The PHA Dose-Response Curve: Validation of the Use of Logarithmic Graph Paper by Computer Analysis Results

Ziegler, John B.; Hansen, P J; Davies, Wayne A.; Penny, Ronald

doi:10.4049/jimmunol.113.6.2035

The Journal of Immunology

1974

DOI: 10.4049/jimmunol.113.6.2035

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The PHA Dose-Response Curve: Validation of the Use of Logarithmic Graph Paper by Computer Analysis Results

John B. Ziegler

P J Hansen

Wayne A. Davies

et al.

Abstract: Phytohemagglutinin in concentrations of 0 to 800 µg/ml was used to stimulate peripheral blood lymphocytes of normal donors and responses were determined using tritiated thymidine incorporation into DNA after 68 hr incubation. A computer program was used to rank responses at each dose of phytohemagglutinin and to test for a normal distribution by the technique of probit transformation of cumulative frequencies. The procedure was repeated using logarithms, square roots, and cube roots of ranked values. Results i… Show more

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Cited by 22 publications

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Identification of T cell subpopulations binding phytohemagglutinin: functional characteristics

Callard

Basten

1978

Eur J Immunol

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Lymphocytes binding 1251-labeled phytohemagglutinin ( i251-PHA) were directly quantitated by autoradiography. The distribution of grain counts proved t o be trimodal indicating the presence of three distinct subpopulations of PHA-binding cells. Low intensity binders (median grain count of 8-12 grains/cell) predominated in spleen and lymph node, included B and T cells, and responded to the mitogen, lipopolysaccharide (LPS). High intensity binders (median grain count greater than 60 grains/cell) occurred at low frequency in every lymphoid organ, were T cells, and included mixed lymphocyte culture (MLC)-reactive but not PHA-reactive cells as judged by preferential sensitivity to suicide with lz5I-PHA. Medium intensity binders (median grain count of 40-45 grains/cell) predominated in the thymus, formed a subpopulation in the spleen and lymph nodes of normal mice, but were absent from bone marrow of normal mice and the spleen of nude mice. In addition, their numbers were markedly reduced following adult thymectomy and cortisone treatment indicating that they were most likely T cells of recent thymic origin.PHA-reactive cells were considered t o belong to the medium intensity PHAbinding subpopulation. This conclusion was based on the following: first, medium intensity binders, like PHA-reactive cells, were T cells; second, they were contained in the same subpopulation of T cells as PHA-reactive cells. That is, they were of recent thymic origin (T, cells) according to studies of tissue distribution, cortisone sensitivity and effect of adult thymectomy; third, 'suicide' of the response t o PHA as well as to allogeneic cells but not LPS was achieved by prior incubation of cells with high doses of soluble '2SI-PHA, whereas lower doses resulted only in abrogation of the MLC response. In other words, PHA-reactive cells resided in the medium, rather than high intensity subpopulation of binding cells. By analogy with suicide of antigen-binding cells, this observation implies that PHA binds preferentially to those cells responding to it, and confirms the validity of using mitogen activation as a model for antigen-induced triggering. On the other hand, binding of PHA t o a subpopulation of T cells casts doubt on the value of the PHA response as a measure of overall T cell function. Support for this conclusion came from the failure of PHA suicide to abrogate effector T cell activities such as help and suppression.

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Identification of T cell subpopulations binding phytohemagglutinin: functional characteristics

Callard

Basten

1978

Eur J Immunol

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Immune function in aged mice III. Role of macrophages and effect of 2‐mercaptoethanol in the response of spleen cells from old mice to phytohemagglutinin, lipopolysaccharide and allogeneic cells

Callard

1978

Eur J Immunol

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Spleen cells from old mice responded less well to phytohemagglutinin (PHA), lipopolysaccharide (LPS), lipid A and allogeneic cells than those from young mice. The defect in each case resided with the nonadherent responding lymphocyte population rather than adherent accessary cells (macrophages). This conclusion was reached by showing that macrophages from old mice functioned normally in each of their known roles in lymphocyte responses in vitro; namely, presentation of antigen or mitogen to responding lymphocytes, support of lymphocyte responses and regulation of responses to mitogens and antigens. Normal presentation function was demonstrated by the comparable ability of peritoneal exudate cells from old and young mice to furnish the macrophage requirement for PHA responsiveness in vitro. This conclusion was confirmed by the finding that old, nonadherent cells reconstituted with macrophages from young mice, responded to PHA like unfrationated old cells. Old macrophages were also shown to be normal in their ability to provide the 2‐mercaptoethanol (2ME) replaceable support function to young lymphocytes in vitro. Furthermore, replacement of macrophage supportive activity with 2ME did not rejuvenate the response of old cells to PHA, LPS, lipid A or alloantigens. Finally, no evidence of adherent cell suppression of responses of old cells could be obtained by two different approaches. First, although depletion of adherent cells increased, to a similar degree, the responses of both old and young cells to allogeneic stimulation, this procedure did not reduce the difference in responsiveness between them. That is, the decreased response of old cells in a mixed lymphocyte culture (MLC) could not be explained by excessive adherent cell suppression. Second, no evidence for suppression could be obtained in co‐cultures of old and young cells responding to PHA, LPS or allogeneic cells. Taken together, these results suggest that macrophage function, in contrast to lymphocyte function, does not decline with age. It is suggested that this difference may result from the different half‐lives of macrophages and lymphocytes in the intact animal. In contrast to the above results, old spleen cells acting as stimulators in an MLC were not always less effective than young cells. Furthermore, any difference that did exist between young and old cells was largely ablated by depletion of adherent cells. This result suggests that adherent cells from old mice may determine the ability of old cells to stimulate allogeneic lymphocytes in an MLC. It is not known whether this adherent cell is a macrophage or some other adherent (perhaps regulatory) cell.

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Different metastatic modes of malignant melanoma implanted in the ear of young and old mice

Hirayama

Sato

Hirokawa

et al. 1984

Cancer Immunol Immunother

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An attempt has been made to determine (a) whether aging plays an important role in resistance against metastasis and (b) whether dithiothreitol, an effective in vitro mitogenic potentiator of splenic cells of young and old mice, can modulate the occurrence of pulmonary metastasis. B16-F10 melanoma cells were injected into the outer ear of young and old female C57BL/6 mice; and the growth of the primary tumor, the palpable size of the cervical lymph node, and the number of lung metastases were then determined at various intervals. The ear was amputated when the primary tumor reached 4 mm in mean diameter. The following results were obtained. (a) The growth rate of the primary tumor in young mice is comparable to that in old mice. (b) Enlargement of the cervical lymph node occurs earlier in old than in young mice. (c) Old mice are more vulnerable to pulmonary metastases, but small metastasized pulmonary colonies are more prominent in old than in young mice. (d) Dithiothreitol (100 micrograms) injected every 2 days after the inoculation of tumor cells is effective in reducing the incidence of pulmonary metastases in old mice.

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The PHA Dose-Response Curve: Validation of the Use of Logarithmic Graph Paper by Computer Analysis Results

Cited by 22 publications

References 0 publications

Identification of T cell subpopulations binding phytohemagglutinin: functional characteristics

Identification of T cell subpopulations binding phytohemagglutinin: functional characteristics

Immune function in aged mice III. Role of macrophages and effect of 2‐mercaptoethanol in the response of spleen cells from old mice to phytohemagglutinin, lipopolysaccharide and allogeneic cells

Different metastatic modes of malignant melanoma implanted in the ear of young and old mice

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