CD34+ progenitor cells from asymptomatic patients are not a major reservoir for human immunodeficiency virus-1

Controversy exists as to whether hematopoietic progenitor cells are infected by human immunodeficiency virus-1 (HIV-1) in vivo. Most studies have focused on patients with acquired immunodeficiency syndrome (AIDS)/AIDS-related complex, and little data are available on asymptomatic patients with well preserved CD4+ T-cell counts. To determine if CD34+ hematopoietic progenitor cells are infected early in the course of HIV-1 disease, we evaluated 10 asymptomatic HIV-1 seropositive (HIV-1+) patients. The CD34+ cell fraction was purified by a two-step procedure consisting of both affinity chromatography and fluorescence-activated cell sorting that resulted in a median purity of over 99%. Using conventional and nested polymerase chain reaction (PCR) assays, we evaluated the presence and frequency of HIV-1 proviral DNA. Both bone marrow mononuclear cells and CD34- cells from all 10 patients were strongly positive for the HIV-1 pol and/or gag gene sequences. In contrast, sorted CD34+ cells from only two of 10 patients were positive, and the number of copies of proviral DNA in these samples was estimated to be from 2 to 5 per 250,000 cells. To test the in vitro functional capacity of CD34+ progenitors, these cells were assayed in both methylcellulose and long-term stromal culture. We found no significant reduction in the number of colony-forming unit-erythroid (CFU-E), burst-forming unit-erythroid (BFU-E), or colony-forming unit- granulocyte macrophage (CFU-GM) colonies, or in the frequency of cobblestone area forming cells from limit dilution analysis in HIV-1+ asymptomatic patients. Pooled methylcellulose colonies generated from CD34+ cells were HIV-1- in nine of 10 samples. All progeny from long- term cultures of CD34+ cells were HIV-1-. We conclude that the CD34+ hematopoietic progenitor compartment is not infected in the majority of asymptomatic HIV-1+ patients, and that these cells may represent a suitable target for strategies designed to protect developing CD4+ T cells from infection.     

Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia-lymphoma in a patient infected with human immunodeficiency virus type 1 (HIV-1)

A patient had adult T-cell leukemia-lymphoma in the unusual setting of coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type I (HTLV-I). The leukemic cells were CD4 positive and showed clonal genetic rearrangement of the T-cell receptor complex. Cytogenetic analysis showed three clonal karyotypic abnormalities: trisomy 3 and two translocations [t(1;15), (X;1)]. The patient was seropositive for HIV and HTLV-I; HTLV-I and HIV-1 DNA sequences were detected in peripheral blood leukocytes by the polymerase chain reaction. The HTLV-I sequences were detected in a relatively high proportion of mononuclear cells (at least 1 in 30 cells), whereas HIV-1 sequences were detected in a smaller proportion of cells (at least 1 in 3000 cells). Clinical remission was achieved after chemotherapy. There was a decrease in the proportion of HTLV-I positive mononuclear cells (at least 1 in 1000 cells), whereas the proportion of HIV-1 positive cells was relatively unchanged (at least 1 in 1000 cells). Adult T-cell leukemia-lymphoma in the setting of HIV coinfection may become increasingly common because asymptomatic retroviral coinfections are frequent.     

Integrated human T-cell leukemia virus II genome in CD8 + T cells from a patient with "atypical" hairy cell leukemia: evidence for distinct T and B cell lymphoproliferative disorders

