High CCR5 Density on Central Memory CD4+ T Cells in Acute HIV-1 Infection Is Mostly Associated with Rapid Disease Progression

CD4+ central memory T cells play a critical role in the pathogenesis of simian immunodeficiency virus disease, and the CCR5 density on the surface of CD4 T cells is an important factor in human immunodeficiency virus (HIV)-1 disease progression. We hypothesized that quantifying central memory cells and CCR5 expression in the early stages of HIV-infection could provide useful prognostic information. We enrolled two different groups of acute HIV-infected subjects. One group progressed to CD4 T cell numbers below 250 cells/µl within 2 years (CD4 Low group), while the other group maintained CD4 cell counts above 450 cells/µl over 2 years (CD4 High group). We compared the CCR5 levels and percentage of CD4 subsets between the two groups during the 1st year of HIV infection. We found no differences between the two groups regarding the percentage of naïve, central memory and effector memory subsets of CD4 cells during the 1st year of HIV-1 infection. CCR5 levels on CD4+ CM subset was higher in the CD4 Low group compared with the CD4 High group during the 1st year of HIV-1 infection. High CCR5 levels on CD4 central memory cells in acute HIV infection are mostly associated with rapid disease progression. Our data suggest that low CCR5 expression on CD4 central memory cells protects CD4 cells from direct virus infection and favors the preservation of CD4⁺ T cell homeostasis.     

Normal Hematopoietic Stem Cells within the AML Bone Marrow Have a Distinct and Higher ALDH Activity Level than Co-Existing Leukemic Stem Cells

Persistence of leukemic stem cells (LSC) after chemotherapy is thought to be responsible for relapse and prevents the curative treatment of acute myeloid leukemia (AML) patients. LSC and normal hematopoietic stem cells (HSC) share many characteristics and co-exist in the bone marrow of AML patients. For the development of successful LSC-targeted therapy, enabling eradication of LSC while sparing HSC, the identification of differences between LSC and HSC residing within the AML bone marrow is crucial. For identification of these LSC targets, as well as for AML LSC characterization, discrimination between LSC and HSC within the AML bone marrow is imperative. Here we show that normal CD34+CD38– HSC present in AML bone marrow, identified by their lack of aberrant immunophenotypic and molecular marker expression and low scatter properties, are a distinct sub-population of cells with high ALDH activity (ALDH^bright). The ALDH^bright compartment contains, besides normal HSC, more differentiated, normal CD34+CD38+ progenitors. Furthermore, we show that in CD34-negative AML, containing solely normal CD34+ cells, LSC are CD34– and ALDH^low. In CD34-positive AML, LSC are also ALDH^low but can be either CD34+ or CD34–. In conclusion, although malignant AML blasts have varying ALDH activity, a common feature of all AML cases is that LSC have lower ALDH activity than the CD34+CD38– HSC that co-exist with these LSC in the AML bone marrow. Our findings form the basis for combined functionally and immunophenotypically based identification and purification of LSC and HSC within the AML bone marrow, aiming at development of highly specific anti-LSC therapy.     

Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

Background and Purpose

Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor.

Methodology/Principal Findings

Human lung cancer cells variously express a tumor antigen, Wilms'' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402⁺ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8⁺ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1_235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3⁺ T cells both in vitro and in vivo. Double gene-modified CD3⁺ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3⁺ T cells.

Conclusion/Significance

Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer reactivity mediated by CD8⁺ T cells double gene-modified to express WT1-specific TCR and CCR2 not only via CCL2-tropic tumor trafficking, but also CCL2-enhanced WT1-responsiveness.     

CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127^lowPD-1^highTIM-3^high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.     

