellular factors. Productive infection of human T lymphocytes by HIV-1 is dependent upon the activation status of the target cells. In general, short-term mitogenic stimulation of CD4 T cells is used to enhance infection of peripheral blood mononuclear cells (PBMC) in vitro. Recently, we demonstrated that adoptive transfer of human PBMC into lethally irradiated BALB/c mice, radioprotected with severe combined immunodeficiency (SCID) mouse bone marrow, leads to marked T-cell activation and proliferation. In the present study, we investigated the effect of such xenoactivation of human T cells on their susceptibility to HIV-1 infection. Human cells that were recovered from human/Balb radiation chimeras supported efficient replication of laboratory strains of HIV-1, as well as of HIV-1 clinical isolates. The multiplicity of infection required to attain effective virus replication in the recovered xenoactivated human cells was 10- to 100-fold lower than that needed for infection of short- or long-term phytohemagglutinin (PHA)-stimulated blasts or of various T-cell lines. Analysis of human cell surface activation markers has indicated that xenoactivation in the mouse, in contrast to in vitro stimulation with PHA, is associated with a marked downregulation of CD25 (interleukin 2 receptor). Our results demonstrate that human cells recovered from human/Balb radiation chimeras, which are hypersensitive to HIV-1 infection, differ from in vitro-stimulated cells in their activation status. Therefore, this system could be used to study host factors that participate in HIV-1 infection and replication in vitro and in vivo.

A polymer containing a repeating peptide sequence can stimulate T-cell-independent IgG antibody production in vivo.

The immunogenicity of a CD4 peptide sequence 303-315 (V4 domain) was determined for three forms of the peptide: (a) a polymer consisting of repeating peptide units, (b) a peptide linked to a large protein carrier, chicken serum albumin, and (c) unmodified free peptide. Two in vivo systems were used: a T-cell-competent BALB/c mouse and a T-cell-deficient nude mouse. In BALB/c mice the IgG antibody responses to the peptide-CSA conjugate were at least 100-fold greater than the response to the polymer, and the free peptide gave no response. However, in contrast to the normal BALB/c mice, in the nude mice the polymer produced the only significant response. Nude mice immunized with the polymer produced a predominantly IgG response with almost equal amounts of IgG1 and IgG 2a + 2b. This IgG subclass distribution was very similar to that obtained in the BALB/c for the peptide-CSA conjugate. Positive sera from both groups were able to react with immobilized CD4 molecule on Western blots, confirming the specificity of both the T-cell-independent as well as T-cell-dependent response. These results suggest that repeating epitopes on the same peptide polymer molecule may cause cross-linking of antigen-specific immunoglobin receptors on B-cells, and thus behave as a relatively T-cell-independent immunogen which can induce isotype switching.

CD4-derived synthetic peptide blocks the binding of HIV-1 GP120 to CD4-bearing cells and prevents HIV-1 infection.

The T cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. In order to identify primary sequences within the CD4 molecule that may be involved in the binding of the HIV-I envelope, we synthesized various peptides corresponding to the V1, V2, V3, and V4 domains of CD4. We tested the ability of these peptides to block the binding of purified HIV-I gp120 to CD4+ human lymphoblastic leukemia cells (CEM) using fluorescence-activated cell sorting. One of these peptides, corresponding to CD4 amino acids (74-95), when preincubated with gp120, blocked its subsequent binding to CEM cells by 80%. A truncated form of this peptide (81-95), was found to be as efficient as the longer peptide (74-95) in inhibiting the binding of gp120 to CEM cells. The same peptide did not block the binding of OKT4A or Leu3A anti-CD4 monoclonal antibodies, which were previously shown to block HIV-I binding to CD4. The peptides were also tested for their ability to block HIV-I infection of a T cell line in vitro. Only CD4 peptide (74-95) and the shorter fragment (81-95) succeeded in protecting T cells against infection with different HIV-I strains. All the other peptides examined had no effect on gp120 binding to CEM cells and did not block syncytia formation. Goat polyclonal antibodies against the CD4 peptide (74-95) gave modest interference of gp120 binding to CEM cells. These data suggest that the CD4 region (74-95) participates in the CD4-mediated binding and/or internalization of HIV-I virion.

Chemically induced CD4 mutants of a human T cell line. Evidence for dissociation between binding of HIV I envelope and susceptibility to HIV I infection and syncytia formation.