We previously reported isolation of human T-cell leukemia virus II (HTLV-II) from a second patient (N.R.A.) with atypical hairy cell leukemia. Follow-up analysis of the characteristics of the patient's HTLV-II infection over a 2-year period has revealed that the patient had two coexistant lymphoproliferative disorders. Oligoclonally integrated HTLV-II was detected in DNA extracted from the patient's peripheral blood mononuclear cells on separate occasions greater than 1 year apart, similar to integration of HTLV-I seen in adult T cell leukemia/lymphoma. Although integrated provirus was readily detected, no HTLV-II viral RNA expression was seen in fresh peripheral blood lymphoid cells. Although the patient's peripheral blood consistently contained a majority of atypical lymphoid cells with a T cell antigenic phenotype, he ultimately developed extensive pleural, hepatic and soft tissue infiltration with malignant Tac+, tartrate-resistant, acid phosphatase-positive (TRAP+) B cells of clonal origin. To further characterize the role of HTLV-II, the patient's peripheral blood mononuclear cells were fractionated into four enriched subpopulations at autopsy. Oligoclonally integrated HTLV-II was detected in DNA from a T cell-enriched fraction and a CD8+ T cell-enriched fraction, but not in a CD4+ T cell-enriched fraction, a non-T cell fraction, or in B cells obtained from the malignant pleural effusion. We conclude that the patient harbored two distinct lymphoproliferative disorders, a TRAP+, Tac+ B cell malignancy consistent with hairy cell leukemia that did not contain HTLV-II and a Tac-, CD8+ lymphoproliferative syndrome with oligoclonally integrated HTLV-II.     

Prevalence of HTLV-I-associated T-cell lymphoma.

In order to assess the prevalence rate of HTLV-1-associated T-cell lymphomas and human retrovirus infection in general, approximately 21,000 individuals representing various patient populations, retroviral risk groups, and blood donors were examined for HTLV-I, HTLV-II, HIV-1, or HIV-2 infection using serologic and PCR assays. The prevalence rates among volunteer blood donors were 0.02% and 0% for HTLV and HIV, respectively. Significantly increased HTLV prevalence rates were observed among paid blood donors, African American health care clinic patients, Amerindians, recipients of HTLV-positive cellular blood products, intravenous drug users, sexual contacts and family members of HTLV-positive people, and patients with primary thrombocytosis and other-than-low-grade non-Hodgkin's lymphoma (NHL). Among some of these groups there were significant differences in the prevalence of HTLV-I versus HTLV-II. The eight HTLV-positive NHL patients all had mature, high-grade, CD4+ T-cell lymphomas with clonally integrated HTLV-I, for a prevalence of 4% among other-than-low-grade NHL patients. Seven of the eight died from their disease within 2 years despite treatment. Interestingly, two groups at risk for HTLV infection, namely needle stick victims and recipients of HTLV-infected and/or pooled plasma products, showed no evidence for infection. Significantly increased HIV-1 prevalence was observed among paid blood donors, African Americans, homosexuals, female prostitutes, hemophiliacs, and other-than-low-grade NHL patients. Only one patient was infected with HIV-2. Of the nine HIV-positive, other-than-low-grade NHL patients, seven HIV-1 positives had B-cell lymphomas, one HIV-1 positive had an HTLV-I-positive CD4+ T-cell lymphoma, and one infected with HIV-2 had a CD4+ T-cell lymphoma that was HTLV negative. The data indicate that HTLV-I lymphoma, while uncommon, is not necessarily rare among other-than-low-grade NHL cases in the United States and, given its poor prognosis, should probably be studied separately in clinical trials.     

Preferential infection of CD4+ memory T cells by human immunodeficiency virus type 1: evidence for a role in the selective T-cell functional defects observed in infected individuals.

CD4+ T cells of patients with AIDS exhibit a qualitative defect in their ability to respond to soluble antigen while their responses to mitogens remain normal. CD4+ T cells can be broadly divided phenotypically into "naive" [CD45RA+ (2H4+)] and "memory" [CD29+ (4B4+) or CD45RO+ (UCHL1+)] cell subpopulations, which represent distinct maturation stages. To determine the human immunodeficiency virus type 1 (HIV-1) infectability of memory and naive CD4+ T-cell subsets in vitro and to determine the in vivo preference of HIV-1 in these subpopulations, we obtained highly purified CD4+ T-cell subsets from normal and HIV-1-infected individuals and studied them by viral cultivation, quantitative polymerase chain reaction, and functional assays. Polymerase chain reaction studies demonstrated that the memory cell subset of CD4+ T cells is preferentially infected (4- to 10-fold more than naive T cells) by HIV-1 in vitro, and these memory cells are the principal reservoir for HIV-1 within CD4+ T cells obtained from infected individuals. Functional abnormalities attributable to CD4+ T cells in HIV-infected individuals (failure to respond in vitro to soluble antigen or to anti-CD3 monoclonal antibodies) were shown to reside primarily within these memory cells. Thus, the present study suggests that the selective functional defects present in the memory CD4+ T-cell subset of HIV-infected individuals may be a direct result of the preferential infection and consequently greater viral burden within these cells.     

