In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines.

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.

Regulated expression of telomerase activity in human T lymphocyte development and activation.

Telomerase, a ribonucleoprotein that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells, and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. In the present study, telomerase activity was analyzed in normal human T lymphocytes. It was found that telomerase is expressed at a high level in thymocyte subpopulations, at an intermediate level in tonsil T lymphocytes, and at a low to undetectable level in peripheral blood T lymphocytes. Moreover, telomerase activity is highly inducible in peripheral T lymphocytes by activation through CD3 with or without CD28 costimulation, or by stimulation with phorbol myristate acetate (PMA)/ionomycin. The induction of telomerase by anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation required RNA and protein synthesis, and was blocked by herbimycin A, an inhibitor of S pi protein tyrosine kinases. The immunosuppressive drug cyclosporin A selectively inhibited telomerase induction by PMA/ionomycin and by anti-CD3, but not by anti-CD3/CD28. Although telomerase activity in peripheral T lymphocytes was activation dependent and correlated with cell proliferation, it was not cell cycle phase restricted. These results indicate that the expression of telomerase in normal human T lymphocytes is both developmentally regulated and activation induced. Telomerase may thus play a permissive role in T cell development and in determining the capacity of lymphoid cells for cell division and clonal expansion.

Telomere length, telomerase activity, and replicative potential in HIV infection: analysis of CD4+ and CD8+ T cells from HIV-discordant monozygotic twins.

To address the possible role of replicative senescence in human immunodeficiency virus (HIV) infection, telomere length, telomerase activity, and in vitro replicative capacity were assessed in peripheral blood T cells from HIV+ and HIV- donors. Genetic and age-specific effects on these parameters were controlled by studying HIV-discordant pairs of monozygotic twins. Telomere terminal restriction fragment (TRF) lengths from CD4+ T cells of HIV+ donors were significantly greater than those from HIV- twins. In contrast, telomere lengths in CD8+ T cells from HIV+ donors were shorter than in HIV- donors. The in vitro replicative capacity of CD4+ cells from HIV+ donors was equivalent to that of HIV- donors in response to stimulation through T cell receptor CD3 and CD28. Little or no telomerase activity was detected in freshly isolated CD4+ or CD8+ lymphocytes from HIV+ or HIV- donors, but was induced by in vitro stimulation of both HIV+ and HIV- donor cells. These results suggest that HIV infection is associated with alterations in the population dynamics of both CD4+ and CD8+ T cells, but fail to provide evidence for clonal exhaustion or replicative senescence as a mechanism underlying the decline in CD4+ T cells of HIV-infected donors.

Differential regulation of HIV-1 fusion cofactor expression by CD28 costimulation of CD4+ T cells.

Activation of CD4(+) T lymphocytes from human immunodeficiency virus-type 1 (HIV-1)-infected donors with immobilized antibodies to CD3 and CD28 induces a virus-resistant state. This effect is specific for macrophage-tropic HIV-1. Transcripts encoding CXCR4/Fusin, the fusion cofactor used by T cell line-tropic isolates, were abundant in CD3/CD28-stimulated cells, but transcripts encoding CCR5, the fusion cofactor used by macrophage-tropic viruses, were not detectable. Thus, CD3/CD28 costimulation induces an HIV-1-resistant phenotype similar to that seen in some highly exposed and HIV-uninfected individuals.

Antiviral effect and ex vivo CD4+ T cell proliferation in HIV-positive patients as a result of CD28 costimulation.

Because stimulation of CD4+ lymphocytes leads to activation of human immunodeficiency virus-type 1 (HIV-1) replication, viral spread, and cell death, adoptive CD4+ T cell therapy has not been possible. When antigen and CD28 receptors on cultured T cells were stimulated by monoclonal antibodies (mAbs) to CD3 and CD28 that had been immobilized, there was an increase in the number of polyclonal CD4+ T cells from HIV-infected donors. Activated cells predominantly secreted cytokines associated with T helper cell type 1 function. The HIV-1 viral load declined in the absence of antiretroviral agents. Moreover, CD28 stimulation of CD4+ T cells from uninfected donors rendered these cells highly resistant to HIV-1 infection. Immobilization of CD28 mAb was crucial to the development of HIV resistance, as cells stimulated with soluble CD28 mAb were highly susceptible to HIV infection. The CD28-mediated antiviral effect occurred early in the viral life cycle, before HIV-1 DNA integration. These data may facilitate immune reconstitution and gene therapy approaches in persons with HIV infection.

Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.

Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards.

Combination measles, mumps, rubella and varicella vaccine.

