Anthony J. Strelkauskas

Abnormalities of Immunoregulatory T Cells in Disorders of Immune Function

We studied a five-year-old girl with several autoimmune disorders and a 16-year-old boy with acquired agammaglobulinemia to determine whether aberrations of immunoregulatory T cells could explain some instances of immunodeficiency or autoimmunity. The normal peripheral blood T-cell population, as defined by specific heteroantiserums, is 20 per cent TH2+ and 80 per cent TH2-. Human suppressor cells are TH2+, whereas helper cells are TH2-. In addition, each subset expresses Ia antigens upon activation. Our patient with autoimmune disease had no demonstrable TH2+ cells, and her lymphocytes could not be induced to suppress. Her circulating T cells were of an activated-helper phenotype, i.e., TH2-,Ia+. In contrast, in the boy with agammaglobulinemia, the T-cell population was predominantly of an activated-suppressor phenotype, i.e., TH2+,Ia+. This patients T cells abrogated both his own and his histoidentical brothers B-cell secretion of immunoglobulins. We conclude that the characterization of T cells may provide insight into the causes of a number of abnormal immune states in man.

Inversion of levels of human T and B cells in early pregnancy

CELL-MEDIATED immunity has been shown to be decreased during pregnancy1,2. Finn et al.3 suggested that the cause may be a decrease in the number of T cells; however, levels of T cells were unchanged in women in the third trimester of pregnancy4. In addition, the normal number of B cells was found in women who had been pregnant for at least 6 months5. Gergely et al.6 also found normal levels of T and B lymphocytes in women between 34 and 36 weeks of gestation. So far no change has been shown in T- and B-cell levels during the latter half of pregnancy.

Functional subsets of human T cells defined by “active” rosette formation☆

Abstract We have confirmed that the number of human T cells forming “active” E rosettes is directly proportional to the ratio of sheep erythrocytes (SRBC) to lymphocytes in the assay mixture and the length of incubation. T cells with high affinity for SRBC were isolated on a Ficoll gradient after active E rosette formation, using a 10:1 ratio of SRBC to lymphocytes and an incubation time of 5 min. Both this subset (A + ) and the remaining T cells (A − ) displayed antigenic heterogeneity, containing cells reactive with autoantibodies from patients with juvenile rheumatoid arthritis (JRA + ) or systemic lupus erythematosus (SLE + ). Furthermore, a second A + subset could be isolated from the A − cells by a second incubation with SRBC under the same conditions. The initially isolated A + subset of T cells was found to contain cells capable of recognizing and killing allogeneic cells, as well as cells with immunoregulatory effects on B-cell immunoglobulin production. JRA − cells, contained within the A + subset, are known to mediate both recognition and killing of allogeneic cells in addition to providing helper function in immunoglobulin synthesis by B cells. The A + subset, functioning in the recognition and killing of allogeneic cells but not providing helper function for B cells synthesizing immunoglobulin, further subdivides the functional capacity of antigenically defined JRA − cells.

Functional abnormalities associated with T lymphocytes from patients with chronic lymphocytic leukemia

Abstract Purified populations of T and B cells were obtained from individuals with chronic lymphocytic leukemia (CLL) by the use of anti-F(ab′) 2 affinity column chromatography techniques in which twice the amount of column material used for normal separations was used for the separation of Ig − and Ig + cells from CLL patients. Furthermore, the flow rate was reduced in order to decrease the possibility that some B cells from CLL patients, containing a low density of surface Ig, would pass through without binding to the coupled Sephadex G-200. Examination of surface Ig, P23,30 antigens, and T-cell antigens in the separated populations indicated that both the Ig − and Ig + populations were highly enriched. These T cells were also tested for their response to mitogenic stimulation as well as their ability to cooperate with leukemic B cells, normal B cells, or B cells from a transformed B-cell line. T cells from leukemic patients were found to be significantly deficient in both the response to mitogens and ability to cooperate with B cells in the synthesis and secretion of immunoglobulin.

Lymphokine Production in T-Lymphocyte Subsets Defined by Active E-Rosette Formation

Human T‐cell subsets, defined by active E rosette formation, were examined for their ability to produce leucocyte migration inhibitory factor (LIF) in response to mitogen or specific antigen. It was determined that T cells enriched for active rosette‐forming (A+) cells produced LIF in response to concanavalin A, whereas T cells depleted of active rosetteforming (A‐) cells did not. Similarly, A+ cells from a tuberculin‐sensitive donor produced LIF in response to tuberculin purified protein derivative, whereas A‐ cells from the same donor failed to produce the mediator. Thus, T‐cell production of LIF in humans appears to be restricted to those T cells capable of active rosette formation.

