G. J. V. Nossal

The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response

Immunization with protein‐containing antigens results in two types of antigen‐specific B cell: antibody forming cells (AFCs) producing antibody of progressively higher affinity and memory lymphocytes capable of producing high affinity antibody upon re‐exposure to antigen. The issue of the inter‐relationship between affinity maturation of memory B cells and AFCs was addressed through analysis of single, antigen‐specific B cells from the memory and AFC compartments during the primary response to a model antigen. Only 65% of splenic memory B cells were found capable of producing high affinity antibody, meaning that low affinity cells persist into this compartment. In contrast, by 28 days after immunization all AFCs produced high affinity antibody. We identified a unique, persistent sub‐population of bone marrow AFCs containing few somatic mutations, suggesting they arose early in the response, yet highly enriched for an identical affinity‐enhancing amino acid exchange, suggesting strong selection. Our results imply that affinity maturation of a primary immune response occurs by the early selective differentiation of high affinity variants into AFCs which subsequently persist in the bone marrow. In contrast, the memory B‐cell population contains few, if any, cells from the early response and is less stringently selected.

Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling.

Abstract DNA labeling by 3 H-thymidine in vitro and antiglobulin- 131 I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow. Bone marrow cells from CBA mice previously injected repeatedly with 3 H-thymidine for 1–84 hr were exposed to 131 I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The 3 H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show 3 H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% 3 H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin- 131 I at a concentration of 1.0 μg/ml became labeled with 3 H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in 3 H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions. The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.

IDENTIFICATION OF HUMAN T AND B LYMPHOCYTES IN NORMAL PERIPHERAL BLOOD AND IN CHRONIC LYMPHOCYTIC LEUKÆMIA

Abstract A sandwich radioimmunolabelling technique has been used to demonstrate two populations of human peripheral blood lymphocytes: one, comprising an average of 34% of lymphocytes, carried a high density of surface immunoglobulin, while the other 66% had a much lower density. Human thymocytes had little detectable surface immunoglobulins. By extension from studies in mice, it is suggested that these two groups represent B and T lymphocytes respectively. Lymphocytes from 3 patients with chronic lymphocytic leukaemia had labelling characteristics of B cells but labelled less heavily than the majority of normal B cells. Almost no immunoglobulin was detected on the surface of blast cells from a patient with acute lymphoblastic leukaemia.

Differentiation of lymphocytes in mouse bone marrow. I. Quantitative radioautographic studies of antiglobulin binding by lymphocytes in bone marrow and lymphoid tissues.

Abstract The density of surface immunoglobulin on small lymphocytes in the bone marrow and other lymphoid tissues has been compared by radioautographic measurements of antiglobulin binding. Cell suspensions from CBA mice were exposed to 125 I-labeled rabbit anti-mouse globulin in a wide range of concentrations for 30 min at 0 °C. With increasing concentration of antiglobulin- 125 I the percentage of labeled antiglobulin-binding small lymphocytes in spleen and lymph node suspensions reached well-defined plateau levels. Very few normal or cortisone-resistant thymus cells were labeled under identical conditions. Bone marrow small lymphocytes showed a linear increment in labeled cells throughout the antiglobulin- 125 I dose range, their labeling intensity varied widely, and approximately one half remained unlabeled at high antiglobulin- 125 I concentrations. In 6 wk-old congenitally athymic mice the bone marrow small lymphocyte labeling pattern resembled that in CBA mice, while nearly all (91–97%) small lymphocytes in lymph nodes, thoracic duct lymph and blood, and 75% of those in the spleen, became labeled under plateau conditions. Treatment of cells from 10 wk-old CBA mice with AKR anti- θ C3H serum and complement resulted in almost complete (93%) antiglobulin-labeling of residual small lymphocytes from the spleen but had little effect on bone marrow lymphocyte labeling. Under germfree conditions the proportion of antiglobulin-binding small lymphocytes was slightly elevated in all lymphoid tissues of CBA mice. The results demonstrate that many of the small lymphocytes in mouse bone marrow have readily detectable surface immunoglobulin molecules which vary considerably in density from cell to cell, while others neither have detectable surface immunoglobulin, nor are they θ -bearing, thymus-dependent or recirculating cells. The concept of bone marrow small lymphocytes as a maturing cell population is discussed.

STUDIES ON THE RATE OF SEEDING OF LYMPHOCYTES FROM THE INTACT GUINEA PIG THYMUS

There has been much discussion in the literature recently about the possible importance of cell emigration from the thymus, particularly during the first days of life. However, there is no decisive evidence in the literature to prove that thymic lymphocytes ever leave the thymus in an intact animal. The present study is an attempt to prove formally that thymic cells do seed out into the other lymphoid organs, and to determine the importance of such emigration in immunological processes.

