Antigen Binding Cells in Embryonic Chicken Bursa and Thymus

THE autoradiographic detection of the binding of ¹²⁵I-labelled antigen to a proportion of normal lymphocytes is now well established and characterized^1–4. Such cells are thought to possess patches of immunoglobulin surface receptors which combine specifically with an appropriate antigen⁵. Evidence for the specificity of the antigen binding includes the capacity of specific anti-L and anti-µ chain antisera to block the receptor sites⁶ and the demonstration that at least the more heavily labelled antigen binding lymphocytes in mouse spleen are immunologically competent⁷.     

Properties of Educated T Cells for Rosette Formation and Cooperation with T Cells

IT has been shown that the humoral antibody response in mice to many antigens requires cooperation between thymus derived lymphocytes (T cells) and bone marrow derived lymphocytes (B cells)^1,2. The B cells are the direct precursors of antibody secreting cells and, although T cells react specifically with antigen, their role is unknown^2–6.     

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.     

Specific Collaboration between T and B Lymphocytes across a Cell Impermeable Membrane <Emphasis Type="Italic">in vitro</Emphasis>

ANTIBODY production to many antigens including heterologous erythrocytes^1–3, serum proteins^4,5 and hapten protein conjugates^6,7, occurs as a result of an interaction between antigen and thymus-derived (T) and non-thymus-derived (B) lymphocytes. Although the specificity of the antibody response is determined by T as well as B cells^8–10, T cells do not actively secrete any of the known classes of immunoglobulin molecules¹¹. Their function seems rather to initiate the sequence of events whereby antigen is presented to B cells in an immunogenic form capable of stimulating antibody synthesis¹².     

Binding sites for measles virus antigens on human B and T lymphocytes

The present study examined the differences in the binding of measles virus antigens to human peripheral blood lymphocyte (PBL) subpopulations. PBL binding sites for measles antigens were detected by an assay involving the rosetting of PBL to measles-infected HeLa cells (HeLa-K11). Three approaches were employed to examine whether measles virus antigen binding sites were present on restricted subpopulations of PBL. First, no significant difference in the proportion of HeLa-K11 forming clusters was observed with unfractionated cells in comparison with enriched B- or T-lymphocyte suspensions. Second, the profile of lymphocyte surface markers before and after adherence of PBL suspensions to HeLa-K11 cells was measured. No difference in the proportion of PBL forming E-rosettes or lymphocytes with Fc-IgG receptors, surface immunoglobulin, or complement receptors was observed. Finally, the percentage of B (Raji, B-35M, Bristow-7B) and T (Molt-3) cell human lymphoid cells which adhered to HeLa-K11 versus noninfected HeLa cells was compared. In all cases, a highly significant adherence of the lymphoid cell suspensions to HeLa-K11 cells was observed in comparison with uninfected HeLa cells. This is the first direct demonstration of binding sites for measles virus antigens present on both human B and T lymphocytes.     

Selective Cytotoxicity of Anti-Kappa Serum for B Lymphocytes

MOUSE lymphocytes can be divided into two distinct populations according to the density of immunoglobulin determinants on their surface. Lymphocytes with a high density of immunoglobulin are marrow-derived, nonthymus-processed, B cells, whereas lymphocytes with little or no immunoglobulin are thymus-derived, T cells^1–3. Since more than 95% of mouse immunoglobulin light chains are of the kappa type⁴, treatment of lymphocyte suspensions with an appropriate dilution of rabbit anti-mouse kappa serum and complement should be cytotoxic for only B lymphocytes. This prediction was tested by using lymphocyte populations enriched for either T or B cells or containing the two cell types in a known proportion.     

Studies on Fc receptor function. I. IgG-mediated inhibition of B lymphocyte activation by T-dependent and T-independent antigens.

Mouse aggregated IgG, when continuously present in cultures of mouse spleen cells immunized with sheep erythrocytes, causes a dose dependent inhibition of the generation of plaque forming cells with a maximum of about 90% at 400 μg IgG/culture. Unaggregated IgG induces a similar inhibition, whereas treatment with mouse albumin or F (ab¹)₂, under the same conditions, does not affect the generation of plaque forming cells.It has been reported that unaggregated IgG binds poorly to Fc receptors of B lymphocytes and thus should not be expected to inhibit PFC generation if the effect is at the level of B lymphocyte Fc receptors. Competitive binding experiments were carried out and showed that aggregated and unaggregated IgG compete similarly with ¹²⁵I-labeled aggregated IgG for binding to Fc receptors of mouse spleen cells.The same inhibition of PFC can be induced by aggregated IgG in cultures of B lymphocytes immunized with the T-independent antigen DNP-Ficoll. When IgG is absorbed extensively with sheep erythrocytes and added to cultures immunized with sheep erythrocytes, PFC generation is inhibited to a level comparable to that of nonabsorbed IgG.These results suggest that IgG binding to Fc receptors leads to a severe inhibition of the induction of PFC by both T-dependent and T-independent antigens. This and other work from our laboratory indicate that this effect may be at the level of B lymphocyte Fc receptors.Taken together with reports from several laboratories, the data presented here suggest that Fc receptors may have a regulatory role on the activation of B lymphocytes by antigens or mitogens.     

