Antigenic stimulation of lymphocytes I. Calorimetric exploration of metabolic responses

The rate of evolution of beat, an index of overall metabolic activity, has been measured for lymphocytes undergoing simple culture as well as in the presence of the specific antigen, DNP-BSA (dinitrophenylated bovine serum albumin), of the mitogenic lectin Con A (concanavalin A), and of inhibitors of various aspects of cell culture. Unstimulated lymphocytes evolve heat initially at a decreasing rate which stabilizes after 3 days of culture. Cells stimulated with antigen increase thermogenesis after 2 days of exposure to a maximum at 4–5 days, on a schedule that parallels increases in DNA synthesis. Cells stimulated with Con A show early increases in heat output which precede the onset of increased DNA synthesis. Inhibition of DNA synthesis alone does not significantly inhibit heat output, whereas inhibition of protein and RNA synthesis has a profound effect, as does interference with the structure of microfilaments with cytochalasin B. The completely synthetic multivalent high molecular weight antigen, PEO-DNP₈₀ (dinitrophenylated polyethylene oxide) is capable of abolishing the response of the cells to DNP-BSA and to Con A as well as of directly inhibiting the output of heat by the cells.     

Analysis of lymphocytes reactive to histocompatibility antigens. II. Exponential alloantigen-dependent lymphocyte growth in vitro

Rat thoracic duct lymphocytes were maintained in continual blast transformation and cell division by repeated in vitro stimulation with allogeneic cells. This resulted in increases in responder cell numbers of up to 10,000-fold in 10-day periods. Growth of responder lymphocyte populations was dependent upon cell density, culture medium nutrients, and the presence of antigen in the form of allogeneic cells. A titration assay for mixed lymphocyte interactions (MLI) was used to relate absolute growth of cells in preparative cultures to [³H]thymidine incorporation in analytical MLI. Growth of lymphocyte populations derived by repeated stimulation with cells bearing a single foreign MHC haplotype was supported to lesser, variable degrees by stimulation with unrelated “third party” stimulator cells. The extent of this operational cross-reactivity was assessed by parallel line analysis of MLI titrations of responder lymphocytes enriched for specific alloreactivity.     

Antigenic differences between tumor-associated fetal antigens and rat histocompatibility antigens

Certain molecular properties of purified tumor-associated fetal antigens (TAFA) were analyzed by sequential immune precipitation (SIP), isoelectric focusing, and high-pressure liquid chromatography (HPLC). The antigenic relatedness of rat histocompatibility antigens and the various TAFA were determined by SIP. SIP of chloramine-T-labeled purified TAFA or lactoperoxidase-iodinated tumor cell membranes, in the presence of rat alloantisera and monospecific rabbit anti-TAFA sera demonstrated no antigenic cross-reactivities or similarities between H-antigens and TAFA. TAFA were also compared with histocompatibility antigens for isoelectric point optima and molecular weight. Rat H-antigens had isoelectric points in the 7.0–8.5 pH range, whereas all TAFA focused at pH 5.0–6.5 or above pH 8.0. Molecular weights were determined by HPLC. TAFA-I and TAFA-III had molecular weights of 16,000–17,500 daltons, whereas TAFA-II had a molecular weight of 12,000. The antigens were not coprecipitated by the rat alloantisera. Each TAFA was also isolated (via immune precipitation) from NP-40-solubilized tumor cell membranes. These TAFA were identical to the chloramine-T-labeled TAFA which had been previously extracted and purified from rat fibrosarcomas and osteosarcomas. These studies demonstrated that although TAFA and H-antigens cocap on embryonic and transformed cell membranes, these determinants are different molecules; they are not covalently linked on cell membranes; and TAFA are not cleavage products of normal NBR H-antigens.     

Immunogenicity of surface and capsular antigens of Burkholderia

Study showed that five (C3, C6, C9, C10, C11) out of ten chromatographic fractions of surface and capsular antigens of B. mallei significantly stimulated cell-mediated immunity that manifested in activation of delayed hypersensivity reactions (DHS) and phagocyteability of noncapsulated avirulent strain of B. mallei with added surface and capsular antigenic complexes. Other fractions did not stimulate cell-mediated immunity, furthermore, fraction C8, which contained capsular biopolymer with mass of 200 kD (Ar8), was characterized by immunosuppressive effect on DHS and phagocytosis. Observed stimulation of cell-mediated immunity by fractions referred above has been confirmed by assessment of their protective effects on the model of experimental melioidosis in white rats. Relationship between markers of humoral and cell-mediated immunity, including markers of specific response, was not observed.     

Antigenic analysis of purified surface antigens of filarial nematode Setaria digitata.