Nadir CD4+ T-cell count and numbers of CD28+ CD4+ T-cells predict functional responses to immunizations in chronic HIV-1 infection

OBJECTIVE: To ascertain whether delaying the initiation of highly active antiretroviral therapy (HAART) compromises functional immune reconstitution in HIV-1 infection in persons who regain 'normal' CD4 T-cell counts after suppressive antiretroviral therapies. DESIGN: Prospective open-label study carried out at two University-affiliated HIV-outpatient clinics in the USA. SUBJECTS AND METHODS: Response to immunization was used as a model for in vivo functional immune competence in 29 HIV-1 infected patients with CD4 T-cell counts > 450 x 106 cells/l and HIV-RNA < 400 copies/ml for > 12 months after HAART and nine HIV-1 seronegative controls. After immunization with tetanus toxoid, diphtheria-toxoid, and keyhole limpet hemocyanin, immune response scores (IRS) were calculated using postimmunization antibody concentrations, lymphocyte proliferation, and delayed-type hypersensitivity responses to vaccine antigens. RESULTS: Despite normal numbers of circulating CD4 T-cells, the CD4 T-cell nadir before HAART initiation predicted the immune response to immunization (rho = 0.5; P < 0.005) while current CD4 T-cell count did not. Likewise, CD4 T-lymphocyte expression of the co-stimulatory molecule CD28 was also an independent predictor of response to immunization (rho = 0.5; P < 0.005). CONCLUSIONS: Even among persons who controlled HIV replication and normalized CD4 T-cell counts with HAART, pretreatment CD4 T-cell count and numbers of circulating CD4+CD28+ T-cells at immunization, but not current CD4 T-cell count, predict the ability to respond to vaccination. Delaying the initiation of HAART in chronic HIV-1 infection results in impaired functional immune restoration despite normalization of circulating CD4 T-cell numbers.     

Relationship of CD4+CD25+ regulatory T cells to immune status in HIV-infected patients

OBJECTIVE: To determine whether the frequencies of CD4+CD25+ regulatory T cells (T Reg) were related to immune status in HIV-infected patients. METHODS: Peripheral blood CD4 T-cell populations were examined for T-helper 1 cells (Th1), T-helper 2 cells (Th2), and T Reg by intracellular staining for interferon (IFN)-gamma and interleukin (IL)-4, and surface staining for CD25, respectively. The immunoregulatory properties of T Reg were assessed by measurement of the inhibitory effects of isolated CD4+CD25+ T Reg on CD4+CD25- T-cell proliferation. RESULTS: Isolated CD4+CD25+ T Reg from both HIV-infected patients and healthy controls strongly expressed CD45RO, HLA-DR, and FoxP3. HIV-infected patients with detectable plasma HIV-1 RNA showed a statistically significant increase in CD4+CD25high T Reg frequencies (P < 0.05) compared to healthy controls, with T Reg frequency inversely proportional to CD4 T-cell count (P < 0.01). However, in HIV-infected patients with undetectable plasma HIV-1 RNA, CD4+CD25high T Reg frequencies were not increased and were not related to CD4 T-cell counts. In both HIV-infected patient groups, T Reg frequency was inversely related to Th1 frequency (detectable HIV-1 RNA: P < 0.05; undetectable: P < 0.001), but positively related to Th2 frequency (detectable HIV-1 RNA: P < 0.01; undetectable: P < 0.001). T Reg activity was lower in patients with detectable plasma HIV-1 RNA than in patients with undetectable plasma HIV-1 RNA. CONCLUSIONS: Increased T Reg frequencies in peripheral blood were related to low peripheral blood CD4 T-cell counts and polarization toward Th2 immune responses in HIV-infected patients.     

