Quiescent CD4 Research Articles

CD3-T cell receptor modulation is selectively induced in CD8 but not CD4 lymphocytes cultured in agar

The CD3-T cell receptor (TcR) complex is central to the immune response. Upon binding by specific ligands, internalized CD3-TcR molecules increase, and either T cell response or unresponsiveness may ensue depending on the triggering conditions. Using semi-solid agar culture, we have shown previously that quiescent CD4 but not CD8 lymphocytes generate clonal colonies under phytohaemagglutinin stimulation. Here we have demonstrated that the agar induces selective CD3-TcR modulation in the CD8 and not in the CD4 subset. CD8 lymphocytes preactivated in liquid culture and recultured in agar with exogenous recombinant interleukin-2 generate colonies with a modulated CD3-TcR surface expression. The peptides composing the CD3-TcR complex are synthesized in CD8 colonies as well as in CD4; however, the CD3 gamma chain is phosphorylated at a higher level in CD8 colonies. A component of the agar polymer, absent in agarose, appears to be the ligand that induces differential CD3-TcR modulation in the CD8 subset. In contrast to agar culture, CD8 colonies can be derived from quiescent CD8 lymphocytes in agarose. These CD8 colonies express unmodulated CD-TcR. CD3-TcR modulation with anti-CD3 monoclonal antibody prior to culturing in agarose inhibits the colony formation. We conclude that given triggering conditions can result in both CD3-TcR modulation and inhibition of the proliferative response selectively in the CD8 lymphocyte subset and not in the CD4.

Resistance Risk Management in HIV Therapy Switching with Explicit Quiescent T-Cell Modeling

Highly Active Antiretroviral Therapy (HAART) has proven remarkably effective in controlling the development of HIV. However, drug resistance may compromise these benefits. During the use of HAART, drug-resistant strains can develop and become the dominant species. Because the number of independent treatment regimens is limited, once resistance to all available drug classes arises, the patient will die. Drug resistance is therefore a critical problem for HIV treatment. In this paper, we explicitly model one known reservoir compartment, the quiescent infected CD4+ T cells, and explore the effects of this reservoir on a drug-switching strategy designed to minimize the further development of drug-resistant virus.

TLR2 Signaling Renders Quiescent Naive and Memory CD4+ T Cells More Susceptible to Productive Infection with X4 and R5 HIV-Type 1

It has been recently demonstrated that circulating microbial products are responsible for a systemic immune activation in individuals infected with HIV-type 1. Bacterial products carry structural conserved motifs recognized by TLRs. Some TLR members are expressed in primary human CD4+ T cells but the precise functional role played by these pattern recognition receptors is still imprecise. In this study, we report that engagement of TLR2 in quiescent naive and memory CD4+ T cells leads to the acquisition of an effector-like phenotype. Interestingly, engagement of TLR2 renders both cell subsets more susceptible to productive infection with X4 virions and a higher virus production was seen with R5 viruses. It can be proposed that exposure of resting CD4+ T cells to pathogen-derived products that can engage TLR2 induces the acquisition of an effector-like phenotype in naive and memory CD4+ T lymphocytes, a phenomenon that might result in an acceleration of virus replication, immune dysregulation, and HIV-type 1-mediated disease progression.

Centrosomal pre-integration latency of HIV-1 in quiescent cells

Human immunodeficiency virus type 1 (HIV-1) efficiently replicates in dividing and non-dividing cells. However, HIV-1 infection is blocked at an early post-entry step in quiescent CD4+ T cells in vitro. The molecular basis of this restriction is still poorly understood. Here, we show that in quiescent cells, incoming HIV-1 sub-viral complexes concentrate and stably reside at the centrosome for several weeks. Upon cell activation, viral replication resumes leading to viral gene expression. Thus, HIV-1 can persist in quiescent cells as a stable, centrosome-associated, pre-integration intermediate.

Microarray Analysis Reveals CCL24/Eotaxin-2 as an Effector of the Pathogenetic Effects Induced by HIV-1 Nef

