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Highly active antiretroviral therapy often results in potent and durable suppression of human immunodeficiency virus (HIV) replication, elevation of CD4 cell counts, decreased incidence of opportunistic infections, and increased survival in HIV-infected patients, including those with advanced disease (6, 7). Immunologic studies in these patients (1, 4) show improvement of some immune functions, but not others, raising the question of whether restoration of this immune function will be complete. Such studies are sometimes performed on cryopreserved peripheral blood mononuclear cells (PBMC). Our understanding of the extent, timing, and determinants of the immune reconstitution will be expanded if assays can be reliably performed on frozen and thawed cells from well-characterized patients.

To identify biological and/or technical factors that influence functional assays performed on cryopreserved PBMC, we analyzed the results of the cryopreservation quality control (QC) program established for the immunology component of protocol AIDS Clinical Trial Group (ACTG) 360, "A Longitudinal Study of the Predictive Value of Quantitative CMV Viremia Assays for CMV Disease in Persons with AIDS". This is an ongoing study which enrolled 403 patients at 27 AIDS Clinical Trials Units (ACTU). PBMC from all subjects were cryopreserved every 4 months at ACTU sites. Personnel at the ACTU were asked to freeze the cells following the ACTG cryopreservation protocol, which included separation of PBMC on Ficoll-Hypaque gradients, washing the cells, and resuspending them at 10⁷ PBMC/ml in cold fetal calf serum with 10% dimethyl sulfoxide. Working on ice, we aliquoted the cell suspension into cryovials at 0.5 ml/vial (5 × 10⁶ cells/vial), gradually brought the suspension to a temperature less than or equal to 70°C over 24 h by using Mr. Frosty devices (Curtis Matheson Scientific) or controlled-rate freezers, and then transferred the frozen aliquots to liquid nitrogen tanks. For testing, the cryovials were shipped every 6 months on dry ice to the University of Colorado Health Sciences Center. The QC protocol was performed on randomly selected samples from each participating ACTU. Cells were thawed by quickly bringing them to 4°C followed by slow addition of cold RPMI medium containing 10% human AB serum. Cells were washed and counted in 0.5% trypan blue to assess numbers of viable cells. The total number of cells in each vial and the percentage and absolute number of viable cells were recorded; functional assays were performed if at least 2 × 10⁶ viable cells were recovered.

Viability. The first QC protocol analyzed 25 samples processed at 21 ACTU; these sites had variable experience with the cryopreservation of PBMC but had judged themselves capable of performing this task (Table 1). The peripheral blood CD4 cell numbers corresponding to the samples utilized in this QC protocol ranged from 8 to 564 cells/µl. The percentages of viable cells in these samples varied from 1 to 91% with a median of 76%. The number of viable cells per vial ranged from 1 × 10⁶ to 17.5 × 10⁶. In an effort to improve the subsequent quality of the frozen PBMC, the results of the first QC protocol were distributed to the ACTU and were discussed together with potential pitfalls of the cryopreservation procedure via conference calls.

Functional assays. Functional assays were performed on 45 cryopreserved samples with 2 × 10⁶ cells. These consisted of lymphocyte proliferation assays (LPAs) for cytomegalovirus (CMV) and pokeweed mitogen (PWM) and/or responder cell frequency (RCF) for CMV. The LPAs were performed as previously described (8): triplicate wells containing 10⁵ cells each in RPMI medium containing 10% human AB serum were incubated for 6 days in the presence of CMV antigen and control, PWM, or plain medium. Proliferation was measured by [³H]thymidine incorporation during a 6-h pulse. Stimulation indices (SIs) were calculated by the ratio between median counts per minute in antigen- or mitogen-stimulated wells and median counts per minute of the appropriate controls, i.e., mock-infected cell control for CMV and plain medium for PWM. Positive responses were defined as SI greater than or equal to 3 for CMV and greater than or equal to 5 for PWM. The RCF measured the frequency of CMV-specific memory CD4 cells by adding a limiting dilution step to the LPA (9). Briefly, 24 replicate cultures containing 100,000, 50,000, 25,000, 12,500, and 6,250 PBMC per well were stimulated with CMV and mock-infected control antigens for 8 days (2). On the last day of culture, cells were pulsed with [³H]thymidine for 6 h and harvested, and incorporated radioactivity was counted in a scintillation counter. The RCF was calculated as described by Henry et al. (3): responder wells were defined as those whose counts per minute exceeded the mean counts per minute plus 3 standard deviations of the control cultures at the same cell concentration. The percentage of nonresponder wells was plotted on a log scale against the number of cells per well plotted on a linear scale, and the RCF was interpolated at the 37% nonresponder well frequency. An SI was also calculated by dividing mean counts per minute in CMV-stimulated wells by mean counts per minute in control wells at 10⁵ cells/well. Among 45 specimens used in functional assays in either of the two QC protocols, positive proliferative responses to CMV or PWM were significantly associated with increased viability of PBMC in each sample (P = 0.05 and 0.002, respectively, Spearman rank correlation) (Fig. 1). Outlier results were confirmed by review of the raw data. The associations persisted when the data were analyzed excluding possible outliers.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   CMV and PWM proliferative results as functions of viability. Data were derived from 44 CMV () and 19 PWM () assays. The continuous and dashed lines indicate smoothed regression curves (moving average) for CMV and PWM, respectively. The outlier result (CMV log SI of 1.48 with a viability of 17%) was confirmed as a valid result by analysis of the raw data.