Acquired immunodeficiency syndrome-associated T-cell lymphoma: evidence for human immunodeficiency virus type 1-associated T-cell transformation.

The majority of lymphomas in the setting of acquired, iatrogenic, or congenital immunodeficiencies are B-cell lymphoproliferations. We describe a rare T-cell lymphoma in a fulminantly ill patient infected with human immunodeficiency virus type 1 (HIV-1). The T-cell nature of the process was defined genotypically (monoclonal T-cell receptor beta-chain [CT beta] rearrangement) and phenotypically (CD45RO+, CD4+, CD5+, CD25+, CD8-, CD3- and negative for a variety of B-cell and monocyte markers). The CD4+, CD25+ (interleukin-2 receptor [IL-2R]) phenotype with production of IL-2 and IL-2R RNA is analogous to human T-lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia/lymphoma (ATLL); however, no HTLV-1 could be detected. Southern blot analysis did demonstrate monoclonally integrated HIV-1 within the tumor genome. Furthermore, the tumor cells were producing HIV p24 antigen as shown by immunohistochemistry. This is the first case of acquired immunodeficiency syndrome (AIDS)-associated non-Hodgkin's lymphoma in which HIV-1 infection may have played a central role in the lymphocyte transformation process.     

Kinetics of the T-cell receptor CD4 and CD8 V beta repertoire in HIV-1 vertically infected infants early treated with HAART.

OBJECTIVES: To determine the kinetics and the relationship between the T-cell receptor V beta (TCRBV) complementary determining region 3 length, the CD4 T-cell count and HIV viral load changes in HIV-1 infected infants treated early with highly active antiretroviral therapy (HAART) during 1 year of follow-up. DESIGN: Two HIV-1 vertically infected infants, two HIV-1 vertically exposed uninfected and two healthy controls were analysed by spectratyping. Evaluation of viral load, CD4 naive and memory cell counts and a proliferation test were also carried out. METHODS: Twenty-six families and subfamilies of the TCR on CD4 and CD8 T cells were analyzed by spectratyping. Flow cytometric analysis on peripheral blood mononuclear cells for CD4CD45Ra, CD4CD45Ro, CD8CD38, proliferation tests and plasma viral load measurements were performed at baseline, 1, 6 and after 12 months of therapy. RESULTS: HAART induced a marked reduction of viral load in both HIV-1 infected infants and an increase to normal CD4 T-cell count in the symptomatic infant. At baseline the TCRBV family distribution in the majority of CD8 and a few of the CD4 T cells was highly perturbed, with several TCRBV families showing a monoclonal/oligoclonal distribution. During HAART a normalization of the TCR repertoire in both CD8 and CD4 subsets occurred. TCR repertoire normalization was associated with a good virological and immunological response. CONCLUSION: These results suggest that complete and early virus replication control as a result of early HAART leads to a marked reduction of T-cell oligoclonality and is an essential prerequisite to the development of a polyclonal immune response in HIV-1 infected infants.     

Progressive spastic myelopathy in a patient co-infected with HIV-1 and HTLV-II: autoantibodies to the human homologue of rig in blood and cerebrospinal fluid.