Cytotoxic T lymphocytes of the rat are predominantly restricted by RTL1.A and Not RT1.C-determined major histocompatibility class I antigens

Two types of biochemically defined class I major histocompatibility complex (MHC) antigens are found in the rat, RTLA antigens that are ubiquitously expressed and RTLC antigens which so far are detectable only on certain cell types, notably B and T lymphocytes. It is shown that the cytotoxic T lymphocyte response to minor H antigens of the LEW strain, including the H-Y antigen, and to TNP-modified syngeneic lymphoid cells is restricted by RTLA but not RTLC gene products. This conclusion is based on bulk culture assays including cold target inhibition tests and limiting dilution experiments using recombinants between the RT1 ^a and RT1 ^u haplotypes. The possibility that class I MHC antigens exist which have no major restriction function is discussed.     

Modulation of the adherence of human lymphocytes to measles-infected cells by prostaglandin E1: A differential effect on lymphocyte subpopulations

Prostaglandins of the E series (PGE) may serve as important regulators of human immune responsiveness. The present study was designed to examine the possibility that PGE may effect human lymphocyte function by the modulation of surface receptors. The presence of surface binding sites on human lymphocytes for measles virus antigens was studied using a rosette adherence assay. We observed that the addition of PGE₁ increased the proportion of measles-infected cells (Hela-Kll) with adherent lymphocytes (75% increase at 3 × 10⁻⁶ M PGE₁). PGE was observed to enhance the adherence of purified normal peripheral T cells (87%) and T lymphoid cells (Molt 3) (27%). In contrast, no significant change in normal peripheral B cell or B lymphoid cell (Raji) adherence was observed with the addition of PGE. These results are consistent with a selective modulation of surface measles virus binding sites by PGE₁ on T and not B lymphoid cells.     

Functional significance of the complement receptors on B lymphocytes

The functional role of complement receptor (CR⁺) lymphocytes in antibody responses was investigated. Initially it was found that in the spleens of 6–8-week-old CBA/H mice only approximately 40% of the B cells were CR⁺. The CR⁺ and CR^? splenocytes were then separated by a recently described fractionation procedure (Parish, C. R., et al., Eur. J. Immunol.4, 808, 1974) and assayed alone or in combination for their ability to transfer a range of antibody responses to irradiated recipients. All of the antigens studied, irrespective of their structure or T-cell dependence, were capable of activating CR⁺ B cells to synthesize antibody. However, only repeating determinant antigens, such as horse red blood cells (HRBC) and dinitrophenyl-polymerized flagellin (DNP-POL), were capable of activating CR^? B cells, the soluble antigen DNP-flagellin (DNP-MON) being unable to trigger these cells. Repeating determinant nature rather than T-cell dependence appeared to be the factor that determined whether an antigen could provoke the CR^? B cells to synthesize antibody, as HRBC and DNP-POL differ widely in their T-cell dependence. The same phenomenon was observed with direct and indirect PFC responses and also with primary and secondary antibody responses. In addition, there was no evidence for collaboration between CR⁺ and CR^? B cells in the induction of antibody responses to the T-dependent antigens, HRBC and DNP-MON. Furthermore, no CR⁺ helper T cells were detected.It is postulated that complement receptors facilitate T-B interaction by stabilizing the union of soluble antigens with antigen-specific receptors on CR⁺ B cells. In contrast, repeating determinant antigens can avidly bind to antigen-specific receptors on B lymphocytes without involvement of the complement receptors.     