Human immunodeficiency virus (HIV)-1 Nef is a regulatory protein critically involved in AIDS pathogenesis. We previously demonstrated that extracellular Nef is efficiently internalized by human primary monocyte-derived macrophages (MDMs), thereby activating a number of transcription factors including STATs, MAPKs, IRF-3, and NF-kappaB. Such an activation state leads to the release of inflammatory factors whose paracrine effects deserve deep consideration. Here, we demonstrate that quiescent CD4 lymphocytes undergo cell activation when cultivated in supernatants from autologous MDMs treated with extracellular wt Nef but not with its counterpart mutated in the (72)PxxP(75) polyproline domain. Of a pathogenetic relevance, this effect coupled with the sensitization of quiescent CD4 lymphocytes to HIV-1 infection. By microarray assay, we found that the CCL24/eotaxin-2 gene was up-regulated in MDMs treated with wt Nef but not with the (72)AxxA(75) mutant. In addition, the higher transcription activity correlated with a significant increase of the CCL24/Eotaxin-2 release. Finally, we observed that anti-CCL24/eotaxin-2 antibodies efficiently neutralized the stimulatory effect on CD4 lymphocytes of supernatants from MDMs treated with extracellular Nef. Overall, these data support the idea that CCL24/eotaxin-2 is part of the mechanism of CD4 lymphocyte activation paracrinally induced by Nef.

The regulatory role of naturally occurring CTLA-4 splice variants:the suppressive function of short splice variant 1/4 WT CTLA-4 (B91)

Abstract Cytotoxic T lymphocyte Antigen 4 (CTLA-4) is an important cell surface molecule that inhibits T cell activation. Four splice variants of CTLA-4 have been identified, indicating the regulatory complexity of CTLA-4. In this study, we characterized the biology of the shortest splice variant 1/4WT CTLA-4 and its functional correlation to full length (FL CTLA-4). 1/4WT CTLA-4 mRNA was found in quiescent CD4+ T cells and upregulated after immunization with MOG35-55 peptide. The kinetic expression of 1/4WT was opposite to FL CTLA-4. To test functions of 1/4WT CTLA-4, we transduced CD4 T cells with a retroviral vector coexpressing 1/4WT CTLA-4 and the green fluorescent protein (GFP). FL CTLA-4 was downregulated on the cell surface by 1/4WT overexpression, which consequently enhanced antigen specific T cell responses by enhancing T cell proliferation and cytokine production in vitro. This functional correlation between 1/4WT and FL CTLA-4 had profound influence on effector CD4 T cell function. Mice adoptively transferred with MOG35-55 specific CD4 T cells containing ectopically expressed 1/4WT CTLA-4 showed dramatically exacerbated EAE disease, increased CD4 T cell proliferation, and increased IFN gamma and IL-17 production. These data suggest that 1/4WT CTLA-4 plays an important role in regulating T cell activation by affecting expression and/or function of FL CTLA-4.

Immediate Activation Fails To Rescue Efficient Human Immunodeficiency Virus Replication in Quiescent CD4+T Cells

Unlike activated T cells, quiescent CD4+ T cells have shown resistance to human immunodeficiency virus (HIV) infection due to a block in the early events of the viral life cycle. To further investigate the nature of this block, we infected quiescent CD4+ T cells with HIV-1(NL4-3) and immediately stimulated them. Compared to activated (prestimulated) cells, these poststimulated cells showed slightly decreased viral entry and delays in the completion of reverse transcription. However, the relative efficiency of integration was similar to that of prestimulated cells. Together, this resulted in decreased expression of tat/rev mRNA and synthesis of viral protein. Furthermore, based on cell cycle staining and BrdU incorporation, poststimulated cells expressing viral protein failed to initiate a second round of their cell cycle, independently of Vpr-mediated arrest. Together, these data demonstrate that the early stages of the HIV life cycle are inefficient in these poststimulated cells and that efficient replication cannot be induced by subsequent activation.

Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells.

Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. We treated human quiescent or proliferating CD8+ cells with 50 microM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable.

A novel in vitro system to generate and study latently HIV-infected long-lived normal CD4+ T-lymphocytes

Studies of mechanisms of HIV-latency and its reactivation in long-lived resting CD4+ T-lymphocytes in patients have been limited due to the very low frequency of these cells (∼ 1–10 cells per 10 6 CD4+ T-cells). To circumvent this obstacle, an in vitro culture system for post-activation long-term survival of normal CD4+ T-cells in a quiescent (non-cycling) state was developed and used to generate latently infected, long-lived quiescent CD4+ T-cells from HIV-infected, activated normal CD4+ T-lymphocytes. This yielded a frequency of ∼ 5 × 10 4 latently infected cells per 10 6 cells in culture, which is ∼ 10 3- to 10 4-fold higher than that available from patients. Moreover, 5–10% of long-term surviving non-cycling T-cells were found to make infectious HIV continuously at low levels, showing that HIV production from infected T-cells does not require full cellular activation. This model system should facilitate studies of long-lived, latently infected and persistently HIV-producing quiescent normal CD4+ T-lymphocytes.

