Homing of Immunologically Committed Lymph Node Cells to Germinal Centres in Rabbits

IT is well established that B-lymphocytes are the precursors of antibody forming cells^1,2. Germinal centres of peripheral lymphoid tissues are considered to represent foci of proliferating B-lymphocytes arising in response to antigen injection, particularly since neonatal bursectomy but not thymectomy results in an absence of these centres in adult chickens³.     

Expression of 18.6/CD23 Antigen on Human Lymphoid Progenitor Cell Lines and Phorbol 12-Myristate 13-Acetate (PMA)-Induced Microglia-Shaped Cells

The nature of lymphoid progenitors and factor(s) determining commitment to either the T- or B-lymphocyte pathway are poorly understood in the human system. In this study, we generated a monoclonal antibody (MoAb), 18.6, that recognizes a cell surface antigen on a human lymphoid progenitor cell line (FL4.4). MoAb 18.6 reacted with lymphoid progenitor lines, B lymphoid cell lines, and myelomonocytic cell lines. It did not react with any T cell or erythroid leukemic cell lines. Two color FACS analyses of normal lymphoid tissues showed that MoAb 18.6 reacted with a majority of CD20⁺ mature B cells and a minority of CD64⁺ monocytes. Molecules of 3 different sizes with MW of 34, 45, and 68 Kd were precipitated with MoAb 18.6 from the lymphoid progenitor cell line. The 18.6 antigen was not expressed on a fetal liver-derived lymphoid progenitor-like cell line, FL1.4, which has the capacity to differentiate into microglia-shaped cells upon PMA-stimulation. Stimulation of FL1.4 cells with PMA induced expression of the 18.6 antigen within 24 hr and the microglia-shaped cells stained positively with MoAb 18.6. Finally, cloning of a cDNA that encoded the 18.6 antigen revealed that the 18.6 antigen is identical to the CD23 antigen. Taken together, these data suggest that the 18.6/CD23 antigen is expressed on lymphoid precursors at a very early stage of differentiation.     

Reversible Blocking Effect of Anti-mouse Immunoglobulin Serum on the Induction of Immunity and Tolerance <Emphasis Type="Italic">in vitro</Emphasis>

THE induction of blast transformation by incubating lymphocytes with anti-immunoglobulin¹ and anti-allotype² sera has suggested that these cells have immunoglobulin on their surface. This hypothesis was directly verified by the demonstration of immunoglobulin on living mouse lymphoid cells by Raff et al.³. There is much evidence to indicate that immunocompetent cells have surface receptors for antigen. This idea is based on the finding that lymphocytes can bind radioactively labelled antigen to their surface^4,5 and that specific immune unresponsiveness occurs if lymphoid cells are exposed to either highly radioactive antigen⁶ or haptens capable of forming covalent bonds with proteins^7,8. The immunoglobulin nature of these antigen receptors is suggested by recent work showing that the binding of radioactively labelled antigen can be blocked by anti-immunoglobulin sera^5,9. Reports that the adoptive immune response of mouse spleen cells can be inhibited by anti-mouse immunoglobulin sera (AMS)^9,10 suggest that the interaction of antigen with the immunoglobulin receptor sites is a crucial step in the induction of the antibody response. We report here that the inhibitory action of AMS on the immune response is potentially reversible and that the induction of immune tolerance to polymerized flagellin (POL) in vitro may be blocked in the presence of AMS.     

An Improved Method for Preparing Anti-B Lymphocyte Serum

THE preparation of a heterologous antiserum specific to the bursa equivalent (B) lymphoid cells of the mouse requires extensive absorption with mouse tissues, especially thymocytes, to obtain specificity. The cell surface antigen(s) against which the serum reacts has been termed mouse specific bone marrow derived lymphocyte antigen(s) (MBLA)¹. The specificity of anti-MBLA for the B lymphoid population has been demonstrated by cytotoxicity tests, by its ability to inhibit a portion of antigen binding cells and by adoptive immune responses of antiserum purified lymphoid cells (refs. 1 and 2 and unpublished results of E. Möller and myself).     

Fractionation of Specific Antigen-reactive Cells in an <Emphasis Type="Italic">in vitro</Emphasis> System of Cell-mediated Immunity

ACCORDING to present concepts the diversity of antibodies is determined by a similar diversity of the precursors of antibody-producing cells. The existence of a diversified cell population in the lymphoid organs was most directly demonstrated by specific adherence of antigen-reactive cells on antigen columns. Antigen-binding cells were specifically eliminated from lymphoid cell populations of both preimmunized^1,2 and non-immunized donors^3–5. The non-bound cells were incapable of producing antibody to the antigen applied on the column, yet they could produce antibody to non-related antigens. Plaque forming cell precursors, plaque forming cells and memory cells towards various antigens were separated^1–5. In all these cases the cells which specifically adhered to the antigenic column were most probably bone marrow-derived lymphocytes (B cells). On the other hand, no such specific adherence was achieved with thymus-derived lymphocytes (T cells), such as those involved in carrier recognition during immunization with hapten carrier conjugates⁶ and in cell-mediated immunity.     