Antigen Experience Shapes Phenotype and Function of Memory Th1 Cells

Primary and secondary (boosted) memory CD8 T cells exhibit differences in gene expression, phenotype and function. The impact of repeated antigen stimulations on memory CD4 T cells is largely unknown. To address this issue, we utilized LCMV and Listeria monocytogenes infection of mice to characterize primary and secondary antigen (Ag)-specific Th1 CD4 T cell responses. Ag-specific primary memory CD4 T cells display a CD62L^loCCR7^hi CD27^hi CD127^hi phenotype and are polyfunctional (most produce IFNγ, TNFα and IL-2). Following homologous prime-boost immunization we observed pathogen-specific differences in the rate of CD62L and CCR7 upregulation on memory CD4 T cells as well as in IL-2+IFNγco-production by secondary effectors. Phenotypic and functional plasticity of memory Th1 cells was observed following heterologous prime-boost immunization, wherein secondary memory CD4 T cells acquired phenotypic and functional characteristics dictated by the boosting agent rather than the primary immunizing agent. Our data also demonstrate that secondary memory Th1 cells accelerated neutralizing Ab formation in response to LCMV infection, suggesting enhanced capacity of this population to provide quality help for antibody production. Collectively these data have important implications for prime-boost vaccination strategies that seek to enhance protective immune responses mediated by Th1 CD4 T cell responses.     

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8⁺CD44^high) T cells displaying a CD25⁻NKG2D⁺ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.     

Adoptive transfer of costimulated CD4+ T cells induces expansion of peripheral T cells and decreased CCR5 expression in HIV infection.

To study the safety and feasibility of T-cell reconstitution in HIV-infected individuals, we adoptively transferred activated autologous CD4+ T cells. Polyclonal peripheral blood CD4+ cells were costimulated ex vivo and subjects were given infusions of up to 3 x 1010 activated CD4+ cells. Dose-dependent increases in CD4+ cell counts and in the CD4:CD8 ratio were observed. Sustained increases in the fraction of cytokine-secreting T cells and decreases in the percentage of CD4+CCR5+ cells were noted in vivo, suggesting enhanced function and resistance to HIV infection. The frequency of CD4+Ki-67+ cells increased whereas CD4+ T cells containing T cell-receptor rearrangement excision circles (TRECs) decreased. These findings indicate that expansion of the peripheral T-cell pool mediated the increase in CD4 counts and suggest that approaches to reconstitute CD4 helper cell activity and decrease CCR5 expression may augment natural immunity to HIV infection.     

Generation of an HIV-1-resistant immune system with CD34(+) hematopoietic stem cells transduced with a triple-combination anti-HIV lentiviral vector

HIV gene therapy has the potential to offer an alternative to the use of current small-molecule antiretroviral drugs as a treatment strategy for HIV-infected individuals. Therapies designed to administer HIV-resistant stem cells to an infected patient may also provide a functional cure, as observed in a bone marrow transplant performed with hematopoietic stem cells (HSCs) homozygous for the CCR5-Δ32-bp allele. In our current studies, preclinical evaluation of a combination anti-HIV lentiviral vector was performed, in vivo, in humanized NOD-RAG1(-/-) IL2rγ(-/-) knockout mice. This combination vector, which displays strong preintegration inhibition of HIV-1 infection in vitro, contains a human/rhesus macaque TRIM5α isoform, a CCR5 short hairpin RNA (shRNA), and a TAR decoy. Multilineage hematopoiesis from anti-HIV lentiviral vector-transduced human CD34(+) HSCs was observed in the peripheral blood and in various lymphoid organs, including the thymus, spleen, and bone marrow, of engrafted mice. Anti-HIV vector-transduced CD34(+) cells displayed normal development of immune cells, including T cells, B cells, and macrophages. The anti-HIV vector-transduced cells also displayed knockdown of cell surface CCR5 due to the expression of the CCR5 shRNA. After in vivo challenge with either an R5-tropic BaL-1 or X4-tropic NL4-3 strain of HIV-1, maintenance of human CD4(+) cell levels and a selective survival advantage of anti-HIV gene-modified cells were observed in engrafted mice. The data provided from our study confirm the safety and efficacy of this combination anti-HIV lentiviral vector in a hematopoietic stem cell gene therapy setting for HIV and validates its potential application in future clinical trials.     

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background

Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multi-potency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34⁺ HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin.

Methodology/Principal Findings

Using commercially available G-CSF mobilized peripheral blood (PB) CD34⁺ cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, pre-stimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin.

Conclusions/Significance

This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34⁺ cells.     

