Effect of thymus cell injections on germinal center formation in lymphoid tissues of nude (thymusless) mice

Nude mice, partially backcrossed to Balb/c or DBA/2, were injected iv with 5 × 10⁷ thymus cells from the respective inbred strain. The response of these mice to immunization with Brucella abortus antigen was studied, with respect to both antibody production and the formation of germinal centers in their lymphoid tissues. The results were compared to those obtained with nude mice to which no thymus cells were given, as well as to Balb/c, DBA/2, or +/? litter mate controls.Nude mice formed less 19S as well as 7S antibody than did litter mate controls and completely lacked germinal centers in lymph nodes and gut-associated lymphoid tissue. Those nude mice which had been injected with thymus cells made a much better secondary response, both for 19S and for 7S antibody, and had active germinal centers in their lymph nodes as early as 3 wk after thymus cell injection. Intestinal lymphoid tissue in nude mice showed only slight reconstitution of germinal center activity several months after thymus cell injection and none at earlier times. Irradiated (3000 R) thymus cells appeared as effective as normal cells in facilitating germinal center appearance and 7S antibody production in the nude mice.     

High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions

Celiac disease is an immune-mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2) are generated in response to the exogenous antigen gluten in individuals who express human leukocyte antigen HLA-DQ2 or HLA-DQ8 (ref. 3). We assessed in a comprehensive and nonbiased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire of ex vivo-isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are markedly expanded within the duodenal mucosa in individuals with active celiac disease. TG2-specific antibodies were of high affinity yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts, the TG2-specific ASCs had not recently proliferated and were not short-lived ex vivo. Altogether, these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favor massive generation of autoreactive B cells. TG2-specific antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naive IgD- and IgM-expressing B cells, thus possibly explaining why the antibody response to TG2 bears signs of a primary immune response despite the disease chronicity.     

Cellular immune response to viral infection: in vitro studies of lymphocytes from mice infected with Sindbis virus

The development and evanescence of cell-mediated immunity to Sindbis virus infection in the mouse was studied using in vitro lymphocyte transformation. Adult mice were inoculated subcutaneously with Sindbis virus, a group A arbovirus, and cells from the draining lymph nodes and spleen were examined temporally for their ability to incorporate ³H-Tdr in the presence of Sindbis virus antigen in vitro. Lymphocyte transformation was shown to be specific and dose-related. Better stimulation was obtained with live virus antigen than with inactivated virus antigen. Specific ³H-Tdr incorporation was markedly reduced when lymph node cells were pretreated with anti-θ and complement, but anti-mouse immunoglobulin also reduced the response. Specifically sensitized cells were present in the draining lymph nodes 3–4 days after primary Sindbis virus infection, peaked at 6 days, and returned to control levels by 16 days. The response in the spleen appeared later and disappeared later. Neutralizing antibody appeared by Day 4, rose rapidly, and plateaued at a high level. The secondary cellular response differed from the primary response by being somewhat earlier and being elicitable with an amount of inactivated virus antigen which was insufficient to produce a primary response.     

Virus-mediated induction in human lymphocytes of antibody-independent cytotoxicity (VDCC) and enhancement of antibody-dependent cytotoxicity (ADCC) against natural killer-resistant tumor target cells

The effect of Parotis virus on the in vitro cytotoxicity of human lymphocytes against NK-resistant mouse mastocytoma cells was studied. In the ⁵¹Cr-release assay, treatment of lymphocytes with virus induced a rapid cytotoxicity in the absence of anti-P8 15 antibody (virus-dependent cellular Cytotoxicity, VDCC) and strongly enhanced antibody-dependent cytotoxicity (ADCC). At the effector cell level, virus treatment was found to increase the frequency of target-binding cells (TBC) as well as the proportion thereof mediating VDCC and/ or ADCC, indicating recruitment of active effector cells. The recruited cells were heterogeneous but contained a major fraction bearing the T-cell-associated antigen T3. Virus was found to decrease rather than to increase the recycling capacity of the cytotoxic lymphocytes, suggesting that VDCC induction and ADCC enhancement were due to a virus-mediated improvement of effector cell-target cell interactions. VDCC and ADCC enhancement may be of protective importance in early phases of virus infection as well as for the production of nonspecific tissue injuries associated with viral disease.     

Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells

The expression and stability of the proliferation-associated nuclear antigen detected by Ki-67 antibody have been investigated in human promyelocytic leukaemic HL-60 cells in relation to their progression through the cell cycle. Expression of this antigen was minimal in late G1 and early S phase cells. The antigen accumulated in the cells predominantly during S phase, and its rate of increase per cell accelerated during the second half of this phase. The accumulation of Ki-67 antigen during S exceeded the increase in DNA content, and thus the Ki-67/DNA ratio rose 80% from late G1 to G2 + M. This antigen rapidly disappeared from post-mitotic cells. The half-life of this protein estimated in post-mitotic cells during stathmokinesis induced by vinblastine appeared to be shorter than 1 h. This rapid turnover should be compared with the relatively long (6-8 h) duration of G1 of the studied cells. In cells in which de novo protein synthesis was inhibited by 0.1 microgram/ml cycloheximide, the half-life of the Ki-67 antigen was also found to be about 1 h regardless of the cell position in the cell cycle. Thus, the data suggest that variations in the level of this protein during the cell cycle are a consequence of its different synthesis rate rather than phase-specific changes in the rate of its degradation. Because the late G1 and very early S phase cells express the antigen at levels only slightly above background, it is possible that, when using Ki-67 antibody as a marker of the cell growth fraction, some late G1 cells can be erroneously classified as non-cycling cells.     

Autologous or syngeneic mixed lymphocyte reaction in bone marrow and thymic chimera mice

Unfractionated peripheral blood mononuclear (UM) cells from adult donors of known serological status wtih respect to Epstein-Barr (EB) virus were exposed to four or more successive in vitro stimulations with irradiated cells of the autologous EB virus-transformed cell line at a responder: stimulator ratio of 4:1, and effector UM and T cells were prepared after each stimulation. Ten out of fourteen seropositive donors and all four seronegative donors thus tested showed at best moderate cell proliferation over two or three stimulations only and a cytotoxic response which became dominated by non-E-rosette-forming cells active against the K562 cell line but not against EB virus-transformed lymphoblastoid lines. Cocultures from three other seropositive donors gave stronger proliferative responses and yielded effector cells dominated by a polyclonal E-rosette-forming population cytotoxic to the autologous and to certain allogeneic (both HLA-related and -unrelated) EB virus-transformed cell lines as well as to some but not all EB virus genome-negative hemopoietic cell lines of the kind sensitive to natural killer cells. With one other seropositive donor, this same repeated stimulation induced a quite different type of cytotoxic response, selectively amplifying an effector T-cell population which appeared on the basis of target cell specificity and of sensitivity to monoclonal antibody blocking to be both EB virus-specific and HLA-A and B antigen restricted in its function.     

On the mechanism of early recovery of specifically depleted lymphoid cell populations by nonspecific activation of T cells.

Specific depletion from normal CBA mouse spleen cells of those bound on pigeon erythrocyte (PRBC) immunoabsorbent columns before transfer of the depleted population into irradiated syngeneic recipients resulted in elimination of the anti-PRBC responsiveness as assessed by rosette (RFC) and hemolytic plaque (PFC) formation. The anti-sheep erythrocyte (SRBC) responses of cell populations treated in the same manner remained unimpaired. When, however, these populations were stimulated with both PRBC and muramyl dipeptide (MDP), an early recovery of specific anti-PRBC responsiveness was produced. PFC response in particular, suddenly increased between the fourth and fifth day after transfer and stimulation thus exhibiting a doubling time of only 4 to 6 hr. This effect of MDP was T-cell dependent since treatment of the depleted population with anti-θ antigen serum and complement hindered early recovery. Depleted populations stimulated with PRBC alone resumed their T-dependent RFC (but not PFC) responsiveness after the eighth day. In spite of the existence of these educated T cells, a second stimulation on the tenth day with PRBC was unable to elicit a specific PFC response. On the other hand stimulation with MDP alone on the day of cell transfer (Day 0) followed by stimulation with PRBC on Day 10 resulted in a specific PFC response on Day 15. Thus, MDP appeared to do more than simply promote education of T cells by antigen. In vitro cultures of depleted populations also recovered their specific reactivity when stimulated by antigen and MDP.     