OBJECTIVE: Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) are closely related human retroviruses. HTLV-I has been implicated in a chronic progressive myelopathy, known as tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM). We sought to determine whether autoantibodies to brain antigens were present in the cerebrospinal fluid (CSF) of a patient with chronic progressive spastic myelopathy with evidence of both HIV-1 infection and HTLV-I/II seropositivity. DESIGN: A 54-year-old bisexual man with clinical features of HAM/TSP of over 20 years' duration was followed. METHODS: We applied discriminatory DNA amplification (polymerase chain reaction) to distinguish HTLV-I from HTLV-II and to verify co-infection with HIV-1. The patient's CSF was used to screen a human brain cDNA expression library to identify antibodies directed against brain antigens. Autoreactive bacteriophage clones were isolated and sequenced. RESULTS: The patient was found to be co-infected with both HIV-1 and HTLV-II, but not with HTLV-I. HTLV-II proviral levels in the peripheral blood remained relatively constant, despite therapy with zidovudine. Prominent oligoclonal banding of immunoglobulins was present in the patient's CSF. A single repeatedly reactive cDNA clone was identified, by screening with CSF antibody, sequenced, and found to be the human homologue of the rat insulinoma gene, rig. CONCLUSIONS: HTLV-II infection may predispose to development of a HAM/TSP-like illness. Autoimmune mechanisms, such as autoantibody formation, may play a role in pathogenesis.     

Selective stimulation of CD4+ versus CD8+ T-cell subsets in symptomatic and asymptomatic HIV-1-infected individuals.

To analyze the proliferative capacity of CD4+ or CD8+ T-cell subsets of individuals infected with human immunodeficiency virus type 1 (HIV-1) and to optimize the in vitro conditions for virus replication, CD4+ or CD8+ cells of HIV-1-infected patients were selectively activated inside the whole peripheral blood mononuclear cell (PMNC) population by dual antibody stimulation. To do so PMNC of HIV-1-infected individuals were stimulated with the per se nonmitogenic anti-CD3 antibody fragment BMA030 F(ab)2 crosslinked through goat antimouse antibodies with an anti-CD4 or an anti-CD8 antibody, which lead to selective proliferation of either the CD4+ or the CD8+ T-cell subset. In the presence of monocyte supernatant and recombinant interleukin-2 (rIL2) CD4+ cells of HIV-1 patients responded normally upon such stimulation as their proliferation correlated (r = 0.9) to the percentage CD4+ cells present in the PMNC population. Selective stimulation and proliferation of CD8+ cells could, however, only partially be elicited by dual antibody stimulation, even in the presence of rIL-2 and monocyte supernatant. Their proliferative response did not correspond (r = 0.1) to the percentage CD8+ cells present in the PMNC culture. A positive correlation (r = 0.7) was detected only between percentage CD8+ HLA-DR- cells and proliferation. This confirmed previous studies showing that the defective in vitro proliferative response of peripheral blood lymphocytes of HIV-infected individuals to mitogens, which is usually interpreted being due to a CD4 cell defect, is actually due to a failure of CD8+DR+ cells to proliferate.(ABSTRACT TRUNCATED AT 250 WORDS)     

HIV-1感染者CD73~+CD8~+ T细胞减少与T细胞异常活化的相关性研究

目的探讨HIV-1感染者外周血CD8~+T细胞上CD73的表达特点及其与T细胞异常活化和疾病进展的关系。方法研究入选65例HIV-1感染者和27例健康对照。通过流式细胞术检测研究对象外周血CD73~+CD8~+T细胞的频率和绝对计数,并将患者CD73~+CD8~+T细胞绝对计数和频率与其CD4~+T细胞计数、HIV-1载量以及CD38~+CD8~+T细胞频率进行相关性分析。结果与健康对照相比,HIV-1感染者外周血CD73~+CD8~+T细胞绝对计数和频率均明显降低(P均0.05);HIV-1感染者外周血CD73~+CD8~+T细胞绝对计数和频率与CD4~+T细胞计数呈正相关(r=0.555,P=0.001;r=0.342,P=0.005),与CD38~+CD8~+T细胞频率呈负相关(r=-0.384,P=0.002;r=-0.387,P=0.001);HIV-1感染者CD73~+CD8~+T细胞的绝对计数与HIV-1载量呈弱负相关(r=-0.261,P=0.035)。结论 HIV-1感染者外周血CD73~+CD8~+T细胞的减少不但与患者T细胞的活化程度呈显著负相关,而且与AIDS疾病进展相关。     