Accelerated Immunodeficiency by Anti-CCR5 Treatment in HIV Infection

In 50% of progressing HIV-1 patients, CXCR4-tropic (X4) virus emerges late in infection, often overtaking CCR5-tropic (R5) virus as the dominant viral strain. This “phenotypic switch” is strongly associated with rapidly declining CD4⁺ T cell counts and AIDS onset, yet its causes remain unknown. Here, we analyze a mathematical model for the mechanism of X4 emergence in late-stage HIV infection and use this analysis to evaluate the utility of a promising new class of antiretroviral drugs—CCR5 inhibitors—in dual R5, X4 infection. The model shows that the R5-to-X4 switch occurs as CD4⁺ T cell activation levels increase above a threshold and as CD4⁺ T cell counts decrease below a threshold during late-stage HIV infection. Importantly, the model also shows that highly active antiretroviral therapy (HAART) can inhibit X4 emergence but that monotherapy with CCR5 blockers can accelerate X4 onset and immunodeficiency if X4 infection of memory CD4⁺ T cells occurs at a high rate. Fortunately, when CXCR4 blockers or HAART are used in conjunction with CCR5 blockers, this risk of accelerated immunodeficiency is eliminated. The results suggest that CCR5 blockers will be more effective when used in combination with CXCR4 blockers and caution against CCR5 blockers in the absence of an effective HAART regimen or during HAART failure.     

Tax-inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to CCR4-expressing CD4+ T cells

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.     

The Influence of Immunization Route,Tissue Microenvironment,and Cytokine Cell Milieu on HIV-Specific CD8+ T Cells Measured Using Fluidigm Dynamic Arrays

Thirty different genes including cytokines, chemokines, granzymes, perforin and specifically integrins were evaluated in Peyer''s patch-KdGag_197–205-specific CD8+ T cells (pools of 100 cells) using Fluidigm 48.48 Dynamic arrays following three different prime-boost immunization strategies. Data revealed that the route of prime or the booster immunization differentially influenced the integrin expression profile on gut KdGag_197–205-specific CD8+ T cells. Specifically, elevated numbers of integrin αE and αD expressing gut KdGag_197–205-specific CD8+ T cells were detected following mucosal but not systemic priming. Also, αE/β7 and αD/β2 heterodimerization were more noticeable in an intranasal (i.n.)/i.n. vaccination setting compared to i.n./intramuscular (i.m) or i.m./i.m. vaccinations. Moreover, in all vaccine groups tested α4 appeared to heterodimerize more closely with β7 then β1. Also MIP-1β, RANTES, CCR5, perforin and integrin α4 bio-markers were significantly elevated in i.n./i.m. and i.m./i.m. immunization groups compared to purely mucosal i.n./i.n. delivery. Furthermore, when wild type (WT) BALB/c and IL-13 knockout (KO) mice were immunized using i.n./i.m. strategy, MIP-1α, MIP-1β, RANTES, integrins α4, β1 and β7 mRNA expression levels were found to be significantly different, in mucosal verses systemic KdGag_197–205-specific CD8+ T cells. Interestingly, the numbers of gut KdGag_197–205-specific CD8+ T cells expressing gut-homing markers α4β7 and CCR9 protein were also significantly elevated in IL-13 KO compared to WT control. Collectively, our findings further corroborate that the route of vaccine delivery, tissue microenvironment and IL-13 depleted cytokine milieu can significantly alter the antigen-specific CD8+ T cell gene expression profiles and in turn modulate their functional avidities as well as homing capabilities.     

Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8⁺ T cells and gene targeting of a GFP transgene cassette in >40% of CD8⁺ and CD4⁺ T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.     

Frozen Cord Blood Hematopoietic Stem Cells Differentiate into Higher Numbers of Functional Natural Killer Cells In Vitro than Mobilized Hematopoietic Stem Cells or Freshly Isolated Cord Blood Hematopoietic Stem Cells

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34⁺) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34⁺) and frozen PBCD34⁺ to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34⁺ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34⁺ cultures. NK cells generated from CBCD34⁺ and PBCD34⁺ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34⁺-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34⁺-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34⁺ for the production of NK cells in vitro results in higher cell numbers than PBCD34⁺, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.     

