Suppression of rabbit lymphocyte functions by antibodies specific for allotypic membrane determinants

Regulation of immunoglobulin synthesis and secretion was analyzed by exposing spleen cells of b4b4 rabbits to anti-b4 for 24 hr in culture. As noted previously, no lymphocytes with membrane-bound b4 were found immediately after pulse treatment, but substantial regeneration of membrane Ig (mIg) occurred on further culture in antibody-free medium. Splenocytes cultured either in the presence or absence of anti-b4 showed a marked loss of Ig-secreting cells (ISC) after 24 hr in culture but recovered and exhibited peak numbers of ISC on Day 2. However, ISC formation in cultures of antibody-treated cells was significantly suppressed and thereafter declined at a more rapid rate than in control cultures. Polyclonal B cell activators from Nocardia and from gram-negative bacteria stimulated ISC formation in cultures of normal spleen cells, but responsiveness to these activators was depressed following antibody treatment. Antibody-induced suppression of Ig synthesis was attributed to interference with differentiation of B lymphocytes to the secretory stage.     

In vitro immunoglobulin allotype synthesis by spleen cells of heterozygous rabbits. Specific suppression by anti-allotype antibody

The production of immunoglobulin (Ig) bearing the b4 and b5 allotypic markers by b⁴b⁵ heterozygous spleen cells cultured in vitro was assessed by means of a sensitive and reproducible radioimmunoassay. Ig synthesis was demonstrated by the increasing amounts of the b4 and b5 allotypes appearing with time in the supernatant fluids. To determine the effect of anti-b4 or anti-b5 antibody on the synthesis of the b4 and b5 allotypes, spleen cells from b⁴b⁵ heterozygous rabbits were incubated for 24 hr in the presence of anti-b4 or anti-b5 and then washed and cultured for an additional 4 days. Anti-b4 suppressed the production of the b4 allotype with no effect on b5 production, whereas anti-b5 suppressed the production of b5 allotype with no effect on b4 production. This suppression of allotype synthesis in vitro presumably results from an antigen-antibody reaction occurring on the surface of lymphoid cells by a mechanism which may be similar to that which brings about allotype suppression in vivo for fetal and newborn rabbits.     

In vitro studies of the rabbit immune system. VI. Rabbit anti-mouse cytotoxic T effector cells are inhibited by anti-rabbit T cell serum in the absence of complement.

Xenogeneic rabbit anti-mouse cell-mediated cytotoxic activity could be generated by culturing lymphoid cells from mesenteric lymph nodes (MLN), spleen, or peripheral blood of rabbits primed 2 to 8 weeks earlier with mouse tumor or spleen cells. MLN cells, which provided the best source of activity after being cultured with 5 to 10 X 10(6) mitomycin C-treated mouse spleen cells for 4 to 6 days, produced 30 to 90% specific isotope release after 4 to 7 hr incubation with 15Cr-labeled tumor target cells. Xenogeneic cytotoxic activity was primarily H-2 specific and could not be blocked by immune complexes but was abrogated by treatment with goat anti-rabbit thymocyte serum plus complement (ATS + C) before or after culture. Therefore, the activity appeared to be mediated by cytotoxic T lymphocytes (CTL). Furthermore, ATS without C abrogated cytotoxic activity when included in the CTL assay at concentrations of 5 to 15 microliter/10(7) effector cells. The inhibitory activity of ATS was directed to the rabbit effector population and could be absorbed completely by rabbit thymocytes. Antisera to mouse T cells with comparable cytolytic activity in the presence of C did not inhibit murine allogeneic CTL.     

RNA conversion of lymphoid cells to synthesize allogeneic immunoglobulins in vivo