Immunity to herpes simplex virus type 2 (HSV-2). I. Development of virus-specific lymphoproliferative and leukocyte migration inhibition factor responses in HSV-2-infected guinea pigs

The development of virus-specific cell-mediated immune (CMI) memory and effector response was studied in strain 13/N guinea pigs infected with herpes simplex virus type 2 (HSV-2) (G). The indirect leukocyte migration inhibition factor (LIF) and the lymphocyte transformation (LT) assays, chosen as probable indicators of effector and memory responses, respectively, were performed simultaneously on spleen cells (SC) obtained at varying times after infection and cultured in the presence of uv-inactivated HSV-2 (G) antigen. Kinetic and dose-response analyses revealed: (i) a time-dependent increase in the magnitude and antigen sensitivity of the LT response as well as a time-dependent decrease in the in vitro “doubling time,” both suggestive of immune maturation, and (ii) a biphasic pattern of LIF production in vitro consisting of an “early” component generated within the first 24 hr in culture, and a “late” component detected between 3 and 6 days in culture. “Late” LIF production correlated well with the lymphoproliferative response and appeared to require the presence of glass-adherent cells and active cell division.     

Antigen transport II. The significance of the localization of antigen-laden cells in the spleen

Previous studies have demonstrated that macrophage-like cells transporting antigen, e.g., human serum albumin (HSA) appear in thoracic duct lymph and blood shortly after antigen injection. The in vivo migration of these antigen-laden (Ag-L) cells from the blood stream was examined systematically by transferring Ag-L cells bearing ¹²⁵I-labelled HSA into syngeneic rats. There was no evidence autoradiographically that Ag-L cells migrated into lymph nodes, but the localization in the spleen followed a defined pattern: within the first hours after transfer, a majority of radiolabelled cells were identified in the marginal zone; by 3 hr and up to 4 days later, 60–80% of labelled cells were resident in the red pulp; Ag-L cells failed to migrate into the white pulp in significant numbers. Ag-L cells which had localized to the spleen, when examined 3 and 18 hr after transfer using combined autoradiography and immunoperoxidase staining, did not express la determinants in situ. The ability of Ag-L cells to stimulate an adoptive secondary response was tested in splenectomized, irradiated recipients receiving HSA-specific memory cells. Removal of the spleen before transfer severely reduced the antibody response evoked by Ag-L cells transporting HSA, thus indicating the functional importance of antigen transport to the spleen. Since Ag-L cell migration was primarily into the red pulp, we have considered whether the red pulp may provide a relevant microenvironment for lymphocyte/ antigen interaction.     

Characteristics of antibodies to the epidermal growth factor receptor-kinase

Polyclonal antibodies to different antigenic forms of the epidermal growth factor (EGF) receptor-kinase from human A-431 cells have been produced, and their properties have been characterized and compared. Biochemically active receptor-kinase purified by affinity chromatography was employed as one type of antigen. Denatured receptor-kinase prepared by sodium dodecyl sulfate-gel electrophoresis of the affinity-purified receptor was used as the second type of antigen. Animals immunized with either type of antigen produced antibody capable of immunoprecipitating the receptor-kinase molecule. Antibodies produced in response to the biochemically active antigenic form of the receptor-kinase are capable of blocking ¹²⁵I-EGF binding to the receptor and inhibited EGF-stimulated biological responses. These antisera are not species specific in their ability to inhibit growth-factor binding to the EGF receptor of various mammalian cells. However, these rabbit antisera were unable to inhibit ¹²⁵I-EGF binding to rabbit cells. Although antisera produced in response to the denatured receptor-kinase molecule are not able to block ¹²⁵I-EGF binding or EGF-stimulated biological responses, they are particularly efficient for the immunoprecipitation of solubilized ¹²⁵I-EGF:receptor complexes. None of the antisera contain antibodies capable of interfering with basal receptor-kinase phosphorylation activity. Although each of the antisera immunoprecipitated this kinase activity, none of the antisera contained antibody which served as a phosphorylation substrate for the EGF receptor-kinase in contrast to the immunoglobulins present antisera to the src gene product of the Rous sarcoma virus.     