Induction of Long Term Lymphocyte Lines from Delayed Hypersensitive Human Donors using Specific Antigen plus Epstein–Barr Virus

THE availability of homogeneous populations of human and murine myeloma cells has provided a unique opportunity for investigating the mechanism of immunoglobulin formation¹. Continuous lines of cultured lymphoid cells producing specific antibody or manifesting delayed hypersensitivity would be even more useful in studying the molecular events of the immune response. Human lymphoid cell lines have been established in long term culture using Epstein–Barr virus (EBV)^{2, 3} or phyto-haemagglutinin⁴ but antigen alone has not been effective⁵. The purpose of the work reported here was selectively to establish antigen-sensitive cells in culture by stimulating peripheral white cells from delayed hypersensitive donors with antigen in vitro and then exposing the cells to EBV. This combination of antigen and virus was chosen because of the following considerations: (1) some RNA and DNA viruses do not replicate in resting lymphocytes but can infect antigen-sensitive lymphocytes which have been stimulated in vitro with mitogens or specific antigen^{6, 7}; (2) polyoma virus transforms cells in the G₂ phase of the cell cycle more effectively than in G₁ (ref. 8). These observations suggested that combined exposure to antigen and EBV might result in the establishment of cell lines enriched for antigen-sensitive or antibody-forming cells.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Optimal population of FoxP3+ T cells in tumors requires an antigen priming-dependent trafficking receptor switch

FoxP3⁺ T cells populate tumors and regulate anti-tumor immunity. The requirement for optimal population of FoxP3⁺ regulatory T cells in tumors remains unclear. We investigated the migration requirement and stability of tumor-associated FoxP3⁺ T cells. We found that only memory, but not naïve, FoxP3⁺ T cells are highly enriched in tumors. Almost all of the tumor-infiltrating FoxP3⁺ T cells express Helios, an antigen associated either with thymus-generated FoxP3⁺ T cells or activated T cells in the periphery. The tumor-infiltrating FoxP3⁺ T cells largely lack CD62L and CCR7, two trafficking receptors required for T cell migration into secondary lymphoid tissues. Instead, the tumor infiltrating FoxP3⁺ T cells highly express memory/tumor-associated CCR8 and CXCR4. Antigen priming is required for induction of this trafficking receptor phenotype in FoxP3⁺ T cells and only antigen primed, but not antigen-inexperienced naive, FoxP3⁺ T cells can efficiently migrate into tumors. While the migration of FoxP3⁺ T cells into tumors was a readily detectable event, generation of induced FoxP3⁺ T cells within tumors was unexpectedly inefficient. Genetic marking of current and ex-FoxP3⁺ T cells revealed that tumor-infiltrating FoxP3⁺ T cells are highly stable and do not readily convert back to FoxP3⁻ T cells. Taken together, our results indicate that population of tumors with thymus-generated FoxP3⁺ T cells requires an antigen priming-dependent trafficking receptor switch in lymphoid tissues.     

Root Cap and Georeaction

CIRCULATING lymphoid cells suffer alterations in a localized disease, chronic viral keratitis; we have established long term lymphoctye cultures from the blood of such patients¹. We have used capillary migration technique to study cellular immunity in the disease. Leucocytes from patients were challenged with corneal antigen and with the virus which had initiated the infection^2,3. Migration inhibition is specific for the antigen to which the animal is sensitive and on contact with antigen, sensitized lymphocytes produce migratory inhibitory factor (MIF).     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Induction of Immunity and Tolerance to the Dinitrophenyl Determinant <Emphasis Type="Italic">in vitro</Emphasis>

THE immune response in dissociated lymphoid cell cultures offers an opportunity to investigate the interaction of antigen with the surface receptors of immunocompetent cells. Using polymerized flagellin of Salmonella adelaide (POL), evidence was obtained that in vitro processes as different as immunity and tolerance both depend on the direct interaction between antigen and antigen-sensitive cells^1–4. The use of chemically defined determinants in place of natural antigens could simplify the study of the molecular mechanisms underlying immunity and tolerance. Systems used in the past to induce immunity to defined determinants in vitro involved either a particulate antigen⁵ or spleen fragment cultures⁶ and were therefore unsuitable for the detailed study of the interactions occurring on the surface of lymphoid cells. A new system had to be devised. Here I describe the induction of a primary immune response to a hapten–protein conjugate in dissociated spleen cell cultures and the immune tolerance to a chemically defined determinant in vitro.     

Induction of immunity and specific unresponsiveness in vitro by cell-bound antigen: I. Symmetry in enhancement of both responses

Pulse treatment of lymphoid cells from rabbits with solubilized antigens from T2 phage results in the firm binding of small but highly active amounts of antigen. Binding of phage antigens to viable, nonviable, or disrupted cells enhances their ability to evoke antibody formation or specific unresponsiveness in the primary in vitro response of rabbit spleen cells. Transfer of sonicate containing the equivalent of 10₂ to 10₃ antigen-pulsed cells carrying 10^?8 to 10^?7 μg phage protein nitrogen into spleen cell cultures regularly evokes antibody formation, while introduction to such cultures of 10^?3 μg phage protein nitrogen in cell-bound form evokes unresponsiveness. These findings indicate a 10- to 100-fold amplification of tolerogenic and immunogenic activities of cell-bound over soluble T2 antigen.     