Ribonucleic acid extracts (“5 day immune” and “nonimmune”-RNA) obtained from lymph nodes and spleens of rabbits homozygous for the b⁴ or b⁵ allele of light chain immunoglobulin allotypes were injected iv into nonimmunized rabbits homozygous for the alternate allele. The recipient rabbits were then given multiple iv injections of sheep red blood cells (SRBC). The spleens were assayed 13, 21, and 37 days following the RNA injection for “direct” IgM and “indirect” IgG plaque forming cells (PFC) specific for SRBC. The b4 or b5 light chain allotype and the a1, a2, and a3 heavy chain allotype of the antibody in the plaques was identified by radioautography and by inhibition of plaque formation using anti-allotype antibodies. The b light chain allotype of the RNA donor was identified in 22–32% of the IgM plaques and in 25–42% of the IgG plaques. The allotype of the host rabbit b light chain allotype was identified in 56–67% of the IgM plaques and in 57–71% of the IgG plaques. Likewise the a heavy chain allotype of the RNA donor was identified in 10–19% of the IgM plaques and in 12–19% of the IgG plaques. The allotype of the host rabbit a heavy chain allotype was identified in 51–60% of the IgM plaques and in 55–63% of the IgG plaques. The concentrated lysates of spleen and lymph node cells were also analyzed for immunoglobulins of each light chain allotype by immunodiffusion with radiolabeled antibody. The allotype of both the RNA donor rabbit and host rabbit were found in most of the lysates of lymphoid tissues and in some of the IgG isolated from the serum and concentrated.     

In vitro allotype suppression of spleen cells from immunized rabbits

We tested whether rabbit immune lymphocytes could be suppressed by anti-allotype antibody (Ab) in vitro as shown for normal lymphocytes. Spleen cells (SpC) from rabbits heterozygous at the b locus (b⁴b⁵) of immunoglobulin (Ig) κ chains were treated with IgG preparations of anti-b4 or anti-b5 Ab in vitro for 24 hr (day 1). After this treatment, the SpC were washed and recultured in medium to day 5. The secreted b4- and b5-Ig were quantitated by a radioimmunoassay. SpC from rabbits injected once with sheep red blood cells (SRBC) were allotype-suppressed. Thus, these SpC treated with anti-b4 Ab secreted normal amounts of b5-Ig but secreted much lower amounts of b4-Ig. Similarly, SpC treated with anti-b5 Ab secreted normal amounts of b4-Ig but secreted no detectable b5-Ig. In contrast, SpC from rabbits injected several times with SRBC (hyperimmunized) could not be allotype-suppressed. Hence, the susceptibility of primary immune cells and the resistance of hyperimmune cells to suppression appear to depend on the stage of B-lymphocyte differentiation, presumably because of loss of surface Ig or perhaps because of other changes in the cells as they differentiate during the immune response.     

Comparative suppressive effects of anti-allotype antibodies on spleen cells of immature and adult rabbits

The suppressive effects of anti-allotype antibody on splenic lymphocytes of adult (>3 months of age) and newborn (1-week-old) rabbits were compared in vitro. An approximately 10-fold lower concentration of anti-b4 sufficed to modulate completely membrane-bound b4 immunoglobulin (Ig) when newborn cells were pulse-treated for 2 or 24 hr than when adult cells were tested. In contrast to splenic lymphocytes from adults, which regenerated most of the original proportion of b4⁺ cells in culture following treatment with up to 300 μg of anti-b4 for 24 hr (4), immature lymphocytes were susceptible to irreversible modulation of membrane-bound Ig even when lower concentrations of anti-b4 were used. However, under conditions permitting reversible modulation of membrane b4, lymphocytes of newborns regenerated the membrane product more rapidly than did adult cells. Both mature and immature splenocytes were shown to be capable of some level of b4 synthesis even in the continuing presence of anti-b4. No evidence for susceptibility to complement-mediated killing of newborn spleen cells by anti-allotypic antibodies was obtained. The observations reported here support the concept that the greater sensitivity of B lymphocytes from the newborn to succumb to irreversible suppressive effects by anti-allotype antibody plays a significant role in the restriction for induction of allotype suppression to the perinatal period.     

Production of anti-human thyroglobulin and anti-thyroid hormone antibodies in rabbits immunized with homogenate of human papillary cancer tissue

Papillary cancer tissue of the thyroid gland removed from each of three patients was homogenized in phosphate buffer followed by centrifugation. Each of three rabbits was immunized with each of the supernatants (TC-1, TC-2, TC-3). These rabbits were immunized on days 0, 7, 14, and 21, and serum from each rabbit, obtained 4 weeks after the first immunization, was examined for the presence of anti-human thyroglobulin (HTg), anti-thyroxine (T4), and anti-triiodothyronine (T3) antibodies. Production of anti-HTg antibodies was observed in all three rabbits. In addition, despite the low content of iodine, T3, and T4 in thyroglobulin that had been purified from the papillary cancer tissues (p-HTg), production of anti-T4 and anti-T3 was observed in two of the three rabbits, and the other immunized with TC-1 showed anti-T4 but no anti-T3 antibodies. The significance of the production of anti-thyroid hormone antibodies in rabbits with respect to the antigenic structure of p-HTg with low content of iodine and thyroid hormone is discussed.     