In vitro generation of K562 killers in human T-lymphocyte subsets

Group A streptococcal pyrogenic exotoxin (SPE) is a potent modulator of the immune system when used experimentally in mice. Typically, a late burst of plaque-forming cells (PFC) follows an early suppression of the antibody response in appropriately immunized and SPE-treated mice or their spleen cells in vitro. This altered response to antigen caused by SPE is termed a deregulated antibody response. The site of action of SPE was studied by use of cellular reconstruction and complementation experiments using the separated subpopulations of immunocytes which are required for full expression of mouse spleen PFC responses to sheep erythrocytes or to trinitrophenylated (TNP) rabbit erythrocytes in vitro. The SPE site was thus localized to the T-cell subpopulation. Recently SPE has been purified to a very high degree, making it possible to ascertain that SPE alone generates the deregulation of the immune system as described before and to limit the role of nondefined components of cruder preparations of SPE. A purified horse anti-scarlet fever antitoxin which recognizes highly purified SPE as being homogeneous also recognized a single component of crude SPE by agar-gel analysis. A rabbit anti-SPE immunoglobulin raised against crude SPE and absorbed with killed, strain NY5, Group A streptococci recognized the pure SPE and a major component of the homologous crude SPE similarly. Both of these antisera neutralized the capacity of SPE to deregulate the in vitro PFC response to TNP almost completely. A third antiserum raised in rabbits against a NY5 Group A streptococcal whole cell vaccine recognized a different component of crude SPE and totally failed to recognize pure SPE. This antiserum also recognized a purified Group A streptococcal peptidoglycan as being related to components contained in the crude SPE preparation. This antiserum, however, totally failed to neutralize the capacity of SPE to deregulate the PFC response to TNP. These results show that SPE-A is the active component of cruder preparations of SPE which deregulates PFC responses.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Selective survival and expression of B-lymphocyte memory cells during long-term serial transplantation

The expression of individual clonal products during long-term in vivo culture was investigated using a rabbit model system of bone marrow transplantation. RLA (MHC) matched rabbits were deliberately mismatched for kappa light chain immunoglobulin allotype to facilitate identification of antibodies as being of donor or recipient origin. Recipients of cells from antigen-primed donors responded to antigen stimulation with antibody of donor origin, showing that cells were effectively triggered for antibody production in the recipient. Isoelectric focusing followed by affinity immunoblotting of the expressed antibodies showed that the responding B-cell clonotype repertoire remained virtually unchanged throughout the extensive cell transfer protocol used. These results suggest that B-memory-cell stimulation, rather than stem cell differentiation, was responsible for the observed response patterns. There was no detectable increase in the heterogeneity of the donor-derived antibody response with time and no new clonotypes appeared which were not present in the cell donor. Unlike previous studies, early stimulation with antigen was not required for successful engraftment and memory cell establishment. However, our data suggest that the timing of antigenic challenge may determine which of the donor-derived clones will dominate a response after antigen challenge of the recipient.     

Evaluation of Borrelia burgdorferi BbHtrA Protease as a Vaccine Candidate for Lyme Borreliosis in Mice

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA^S226A). Although inoculation with either BbHtrA or BbHtrA^S226A produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.     

Antibody formation by one or both lymphoid populations present in chimeric marmosets

Development of an isoimmune serum capable of identifying a specific leukocyte antigen in the marmoset, Saguinus fuscicollis illigeri, permitted detection of lymphoid cell chimerism in this species by the cytotoxic test. This reagent was then used to identify the cell population in the chimera responsible for antibody production against a test antigen, sheep red blood cells. Primary in vitro antibody formation as measured by plaque-forming cells with blood leukocytes or splenic lymphocytes of six animals chimeric for the leukocyte antigen, MLA-1, revealed an immune response by both cell types of the chimeric population from three animals and a response by only one cell type in the other three.     