Anti-human immunodeficiency virus noncytolytic CD8+ T-cell response: a review

The CD8+ T-cell immune response for human immunodeficiency virus (HIV) is divided into a cytolytic and noncytolytic mechanism. The mechanism of cell-mediated cytotoxic immunity for the partial control of human immunodeficiency virus type 1 (HIV-1) replication in infected individuals is well-characterized, and the direct killing of virus-infected cells by antigen-specific cytotoxic T-lymphocytes (CTL) is widely correlated with disease outcome. However, the mechanism of the noncytolytic component is not well understood. In part, this is because the main inhibitory factor or factors called CD8+ T-cell antiviral factor (CAF), have not yet been purified. In addition, results between the investigators are difficult to compare because of technical differences between laboratories, including the use of different in vitro cell expansion and stimulation methods for the CD8+ T cells, the necessity of sequential biochemical purification steps with restricted amounts of material, the complex analysis and interpretation of gene expression arrays, the use of different HIV strains, and the use of different short- or long-term inhibition assays using primary or immortalized target cells. Nevertheless, the diminishing efficacy of highly active antiretroviral therapy (HAART) because of the development of resistant HIV and the persistence of latent HIV provides a strong rationale for an immune therapy approach using antiviral factor(s) of the CD8+ T-cell noncytolytic immune response.     

Mobilization of CD34+ progenitor cells by granulocyte colony- stimulating factor in human immunodeficiency virus type 1-infected adults

We conducted a clinical trial to determine the feasibility of growth factor mobilization of CD34+ progenitor cells in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Eight asymptomatic, HIV-1- infected adults (median CD4+ T-cell count, 415 cells/microL), received 480 micrograms/d of granulocyte colony-stimulating factor (G-CSF) for 6 days without evidence of viral activation. Despite concerns that HIV-1 might inhibit hematopoiesis, CD34+ cells were successfully mobilized to the periphery of all donors, independent of the baseline CD4+ T-cell count, and the status of antiretroviral therapy. Leukapheresis was performed on day 6, and yielded a median of 194 x 10(6) CD34+ cells per leukapheresis (n = 7). CD34-enriched cells from the leukapheresis were predominantly myeloid-committed, but between 0.2% and 1.7% were primitive CD34+/CD38- progenitors. A median of 21.7% of the mobilized CD34+ cells were dimly positive for CD4. Consequently, CD34(+)-enriched cells were purified on the cell sorter (mean purity, 97.7% +/- 2.4%; n = 7), and examined for HIV-1 DNA. Purified CD34+ cells from two of seven donors were polymerase chain reaction (PCR)-positive for HIV-1, but only from one of three samples from each donor. We conclude that G- CSF can safely mobilize CD34+ progenitor cells in HIV-1-infected subjects, and that these cells are suitable for consideration in gene- transfer strategies.     

Human T-cell lymphotropic virus I and adult T-cell leukemia: report of a cluster in North Carolina