Induction of T-suppressor cells by immunization with allogenic spleen cells in the H-2 system of mice]

The nature of suppressor cells induced by immunization with the allogenic spleen cells and inhibiting the DNA synthesis activation in the mixed lymphocyte culture was studied. Suppressor cells are resistant to mitomycin C and carrageenan. They are not inactivated by the treatment with rabbit anti-B- and anti-Ig- as well as with mouse antibodies (anti-Mls serum) against B lymphocytes in the presence of complement but eliminated by rabbit anti-lymphocyte and anti-T globulins and by mouse anti-theta serum. These T suppressor cells are concentrated in the large lymphocyte fraction in the ficoll gradient. Their blocking of the DNA synthesis activation is evidently non-specific.     

Selective suppression of allotype expression induced in vitro: maintenance of suppression following adoptive transfer

The question of the ultimate fate of lymphocytes subjected to treatment with anti-allotype antibody (Ab) has been investigated by means of an adoptive transfer system that uses noninbred rabbits matched for major histocompatibility antigens and mismatched for allotype. Suppression of b4 immunoglobulin (Ig) production was induced by incubating lymphocytes from b4b5 rabbits with anti-b4 in culture. Transfer of b4-suppressed cells to newborn recipients of allotype b6b6 resulted in stable chimerism of mixed donor-recipient allotypes, in which b4 Ig production remained suppressed. In recipients of non-Ab-treated cells, b4 Ig production predominated over b5, as had been the case in the intact donor. No evidence for stimulation of b4 Ig synthesis was seen, even when lymphocytes and serum from 1-week-old recipients were examined. When lymphoid cells of antigen-primed b4b5 donors were treated with anti-b4 in vitro and transferred, Ab production of the b4 type was specifically suppressed, with compensatory over-production by Ab-forming cells of the b5 type. The results reported here indicate that although anti-allotype Ab is not directly cytotoxic, a significant proportion of the b4-committed cells were irreversibly inactivated as a result of Ab pulse treatment.     

Allelic allotype inclusion and exclusion among rabbit Ig-bearing lymphocytes of peripheral blood and lymphoid organs

Mononuclear cells isolated from peripheral blood, appendix, sacculus rotundus, mesenteric lymph nodes, and spleen of b⁴b⁵ heterozygous rabbits were examined for surface Ig allotypes of the b locus. Ig allotype-bearing cells were detected as cells binding erythrocytes or bacteria coated with monospecific anti-b4 or anti-b5 antibody (Ab). Rosetting the cells with Ab-coated erythrocytes indicated that many peripheral blood lymphocytes, but relatively few appendix cells, bore both the b4 and b5 allotypes. Lymphocytes bearing both the b4 and b5 allotypes were also detected by incubating the cells with a mixture of Escherichia coli coated with anti-b4 Ab and Gaffkya tetragena coated with anti-b5 Ab. The percentage of Ig-positive lymphocytes binding both bacteria was 22–31% in the peripheral blood, 4–6% in the appendix, 3–5% in the sacculus rotundus, 4–10% in the mesenteric lymph nodes, and 5% in the spleen. Thus, the percentage of double-bearing lymphocytes was higher in the blood than in the appendix, sacculus rotundus, mesenteric lymph nodes, or spleen. The b4b5-bearing cells in the blood were not cells with adsorbed cytophilic Ab, since these cells still bore both the b4 and b5 allotypes after pronase digestion and Ig regeneration. These double-bearing lymphocytes, i.e., cells exhibiting allelic allotype inclusion, are probably less differentiated cells.     