Differential partitioning and trafficking of GM gangliosides and cholesterol-rich lipid rafts in thymic and splenic CD4 T cells

The GM gangliosides and cholesterol components of plasma membrane lipid rafts play an important role in the recruitment and signaling of protein receptors in eukaryotic cells. Herein, we have analyzed at the single-cell level the partitioning and intracellular trafficking of GM gangliosides and cholesterol in quiescent (CD4 +CD69 −) and CD3-activated (CD4 +CD69 +) thymic and splenic T cells. First, regardless the gender and the quiescent or activated status of T cells, the GM and cholesterol content in cytosol and plasma membrane as well as the expression levels of GM synthase, Sphingomyelin phosphodiestarase 2 and HMG Co-A reductase genes involved in GM and cholesterol synthesis were constantly lower in CD4 thymocytes than in CD4 splenocytes. Second, we detected variations in the balance between GM and cholesterol in plasma membrane depending on aging, and found that deprivation of cellular cholesterol does not necessarily affect the GM content in both quiescent CD4 thymocytes and splenocytes. Third, CD3 stimulation up-regulated the GM and little if any the cholesterol content in both thymic and splenic CD4 T cells, suggesting a cross talk between the CD3 signaling and GM but not cholesterol biosynthesis pathway. Fourth, partitioning and trafficking of GM to the plasma membrane depended on the transport of ceramide precursors from endoplasmic reticulum to Golgi network, as well as on the synthesis, glycosylation and vesicular assembly in trans-Golgi, and less on the cytoskeleton architecture in both quiescent and activated CD4 thymic and splenic T cells. Together, these findings suggest that the differential partitioning and intracellular trafficking of GM and cholesterol in thymic and splenic CD4 T cells may account for the stage of functional maturation.

Latent HIV-1 reservoirs in children: considerations for therapy.

Understanding the genetic properties and clinical relevance of latent HIV-1 reservoirs in children is critical for guiding pediatric treatment strategies, especially as antiretroviral drugs gain widespread use in resource-constrained settings in which the inherent risk of early infection with drug-resistant HIV-1 is most challenging. Latent reservoirs of HIV-1 prevent virus clearance in children on highly active antiretroviral therapy. Effective highly active antiretroviral therapy, however, fixes the genetic composition of the resting CD4 T-cell reservoir, which contains only founder virus in children treated during primary infection. This reservoir otherwise stores both archival variants and drug-resistant variants arising during treatment failure. The persistence of HIV-1 in quiescent CD4 T cells can facilitate immune escape, because genetic evidence suggests that activation of these cells feeds plasma viremia. Ultrasensitive molecular assays can detect low levels of virus in plasma even in children on effective highly active antiretroviral therapy. Importantly, however, this continuous low-level production of plasma virus does not appear to promote antiretroviral drug resistance in the majority of children on effective antiretroviral therapy. The development and persistence of latent HIV-1 reservoirs occur in children despite effective treatment during primary infection. Making a wide variety of antiretroviral drugs in formulations suitable for dosing in children is necessary to minimize infection and to overcome multi-drug-resistant HIV-1 reservoirs in children. The long-term suppression of HIV-1 in children is an achievable goal.

Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS)

A major goal in genomics is to understand how genes are regulated in different tissues, stages of development, diseases, and species. Mapping DNase I hypersensitive (HS) sites within nuclear chromatin is a powerful and well-established method of identifying many different types of regulatory elements, but in the past it has been limited to analysis of single loci. We have recently described a protocol to generate a genome-wide library of DNase HS sites. Here, we report high-throughput analysis, using massively parallel signature sequencing (MPSS), of 230,000 tags from a DNase library generated from quiescent human CD4+ T cells. Of the tags that uniquely map to the genome, we identified 14,190 clusters of sequences that group within close proximity to each other. By using a real-time PCR strategy, we determined that the majority of these clusters represent valid DNase HS sites. Approximately 80% of these DNase HS sites uniquely map within one or more annotated regions of the genome believed to contain regulatory elements, including regions 2 kb upstream of genes, CpG islands, and highly conserved sequences. Most DNase HS sites identified in CD4+ T cells are also HS in CD8+ T cells, B cells, hepatocytes, human umbilical vein endothelial cells (HUVECs), and HeLa cells. However, approximately 10% of the DNase HS sites are lymphocyte specific, indicating that this procedure can identify gene regulatory elements that control cell type specificity. This strategy, which can be applied to any cell line or tissue, will enable a better understanding of how chromatin structure dictates cell function and fate.

IL-7 Differentially Regulates Cell Cycle Entry and Exit of Naive and Memory CD4+ T Lymphocytes: Effects on HIV-Mediated Infection.

