Hematopoietic stem cells and HIV infection.

(See the major article by McNamara et al on pages 1807–16.) New emphasis has been placed on curing human immunodeficiency virus (HIV) infection. Before we can eradicate HIV, we need to understand which cells are infected in vivo and which contribute to reservoirs. The best-characterized latent reservoir is resting CD4+ T cells. However, there is evidence that other reservoirs exist, as well [1–3]. Recent data suggest that hematopoietic stem cells and/or progenitors (HPCs) may contribute to the reservoir [4], but other investigators could not detect infected HPCs in vivo [5, 6]. It is challenging to determine whether HPCs are infected, because T cells (which are likely infected at higher levels) may contaminate HPC preparations [5, 6]. A related, more-difficult challenge is to determine whether true hematopoietic stem cells (HSCs) are infected by HIV in vivo. Resolving these issues will have important implications for HIV cure efforts, including transplantation approaches. In this issue of the Journal, McNamara et al [7] address whether HPCs are detectably infected in vivo and, if so, whether T-cell contamination explains their results. Their data suggest that HPCs are only infected in a subset of HIV infected individuals—those who have been infected for a relatively short time before initiating antiretroviral therapy (ART). This finding may help resolve conflicting data from other groups who did not detect HPC infection [5, 6]. McNamara et al [7] positively selected for immature HPCs, using CD133. The authors chose this simple approach because it maximized cell yield, allowing them to separate a subset of HPCs away from contaminating T cells. By taking this approach, they successfully limited the number of T cells in the CD133+ fraction to <1%, while T cells were enriched in the CD133– fraction (36%–82%). They then measured, by limiting-dilution polymerase chain reaction analysis, the amounts of HIV DNA in the CD133+ and CD133– fractions. In 4 of 11 patients, they detected signal in the CD133+ but not the CD133– fraction. In 5 of 11 subjects, a Fisher exact test indicated that the signal in the CD133+ fraction was unlikely to be due to contaminating T cells or CD133– cells. Despite these in vivo data, an argument against HSC infection is that lineages such as B cells and neutrophils are not infected with HIV. One potential explanation may be that infected HPCs die as they differentiate into progenitors. In vitro work by Carter et al [8] suggested that HPCs infected with a gutted reporter virus (that did not encode HIV proteins) develop into progenitors more efficiently than cells infected with a Δenv virus. A wild-type virus expressing Env should be still more toxic. Consistent with the idea that HIV infection might kill progenitors, bone marrow mononuclear counts correlated with years of infection in patients with high viral loads [9]. However, even if cytopathic events contribute to HSC death, a portion of the viruses may be mutated such that they cannot express HIV proteins. These proviruses should be detectable in downstream lineages (ie, B cells); more vigorous studies should be pursued to determine whether these cells contain defective proviruses at very low levels. If HPCs are infected, why could other groups not repeat this finding? Perhaps HPC infection is only detectable early in HIV disease. Notably, the patient population with HPC infection is very different from the patient population in the study by Durand et al [10]. The patients (without infected HPCs) studied by Durand et al received a diagnosis of HIV infection on average 18 years (median, 21 years) prior to the study, while the patients with infected HPCs in the study by McNamara et al received a diagnosis more recently, on average 7 years (median, 6 years) prior to the study. Similarly, the patients without infected HPCs in the study by McNamara et al also received a diagnosis on average 18 years (median, 21 years) prior to the study. Other, related interpretations are possible. Perhaps, the time during which a patient is not receiving ART determines whether HPCs can be detectably infected. It is possible that after many years of HIV infection, the majority of HPCs have already been infected and died, such that the level of remaining HIV-susceptible HPCs is too low to detect. It is also possible that the results are donor related; for example, some humans could have more HPCs susceptible to HIV infection, perhaps related to the level of CD4 on their surface. Whether true human stem cells express CD4 has not been fully answered, but it is an important question. A lack of CD4 may explain why no infection was detected by Josefsson et al, who selected for CD4– HPCs [6]. Because HIV gp120 binding and HIV infection of enriched HPCs is inhibited by anti-CD4 in vitro [9, 10] and because CD34+CD4+ sorted cells are enriched for pluripotency in long-term culture assays [10], at least a subset of HPCs may express low levels of CD4. Thus, CD4, if present, is so at low levels, meaning there should be lower levels of infection in HPCs than in CD4+ T cells [11]. Limitations of the report by McNamara et al [7] include the small number of patients studied and, because of the available volume of bone marrow aspirate, the small number of genomes assayed in both the CD133+ and CD133– fractions. Initially, it appears a bit surprising that they did not detect any signal in the CD133– fraction in several subjects, since 36%–72% of these cells expressed CD3. However, this is possible because of the small number of genomes assayed and the short time course of infection in the patients with infected HPCs. Moreover, data are accumulating that the T-cell reservoir increases as the interval during which ART is not received increases [12]. In other words, the level of infection among contaminating T cells rises and becomes more problematic over time, just as the level of infection among HPCs may be decreasing. Thus, the short course of infection likely allowed detection of HPC infection. It is also possible that many more HIV-infected individuals have infected HPCs, but we are only able to detect this in a subset of patients who have a small T-cell reservoir. Enhancements in cell sorting technology may achieve the necessary purities to overcome this issue in the future. The most important outstanding question is whether HPCs are an HIV reservoir. Because many patients do not have detectable infection in these cells [5, 6], it seems likely that HPCs at most may be a reservoir in a subset of infected individuals. Furthermore, as yet, no infectious virus has been cultured from exogenously stimulated HPCs from patients (again, likely because of the small volumes obtained from bone marrow aspirates, which is a substantial hurdle). Additionally, comparison of the data from an earlier report by Carter et al [8] with data from the current study (which includes later measurements of the same patients) suggests that the level of HPC infection may decrease over time during ART. Clearly, more longitudinal studies need to be performed to define which patients have infected HPCs and to determine the half-life of these HPCs in patients receiving ART. If the half-life of the HPC reservoir were short, then autologous HPC transplantation in an ART-treated individual could be a feasible approach to eradication if the T cells could be adequately removed. If only a subset of patients were infected, identification of this subset would also be important in eradication trials. Other important issues include the tropism of virus in infected HPCs in vivo. McNamara et al suggest that they will be CXCR4-tropic viruses, since earlier progenitors are infected more efficiently with CXCR4-tropic viruses [4]. This may explain why patients do less well once a CXCR4-tropic virus predominates. Notably, sequencing proviruses from patient HPCs will provide information on tropism and the frequency of defective forms in HPCs, which may differ from the frequency in T cells. Additionally, while CD133– cells are enriched for HSCs, proving that HIV-infected HPCs in vivo include true HSCs with long-term renewal and engraftment capability will also be important. If only downstream progenitors are infected, their half-lives will be shorter, and the cells may die out. A more conclusive approach will likely require serial transplantation of infected cells (with reporter HIV) in NSG mice [13]. McNamara et al provide insight into the infection of HPCs in vivo and provide evidence suggesting that HPC infection occurs in a subset of patients and is not a result of T-cell contamination [7]. While it will be important to know whether HPCs are a reservoir for HIV, further studies to establish if, when, and at what level HPCs are infected should also be pursued to both understand HIV pathogenesis and help in the design of eradication approaches.