The surface antigens of S. digitata were isolated by treatment with Triton X-100. In non SDS-PAGE the surface antigen preparation resolved into more than 6 protein bands. Electroelution of gel slices corresponding to the protein bands with relative mobilities 0.09, 0.32, 0.41, 0.53, 0.61 and 0.76 gave 6 purified surface antigen fractions (SAF). Analysis of SAFs by SDS-PAGE showed that the proteins with molecular weights 17, 29 and 36 KD were the three major polypeptides and different combination of these gave rise to the 6 native surface proteins. The 29 KD protein existed as a monomer and as cross-linked with the 17 and 36 KD proteins. All surface antigen fractions showed antigenicity, where as 29 KD protein remained as a high avidity surface antigen.     

Mitogens and T-independent antigens stimulate T lymphocytes to secrete La antigens.

Previous reports from this laboratory suggest that certain I region-associated (Ia) antigens can be detected in normal mouse serum. It was found that, when mitogens are injected into mice, they produce substantial increases (up to 125-fold) in the levels of these Ia antigens in mouse serum. Similar increases were obtained when either T- or B-cell mitogens were injected. Furthermore, in vitro and in vivo studies demonstrated that the mitogens stimulated T cells to secrete Ia antigens. It appears likely, however, that the Ia antigens detected in these studies may differ from the conventional Ia glycoproteins found on the surface of B lymphocytes.All T-independent antigens tested also augmented the concentrations of Ia antigen in serum, the increases depending on the T-independent antigen injected and ranging from 3- to 125-fold. In contrast, T-dependent antigens, unless injected in large amounts, were unable to produce detectable changes in the serum levels of Ia antigen. These data indicate that an inverse relationship exists between the T dependence of an antigen and its ability to stimulate T cells to secrete Ia antigens. On the basis of this conclusion it is proposed that all antigens are T dependent and merely vary in the efficiency with which they activate T cells to release helper factors.     

Induction of cell-mediated immunity to chemically modified antigens in guinea pigs. II. The interaction between lipid-conjugated antigens, macrophages, and T lymphocytes.

Protein antigens covalently conjugated with lipid groups (dodecanoic acid) have previously been shown to stimulate strong delayed-type hypersensitivity (DTH) without the aid of adjuvants. The present experiments show that lipid-conjugated bovine serum albumin (L-BSA) is taken up in vitro by macrophages (Mpsi) 25- to 50-fold more than unconjugated BSA or aminidated BSA, neither of which induces DTH. Macrophages that take up 125I-labeled L-BSA in vitro stimulate DTH even more efficiently, when injected into syngeneic guinea pigs, than does soluble L-BSA. Tracer studies on the fate of radiolabeled BSA and L-BSA showed that much more L-BSA than BSA was retained by draining lymph nodes. Autoradiography demonstrated that 125I-L-BSA is rapidly taken up by Mpsi in the medullary sinuses of the lymph nodes. Some of this antigen is then transported into the paracortex, a region in which T lymphocytes predominate. The capacity of lipophilic antigens to stimulate cell-mediated immune responses may be caused by their increased uptake by Mpsi, resulting in more efficient presentation to immunocompetent T lymphocytes. The anatomical site of this Mpsi-T cell interaction may be within the sinusoids or paracortex of the draining lymph nodes.     

Antigenic expression of B-cell chronic lymphocytic leukemic lymphocytes

A flow cytometric analysis of lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL) samples and in monocyte-depleted and T-cell-depleted normal peripheral blood (B-PBL) samples was undertaken using 129 reagents from the blind panel (BP) and 72 reagents from the cluster designation (CD) panel obtained from the Fourth International Leucocyte Differentiation Conference and Workshop, B-Cell Section. After determining the average mean channel fluorescence and the average percentage of positive cells for the B-CLL and the normal B-PBL preparations, a combined ratio and difference analysis was performed for each monoclonal antibody reactivity. This analysis confirmed the intense expression of class II antigens on B-CLL and the preferential expression of CD19, CD20, CD23 and CD24 antigens. In addition, three new clustered and three new unclustered antigens were also preferentially expressed on B-CLL lymphocytes. Cluster analysis of these differences suggests the existence of at least three overlapping immunophenotypic subpopulations, composed of CD19, CD20, CD21, CD22, CD23, CD24, CD75, CD76 and CDw78.     

Interaction of peritoneal exudate lymphocytes with histocompatibility antigens.

The effect of allo-immunization on the response of mouse peritoneal exudate lymphocytes (PELs) in mixed lymphocyte culture (MLC) and cell mediated cytotoxicity was described. In characterizing these responses, the results were compared with data obtained in similar experiments with splenic lymphocytes (SpLs). While unimmunized BALB/c SpLs (H-2^d) showed strong reactivity in the one way MLC against irradiated C57BL/6 spleen cells, unimmunized PELs gave a barely detectable response. Subcutaneous (sc) but not intravenous (iv) allo-immunization resulted in a transient but marked increase in MLC reactivity by PELs. Immunization by either route resulted in an augmented MLC by SpLs. Further, sc, but not iv, allo-immunization resulted in the transient appearance of cellular cytotoxic lymphocytes in PELs. It was concluded that PELs were unique among secondary lymphoid populations in that they contained very few histocompatibility antigen reactive cells in the absence of immunization; and as such represented a population committed solely to antigens which the animal had recently experienced.