The IL-7 cytokine plays a major role in the in vivo maintenance of polyclonal naive and memory T-cells, positively regulating the survival, differentiation and proliferation of thymocyte and peripheral T lymphocyte populations. Under conditions of lymphopenia due to HIV infection, it has been shown that increased peripheral IL-7 levels correlate with T cell differentiation. Moreover, a short-term exogenous IL-7 treatment enhances T-cell differentiation as well as expansion of naive and memory T-cells. These properties have lead to the proposal that recombinant IL-7 be used as an adjuvant immune therapy in HIV-infected individuals. Nevertheless, primate studies have shown that after an initial increase in in vivo T cell proliferation, there is a substantial drop in the absolute lymphocyte numbers (Fry et al., 2003; Nugeyre et al., 2003). As the bases for this decrease have not yet been elucidated, it is important to assess the long-term biological effects of IL-7 on quiescent human CD4+ T lymphocyte subsets. Here, we demonstrated that although IL-7-stimulated memory T-cells enter into cycle much more rapidly than their naive counterpart (3 days as compared to 6 days), they also exit the cell cycle much earlier, by day 10 vs. day 18. Importantly, cell cycle exit occurred despite the continuous replenishment of IL-7 and was inversely correlated with IL-7 receptor levels. Specifically, IL-7Rα levels were completely downregulated on both naive and memory T subsets within 12 hours post cytokine treatment but were significantly re-upregulated in memory T cell within 8–10 days. In naive lymphocytes, IL7Rα expression was absent during the first 14 days of continuous IL-7 stimulation after which time it slowly increased, with expression coinciding with cell cycle exit. Importantly, the permissivity of IL-7-stimulated CD4+ lymphocytes to infection with an HIV-1 vector was not related to cell cycle entry per se. After extended IL-7 stimulation of the naive T cell subset, they remained more refractory to HIV-1 vector infection than memory cells even though they demonstrated a higher level of cell cycle progression. Moreover, under conditions mimicking the lymph node environment, cell cycle entry was not required for IL-7 mediated infection. The differential effects of recombinant IL-7 on the cell cycle entry-exit status of naive and memory CD4+ T lymphocytes as well as the relative susceptibilities of these CD4+ subsets to HIV-1 vector infection have important implications for the use of this cytokine as an adjuvant immunotherapy.

Switching from a restricted to an effective CD4 T cell response by activating CD8+ murine dendritic cells with a Toll-like receptor 9 ligand

Freshly isolated quiescent splenic dendritic cell (DC) subtypes differ in their capacity to activate naive CD4 T cells in culture. The CD8+ DC showed a reduced capacity to stimulate T cell proliferation compared to either of the CD8- DC subsets, regardless of antigen and DC dose. In contrast to CD8- DC, the quiescent CD8+ DC did not induce IFN-gamma production from CD4 T cells. The difference between the DC subtypes appeared to be at the level of initial surface molecule interactions, but could not be attributed to differences in expression of MHC class II or B7 family molecules, or to the expression of Fas ligand on DC. However, when activated by inclusion of the Toll-like receptor 9 ligand CpG in culture, CD8+ DC became potent stimulators of both CD4 T cell proliferation and IFN-gamma production. In contrast, similar activation of CD8- DC produced a more modest increase in capacity to stimulate CD4 T cell proliferation and no increase in capacity to stimulate IFN-gamma production. The difference between a quiescent and an activated state is therefore more extreme for CD8+ than for CD8- DC. The especially tight regulation of the activity of CD8+ DC may be essential for the maintenance of self tolerance.

Heparan Sulfate Proteoglycans Mediate Attachment and Entry of Human T-Cell Leukemia Virus Type 1 Virions into CD4 + T Cells

Heparan sulfate proteoglycans (HSPGs) are used by a number of viruses to facilitate entry into host cells. For the retrovirus human T-cell leukemia virus type 1 (HTLV-1), it has recently been reported that HSPGs are critical for efficient binding of soluble HTLV-1 SU and the entry of HTLV pseudotyped viruses into non-T cells. However, the primary in vivo targets of HTLV-1, CD4(+) T cells, have been reported to express low or undetectable levels of HSPGs. For this study, we reexamined the expression of HSPGs in CD4(+) T cells and examined their role in HTLV-1 attachment and entry. We observed that while quiescent primary CD4(+) T cells do not express detectable levels of HSPGs, HSPGs are expressed on primary CD4(+) T cells following immune activation. Enzymatic modification of HSPGs on the surfaces of either established CD4(+) T-cell lines or primary CD4(+) T cells dramatically reduced the binding of both soluble HTLV-1 SU and HTLV-1 virions. HSPGs also affected the efficiency of HTLV-1 entry, since blocking the interaction with HSPGs markedly reduced both the internalization of HTLV-1 virions and the titer of HTLV-1 pseudotyped viral infection in CD4(+) T cells. Thus, HSPGs play a critical role in the binding and entry of HTLV-1 into CD4(+) T cells.