Quantitative Assessment of Intra-Patient Variation in CD4+ T Cell Counts in Stable,Virologically-Suppressed,HIV-Infected Subjects

Objectives

Counts of absolute CD4+ T lymphocytes (CD4+ T cells) are known to be highly variable in untreated HIV-infected individuals, but there are no data in virologically-suppressed individuals. We investigated CD4+ T cell variability in stable, virologically-suppressed, HIV-1 infected adults on combination antiretroviral therapy (cART).

Methods

From a large hospital database we selected patients with stable virological suppression on cART for >3 years with >10 CD4+ T cell measurements performed over a further >2 years; and a control group of 95 patients not on cART.

Results

We identified 161 HIV-infected patients on cART without active HCV or HBV infection, with stable virological suppression for a median of 6.4 years. Over the study period 88 patients had reached a plateau in their absolute CD4+ T cell counts, while 65 patients had increasing and 8 patients had decreasing absolute CD4+ T cell counts. In patients with plateaued CD4+ T cell counts, variability in absolute CD4+ T cell counts was greater than in percent CD4+ T cells (median coefficient of variation (CV) 16.6% [IQR 13.8-20.1%] and CV 9.6% [IQR 7.4-13.0%], respectively). Patients with increasing CD4+ T cell counts had greater variability in absolute CD4+ T cell counts than those with plateaued CD4 T cell counts (CV 19.5% [IQR 16.1-23.8%], p<0.001) while there was no difference in percent CD4+ T cell variability between the two groups. As previously reported, untreated patients had CVs significantly higher than patients on cART (CVs of 21.1% [IQR 17.2-32.0%], p<0.001 and 15.2% (IQR 10.7-20.0%), p<0.001, respectively). Age or sex did not affect the degree of CD4+ variation.

Conclusions

Adults with stable, virologically-suppressed HIV infection continue to have significant variations in individual absolute CD4+ T cell and percent CD4+ T cell counts; this variation can be of clinical relevance especially around CD4+ thresholds. However, the variation seen in individuals on cART is substantially less than in untreated subjects.     

Plasmacytoid Dendritic Cells Accumulate and Secrete Interferon Alpha in Lymph Nodes of HIV-1 Patients

Circulating plasmacytoid dendritic cells (pDC) decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNα), which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN) of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNα compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNα before undergoing cell death. Since IFNα inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4⁺ T cells, thus playing into the mechanisms of AIDS immunopathogenesis.     

Poor Immune Reconstitution in HIV-Infected Patients Associates with High Percentage of Regulatory CD4+ T Cells

CD4⁺ regulatory T cells (Tregs) are essential for the maintenance of the immune system''s equilibrium, by dampening the activation of potential auto-reactive T cells and avoiding excessive immune activation. To correctly perform their function, Tregs must be maintained at the right proportion with respect to effector T cells. Since this equilibrium is frequently disrupted in individuals infected with the human immunodeficiency virus (HIV), we hypothesize that its deregulation could hamper immune reconstitution in patients with poor CD4⁺ T cell recovery under highly active antiretroviral therapy (HAART). We analysed Tregs percentages amongst CD4⁺ T cells in 53 HIV-infected patients under HAART, with suppression of viral replication and distinct levels of immune reconstitution. As controls, 51 healthy individuals were also analysed. We observed that amongst the patients with Nadir values (the lowest CD4⁺ T cell counts achieved) <200 cells/µL, the individuals with high Tregs percentages (≥10% of total CD4⁺ T cells) had the worse CD4⁺ T cell reconstitution. In accordance, the well-described direct correlation between the Nadir value and CD4⁺ T cell reconstitution is clearly more evident in individuals with high Tregs proportions. Furthermore, we observed a strong negative correlation between Tregs percentages and CD4⁺ T cell recovery among immunological non-responder HIV⁺ individuals. All together, this work shows that high Tregs frequency is an important factor associated with sub-optimal CD4⁺ T cell recovery. This is particularly relevant for immunological non-responders with low Nadir values. Our results suggest that the Tregs proportion might be of clinical relevance to define cut-offs for HAART initiation.     