Leishmania Specific CD4 T Cells Release IFNγ That Limits Parasite Replication in Patients with Visceral Leishmaniasis

Visceral leishmaniasis (VL) is associated with increased circulating levels of multiple pro-inflammatory cytokines and chemokines, including IL-12, IFNγ, and TNFα, and elevated expression of IFNγ mRNA in lesional tissue such as the spleen and bone marrow. However, an immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) typically fail to respond to stimulation with leishmanial antigen. Unexpectedly, it was recently shown that Leishmania specific IFNγ, can readily be detected when a whole blood stimulation assay (WBA) is used. We sought to define the conditions that permit whole blood cells to respond to antigen stimulation, and clarify the biological role of the IFNγ found to be released by cells from VL patients. CD4+ T cells were found to be crucial for and the main source of the IFNγ production in Leishmania stimulated whole blood (WB) cultures. Complement, antibodies and red blood cells present in whole blood do not play a significant role in the IFNγ response. The IFNγ production was reduced by blockade of human leukocyte antigen (HLA)-DR, indicating that the response to leishmanial antigens observed in WB of active VL patients is a classical HLA- T cell receptor (TCR) driven reaction. Most importantly, blockade of IFNγ in ex-vivo splenic aspirate cultures demonstrated that despite the progressive nature of their disease, the endogenous IFNγ produced in patients with active VL serves to limit parasite growth.     

Increased proliferation of activated T-lymphocytes in response to a monoclonal antibody (anti-p68).

A series of monoclonal antibodies was produced by immunization of mice with cells of the human promonocytic cell line CM-S; one of these recognized a membrane antigen (MW 68,000) constitutively expressed by these cells. Antigen p68 was also found to be expressed on all granulocytic cells and most mononuclear leukocytes from normal human peripheral blood, but not on hemopoietic precursor cells from bone marrow. Various types of leukemic cells also expressed antigen p68 as did various transformed human cell lines whether derived from hemopoietic cells or from other tissues. Antigen p68 is involved in T-lymphocyte regulation. In fact, the antibody anti-p68 has a strong synergistic effect increasing the proliferative response of peripheral blood T-lymphocytes both in the mixed lymphocyte reaction and when the lymphocytes are stimulated by suboptimal doses of lectin (phytohemagglutinin), tumor promoter phorbol esters, or tetanus toxoid. The anti-p68 antibody synergizes with the active metabolite of vitamin D3, 1,25-dehydroxyvitamin D3, to induce monocyte to macrophage maturation and enhances the function of mature granulocytes stimulated with the granulocyte-macrophage colony-stimulating factor in vitro.     

Toll-like receptor 3 is involved in airway epithelial cell response to nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is the etiological agent most frequently associated with bacterial exacerbations of chronic obstructive pulmonary disease (COPD). The present work shows that NTHi strains induced in primary normal human bronchial epithelial cells (NHBE) a cytokine/chemokine response in which CCL-5 and CXCL-10 were predominant. Production of both cytokines was inhibited by an anti-TLR3 monoclonal antibody (mAb) in a dose-dependent manner, but not by control human IgG4 antibodies, thus suggesting a TLR3-dependency of the NTHi stimulation. BEAS-2B, an immortalized human bronchial epithelial cell line, also showed a similar NTHi-induced response that was inhibited by the anti-TLR3 mAb. A BEAS-2B cell line stably expressing TLR3 siRNA showed significantly reduced cytokine/chemokine responses to NTHi stimulation, confirming the role of TLR3 in the response. These results indicate that TLR3 is a key component in the response of human bronchial epithelial cells to NTHi, and suggest that cognate neutralizing mAbs might be a useful therapeutic tool to regulate the inflammatory response.     

Feedback by early and late primary antisera on the primary and secondary adoptive immune responses of mice to burro erythrocytes

Experiments on antibody feedback inhibition of the immune response have confirmed that control is more effective against a primary response than against a secondary response. The cells producing antibodies in primary and secondary responses are different both in terms of number of IgFC and amount of antibody produced by individual IgFC (plaque size). Late primary anti-burro RBC sera (greater than 200 days), despite low titers, are, on a volume for volume basis, feedback inhibitors at least as good as early (8-12 days) primary antisera on primary responses but are more effective in suppressing secondary responses (B memory cells). Late primary antisera, due to the process of affinity maturation, have a high affinity for antigen. The suppressive effect of early and late antisera is equally removable by absorption with burro erythrocytes: a result which it is thought, decreases the likelihood of feedback by anti-idiotype being involved in the observed suppression. It is suggested that feedback antibody acts (a) in competition with receptors, inter alia removing antigen into immunologically irrelevant pathways, (b) by a process involving the linking of antigen to Fc receptors, and (c) as a blocking antibody coating B cells (Bm) or APC which are already binding epitopes, thus preventing their cooperation with specific helper or other accessory cells.