Liver Is Able to Activate Na?ve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8⁺ T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8⁺ T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8⁺ T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8⁺ T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8⁺ T cell response and accounts for the dysfunctional CD8⁺ T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection.     

Gm-3.2, A new granulocyte/macrophage alloantigen

A monoclonal antibody defining a unique mouse neutrophil cell-surface antigen, Gm-3.2, is described. Gm-3.2 is found on all neutrophils in peritoneal exudates and in bone marrow, and is also present on macrophages activated by thioglycolate but is absent from lymphoid, kidney, liver, heart, and red cells. Gm-3.2 is a differentiation antigen of myeloid cells, as granulocyte/macrophage colony-forming cells are Gm-3.2^– while mature neutrophils are Gm-3.2⁺. Strain distribution pattern analysis shows linkage of the Gm-3 locus to the Ly-4, B2m, H-3 complex on chromosome 2.     

Normal thymic cortical epithelial cells developmentally regulate the expression of a B-lineage transformation-associated antigen

Nonlymphoid, stromal cells in the mouse thymus are believed to be important in T cell maturation and have been proposed to play a central role in the acquisition of major histocompatibility complex (MHC) restriction and self-tolerance by maturing thymocytes. Both cortical and medullary epithelial cells in the thymus express high levels of class II (A) major histocompatibility antigens (MHC Ags). We show here that a specific subset of these A^– epithelial cells express a transformation-associated antigen (6C3Ag) found previously on the surfaces of Abelson murine leukemia virus-transformed pre-B cells and on those bone marrow-derived stromal cell clones which support normal and preneoplastic pre-B cell proliferation. Among solid lymphoid organs, only the thymus contains 6C3Ag¹ cells and within the thymus, this antigen is found exclusively on A^– epithelial cells in cortical regions. It is striking that the expression of the 6C3Ag on thymic epithelium is developmentally regulated, suggesting a role for this lymphostromal antigen in the maturation of the thymic microenvironment.     

Warthin's tumor as a hamartomatous dysplastic lesion: a histochemical and immunohistochemical study.

The etiology of Warthin's tumor was sought by histochemical and immunohistochemical methods using 7 surgically extirpated samples and normal salivary glands as a control for the epithelial component. All the samples exhibited a variety of amyloid deposition in the interfollicular area of the lymphoid component. The interfollicular lymphoid cells were both T-cells and cells of B-cell lineage with an almost 1 to 2 population ratio. Most antigen-positive B-cells were plasma cells that exhibited polyclonality of immunoglobulin. B-cells were also present in the lymphoid mantles and a few were found in the germinal centres. The epithelial component exhibited mucinous and proteinaceous fluid in the lumen and varied immunohistological reactions; being particularly positive to carcinoembryonic antigen, S-100 protein, and B-cell antigen; quite similar to that of normal salivary duct cells. The results suggest that Warthin's tumor may not be a hamartomatous neoplasm at all but a hamartomatous dysplastic lesion.     

DIFFERENT LABELLING PATTERNS IN MOUSE LYMPHOID TISSUES WITH [3H]DEOXYCYTIDINE AND [3H]THYMIDINE

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [³H]CdR, or tritiated thymidine, [³H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [³H]CdR, whereas with [³H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [³H]TdR. Thus, labelling patterns with [³H]CdR differed from those with [³H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [³H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [³H]CdR. This radioactivity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.     

Phenotypic Analysis of a Population of IgA+ Cells in the Follicle-Associated Epithelium of Mouse Peyer's Patches

The follicle-associated epithelium (FAE) selectively transports prions, viruses, pathogenic bacteria, commensal microflora, and even secretory IgA (SIgA)-immune complexes from the intestinal lumen to underlying gut-associated lymphoid tissues like Peyer’s patches. The FAE consists of a single layer of columnar epithelial cells that includes enterocytes and M (microfold) cells, intermingled with dendritic cells (DCs), macrophages, and naïve and memory B and T lymphocytes. In this report we describe a population of IgA⁺ cells that reside within and immediately below the FAE in mouse Peyer’s patches. Immunofluorescence microscopy analysis indicated that the FAE-associated IgA⁺ cells were negative for surface antigen markers specific for B cells (B220), T cells (CD3), DCs (CD11c), and plasma cells (CD138). The IgA⁺ cells were also negative Ki-67 and IRF4, indicating that they are not mature B cells or plasma cells. The IgA⁺ cells were, however, often found in close proximity to DCs, leading us to speculate that the population of IgA⁺ cells in the FAE constitutes an atypical subset of B cells involved in mucosal antigen surveillance and/or immune recall.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.