Antigen-binding receptors on T cells from long-term MLR. Evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (V_H) determinants strongly inhibited vesicle binding by both primary and longterm MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesiclebinding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectively blocked with the anti-V_H reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.Abbreviations used in this paper B10 C57BL/10 - Con A concanavalin A - FcR Fc receptor - FCS fetal calf serum - H heavy chain - Ia I-region associated antigen - Ig immunoglobulin - LPS lipopolysaccharide - Lyt T-lymphocyte differentiation antigen - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - PM plasma membrane - T thymus derived - Tcr T-cell receptor - V variable region of Ig     

Isolation of Surface Immunoglobulin from Lymphocytes from Human and Murine Thymus

CELLS capable of binding radioiodinated antigen occur in lymphocyte populations obtained from foetal and neonatal human thymus¹. In accordance with Ehrlich's hypothesis of 1900 (ref. 2), the receptors for antigen on the surface of these cells probably consist of immunoglobulin. A first step in the attainment of chemical proof of the nature of this receptor is the identification of immunoglobulin on the thymus cells. We now report the isolation of immunoglobulin from the surfaces of lymphocytes obtained from murine and neonatal human thymuses. This isolation and identification was made possible by application of a modification³ of the enzymatic iodination procedure of Marchalonis⁴ which catalysed the iodination of proteins present on the surfaces of living lymphocytes.     

Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers,Thy-1, Lyt-1, and Lyt-2

Using immunofluorescence with a monoclonal anti-Ly-6.2 antibody and FACS analysis we have confirmed that the Ly-6.2 antigen is present on approximately 70% of mature T cells and B cells but on few immature lymphocytes. There is a wide range of antigen density among the Ly-6.2⁺ populations, with the mean density higher on T cells than B cells. Following Con A activation of splenocytes there was a sixfold increase in Ly-6.2 antigen density though approximately 20% of the activated lymphocytes were Ly-6.2^?. The increase in Ly-6.2 density was specific since similar density increases did not occur for the closely linked antigens ThB and H $\text{9}\text{25}$. By panning a predominantly T-cell population for Lyt-2-bearing cells, it was found that Lyt-2⁺ lymphocytes were either negative or dully staining for Ly-6.2. However, activated cells bearing the Lyt-2 antigen were all Ly-6.2 positive. Double-staining experiments showed that T cells which had high Ly-6.2 antigen densities also had high Thy-1 antigen densities. Corticosteroid-resistant thymocytes were highly enriched for Ly-6.2-bearing cells compared to untreated thymocytes and had staining profiles for Ly-6.2 which were similar to peripheral T cells, supporting the idea that steroid treatment selects for a phenotypically mature thymic population.     

Selective Stimulation of IgM Synthesis in Mouse B Lymphocytes by Pokeweed Mitogen

THE division of lymphocytes into thymus-derived (T) cells and bursa-equivalent-derived (B) cells is well established (reviewed in refs. 1–3). The result of antigenic stimulation in the B line of lymphocytes is a differentiation process, involving clonal expansion and ultrastructural changes, to give a specialized population of cells which synthesize and secrete immunoglobulin. In the study of these processes a major problem is the small number of cells involved in response to antigen, usually less than 1% of the total lymphocyte population. Clearly a system for activating large numbers of lymphocytes into immunoglobulin synthesis would offer considerable advantages. This seems to occur when mouse B lymphocytes are stimulated by pokeweed mitogen (PWM). In our experimental conditions, however, IgM is the only immunoglobulin class to be synthesized. The rational basis for our experiments rests on three previous observations: (1) PWM-stimulated lymphocytes develop rough endoplasmic reticulum^4,5 and might therefore be expected to be secreting cells; (2) a small proportion of enlarged (“blast”) lymphoid cells in PWM-treated human blood lymphocyte cultures contain immunoglobulin demonstrated by immunofluorescence⁶ and (3) the recent demonstration that mouse B lymphocytes are activated by PWM⁷.     

Shark lymphocytes: Primitive antigen reactive cells

Shark lymphocytes have been characterized by the presence or absence of surface immunoglobulin (SIg). Thus, SIg⁺ cells are B lymphocytes, and SIg⁻ cells are presumed to include the shark T cell subset, as well as other minor subsets of lymphocytes. Few functional studies have been performed to characterize the nature of either lymphocyte population. To date, there is no information concerning the shark T cell receptor. The majority of adult mammalian T cell receptors are composed of α and β chain heterodimers while a minority use γ and δ chains. The discussion presented here explores the evidence that the majority of shark lymphocytes are analogous to mammalian T and B lymphocytes that appear during early fetal development. The hypotheses considered suggest that shark T cells are similar to γδ T cell receptor (TCR)-bearing mammalian T lymphocytes, and that shark B cells are the primitive equivalent of neonatal and newborn primary B cells.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined ¹. This is particularly true in autoimmune diseases and cancer². Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes ^1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays ⁶. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.     

