Role of thymosin α1 in restoring immune response in immunological nonresponders living with HIV

Human immunodeficiency virus type 1 (HIV-1) infection leads to progressive depletion of CD4 + T cells and functional impairment in people living with HIV (PLWH). The introduction of antiretroviral therapy (ART) has reduced morbidity and mortality in PLWH by suppressing viral replication and promoting CD4 + T cell recovery [1, 2]. However, approximately 20% of PLWH fail to restore CD4 + T cell counts despite persistent viral suppression with ART, leading to a heightened susceptibility to opportunistic infections, non-AIDS-related diseases, and deteriorating quality of life as compared to immunological responders (IRs) who achieve substantial CD4 + T cell count recovery [3‐5]. Currently, no definitive pharmaceutical intervention exists to restore CD4 + T cell count in INRs. The failure to restore CD4 + T cell count following ART has been attributed primarily to a defect in recent thymic immigrants [6]. A cross-sectional study found INRs maintain a lower proportion of thymic immigrant CD4 + T cells and naïve CD4 + T cells, but a higher proportion of effector memory CD4 + T cells and elevated levels of thymic immigrant CD4 + T cell pyroptosis compared with IRs [7]. Therefore, immunomodulatory agents may hold promise for enhancing thymic recovery and immune reconstitution in INRs.

Thymosin α1 (Tα1), a synthetic thymic polypeptide homologous to a natural product isolated from thymosin fraction 5 of calf thymuses, has emerged as a potential candidate [8]. As an agonist of Toll-like receptors 2 and 9, Tα1 can boost thymic output, stimulate various immune cells populations including dendritic cells, natural killer cells, B cells, and T cells, and promote their proliferation and adaptive response crucial for combatin viral, bacterial, and fungal infections, and cancers [9]. After the advent of the first antiretroviral drug, Tα1 was found to elevate CD4 + T cell levels only in combination with IFN-α and zidovudine [10]. However, a contemporaneous study reporte that IL-2 led to significant increases in CD4 + T cells in the presence of zidovudine but does not when combined with Tα1 [11]. With the popularization of ART, a phase II randomized, controlled open-label trial noted changes primarily in signal joint T cell receptor excision circles (sjTREC) levels in peripheral blood mononuclear cells (PBMCs) but not in CD4 + , CD8 + , and CD45RA + T lymphocyte subsets after week 12 of Tα1 treatment in PLWH [12]. Previous investigations have predominantly focused on antiretroviral-naïve PLWH and considered Tα1 as an adjunct to ART. Here we would like to expand the applicability of Tα1 to explore its potential in immune reconstitution in INRs after long-term ART.

The present prospective pilot trial seeks to assess the safety and efficacy of Tα1 in restoring immune response in INRs. After Tα1 treatment, within 24 weeks, parameters related to thymic output, T cell subsets, immune exhaustion, and immunosenescence were evaluated to characterize the immunological profiles of these participants.

In spite of the mounting life expectancy of PLWH after ART treatment worldwide, INRs living with HIV were still suffering from severe conditions due to diminished thymic output, bone marrow hematopoiesis dysfunction, residual virus replication, immune exhaustion, and aberrant immune activation [6, 7]. Impaired thymic function in patients with low CD4 + T cell counts may account for inadequate CD4 + T cell restoration [14]. In this study, Tα1 treatment altered CD4 + T-cell counts and thymic output characterized by PBMC sjTREC at an overall level after initiation. Previous studies have demonstrated a correlation between levels of PBMC sjTREC, recent thymic emigration, long-time CD4 + T count response, and survival in PLWH [13, 15, 16]. However, more in-depth investigations have revealed that the elevation of sjTREC levels could be the consequence of homeostatic proliferation of CD45RA + naïve T cells in the periphery [17, 18]. Combined with the great increase of naïve T cells in this study, the use of sjTREC as a measure of thymic output might carry the risk of producing false-positive results. In contrast, the sj/βTREC ratio, serving as a surrogate for thymic output, remained unaffected by peripheral proliferation and predicted the therapy-mediated recovery of naive and total CD4 + T cells, as well as HIV disease progression [19, 20]. Given the positive correlation between the sj/βTREC ratio and CD4 + naïve T cells as demonstrated (R = 0.504, P = 0.009 in Dion ML, Blood 2007), the findings of increased thymic output in this study still hold merit.

