Immunomodulatory effect of zidovudine (ZDV) on cytotoxic T lymphocytes previously exposed to ZDV

Abstract

In a previous study, zidovudine (ZDV) was shown to cause a concentration-dependent inhibition of antigen-specific cytotoxic T-lymphocyte (CTL) clonal expansion (S. Francke, C. G. Orosz, K. A. Hayes, and L. E. Mathes, Antimicrob. Agents Chemother. 44:1900-1905, 2000). However, this suppressive effect was lost if exposure to ZDV was delayed for 24 to 48 h during the antigen sensitization period, suggesting that antigen-primed CTL may be less susceptible than naive T lymphocytes to the suppressive effects of ZDV. The present study was undertaken to determine if naive T lymphocytes were more sensitive to the suppressive effects of ZDV than T lymphocytes previously exposed to antigen. The 50% inhibitory concentration (IC(50)) values of ZDV were determined on naive and antigen-primed T-cell responses in an alloantigen system. Lymphocyte cultures with continuous antigen exposure (double prime) were more resistant to ZDV suppression (IC(50) = 316 micro M) than were naive lymphocytes (IC(50) = 87.5 micro M). Interestingly, lymphocytes that were antigen primed but deprived of antigen during the final 7 days of culture (prime/hold) were exquisitely sensitive to ZDV suppression (IC(50) = 29.3 micro M). The addition of 80 micro M ZDV during the initial priming of the single-prime (prime/hold) and double-prime cultures did not select for a more drug-resistant cell population. The differences in ZDV sensitivities are likely a reflection of the physiological properties of the lymphocytes related to their activation state.

FIG. 1.

CTL culturing protocol for preparing alloantigen-reactive CTL for analysis of their ZDV sensitivity in the IC₅₀ assay.

FIG. 2.

IC₅₀ assay. The influence of ZDV on generation of alloreactive CTL is shown in a representative assay using naive splenocytes. All wells in 96-well culture dishes received the same number of responder cells (C57BL6) and stimulator cells (irrDBA-2). ZDV was present in replicates of six at the indicated concentrations during the 7-day culture period. CTL were measured by ⁵¹Cr release. The upper horizontal line represents the mean total release, while the lower horizontal line represents mean spontaneous release from control cultures.

FIG. 3.

Drug inhibition concentration (IC₅₀) determination. To define the IC₅₀ of ZDV, data points were fitted to a logistic model by nonlinear regression (see Materials and Methods). The log dose is the log₁₀ of the ZDV concentration (micromolar) used in the assay. A fitted line was plotted on an x-y axis. fa was defined as the fraction of cytolysis affected by drug. The median-effect log concentration was determined from the fa value of 0.5 on the y axis (50% effect dose) to the x axis. The median-effect concentration and 95% confidence limits were then calculated from the anti-log of the extrapolated value.