EFFECT OF LOW-DOSE CYCLOPHOSPHAMIDE ON THE IMMUNE-SYSTEM OF CANCER-PATIENTS - DEPLETION OF CD4+, 2H4+ SUPPRESSOR-INDUCER T-CELLS

  • 15 March 1988
    • journal article
    • research article
    • Vol. 48  (6) , 1671-1675
Abstract
We studied peripheral blood lymphocytes (PBL) from 42 patients with metastatic melanoma undergoing treatment with cyclophosphamide (CY) plus melanoma vaccine to determine whether CY immunopotentiation could be related to depletion of T-cells that function as inducers of suppression. Every 28 days, the patients were given CY, 300 mg/m3 i.v., followed 3 days later by the intradermal injection of autologous, irradiated melanoma cells mixed with Bacillus Calmette-Guerin. PBL were separated by density-gradient centrifugation and cryopreserved until needed for testing. They were stained with monoclonal antibodies directly conjugated to fluorescein isothiocyanate or phycoerythrin and analyzed by two-color flow cytometry. At no time after the initiation of CY plus vaccine were there any significant changes in the percentages of helper-inducer T-cells (CD4+), suppressor-cytotoxic T-cells (CD8+), or the subpopulation of CD8+ cells expressing Leu 15, a marker for suppressor cells. Treatment of melanoma patients with CY plus vaccine resulted in a progressive fall in the proportion of CD4+ T-cells expressing the 2H4 (CD45) antigen, which identifies inducers of suppression. The reduction of CD4+, 2H4+ T-cells did not become apparent until day 28 after the first dose of CY and reached statistical significance only on days 49 (21 days after the second dose) and 105 (21 days after the fourth dose) (mean changes .+-. SE; day 49, -5.4 .+-. 1.4%, P < 0.01; day 105, -9.1 .+-. 2.2%, P < 0.01; t test for nonindependent samples). In contrast, the proportion of CD4+ T-cells expressing the antigen (CDw29), which are true helper cells, increased slightly, although not significantly, following the institution of CY plus vaccine (mean changes; day 49, +2.9 .+-. 2.1%; day 105; +3.6 .+-. 2.4%). Similar results were obtained when absolute numbers of circulating cells, rather than percentages, were analyzed. Thus the number of CD4+, 2H4+ T-cells fell from a mean of 395,000/ml on day 0 to 309,000/ml on day (P < 0.01) to 256,000/ml on day 105 (P < 0.05). The absolute number of CD4+, 4B4+ cells remained unchanged at the same time points. These changes were not due to progression of metastatic disease, since a comparison of patients with progressive metastases with those who were rendered disease free by surgery showed no significant differences in the reduction of the percentage of CD4+, 2H4+ T-cells. Moreover, PBL from 5 of 6 patients who had received vaccine without CY pretreatment and who had progressive metastases, exhibited slightly increased rather than decreased percentages of CD4+ cells expressing 2H4. Leu-8, another proposed marker of suppressor-inducer cells, could not be studied because of its unexpected sensitivity to cryopreservation. There was no evidence that activated suppressor or inducer cells, as defined by expression of the interleukin 2 receptor (tac) or the recently described activation antigen, Ta1, were reduced by CY plus vaccine treatment. Thus, administration of CY appears to result in progressive depletion of the suppressor-inducer subset of PBL which could be at least partially responsible for its immunopotentiating effects.

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