Traditionally, the CD4 + T cell count and the CD4/CD8 T-cell ratio serve as primary indicators for evaluating immune reconstitution in PLWH [21]. We observed a difference in multiple comparisons of CD4 + T cell counts and a gradual, albeit insignificant, increase in the median CD4 + T cell count throughout the treatment period (225.8 vs. 286.0 vs. 238.1 vs. 236.0 vs. 274.0). Although a large gap still existed from satisfactory level (> 350 cells/μL), we had reason to believe that participants achieve an improved immune profile and prognosis. The feasibility of the N/EM T cell ratio as a useful predictive marker of immune reconstitution in chronic HIV patients suggested the role of Tα1 in immune reconstitution [22]. In brief, our results suggest that INRs achieve partial immune reconstitution and probably a better prognosis after treatment.

We confirmed that Tα1 was safe and well tolerated in this group, consistent with previous clinical trials of Tα1 in PLWH [10‐12]. While different immunomodulating compounds like IL-2 were used to restore CD4 + T cell count in HIV infection, they failed to reduce the incidence of opportunistic disease or death because the expanded CD4 + T cells lacked host defense capacity. [23] Encouragingly, recombinant human IL-7 was proven well tolerated and result in a dose-dependent increase of CD4 + T cell and thymic output in patients with poor immune reconstitution [24]. Considering that Tα1 has demonstrated its effectiveness in elevating CD4 + T cells after 12 months [10], a larger and longer cohort study is necessary to further investigate the therapeutic efficacy of Tα1 for INRs living with HIV in the future.

Immunophenotyping analysis revealed the distinct roles played by T cell subsets at various stages of development. The production of de novo naïve T cells in the thymus has been demonstrated to push reconstitution of all T cell subsets [14]. Based on our findings, Tα1 reversed the decrease of proportions of naïve T cells in chronic HIV infection, and reduced the proportions of exhausted PD-1 + T cells caused by sustained abnormal immune activation. Besides, it significantly reduced the proportion of CM CD4 + T cells, which have been identified as primary host cells for HIV reservoir [25]. Fromentin et al. found that CD4 + CM T cells co-expressing PD-1 and CTLA-4 exhibit diminished responsiveness to activating stimuli facilitating HIV latency, and the PD-1/PD-L1 inhibitor Pembrolizumab induced HIV latency reversal in PLWH [26, 27]. HIV persists preferentially in CD4 + T cells expressing multiple immune checkpoint molecules, including PD-1, TIM-3, CTLA-4 and LAG-3. Therefore, combining immune checkpoints to reverse latency is a more effective strategy than using a single latency reversal agent, like vorinostat and bryostatin [28]. In fact, some studies found INRs maintain a lower proportion of thymic migrated cells and naïve T cells, a higher proportion of CD4 + PD-1 + T cells, and a higher level of HIV DNA and CA-RNA compared to IRs [7]. Mechanistically, HIV could evade host immune responses by upregulating the expression of immune checkpoint receptors, like PD-1, CTLA-4, and Tim-3 [29]. Our findings extend one underlying scientific hypothesis that Tα1 treatment exerts a positive effect on the regeneration of T-cell storage, and participated in diminishing the HIV reservoir, which represents the focal point of our forthcoming investigations.

The present study has some limitations. First, the sample size is relatively small. Thus, a larger cohort is required to confirm the safety and efficacy of Tα1 on INRs living with HIV. Second, the absence of a control group, such as a placebo arm. Third, the utilization of sjTREC rather than sj/βTREC ratio as a surrogate for thymic output might introduce some degree of false-positive results. Lastly, longer duration of treatment might be necessary to achieve statistically significant increases in CD4 + T cell counts.

In summary, we first report that Tα1 treatment for INRs living with HIV was well-tolerated. Tα1 treatment improves the CD4 + T cell count at overall level but not at endpoint. Notably, Tα1 increases thymic output, rebuilds the composition of T cell subsets, and partially reverses immune exhaustion. The efficacy of Tα1 treatment in INRs necessitates further evaluation through extended and expanded clinical trials, as well as comprehensive mechanistic investigations.