Production of immunoadsorbents based on cellulose carbonates and their application to the isolation of specific immunoglobulin light chains

The purification of rabbit immunoglobulin molecules expressing kappa (κ) light chains, utilizing the allotypic specificity b4, has been achieved in stages involving isolation of specific antibody, preparation of a solid phase immunoadsorbent of coupled antibody, and subsequent isolation of b4 (κ) IgG. Cellulose trans-2.3-carbonate is shown to be an effective matrix enabling chemical coupling of antibodies and antigens to the support at neutral pH thus preservng immunological activity. The trans-2,3-carbonate derived from microcrystalline cellulose is more effective as a matrix than the trans-2,3-carbonate derived from macroporous cellulose for the chemical coupling of rabbit a1a3/b4 IgG antigen and binding of specific anti-b4 antibody. The microcrystalline celulose carbonate is also more efficient for the coupling of rabbit anti-b4 antibody and the subsequent binding and elution of rabbit b4 (κ) IgG, thus separating immunoglobulin, expressing kappa light chain, from that expressing lambda light chain. The purification technique has potential application in other allotypic systems and antibody- antigen populations.     

Growth inhibition of human T cells by antibodies recognizing the T cell antigen receptor complex

Monoclonal antibodies that bind to the T cell MHC-antigen recognition complex (anti-T3 or anti-Ti) are known to either mimic ligand binding and activate T cells or block ligand binding, leading to an inhibition of T cell activation. In the present experiments, we demonstrate a direct inhibitory effect on the growth of human T cells by anti-T3 or anti-Ti antibodies. The proliferation of human peripheral blood T cells preactivated by exposure to PHA was inhibited in a specific manner by anti-T3. Colony formation in soft agar by REX cells, a leukemic cell line of early T cell phenotype, was completely inhibited by anti-T3 or anti-Ti antibodies, whereas isotype-matched antibodies to a variety of other T cell markers had no effect. Growth of REX cells in suspension culture was not affected by anti-T3 or anti-Ti. A cell line, T3.N1, was established from an agar colony of anti-T3-resistant REX cells. T3.N1 was phenotypically identical to REX except for failure to express any detectable T3 or Ti surface antigen. T3.N1 colony formation in soft agar was not inhibited by anti-T3 or anti-Ti. There was no rise in [Ca2+]i of T3.N1 cells after anti-T3 or anti-Ti exposure. These results indicate that in addition to the well-known positive regulatory effects of ligand binding to the T3/Ti complex, T3/Ti binding can also result in a down-regulatory signal for human T cell growth.     

Alteration of Immune Responses of Rabbits Infected with Bovine Immunodeficiency-Like Virus

Nine 3-month-old rabbits were inoculated with bovine immunodeficiency-like virus (BIV) to study the pathogenesis of BIV and alteration of the immune responses in experimentally infected rabbits. BIV proviral DNA and anti-BIV antibodies were detected from all rabbits inoculated with BIV-infected bovine embryo spleen (BESP) cells. Rabbits inoculated with spleen cells of the BIV-infected rabbit also converted to proviral DNA-positive and BIV-antibody-positive. The blastogenic responses to concanavalin A of peripheral blood mononuclear cells prepared from BIV-infected rabbits were not significantly different from those from uninfected controls at 2 and 4 months post-inoculation (PI). The humoral immune responses against bovine serum albumin (BSA) were depressed in two of four BIV-infected rabbits at 1 to 3 months PI. The antibody responses against sheep red blood cells (SRBCs) were significantly depressed in all BIV-infected rabbits at 2 to 4 months PI. BIV was rescued by cocultivation of spleen cells of infected rabbits with BESP cells. Distinct development of lymphoid follicle was observed in lymph nodes and spleens of uninfected rabbits which received BSA and SRBCs. In contrast, moderate lymphoid cell depletion was observed in BIV-infected rabbits which received the same immunogens.     

