Initial studies
Initial studies conducted after randomization examined five scIL-2 treated patients at high dose (7.5 MIU per dose) and five on low dose (1.5 MIU per dose) for three cycles of scIL-2 over a six month period. These groups were compared to four patients treated with ART who did not receive scIL-2 therapy (controls). Results show that the absolute CD4 cell counts increased in all three groups and that the ending CD4:CD8 ratios were significantly different between groups (Table 1). Those receiving the highest dose of scIL-2 had the biggest increase in the CD4:CD8 ratio.
Similar to what others have observed, there was an increase of CD25, the high affinity IL-2 receptor, on the CD4+ T cells five days after scIL-2 therapy, as shown in Figure 1. The percentages and numbers of CD25+ CD4+ T cells before and after three cycles of scIL-2 therapy in the three groups are shown in Tables 2 and 3. Such an increase in CD25 expressing cells was not observed for CD8+ T cells (Figure 2). Rather, at Day 5 there was a sharp increase in CD8+ CD38+ DR+ bright cells, as shown in Figure 3. The high levels of these cells returned to baseline by Day 30 of each cycle.
Next, we examined whether cells were dying at increased levels both before and after IL-2 therapy by Annexin V staining. At baseline, 7 patients had high levels of CD8+ T cell apoptosis (17.0% ± 6.0) and 7 patients had low levels CD8+ T cell apoptosis (2.2% ± 2.1). Four and 6 patients from each group, respectively, received scIL-2. After five days of scIL-2 therapy there was an increase in CD8 apoptosis which mirrored the amount of the CD38+ DR+ increase, as shown in a representative patient in Figure 4. Very little increase in CD4+ T cell death was seen. We also observed increases in CD38 and DR expression on the CD4+ T cells at 5 days (data not shown). The levels of CD38 and DR returned to normal on both CD4+ and CD8+ T cells after each cycle. There was variability in the amounts of cell death and CD38 and DR increases over the cycles, depending on the person and cycle number with no discernible pattern. In sum, our initial data is characteristic of the data in a number of published papers with larger groups of patients, except that we saw little CD4+ T cell death [7,23].
Follow-up studies
Six years after our initial study, we examined the patients for their immune status using an exploratory analysis. We compared their CD4 and CD8 cell counts and the CD4:CD8 ratios to their baseline parameters. At baseline, only 3 of the 14 patients had CD4:CD8 ratios over 1; five patients had ratios below 0.5. Examination after six years showed that 9 of 12 patients had CD4:CD8 ratios over 1 and only 2 patients had ratios below 0.5.
We next examined possible factors associated with good responses and poor responses to ART in this 6-year period, independently of scIL-2 administration. We found a correlation between the percentage of Annexin V+ CD8+ T cells at baseline and the final CD4 cell count and CD4:CD8 ratio, which indicates the extent of T cell normalization. Patients with high levels of CD8+ T cell apoptosis at baseline (mean 17.0% ± 6.0) did not normalize their CD4 cell counts or CD4:CD8 ratios after six years of follow-up; whereas, patients with low levels of CD8+ T cell apoptosis (mean 2.2% ± 2.1) normalized their CD4 cell counts and CD4:CD8 ratio. The mean CD4 cell counts were 1,209 ± 164 vs 754 ± 320 cells/mm3 and the CD4:CD8 ratios 1.55 vs. 0.70, for the low and high baseline CD8+ T cell apoptosis level groups, respectively (Tables 4 and 5).
To determine when during the follow-up period this effect occurred, we examined CD4:CD8 ratios in the high and low CD8+ T cell apoptosis groups over time and found the results shown in Figure 5. By two years of follow-up, the high and low CD8+ T cell Annexin V positive groups were separated by differences in their CD4:CD8 ratios, which were differentially maintained to the present.
We also examined this small group of patients for predictors of CD4 cell count increase and CD4:CD8 ratio normalization. A summary is shown in Table 6. There were no differences in age, sex, duration of HIV infection or co-infections with HBV or HCV. There was a trend of lower CD4 nadir among patients in the high vs. low CD8 apoptosis groups (
= 299 ± 144 vs. 178 ± 93, respectively; median was 384 versus 203), but this difference was not statistically significant.
Examination of the ethnic distribution of the two groups revealed that there were one white, two Hispanics and four Blacks in the low apoptosis group and two whites and 5 Hispanics in the high apoptosis group. Thus, in this small sample, all four Blacks recovered CD4 numbers better after six years. Similar results were suggested in a previous much larger study, which examined non-whites, most of which were Black (70%) [6].
We next used GEE modeling to exam the outcomes over time. Baseline annexin had a significant inverse relationship with CD4 T cell count and ratio over time (p ≥ 0.02). When age at randomization was entered into the model, baseline annexin remained a significant predictor of both CD4 T cell count over time (p ≥ 0.04) and CD4:CD8 ratio (p ≥ .0.05). A similar finding occurred for age at HIV diagnosis, so that baseline annexin remained a significant predictor of CD4+ cell count over time (p ≥ 0.03) and CD4:CD8 ratio (p ≥ .0.04). When race was entered into the model, annexin was no longer independently related to CD4 T cell and CD4:CD8 ratio over time. When annexin, age and race were entered into the model, age at diagnosis was significant predictor of CD4 T cell number over time (p ≥ 0.03). Thus, this data is similar to previously reported trends with age as a predictor.
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