A rapid method is described for effectively removing immunoglobulin-bearing cells from either primed or unprimed mouse spleen and lymph node cell suspensions. Incubation of cell suspensions in nylon wool columns for 45 min at 37 °C resulted in a 9 to 100-fold depletion of immunoglobulin-bearing cells and a complementary 1.5 to 2-fold enrichment of T cells in the column effluent populations.



The effluent population, derived from passage of spleen cells through these columns, was virtually devoid of B precursor and memory cell activity, but contained all of the helper cell and cytotoxic effector cell precursor activity when compared to unfractionated spleen cells.

A rapid method for the isolation of functional thymus‐derived murine lymphocytes

1) Induction of humoral antibody formation involves the obligatory recognition of two determinants on an antigen, one by the receptor antibody of the antigen-sensitive cell and the other by carrier antibody (associative interaction). 2) Paralysis of antibody formation involves the obligatory recognition of only one determinant by the receptor antibody of the antigen-sensitive cell; that is, a nonimmunogenic molecule (a hapten) can paralyze antigen-sensitive cells. 3) There is competition between paralysis and induction at the level of the antigen-sensitive cell. 4) The mechanisms of low- and high-zone paralysis, and maintenance of the unresponsive state, are identical. 5) High-zone paralysis occurs when both the carrier antibody and the receptor antibody are saturated, so that associated interactions cannot take place. 6) The mechanisms of paralysis and induction for the carrier-antigen-sensitive cell are identical to those for the humoral-antigen-sensitive cell. 7) The formation of carrier-antigen-sensitive cells is thymus-dependent, whereas humoral-antigen-sensitive cells are derived from bone marrow. Since carrier antibody is required for induction, all antigens are thymus-dependent. 8) The interaction of antigen with the receptor antibody on an antigen-sensitive cell results in a conformational change in an invariant region of the receptor and consequently paralyzes the cell. As the receptor is probably identical to the induced antibody, all antibody molecules are expected to be able to undergo a conformational change on binding a hapten. The obligatory associated recognition by way of carrier antibody (inductive signal) involves a conformational change in the carrier antibody, leading to a second signal to the antigen-sensitive cell. 9) The foregoing requirements provide an explanation for self-nonself discrimination. Tolerance to self-antigens involves a specific deletion in the activity of both the humoral- and the carrier-antigen-sensitive cells.

https://science.sciencemag.org/content/sci/169/3950/1042.full.pdf

A Theory of Self- Nonself Discrimination.

Antibody specificity is determined by structural v-genes that code for the amino acid sequences of the variable regions of antibody polypeptide chains. The present hypothesis proposes that the germ-cells of an animal carry a set of v-genes determining the combining sites of antibodies directed against a complete set of a certain class of histocompatibility antigens of the species to which this animal belongs. The evolutionary development of this set of v-genes in phylogeny is traced back to the requirements for cell to cell recognition in all metazoa. The hypothesis leads to a distinction between two populations of antigen-sensitive cells. One population consists of cells forming antibodies against foreign antigens; these lymphocytes have arisen as mutants in clones descending from lymphocytic stem cells which expressed v-genes belonging to the subset (subset S) coding for antibody against histocompatibility antigens that the individual happens to possess. The other population consists of allograft rejecting lymphocytes that express v-genes of the remaining subset (subset A) coding for antibody against histocompatibility antigens of the species that the individual does not possess. The primary lymphoid organs are viewed as mutant-breeding organs. In these organs (e. g. in the thymus), the proliferation of lymphocytes expressing the v-genes of subset S and the subsequent suppression of the cells of these “forbidden” clones, leads to the selection of mutant cells expressing v-genes that have been modified by spontaneous random somatic mutation. This process generates self-tolerance as well as a diverse population of antigen-sensitive cells that reflects antibody diversity. The proliferation in the primary lymphoid organs of lymphocytes expressing v-genes of subset A generates the antigen-sensitive cell population that is responsible for allo-aggression.



The theory explains how a functional immune system can develop through a selection pressure exerted by self-antigens, starting during a period in early ontogeny that precedes clonal selection by foreign antigens. The hypothesis provides explanations for the variability of the N-terminal regions of antibody polypeptide chains, for the dominant genetic control of specific immune responsiveness by histocompatibility alleles, for the relative preponderance of antigen-sensitive cells directed against allogeneic histocompatibility antigens, for antibody-idiotypes, for allelic exclusion, for the precommitment of any given antigen-sensitive lymphocyte to form antibodies of only one molecular species and for the cellular dynamics in the primary lymphoid tissues.

The somatic generation of immune recognition.

https://www.cell.com/cell/pdf/0092-8674(77)90266-5.pdf

Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes.

Separation of mouse spleen cells by passage through columns of sephadex G-10.