Uncoating of HIV-1 requires cellular activation

Uncoating is an essential step in viral replication cycle. Little is known about the mechanism and requirement of HIV uncoating. Using an in vitro uncoating model, we demonstrate here that the uncoating of HIV-1 was efficiently induced by lysate from activated CD4+ lymphocytes, while quiescent CD4+ lymphocyte lysate was unable to uncoat HIV-1 core. The uncoating activity was associated with an induction of in vitro reverse transcription of the viral genome. Using CD4+ lymphocytes that were arrested in cell cycle, we showed that the uncoating activity required transition of cells from G 0/G 1a into G 1b stage. These results strongly suggested a requirement of cell cycle-dependent specific factors for HIV-1 uncoating. The putative HIV-1 uncoating factors could be fractionated from cell lysate by gel filtration chromatography.

Monocyte-Derived Dendritic Cells: A Promising Armament for Immunotherapy in Human Malignancies

Monocyte-Derived Dendritic Cells: A Promising Armament for Immunotherapy in Human Malignancies

Human CD4+ Effector Memory T Cells Persisting in the Microenvironment of Lung Cancer Xenografts Are Activated by Local Delivery of IL-12 to Proliferate, Produce IFN-γ, and Eradicate Tumor Cells

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-gamma. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45(+) leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-alpha. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4(+) effector memory T cell. We conclude that quiescent CD4(+) effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-gamma, leading to tumor cell eradication.

Temporarily controlled HIV-1 replication after intravenous immunoglobulin treatment of Guillain-Barré syndrome

HIV establishes a latent infection in resting CD4(+) T-lymphocytes. A possible strategy to eliminate cellular reservoirs in long-lived, HIV-1-infected quiescent CD4(+) T-lymphocytes might be to add T-cell-activating agents to potent antiretroviral therapy. In this report we describe a patient with Guillain-Barré syndrome treated with high dose intravenous immunoglobulin (IVIG) in addition to antiretroviral therapy. A transiently increased viral load and immunoactivation during the IVIG treatment suggest activation of latently infected cells and increased turnover rate of the latent viral reservoir. HIV replication was controlled with plasma viral load <20 copies/ml, for at least 3 months after antiretroviral treatment interruption. CSF neural markers reflecting degenerative processes in the brain during the symptomatic period and follow-up were also analysed. Very high CSF sulfatide concentrations were found indicating that the pathology involves severe demyelination.We hypothesize that IVIG in this case contributed to an activation of latently infected cells, which led to a transient increase in plasma HIV-1 RNA during the IVIG treatment and a long period of undetectable viral load after antiretroviral treatment interruption. Further, this is the first time, to our knowledge, that detailed CSF findings are described in HIV-1 associated GBS.

Identification of LKLF-regulated genes in quiescent CD4 + T lymphocytes

The transcription factor LKLF (lung Krüppel-like factor) is expressed at high levels in quiescent CD4 + and CD8 + T lymphocytes and provides an important role in establishment and maintenance of the quiescent state. To identify LKLF-regulated genes, we performed microarray analysis using an established Jurkat T cell line containing a tetracycline-inducible LKLF. LKLF induction in this Jurkat T cell line generates a quiescent phenotype that resembles memory CD4 + T lymphocytes. We found that LKLF induction in Jurkat cells resulted in up-regulation (>1.5-fold) of about 100 mRNAs, while it repressed (>1.5-fold) a similar number of mRNAs. A striking feature of the LKLF-stimulated mRNAs was that a significant number of them encode cell surface proteins or proteins implicated in initiating and propagating cellular signaling cascades. The data suggests that LKLF may establish a phenotype that primes quiescent cells for responses to specific extracellular stimuli. The mRNAs encoding CDw52, IL-10Rα and paxillin were among the most highly induced transcripts in Jurkat T cells, and we observed that the encoded proteins are down-regulated following activation of quiescent CD4 + T cells isolated from healthy blood donors. We also examined whether LKLF-induced quiescence in Jurkat cells could silence transcription of integrated HIV-1 proviruses. We found however that LKLF-induced quiescence is not sufficient to repress expression of HIV-1 proviruses in Jurkat T cells, suggesting that the HIV-1 provirus is resistant to LKLF-regulated quiescence.