IL-2 immunotherapy resulted in a significant and sustained rise in both absolute and percentage CD4 T cells (weeks 10 and 48; P < 0.01) and CD4/CD8 cell ratio (weeks 10 and 48; P < 0.05), whereas CD8 count remained stable up to 48 weeks (P = 0.05) (Table 1; Fig. 2a–d). At week 48, CD4 T cells displayed a more relevant recovery than CD8 T cells in percentage terms (P < 0.05), with a tendency toward higher rescue also in absolute count (P = 0.069) (Fig. 2a–d). Conversely, HAART controls displayed a constrained yet parallel CD4 and CD8 recovery, reaching significance only in absolute value at 48 weeks (CD4 cells P < 0.01; CD8 cells P < 0.05), with no significant changes in CD4/CD8 ratio at all time points (P > 0.05) (Fig. 2a–d). At week 48, IL-2 patients experienced significantly higher recovery than the HAART-alone controls in CD4 cells (P < 0.01), but not in CD8 cells (P > 0.05) (Fig. 2a–d).
Since peripheral T cell pool is maintained by both proliferation and renewal, the contribution of cell turnover and neothymopoiesis to both subsets was examined. At baseline, overall Ki67 expression was significantly higher in CD8 cells (4%) than in CD4 cells (1.6%) (P < 0.01), confirming data describing relatively higher turnover rates within the CD8 subset [15,16] as a possible on-going response to low-level viral replication despite successful HAART [17,18]. IL-2 induced a significant rise in Ki67+CD4+ at week 10, from 2.1% to 3.4% (P < 0.05), but not in Ki67+CD8+ (P > 0.05). At IL-2 discontinuation, both CD4 and CD8 T cell turnover progressively dropped to below pretreatment values (Fig. 2e) . However, in HAART-alone controls, the frequency of proliferating CD4 and CD8 were closely matched at all time points (Fig. 2f).
To evaluate the role played by IL-2 on CD8 T cell dynamics further, the CD8 T cell turnover data was integrated by studying the CD8+CD38+ (activated) lymphocytes . At baseline, IL-2-receiving patients and controls displayed comparable proportions (P > 0.05) (Table 1). Similar to the effect on CD8 cell turnover, IL-2 did not induce an elevation in the percentage of CD8+CD38+ T lymphocytes (week 10; P > 0.05), which indeed progressively decreased, reaching significance by week 34 (week 34 P < 0.05; week 48 P < 0.01) [21,22]; no changes were observed in the HAART controls (week 48; P > 0.05) (Table 1).
Analysis of individual CD4 subsets showed a discrepant pattern of recovery between IL-2-patients and HAART-alone controls. In particular, IL-2 immunotherapy induced a significant and sustained expansion of both CD4+CD45RA+CD62L+ (naive) (week 10 and 48; P < 0.05) and CD4+CD45RA− (memory) (week 10 and 48; P < 0.05) subsets (Table 1), whereas HAART alone resulted in a preferential rescue of memory CD4 cells (week 48; P < 0.05) with no significant changes in the naive compartment (week 48; P > 0.05) (Table 1). At week 48, IL-2 patients experienced higher naive and memory CD4 recovery compared with controls (P < 0.01) (Table 1).
Having demonstrated that IL-2 immunotherapy induces a preferential expansion of CD4 T cells [11-13], a large proportion of which bear a naive phenotype, the impact of neothymic output on both CD4 and CD8 subsets was analysed. As with other parameters, CD4 and CD8 T cells displayed a dichotomy in TREC content, with a temporary tendency to increase at week 10 in CD4 cells (from 4.1 × 106 to 6.5 × 106 copies/μl; P = 0.08) and no changes within CD8 pool (P > 0.05) (Fig. 2g). As TREC signal is affected by proliferative dilution [23,24], the concurrent rise in CD4 TREC and turnover strongly suggests a more substantial actual increase in CD4 neothymopoiesis. No significant CD4 and CD8 TREC changes were seen in HAART controls at all time points (P > 0.05) (Fig. 2h). Overall, at week 10, IL-2-receiving patients experienced a more relevant CD4 TREC increase than did HAART controls (P < 0.05), whereas no significant differences were shown in CD8 TREC (P > 0.05) (Fig. 2g,h).
This study investigated the long-term kinetics of CD4 and CD8 T cells in a cohort of HIV-positive patients treated with low-dose prolonged intermittent IL-2 immunotherapy compared with patients receiving HAART alone. The results show different IL-2-driven dynamics in CD4 and CD8 subsets, clearly demonstrating that IL-2 has a differential impact on these T lymphocyte subsets, with a preferential action on CD4 T cells. In particular, we confirm previous data pointing to enhanced proliferation of existing cells as the mechanism responsible for the IL-2-driven CD4 reconstitution [25,26]. Moreover, our data suggest a possible temporary boost of neothymopoiesis: the actual increase may be masked somewhat here by the increased proliferation. The disproportionate rise of proliferating CD4 versus CD8 cells  could be explained by both in vitro higher expression of IL-2 receptor mRNA in CD4 than in CD8 T cells upon activation , and the more significant in vivo IL-2-driven upregulation of IL-2 receptor α-chain (CD25) on CD4 [28,29]. By showing a preferential impact of IL-2 on CD4 neothymic synthesis, our data add to the evidence that successful CD4 reconstitution is dependent upon neothymopoiesis [7,8] and suggests a possible role of IL-2 in selectively reconstituting T helper activity to neoantigens , given that TREC increases have indeed been correlated with improved neoantigen immune function . Moreover, our data show that IL-2 does not affect CD8 cell turnover and thymopoiesis, thus implying that CD8 T cells follow different homeostatic dynamics [7,8], possibly peripheral redistribution. The broad examination of T lymphocyte dynamics during IL-2 immunotherapy provides a more detailed definition of the protean interactions between specific T lymphocyte subsets during immune reconstitution; this will be of great value in the design of immune-based strategies in the treatment of HIV infection.
We are grateful to Shane Fodgen, Greta Di Felice and Anna Cristina Sacchetta for critical reading of the manuscript and valuable advice; Maura Mezzetti (Istituto dei Metodi Quantitativi, Università ‘Luigi Bocconi', Milan) for assistance with statistical analysis; Chiron Italia for valuable advice; Bianca Ghisi for excellent typing assistance; all patients participating in the study; and Patrizia Franza, Maria Tomminello and all the staff at the Institute of Infectious Diseases and Tropical Medicine, ‘L. Sacco’ Hospital, who cared for the patients.
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