Incidence of cells simultaneously secreting IgM and IgG antibody to sheep erythrocytes

Abstract The open carboxymethylcellulose (CMC) hemolytic plague technique has been used to study the question of whether plaque-forming cells (PFC) exist which can secrete both IgM and IgG antibody. Using a micromanipulation transfer technique, PFC were exposed serially to three types of plaque-revealing monolayers. One of these revealed chiefly IgM plaques; the second chiefly IgG plaques, and the third achieved discrimination between the true IgM-IgG double producers and certain atypical categories of PFC. Preliminary work was done to study the efficiency of serial cell transfer. It was found that 93% of both IgM PFC and IgG (enhanced) PFC could form three plaques. In fact, most cells could be transferred successfully more frequently. IgM PFC could make up to 30 successive plaques and IgG PFC up to 10 successive plaques. Experiments involving 3 different monolayers (direct, anti-μ antibody-containing, and anti-γ antibody-containing) were performed to search for cells capable of (a) forming IgG antibody of sufficient hemolytic activity to produce a plaque without enhancing serum; (b) forming IgM antibody of such potency as to break through the inhibitory capacity of anti-μ reagents; (c) forming non-complement-fixing IgM. Cells of categories (a) and (b) were easily found and distinguished from each other. Cells of category (c) were not found by us. The serial transfer approach outlined above was next used with the modification that the anti-γ antibody-containing monolayer was replaced by one containing a serum mixture capable of inhibiting most IgM PFC and of enhancing IgG PFC. Cells capable of making plaques in both this “enhancing-inhibitory” monolayer and in a direct monolayer, but not in the category of “breakthrough IgM PFC” or “lytic IgG PFC” were considered as true double-producers of IgM and IgG. Of 900 selected antibody-forming cells examined in either the primary or the secondary response of mice to SRBC, 14 (1.5%) were double producers. The present status of the hypothesis of an IgM to IgG transition in expanding immunocyte clones is briefly considered.

Immunological tolerance in germinal centres

Abstract The ability to avoid autoimmunity is a cardinal feature of the immune system. In healthy individuals, this is achieved by several mechanisms that exclude self-reactive lymphocytes from mature lymphocyte repertoires. Here, Bali Pulendran, Rosemary van Driel and G.J.V. Nossal review recent studies indicating that several mechanisms of self-tolerance operate in germinal centres.

T-Cell-Mediated Immune Responses Induced in Vitro: A Probe for Allograft and Tumor Immunity

The specific immune responses of vertebrates have long been divided into those mediated hy humoral antibodies and those dependent on sensitized lymphoid cells. A major new chapter in immunobiology has been opened up by the realization that these two great limbs of the immune response are due to two types of lymphocytes, thymus-derived or T lymphocytes being responsible for cell-mediated immunity and bone marrow-derived or bursalequivaient B lymphocytes acting as progenitors of antibody-secreting cells (Warner, Szenberg & Burnet 1962, Miller & Mitehell 1969, Davies 1969, Claman & Chaperon 1969). Understanding of molecular and eellular mechanisms is far more advanced for the B than it is for the T lymphocyte system. This is due essentially to three factors. First, antibody-forming cells secrete their product, antibody, into the serum in large amounts, facilitating the progressive unravelling of antibody structure. Secondly, good single cell methods for study

The Global Alliance for Vaccines and Immunization—a millennial challenge

Global immunization of children is an elusive goal of public health officials world-wide. Extraordinary recent funding and organizational initiatives as described by Gustav Nossal have ignited renewed optimism that the stage is finally set to bring this acheivement within our grasp.

Host immunobiology and vaccine development

As the rules of immunoregulation become clearer, the design of vaccines and adjuvants is becoming more scientific. To understand these rules, the interactions between three kinds of cells need to be grasped. Antigen-presenting cells (APCs) initiate the immunoglobulin cascade. The most important of these are dendritic cells, which must first capture antigen, a process aided by particulate matter, the presence of natural or acquired antibodies, or the capacity to activate complement. Then T cells become activated through conjoint action of processed antigenic peptides and APC surface and secreted molecules. T cells mediate inflammation, develop cytotoxic capacity, and help in antibody formation. Whether cells of type 1 or type 2 predominate influences the direction of both cellular and humoral responses. B cells are then activated, leading to antibody formation and often to better antigen presentation. Both T and B cell memory, embedded in long-lived lymphocyte populations, aid heightened immune reactivity when the antigen is re-encountered. The best vaccines stimulate strong memory.