Both memory and CD45RA+/CD62L+ naive CD4(+) T cells are infected in human immunodeficiency virus type 1-infected individuals

Abstract

Cellular activation is critical for the propagation of human immunodeficiency virus type 1 (HIV-1) infection. It has been suggested that truly naive CD4(+) T cells are resistant to productive HIV-1 infection because of their constitutive resting state. Memory and naive CD4(+) T-cell subsets from 11 HIV-1-infected individuals were isolated ex vivo by a combination of magnetic bead depletion and fluorescence-activated cell sorting techniques with stringent criteria of combined expression of CD45RA and CD62L to identify naive CD4(+) T-cell subsets. In all patients HIV-1 provirus could be detected within naive CD45RA+/CD62L+ CD4(+) T cells; in addition, replication-competent HIV-1 was isolated from these cells upon CD4(+) T-cell stimulation in tissue cultures. Memory CD4(+) T cells had a median of fourfold more replication-competent virus and a median of sixfold more provirus than naive CD4(+) T cells. Overall, there was a median of 16-fold more integrated provirus identified in memory CD4(+) T cells than in naive CD4(+) T cells within a given patient. Interestingly, there was a trend toward equalization of viral loads in memory and naive CD4(+) T-cell subsets in those patients who harbored CXCR4-using (syncytium-inducing) viruses. Within any given patient, there was no selective usage of a particular coreceptor by virus isolated from memory versus naive CD4(+) T cells. Our findings suggest that naive CD4(+) T cells may be a significant viral reservoir for HIV, particularly in those patients harboring CXCR4-using viruses.

FIG. 1

Fluorescence-activated cell sorter analysis and sorting of CD4⁺ T cells. CD4⁺-enriched T cells were obtained by negative bead selection and subsequently stained for the expression of CD62L, CD45RA, and CD4. Sorting gates are shown for lymphocytes (left panel), CD4-positive T cells (middle panel), and naive and memory cells (right panel). Cell purities were always assessed immediately and 36 h postsorting and were >95%.

FIG. 2

Quantitative virus isolation from memory and naive CD4⁺ T-cell subsets. Frequencies of memory and naive CD4⁺ T cells carrying replication-competent HIV-1 DNA were determined by activating sorted CD4⁺ T-cell memory and naive subsets on day 0. For each assay, a statistical method developed by Myers et al. (24) was used to calculate copy numbers or infectious units per million cells. Patients are grouped according to the presence of NSI or SI virus isolated in tissue cultures based on MT2 cell assays. Note the log scale of the y axis.

FIG. 3

Quantitation of total proviral HIV-1 DNA in memory and naive CD4⁺ T-cell subsets. The number of copies of total HIV-1 proviral DNA was determined in freshly sorted memory and naive CD4⁺ T-cell subsets. Patients are grouped according to the presence of NSI or SI virus isolated in tissue cultures based on MT2 cell assays. Note the log scale of the y axis.

FIG. 4

Quantitation of integrated HIV-1 DNA in memory and naive CD4⁺ T-cell subsets. The number of copies of integrated HIV-1 proviral DNA was determined in freshly sorted memory and naive CD4⁺ T-cell subsets. Integrated provirus was undetectable in patient 11 in either CD4⁺ T-cell subset with an input DNA of 2 μg in the assay. Data for patient 8 were not determined. Patients are grouped according to the presence of NSI or SI virus isolated in tissue cultures based on MT2 cell assays. Note the log scale of the y axis.

FIG. 5

HTA and accompanying HMA of amplified envelopes obtained from cDNA in plasma (P), DNA in ex vivo-sorted memory (M) and naive (N) CD4⁺ T cells, and supernatant viral culture cDNA of memory (C-M) and naive (C-N) CD4⁺ T cells in two representative patients. U, unrelated sample. For each patient, two HTA were performed, with a probe made from virus cultured from memory CD4⁺ T cells (C-M*) (left side of gel) and a probe made from virus cultured from naive CD4⁺ T cells (C-N**) (right side of gel). In patient 5 (a and b), the migration patterns between envelopes from cultured memory and naive CD4⁺ T cells differed, indicating genetic differences between viruses cultured from these two subsets; however, in patient 7 (c and d) migration occurred equally along the length of the gel, indicating the presence of viruses with genetically identical envelopes. In both patients, envelope PCR products from cultured memory CD4⁺ T cells migrated equally with those from plasma virus (left side of gel), whereas there was a retardation of migration in the plasma viruses compared to viruses from naive CD4⁺ T cells with the probe of virus cultured from naive CD4⁺ T cells (C-N**) (right side of gel), thus indicating the closer genetic similarity of viruses in plasma and memory cells than in plasma and naive cells.