The ovarian follicle as a model for the cell-mediated control of tissue growth

Summary Thy-1⁺ cells, producing Thy-1⁺ material, have been demonstrated by the indirect immunoperoxidase technique in the theca of growing ovarian follicles of the rat. OX-2 antigen, known as the minor glycoprotein of rat thymocytes, was detected in granulosa cells of non-growing follicles. Ia⁺ cells of dendritic type and/or activated macrophages were identified in the granulosa of advanced degenerating follicles, and remnants of the zona pellucida exhibited immunoglobulins. In some ovaries immunoglobulins were also bound to the zona pellucida of oocytes of early degenerating antral follicles. Medium-sized antral follicles with degenerating granulosa were occasionally invaded by cells carrying antigens of cytotoxic T lymphocytes or other T lymphocyte subsets, while degenerating large antral follicles were sometimes invaded by cells exhibiting antigen of cells with natural killer function (but not antigens of T lymphocytes). Granulosa cells of some degenerating antral follicles exhibited class-I antigens derived from the major histocompatibility complex. We suggest that cell-mediated control mechanisms of antigen expression and metabolism of tissue cells during their differentiation and degeneration should be considered in addition to the well-documented hormonal dependence of some tissues.     

Receptor-mediated B cell antigen processing. Increased antigenicity of a globular protein covalently coupled to antibodies specific for B cell surface structures

Helper T cell recognition of globular protein antigens requires the intracellular processing of the native molecule by an antigen-presenting cell and subsequent presentation of a peptide fragment, containing the antigenic determinant, on the cell surface where it is recognized by the specific T cell in conjunction with Ia. B lymphocytes can function as antigen-presenting cells and, when antigen is bound by their surface Ig, are greatly enhanced in this capacity. In this report it is demonstrated that pigeon cytochrome c covalently coupled to antibodies directed toward either B cell surface immunoglobulin, class I or class II are effectively processed and presented by B cells to cytochrome c-specific T cells, requiring up to 1000-fold less cytochrome c as compared with cytochrome c alone or cytochrome c coupled to nonspecific immunoglobulin. The potent activity of the cytochrome c-antibody conjugates appears to be due to the ability of B cells to concentrate the antigen when the process becomes receptor mediated rather than to a signal provided to the B cell by the conjugate binding, because cytochrome c was not more effectively presented in the presence of unconjugated antibodies as compared with cytochrome c alone. Furthermore, the binding of the native antigen to B cell surfaces is not alone sufficient for T cell activation, in that the cytochrome c-antibody conjugates require processing and are major histocompatibility complex restricted. The results presented here indicate that surface immunoglobulin is not unique in its ability to facilitate antigen processing and/or presentation and that Ig, class I and class II are capable of transporting the cytochrome c to a cytoplasmic vesicle where proteolysis occurs yielding the required peptide, minimally of 10 amino acids. Cytochrome c coupled to monovalent fragments of anti-Ig-antibodies was nearly as effectively presented as cytochrome c coupled to bivalent antibodies, indicating that phenomena mediated by bivalent binding, such as patching and capping of the surface Ig, were not required for effective antigen presentation. The cytochrome c-antibody conjugates, which allow antigen processing to be initiated by receptor-mediated endocytosis, may provide the necessary tools to unravel the intracellular processes by which protein antigens are processed and presented by B lymphocytes.     

VLR Recognition of TLR5 Expands the Molecular Characterization of Protein Antigen Binding by Non-Ig-based Antibodies

Variable lymphocyte receptors (VLRs) are unconventional adaptive immune receptors relatively recently discovered in the phylogenetically ancient jawless vertebrates, lamprey and hagfish. VLRs bind antigens using a leucine-rich repeat fold and are the only known adaptive immune receptors that do not utilize an immunoglobulin fold for antigen recognition. While immunoglobulin antibodies have been studied extensively, there are comparatively few studies on antigen recognition by VLRs, particularly for protein antigens. Here we report isolation, functional and structural characterization of three VLRs that bind the protein toll-like receptor 5 (TLR5) from zebrafish. Two of the VLRs block binding of TLR5 to its cognate ligand flagellin in functional assays using reporter cells. Co-crystal structures revealed that these VLRs bind to two different epitopes on TLR5, both of which include regions involved in flagellin binding. Our work here demonstrates that the lamprey adaptive immune system can be used to generate high-affinity VLR clones that recognize different epitopes and differentially impact natural ligand binding to a protein antigen.