PURPOSE: Human adult T-cell leukemia-lymphoma is a malignant, proliferative disease of CD4+ lymphocytes associated with infection with human T-cell lymphotropic virus type I (HTLV-I). Following the presentation of a patient who was infected with the virus, we undertook a study of his family members and sexual contacts to see if a cluster of infected persons could be identified. CASE REPORT: A black heterosexual North Carolina native with a history of drug abuse presented with jaundice, and pancytopenia subsequently developed. He then became hypercalcemic and leukemic, with high numbers of circulating, morphologically abnormal CD4+ lymphocytes. RESULTS: As determined by radioimmunoassay and immunoblot analyses, the serum of the index case contained antibodies against core proteins (p19 and p24) of HTLV-I. When cultured in vitro with interleukin-2, the lymphocytes expressed HTLV-I specific core proteins. The virus recovered from these T cells was transmitted to cord blood T cells, which became immortalized for continuous growth in vitro, expressed HTLV-I p19 protein, and displayed characteristic C-type particles by electron microscopy. Studies of family members and sexual contacts, all of whom were black, heterosexual central North Carolina natives, revealed five of 28 whose serum had anti-HTLV-I antibodies as determined by radioimmunoassay and immunoblot. Neither the patient nor the seropositive family/contacts had antibodies against human immunodeficiency virus proteins. Four of the six people with HTLV-I infection had no history of intravenous drug abuse. Three of the five seropositive family/contacts had circulating, morphologically abnormal lymphocytes suggestive of "preleukemic" or "smoldering" human adult T-cell leukemia-lymphoma.     

Exposure of human CD34+ cells to human immunodeficiency virus type 1 does not influence their expansion and proliferation of hematopoietic progenitors in vitro

The susceptibility of highly purified human CD34+ cells to monocytotropic (Ba-L) and lymphotropic (A018-post) strains of human immunodeficiency virus-1 (HIV-1) was examined. Liquid cultures initiated with fresh immunomagnetically purified CD34+ cells using the K6.1 CD34 monoclonal antibody (MoAb) (K6.1/CD34+) were positive for HIV expression 2 weeks after exposure to HIV-1 Ba-L. These cells were initially greater than 90% CD34+ and had undetectable monocyte contamination by flow-cytometric staining and side-scatter analyses, respectively, and undetectable T-cell contamination by CD3 polymerase chain reaction (PCR) analysis. However, secondary CD34+ liquid cultures reselected from the primary liquid cultures 24 hours after HIV exposure by panning with the ICH3 CD34 MoAb (ICH3/CD34+) and maintained for an additional 14 days were negative for HIV expression. The ICH3-unbound cells were positive for both spliced and unspliced HIV RNA when exposed to HIV-1 Ba-L, and were DNA PCR positive when exposed to either monocytotropic or lymphotropic HIV-1. To further test that CD34+ cells were not infectible by HIV-1, we exposed K6.1/CD34+ cells continuously to HIV-1 in a culture system capable of maintaining and expanding primitive CD34+ cells. HIV-exposed K6.1/CD34+ cells proliferated and expanded as efficiently as uninfected cultures. However, when reselected magnetically using the K6.1 CD34 MoAb after expansion for 7 days, bound K6.1/CD34+ cells were again negative for HIV-1 expression, whereas unbound cells were positive for HIV-1 expression. These findings suggest that a sequential CD34+ cell-selection process, in which the two selections are separated by a brief culture period, can yield a population of CD34+ cells that are not infected with HIV-1. This process may be useful in the design of stem or progenitor cell- based transplantation therapies for HIV infection.     

Human immunodeficiency virus DNA is present in a high percentage of CD4+ lymphocytes of seropositive individuals.

Human immunodeficiency virus type 1 (HIV-1) infects predominantly CD4+ cells in human peripheral blood and infection is associated with CD4+ lymphocyte dysfunction in patients with AIDS. To determine the frequency of HIV-1 infection in CD4+ lymphocytes in vivo, peripheral blood CD4+ lymphocytes were isolated by fluorescence-activated cell sorting from HIV-1-infected persons with clinical disease ranging from asymptomatic to AIDS. Using standard and booster polymerase chain reaction analyses, study patients with AIDS and AIDS-related complex (ARC) were found to harbor the HIV-1 genome in at least 10% of CD4+ lymphocytes, and approximately 10-fold less infected cells were found in those with asymptomatic infection. In addition, the peripheral blood mononuclear cells from patients with ARC frequently contained a higher absolute number of HIV-1-infected CD4+ lymphocytes than those with AIDS or asymptomatic infection. It is likely that this high level of infection of CD4+ lymphocytes is the primary cause for the progressive immunologic deficiency observed in patients infected with HIV-1.