Immunosuppression during viral oncogenesis. IV. Generation of soluble virus-induced immunologic suppressor molecules

We describe herein functional attributes and generation of immunologic suppressor activity elaborated in response to oncogenic virus infection. Malignant rabbit fibroma virus-induced immunologic suppressor factor (VISF) is a T cell product produced in peak quantities by spleen cells taken from infected rabbits 7 days after infection in vivo. Its production does not appear to require macrophage participation. VISF is highly labile, 3.5 to 12 kDa, and capable of suppressing both B and T lymphocytic responses. Indomethacin and the cyclic nucleotides cAMP and cGMP inhibit its generation. VISF activity is neither antigen nor species specific. It suppresses murine and leporine immune responses to antigens unrelated to the inducing virus. Comparable suppressor activity may be induced by infecting an apparently non-functional rabbit T lymphoma line, RL-5, with malignant rabbit fibroma virus. VISF is principally a suppressor-inducer factor: in vitro, lymphocytes exposed to VISF do not show decreased immunologic responsiveness until 4 days of culture. VISF induces T suppressor cell activity when normal spleen cells are exposed briefly to VISF. Thus, immunosuppressive consequences of malignant fibroma virus infection are partially mediated by a small, non-specific T cell-derived suppressor lymphokine with unique functional characteristics. Non-specific immunologic dysfunction that often attends virus infections may reflect the activity of such factors in humans as well.     

Neutralizing monoclonal antibodies to hepatitis A virus: partial localization of a neutralizing antigenic site.

BALB/c mice were immunized with purified preparations of hepatitis A virus (HAV) isolated after 21 days of growth in LLC-MK2 cells. The HAV antigen was isolated from CsCl gradients and consisted primarily of the following three proteins as analyzed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining: VP-1 at 33,000 daltons, VP-2 at 29,000 to 30,000 daltons, and VP-3 at 27,000 daltons. The spleen cells isolated from two BALB/c mice, immunized with two inoculations of HAV, were fused with SP 2/0 myeloma cells and grown in hypoxanthine-aminopterin-thymidine medium. Of 270 hybridomas initially screened, 72 were positive for binding HAV by a noncompetitive radioimmunoassay. All 72 were tested for the ability to neutralize the infectivity of HAV in an in vitro cell culture assay that was adapted for microtiter plates and that used detergent-treated virus for improved neutralization sensitivity and newborn cynomolgus monkey kidney cells for rapid growth. Eighteen hybridomas were positive for neutralization; 16 remained stable. Of the 16, 9 were able to compete with labeled polyclonal serum for binding to HAV. The nine competing hybridomas could be separated into two groups which appear to be directed towards two different sites on HAV and could complement each other in the competitive radioimmunoassay against polyclonal sera. Of the original 16 neutralizing hybridomas, 4 were subcloned through two cycles of limit dilutions. All four monoclonal antibodies retained their original neutralizing and competitive properties; three were immunoglobulin G2a, and one was immunoglobulin G1. All four monoclonal antibodies readily precipitate whole 125I-labeled HAV but are not able to recognize the disrupted proteins of the virus (as tested by immune precipitations of heat- and detergent-disrupted virions or Western blot analyses). However, the heterobifunctional cross-linking reagent toluene-2,4-diisocyanate was used to cross-link purified Fab fragments of two different monoclonal antibodies (2D2 and 6A5) to HAV before disruption. This reagent demonstrated a specific reaction of the monoclonal antibodies to the VP-1 of HAV, suggesting this major surface protein contains at least one of the major neutralization sites for HAV.     

Evaluation of the potency of the anti-idiotypic antibody Ab2/3H6 mimicking gp41 as an HIV-1 vaccine in a rabbit prime/boost study

The HIV-1 envelope protein harbors several conserved epitopes that are recognized by broadly neutralizing antibodies. One of these neutralizing sites, the MPER region of gp41, is targeted by one of the most potent and broadly neutralizing monoclonal antibody, 2F5. Different vaccination strategies and a lot of efforts have been undertaken to induce MPER neutralizing antibodies but little success has been achieved so far. We tried to consider the alternative anti-idiotypic vaccination approach for induction of 2F5-like antibodies. The previously developed and characterized anti-idiotypic antibody Ab2/3H6 was expressed as antibody fragment fusion protein with C-terminally attached immune-modulators and used for immunization of rabbits to induce antibodies specific for HIV-1. Only those rabbits immunized with immunogens fused with the immune-modulators developed HIV-1 specific antibodies. Anti-anti-idiotypic antibodies were affinity purified using a two-step affinity purification protocol which revealed that only little amount of the total rabbit IgG fraction contained HIV-1 specific antibodies. The characterization of the induced anti-anti-idiotypic antibodies showed specificity for the linear epitope of 2F5 GGGELDKWASL and the HIV-1 envelope protein gp140. Despite specificity for the linear epitope and the truncated HIV-1 envelope protein these antibodies were not able to exhibit virus neutralization activities. These results suggest that Ab2/3H6 alone might not be suitable as a vaccine.     