This study describes the derivation of a series of mutants from the human leukemic cell line CEM using the frame shift mutagen Ethyl-methanesulfonate followed by negative selection with multiple treatments of OKT4A + C, and sorting into CD4-, CD4-dull, and CD4-intermediate mutants. These mutants express reduced CD4 levels ranging from 0 to 60% of the parental line. The mutants were analyzed by staining with a battery of CD4-specific mAb, by assessing their ability to bind soluble gp120, and by their ability to form syncytia after infection with cell-free HIV I virus and a gp160-vaccinia vector. Two groups of particularly interesting mutants were identified: (1) CD4-dull mutants expressing only 5 to 10% of the wild type surface CD4 density, which nevertheless were infectable by HIV I and produced as many syncytia and reverse transcriptase activity as the parental line after infection with gp160-vaccinia or cell free HIV I. (2) CD4-intermediate mutants (30 to 60% of parental CD4 level), which express CD4-epitopes required for interaction with the HIV I envelope protein, yet are markedly deficient in their ability to form syncytia after gp160-vaccinia or HIV I infection. Two of these mutants did form syncytia after transient reconstitution with a wild type CD4 containing vaccinia vector. Inasmuch as they were found to bind soluble gp120 with the same avidity as other, functionally normal, CD4-intermediate mutants, these human T cell mutants may have a reduced susceptibility to HIV I infection due to the absence of a "fusogenic component" or to a structural alteration in a region of the CD4 molecule not required for binding of the HIV I envelope, but for the subsequent fusion and entry process.

Isotype specificity of antigen-specific helper clone in vivo.

Inoculation of an immortalized clone of radiation leukemia virus (RadLV)-transformed antigen (ovalbumin, OVA)-specific T cells together with the relevant carrier (OVA) into unprimed syngeneic mice results in a preferential increase in the expression of anti-OVA antibodies of the immunoglobulin (Ig)G2b and IgG2a isotypes. Identical boosting of the clone-primed mice further augments the preferential production of anti-OVA antibodies of these two isotypes. The class-related helper activity is not due to nonspecific shift of class expression produced by the injected tumor cells, as a non-helper clone of RadLV-transformed T cells does not change the isotypic pattern of anti-OVA antibodies in the inoculated mice. A carrier-specific activation of the B cells is responsible for the class-restricted function of the helper clone. The isotypic profile of anti-hapten antibodies in mice injected with 2,4-dinitrophenyl (DNP)-bovine serum albumin and OVA-specific helper clone is not altered. On the other hand, mice inoculated with the OVA-specific helper clone and DNP-OVA respond with a preferential elevation of anti-DNP antibodies of the IgG2a and IgG2b isotypes. The preferential class augmentation may result from carrier-specific signals delivered by the helper clone which activate B cells in vivo toward certain CH expression. Alternatively, the observed class pattern may be induced by an isotype noncommited helper clone which triggers selected population of B lymphocytes of defined differentiation status toward secretion of a restricted array of isotypes. Regardless of the mechanism of the clone-dependent class expression, the isotypic profile in most of the experiments clearly demonstrates that an antigen-specific helper clone may be one of the elements which regulates the class of antibodies to be produced in vivo under normal physiologic conditions.

Local and peripheral cell-mediated immune response to influenza virus in mice.

Presentation of different influenza virus antigens generates different immune responses. Intranasal immunization with either live (VA) or formalin-inactivated (VF) A/PR/8/34 (HON1) influenza virus induced local as well as peripheral cell-mediated immune response (CMI), as evidenced by elevation in 3H-thymidine incorporation. Cell-mediated immune response was detected as soon as 24-48 hr following the application of VA and 4-5 days following VF. Cell-mediated immune response in both instances peaked on the 12th day and disappeared between 16 and 20 days after application. Local CMI response was threefold higher after immunization with VA (SI = 28.6) than with VF (SI = 9.4), while VF induced higher peripheral response (32.0 vs 17.7). The mononuclear cell population in the lungs increased, correlating with a rise in the stimulation index (SI). The percentage of IgA surface-bearing B lymphocytes was significantly higher following IN administration of VA, but not following VF instillation. This corroborated the finding that VF failed to induce local antibody response in the lungs in spite of its capacity to stimulate humoral antibody and CMI responses. Mice immunized intramuscularly with both viral preparations developed a fair humoral antibody response without detectable CMI (peripheral or local).

Cellular response in humans following vaccination with Gripax influenza virus.

Cellular response of peripheral blood lymphocytes to influenza antigens was measured in a group of young nurse-student volunteers (17-24 years old), following vaccination with a formol-inactivated trivalent influenza vaccine (Gripax). Cord blood lymphocytes (controls) did not react with any of the antigens. This excluded the possibility of any nonspecific mitogenicity of viral antigens. Viability of the cells was indicated by their responsiveness to phytohemagglutinin (PHA). Prior to immunization antigenic recognition to circulating strains (A/England (H3N2) and B/Hong Kong) was found in about 44% of the vaccinees; recognition of the recent strain A/USSR (H1N1) was found in only 10.5%. Following vaccination, approximately 80% of the subjects exhibited cellular response to all three vaccine strains. This includes the negative subjects, who showed an approximate 70% rate of conversion. There was no correlation between the antibody state and cellular response prior to and following vaccination as gathered from matched data of each participant.