Four separate effects can be demonstrated when lymphoid cell suspensions are passed through columns of siliconed glass beads. (a) A temperature-dependent "active adherence" of phagocytic cells, such as macrophages and polymorphs. (b) A temperature-independent and selective trapping by "physical adherence" of particular classes of lymphoid cells, including certain antibody-forming cells. (c) A "size-filtration" effect that traps larger cells, but only becomes significant with beads below 100 µ in diameter. (d) A selective retention of damaged cells, which occurs with all columns under all conditions tested. An active adherence column technique has been developed to separate phagocytes from lymphocytes while minimizing selection within the lymphocyte population by physical adherence or size filtration. In less than 10 min at 37°C it reproducibly produces a preparation of mouse spleen lymphocytes >500-fold depleted of active macrophages, and approximately 50-fold depleted of active polymorphs, with good over-all cell recoveries and cell viability. The lymphocyte fraction appears fully active in its ability to initiate immune responses to at least two different antigens, but is changed in over-all composition and selectively depleted in certain classes of antibody-forming cells.

https://rupress.org/jcb/article-pdf/48/3/566/1385353/566.pdf

THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : IV. The Separation of Lymphocytes from Phagocytes on Glass Bead Columns, and Its Effect on Subpopulations of Lymphocytes and Antibody-Forming Cells

A QUESTION of current interest is whether the initial stages of an immune response—either the induction of antibody formation or of specific tolerance—may involve the reaction of an antigen with a specific lymphocyte. Naor and Sulitzeanu1 have demonstrated, both in vivo and in vitro, a reaction of 125I-labelled bovine serum albumin with a small proportion (about 1/5,000) of mouse spleen lymphocytes. We have since shown2 that both flagellin and polymerized flagellin (Salmonella adelaide) and haemocyanin (Jasus lalandii) labelled with 125I or 131I react in vitro with certain cells from spleens of rats and mice. Of the strongly reactive cells, almost all are mononuclear with a high nuclear/cytoplasmic ratio and 7–12 microns in diameter and, for any one antigen, comprise about 1/5,000 of the total cell population. These reactive cells adsorb between 4,000–40,000 molecules of labelled protein when allowed to react at 0° C with labelled protein (about 3 × 1012 molecules/ml.) in 10 per cent foetal calf serum. A similar proportion of such reactive cells was observed in cell suspensions from lymph nodes and thoracic duct lymph, while peritoneal exudate contained a higher proportion and thymus a lower proportion of these cells2. The reaction was not inhibited by concentrations of sodium azide which restricted the uptake of labelled antigen by macrophages but was inhibited, using mouse cells, by rabbit anti-mouse globulin serum. Spleen cells from germ-free and conventional mice reacted equally well with 131I-labelled flagellin2. We wished to know whether the ability of these cells to react with antigen was immunologically significant. Experiments were devised to distinguish between the following possibilities: that the reactive cells were (1) cells present because of a prior experience of the animal with a related antigen, (2) antigen-reactive cells3,4 which had not had prior antigenic experience but which were capable of contributing to a specific immune response such as formation and secretion of antibody, and (3) cells coated non-specifically with cytophilic antibody.

Specific Inactivation of Antigen-reactive Cells with 125I-Labelled Antigen

ALTHOUGH the events occurring during the induction of an antibody response are not clearly understood, it is evident that antigen must react with some type of receptor which is either free in serum or present at a cell surface. Various experiments1–3 in which labelled antigen is allowed to react in vitro with lymphocytes suggest that a very small proportion of all lymphocytes does in fact have the ability to recognize and bind a given antigen. The specificity of this binding reaction between antigen and lymphocyte was demonstrated in both adoptive transfer experiments4 and in vivo3. Electron microscopic studies indicate that the reactive lymphocytes possess localized receptor sites for antigen on their surface5. Preliminary studies2 showed that pretreatment of these cells with a polyvalent anti-immunoglotaulin serum inhibited antigen binding and suggested that the antigen receptor site involves some type of immunoglobulin molecule. We now report in more detail on the nature of the receptor sites on lymphocytes and suggest that interaction of antigen with these sites is crucial in the induction of the antibody response.

Blocking of the lymphocyte antigen receptor site with anti-immunoglobulin sera in vitro.

Secretion and Synthesis of Amylase in the Rat Parotid Gland after Isoprenaline

The BSA density-gradient technique for separating mouse spleen cells into partially purified populations has been used to compare the responsiveness of such populations to SRBC using in vivo and in vitro techniques. Two major populations were distinguished, one of which responded very well in vivo with an exponential dose response and poorly in vitro (fraction 3), and another which responded in vivo and in vitro with a linear dose response (fraction 2). A light density, radiation-resistant component was identified which markedly stimulated the response of fraction 3 in vitro, and a density gradient profile was obtained for this cell which did not correspond with a macrophage profile. A high density, radiation-sensitive cell was identified which stimulated the response of PFC precursors in lighter regions of the gradient. The activity of this cell could be replaced using thymus cells. A density profile for the PFC precursor cell was obtained by assaying small numbers of spleen cell fractions in the presence of an excess of the two auxiliary cell types.

/pdf/in-vitro-and-in-vivo-studies-of-the-immune-response-to-sheep-425aeh0cmp.pdf

Pauline Byrt

Papers

THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : IV. The Separation of Lymphocytes from Phagocytes on Glass Bead Columns, and Its Effect on Subpopulations of Lymphocytes and Antibody-Forming Cells

Specific Inactivation of Antigen-reactive Cells with 125I-Labelled Antigen

Blocking of the lymphocyte antigen receptor site with anti-immunoglobulin sera in vitro.

Secretion and Synthesis of Amylase in the Rat Parotid Gland after Isoprenaline

In vitro and in vivo studies of the immune response to sheep erythrocytes using partially purified cell preparations