T11/CD2 activation of cloned human natural killer cells results in increased conjugate formation and exocytosis of cytolytic granules

The T11 (CD2) antigen has been found to be an alternate pathway for antigen-independent activation of resting T cells. T11 triggering also results in activation of NK cells and enhancement of their cytolytic function. The present studies were carried out to further define the mechanisms whereby cytotoxicity is enhanced after T11 activation. A series of clonal human NK cell lines were analyzed after incubation with monoclonal anti-T112 and anti-T113 antibodies specific for different epitopes of the CD2 protein. Anti-T112/3 triggering resulted in increased cytotoxicity against a variety of target cells. Similar results were obtained with F(ab')2 fragments of anti-T112/3, indicating that this effect was not mediated through binding of FcR. The induction of cytotoxicity was found to be associated with increased formation of effector cell-target cell conjugates and with release of secretory granule-localized 35S-labeled proteoglycans. Both enhanced conjugate formation and cytotoxicity could be blocked by anti-lymphocyte function-associated antigen (LFA-1) mAb. Ultrastructural analysis of NK cells after T11 activation demonstrated increased adherence of effector cells to targets and other NK cells as well as a directional reorientation of cytoplasm and intracellular granules toward the area of contact between cells. Discharge of granules occurred into pockets bounded by closely apposed plasma membranes. In the presence of anti-LFA-1 and anti-T112/3, the close apposition and formation of pockets between effector cells and target cells did not occur but the cells exocytosed their intracellular granules. T11 activation of NK cloned cells also resulted in the formation of the homotypic conjugates and autocytotoxicity. As seen with resistant allogeneic targets, autocytotoxicity was mediated by F(ab')2 fragments of T112/3 antibodies and could be blocked by anti-LFA-1 antibody. Ultrastructural analysis of NK cloned cells after T11 activation confirmed the presence of homotypic conjugates with reorientation of effector cells toward one another and discharge of cytolytic granules into pockets formed between NK cloned cells. Taken together, these results indicate that T11-induced cytolytic function of NK cells is, in part, mediated through increased binding of effector cells and targets and that enhanced conjugate formation is at least in part mediated by the LFA-1 antigen. In addition, T11 activation results in the triggering of the cytolytic mechanism of NK cells and the exocytosis of cytolytic granules and their constituents.     

Rabbit collagenase. Immunological identity of the enzymes released from cells and tissues in normal and pathological conditions.

1. The immunological cross-reactivity between rabbit collagenases from a variety of normal and pathological sources was examined. The specific antibody raised against collagenase secreted from normal rabbit synovial fibroblasts gave reactions of complete identity with collagenases secreted from fibroblasts derived from rabbit skin, and from synovium from experimentally arthritic rabbits. 2. The rabbit fibroblast collagenase was immunologically identical with collagenases obtained from the organ culture medium of normal rabbit skin, synovium, ear fibrocartilage and subchondral bone. 3. Collagenases from the culture media of normal rabbit synovium and from hyperplastic synovium of rabbits made experimentally arthritic were identical. 4. The collagenase secreted from rabbit fibroblasts gave a reaction completely identical with that of a collagenase extracted directly from a rabbit carcinoma. 5. IgG (immunoglobulin G) from a specific antiserum to rabbit fibroblast collagenase was a potent inhibitor of the collagenases obtained from the culture media of the various rabbit cells and tissues. 6. Collagenases from human synovium and from mouse macrophages and bone were neither precipitated nor inhibited by antibodies to rabbit collagenase. 7. No immunoreactive material was found in lysates of rabbit polymorphonuclear leucocyte granules with the specific antisera to rabbit fibroblast collagenase. No evidence for inactive forms of rabbit collagenase in lysates of the rabbit synovial fibroblasts could be found, either by double immunodiffusion against the specific collagenase, or by displacement of active enzyme from inhibition by the IgG.     