Protection of Stem Cell-Derived Lymphocytes in a Primate AIDS Gene Therapy Model after In Vivo Selection

Background

There is currently no effective AIDS vaccine, emphasizing the importance of developing alternative therapies. Recently, a patient was successfully transplanted with allogeneic, naturally resistant CCR5-negative (CCR5Δ32) cells, setting the stage for transplantation of naturally resistant, or genetically modified stem cells as a viable therapy for AIDS. Hematopoietic stem cell (HSC) gene therapy using vectors that express various anti-HIV transgenes has also been attempted in clinical trials, but inefficient gene transfer in these studies has severely limited the potential of this approach. Here we evaluated HSC gene transfer of an anti-HIV vector in the pigtailed macaque (Macaca nemestrina) model, which closely models human transplantation.

Methods and Findings

We used lentiviral vectors that inhibited both HIV-1 and simian immunodeficiency virus (SIV)/HIV-1 (SHIV) chimera virus infection, and also expressed a P140K mutant methylguanine methyltransferase (MGMT) transgene to select gene-modified cells by adding chemotherapy drugs. Following transplantation and MGMT-mediated selection we demonstrated transgene expression in over 7% of stem-cell derived lymphocytes. The high marking levels allowed us to demonstrate protection from SHIV in lymphocytes derived from gene-modified macaque long-term repopulating cells that expressed an HIV-1 fusion inhibitor. We observed a statistically significant 4-fold increase of gene-modified cells after challenge of lymphocytes from one macaque that received stem cells transduced with an anti-HIV vector (p<0.02, Student''s t-test), but not in lymphocytes from a macaque that received a control vector. We also established a competitive repopulation assay in a second macaque for preclinical testing of promising anti-HIV vectors. The vectors we used were HIV-based and thus efficiently transduce human cells, and the transgenes we used target HIV-1 genes that are also in SHIV, so our findings can be rapidly translated to the clinic.

Conclusions

Here we demonstrate the ability to select protected HSC-derived lymphocytes in vivo in a clinically relevant nonhuman primate model of HIV/SHIV infection. This approach can now be evaluated in human clinical trials in AIDS lymphoma patients. In this patient setting, chemotherapy would not only kill malignant cells, but would also increase the number of MGMTP140K-expressing HIV-resistant cells. This approach should allow for high levels of HIV-protected cells in AIDS patients to evaluate AIDS gene therapy.     

GB Virus C Infection Is Associated with Altered Lymphocyte Subset Distribution and Reduced T Cell Activation and Proliferation in HIV-Infected Individuals

GBV-C infection is associated with prolonged survival and with reduced T cell activation in HIV-infected subjects not receiving combination antiretroviral therapy (cART). The relationship between GBV-C and T cell activation in HIV-infected subjects was examined. HIV-infected subjects on cART with non-detectable HIV viral load (VL) or cART naïve subjects were studied. GBV-C VL and HIV VL were determined. Cell surface markers of activation (CD38⁺/HLA-DR⁺), proliferation (Ki-67+), and HIV entry co-receptor expression (CCR5+ and CXCR4+) on total CD4+ and CD8+ T cells, and on naïve, central memory (CM), effector memory (EM), and effector CD4+ and CD8+ subpopulations were measured by flow cytometry. In subjects with suppressed HIV VL, GBV-C was consistently associated with reduced activation in naïve, CM, EM, and effector CD4+ cells. GBV-C was associated with reduced CD4+ and CD8+ T cell surface expression of activation and proliferation markers, independent of HIV VL classification. GBV-C was also associated with higher proportions of naïve CD4+ and CD8+ T cells, and with lower proportions of EM CD4+ and CD8+ T cells. In conclusion, GBV-C infection was associated with reduced activation of CD4+ and CD8+ T cells in both HIV viremic and HIV RNA suppressed patients. Those with GBV-C infection demonstrated an increased proportion of naive T cells and a reduction in T cell activation and proliferation independent of HIV VL classification, including those with suppressed HIV VL on cART. Since HIV pathogenesis is thought to be accelerated by T cell activation, these results may contribute to prolonged survival among HIV infected individuals co-infected with GBV-C. Furthermore, since cART therapy does not reduce T cell activation to levels seen in HIV-uninfected people, GBV-C infection may be beneficial for HIV-related diseases in those effectively treated with anti-HIV therapy.