A rapid method for the isolation of human peripheral null lymphocytes

Abstract We describe here a new technique for the isolation of human peripheral null lymphocytes: cells lacking detectable surface immunoglobulin as well as receptors for sheep erythrocytes. The double rosette technique described employs a combined negative selection for Ig+ cells by specific anti-Ig-coated sheep erythrocytes and E rosette selection for T cells. This singlestep procedure facilitates the isolation of null cells from large cell populations and can be completed in less than 2 hr. The isolated null subpopulation represents less than 5% of the total peripheral mononuclear cells and is at least 85–90% pure by sensitive surface marker analysis. The null subpopulation is shown to be highly enriched for effectors of both antibody-dependent cytotoxicity against autologous lymphocytes and spontaneous cytotoxicity against the K562 leukemia blast cell line. Application of this technique will allow further characterization of the effector populations for ADCC and NK as well as differentiation of T and B cell precursors.

Functional characteristics of human T-lymphocyte subsets identified by sera from patients with systemic lupus erythematosus.

Abstract The sera from some patients with SLE can be used to identify and isolate two distinct subsets of normal human T cells. These subsets are designated SLE+ (those which react with anti-T-cell antibodies from these sera) and SLE− (those unreactive with SLE sera). SLE+ T cells enhance the secretion of immunoglobulin by B cells, proliferate poorly to PHA, Con A, soluble antigens, and allogeneic cells. The SLE− subset, in contrast, responds as well as or better than the total T-cell population in proliferative assays but will not enhance the secretion of Ig by B cells. Furthermore, the subset of T cells identified by SLE sera (SLE+) is not identical to that identified with sera from patients with juvenile rheumatoid arthritis (JRA).

Activation of human complement by totally human monoclonal antibodies

A uniquely developed series of totally human monoclonal antibodies (mAbs) were examined for their complement fixing properties in comparison to human myeloma preparations and to commercially available human polyclonal immunoglobulins. C3b and C4b deposition was measured using a kinetic ELISA technique. When the IgG myeloma proteins were tested for classical pathway activation, our findings were similar to those previously described, where IgG1 and IgG3 were more potent activators of the classical pathway than IgG2 and IgG4. However, those same studies determined that IgG2 was the best activator of the alternative pathway followed by IgG1 and IgG3 while IgG4 does not activate complement via either pathway. In our studies of alternative pathway activation, the IgG2 myeloma exhibited strong activation of the alternative pathway, but, at levels lower than the other three IgG subtypes. Using this test system, we examined the complement activating potential of four totally human mAbs that were constructed from the peripheral blood lymphocytes of a colon carcinoma patient in long term remission. We found that our uniquely constructed totally human IgG2 mAbs (A3, E1, F6 and F8) were able to activate complement by both the classical and alternative pathways to varying degrees. In addition, we found that the complement activating ability of the human mAbs was greater than that of the human IgG2 myeloma immunoglobulins or normal human IgG2 preparations. This study represents the first report of complement activation by totally human mAbs and confirms more recent findings which indicate that levels of complement activation by human IgG immunoglobulins cannot be predicted based solely on their subclass identity.

Secretion of human monoclonal antibody after fusion of an immortal T cell line and lymphocytes from the peripheral blood of a patient with colon carcinoma

A series of human hybridomas were derived by the fusion of the immature T cell line, JM, and lymphocytes from the peripheral blood of a colon carcinoma patient in long term remission who was shown to produce anti-tumor antibody. Five of the hybridomas have been selected for further study. These hybridomas express the T cell surface markers CD2, CD3, CD4, and CD8. The human IgG2/kappa antibody produced by these clones has been purified using affinity chromatography and has been used in immunohistiologic staining procedures. Biotinylated monoclonal antibody (MAb) stained colon carcinoma tissue but did not stain normal colon. Positive immunostaining was also seen utilizing colon carcinoma cell lines but not with other cell lines tested. These hybrids were constructed without using the conventional fusion technology which employs 8-azoguanine resistant, HAT sensitive malignant fusion partners. Because the fusion partner was a T cell, we were able to select hybrids on the basis of surface immunoglobulin. This approach was accompanied by stringent selection of patients as lymphocyte donors. Utilizing these unique methods, we have successfully produced hybridomas with T cell surface markers that produce human MAb with reactivity to colon carcinoma.

Human monoclonal antibody JDB1 reacts with cytoplasmic and membrane bound antigens present on a variety of human breast tumor cell lines.

The human monoclonal antibody JDB1 was examined for reactivity to five breast cancer cell lines as well as normal fibroblasts. The JDB1 clone secretes both IgG3 and IgM antibody. In these studies both the total antibody (containing IgG3 and IgM) as well as the purified IgG3 and IgM fractions were examined for tumor cell binding reactivity. Peroxidase staining was observed in the breast cancer lines SW527, MCF-7, T47D, SKBR3, and MDAMB231, while GM179 and GM5758 fibroblast lines were negative for antibody binding. Tumor cells were examined using two techniques: a drop cell technique in which cells were fixed onto slides and also a cover slip assay in which cells were grown onto sterile cover slips and subsequently stained with the human monoclonal antibody using a peroxidase technique. Both cytoplasmic and membrane staining were observed for all of the breast tumor cells tested using both assays. In addition, a cellular ELISA was developed and used to quantitate the binding of these antibodies to tumor cells.