Serologic and mucosal immune response to rotavirus infection in the rabbit model.

We examined the humoral immune response to rotavirus infection in specific pathogen-free rabbits inoculated and challenged orally with rabbit Ala rotavirus (7.5 x 10(5) to 1 x 10(7) PFU). The humoral immune response in both serologic and mucosal samples was monitored by using total antibody enzyme-linked immunosorbent assays (ELISAs), isotype-specific ELISAs, and plaque reduction neutralization assays. Following a primary infection, all rabbits shed virus and serologic and mucosal antibody responses were initially detected by 1 week postinoculation. Intestinal immunoglobulin M was detected by 3 days postinoculation, and secretory immunoglobulin A was detected by 6 days postinoculation. Following challenge, rabbits were protected (no detectable virus shedding) from infection. An anamnestic immune response was observed only with mucosal neutralizing antibodies, and all serologic and mucosal immune responses persisted at high levels until at least 175 days postchallenge (204 days postinoculation). Detection of neutralization responses was influenced by the virus strain used in the neutralization assay; all inoculated rabbits developed detectable serum and intestinal neutralizing antibodies against the infecting (Ala) virus strain. Neutralization activity in both serum and mucosal samples was generally, but not exclusively, homotypic (VP7 serotype 3) after both primary and challenge inoculations with Ala virus. Heterotypic serum neutralization activity was observed with serotype 8 (9 of 12 rabbits) and 9 (12 of 12 rabbits) viruses and may be based on reactivity with the outer capsid protein VP4 or on a shared epitope in the C region of VP7. Comparisons of heterologous (serotype 3) and heterotypic neutralizing responses in mucosal and serologic samples revealed that 43% (21 of 49) of the responses were discordant. In 19 of 49 (39%) of these cases, a heterotypic serologic response was seen in the absence of a heterotypic mucosal response, but in 2 of 49 (4%) instances, a heterotypic mucosal response was seen in the absence of a concomitant serologic response. These results provide insight into factors which may affect detection of heterotypic responses.     

Analysis of the monocyte Fc receptors and antibody-mediated cellular interactions required for the induction of T cell proliferation by anti-T3 antibodies

The induction of human T cell proliferation by antibodies that cross-link T3 antigens is dependent on functional interactions of anti-T3 antibodies with monocyte Fc receptors. In this report, we used a panel of anti-T3 antibodies of differing heavy chain isotype and a variety of other monoclonal antibodies to analyze several features of the antibody-mediated interactions between T cells and monocytes that are required for mitogenesis. Whereas three IgG2a anti-T3 antibodies were mitogenic for cells from all individuals, IgM and IgG2b anti-T3 antibodies did not induce T cell proliferation in any donor and could block the proliferative responses induced by other mitogenic anti-T3 antibodies. Dose-response analyses with four IgG1 anti-T3 antibodies demonstrated donor heterogeneity as reported by other investigators. However, in contrast to these previous reports, high concentrations of IgG1 anti-T3 antibodies were found to be mitogenic for all donors, indicating that this heterogeneity is based on relative rather than absolute defects in low responder monocytes. Cell mixing experiments in which monocytes from two different low responder donors were co-cultured with T cells and IgG1 anti-T3 antibodies did not identify any complementary defects, suggesting that the low responder phenotype results from a relatively restricted polymorphism. To assess the nature of the signals required for inducing T cell proliferation, nonmitogenic anti-T3 antibodies were co-cultured with other pan-T cell antibodies having the IgG2a isotype. The combination of signals from T3 antigen cross-linkage and those independently generated by other IgG2a antibodies bound to monocyte Fc receptors did not induce T cell proliferation. Hence, it appears that the T3 antigen or closely associated structures must be clustered at the monocyte membrane for mitogenesis. Finally, in competitive inhibition experiments, the isotype specificity of monocyte Fc receptors involved in the induction of T cell proliferation was examined. Two distinct Fc receptor sites, one that binds murine IgG2a and IgG3 antibodies and a second that binds murine IgG1 antibodies, were identified. Murine IgM or IgG2b did not appear to bind either of these receptor sites. Taken together, these data indicate that human monocytes have two distinct Fc receptor sites, which must specifically and directly interact with T cell-bound anti-T3 antibodies for mitogenesis.