A comparative clinical trial was conducted in 15- to 17-month-old healthy children to compare an investigational combination measles, mumps, rubella, varicella (MMRV) vaccine v standard measles, mumps, rubella vaccine followed 6 weeks later with the varicella (MMR + V) vaccine. Both the MMRV and MMR + V vaccine schedules stimulated virtually 100% seroconversion for all component viruses. Mean antibody titers were similar for each virus component in the two vaccine groups. Clinical reactivity postimmunization was also similar with 25% to 29% morbilliform rashes, 12% to 25% mild papulovesicular (varicella) rashes, and 12.5% to 18% temperature elevations above 38.3 degrees C (101 degrees F). Antibodies to measles, mumps, and rubella viruses were persistent at 1 year of follow-up in both groups. Varicella antibody was persistent in 8/10 originally seronegative MMRV vaccinees and 5/5 MMR + V vaccine recipients tested. One MMRV vaccine recipient had a household exposure to chickenpox during the year postvaccination that resulted in a subclinical boost in varicella antibody titer. Two children in the MMR + V vaccine group had close varicella exposures; mild varicella (20 lesions) developed in one. There were no known exposures to natural measles, mumps, or rubella. Three of four MMRV vaccinees with low titer antibody to varicella prior to immunization had greater than fourfold increases in antibodies after vaccination. The combination MMRV vaccine is an immunogenic, safe, and cost-effective approach to varicella immunization of healthy children. Continued work is needed to select the appropriate dose of varicella component, to assure higher persistence rate of varicella antibody.

Molecular remission of CML after autotransplantation followed by adoptive transfer of costimulated autologous T cells.

Four patients with chronic myelogenous leukemia (CML) that was refractory to interferon alpha (two patients) or imatinib mesylate (two patients), and who lacked donors for allogeneic stem cell transplantation, received autotransplants followed by infusions of ex vivo costimulated autologous T cells. At day +30 (about 14 days after T-cell infusion), the mean CD4+ cell count was 481 cells/microl (range 270-834) and the mean CD8+ count was 516 cells/microl (range 173-1261). One patient had a relative lymphocytosis at 3.5 months after T-cell infusion, with CD4 and CD8 levels of 750 and 1985 cells/microl, respectively. All the four patients had complete cytogenetic remissions early after transplantation, three of whom also became PCR negative for the bcr/abl fusion mRNA. One patient, who had experienced progressive CML while on interferon alpha therapy, became PCR- post transplant, and remained in a molecular CR at 3.0 years of follow-up. All the four patients survived at 6, 9, 40, and 44 months post transplant; the patient who remained PCR+ had a cytogenetic and hematologic relapse of CML, but entered a molecular remission on imatinib. Autotransplantation followed by costimulated autologous T cells is feasible for patients with chronic phase CML, who lack allogeneic donors and can be associated with molecular remissions.

Anti-CD3/anti-CD28 bead stimulation overcomes CD3 unresponsiveness in patients with head and neck squamous cell carcinoma.

OBJECTIVES: To test whether T-cell CD3 responses are altered in patients with advanced-stage head and neck squamous cell carcinoma (HNSCC) and whether anti-CD3/anti-CD28 (alphaCD3/alphaCD28) bead stimulation could reverse CD3 unresponsiveness. DESIGN: Anti-CD3 (alphaCD3) monoclonal antibody immobilized on tissue culture plastic was used to stimulate lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs) from patients with advanced-stage HNSCC. Proliferation, T-cell phenotype, and cytokines were measured during 8-day in vitro stimulation. Immune-enhancing properties of alphaCD3/ alphaCD28 beads were also tested on LNMCs and PBMCs. Cytotoxicity of bead-activated T cells (ATCs) was measured against autologous and allogeneic HNSCC. RESULTS: Six patients were nonresponders to alphaCD3 stimulation defined by tritium (3H) incorporation of less than 3500 cpm, whereas 11 patients were responders with 3H incorporation of 3500 cpm or more. Responders produced higher levels of interleukin (IL)-12 and interferon gamma (IFN-gamma) after alphaCD3 stimulation than nonresponders. No phenotypic or clinical differences were identified between groups. Stimulation with alphaCD3/alphaCD28 beads enhanced IFN-gamma and IL-2 produced by both groups. Bead ATCs were generated from PBMCs of patient 11 in the responder group and lysed (+/- SD) 100% +/-1% of autologous tumor and 49% +/-1% of allogeneic tumor. Bead ATCs from LNMCs of this patient lysed 58%+/-1% of autologous tumor and 63%+/-1% of allogeneic tumor. CONCLUSIONS: A subpopulation of patients with HNSCC who are nonresponders to alphaCD3 stimulation has been identified, showing reduced proliferation and IL-12 and IFN-gamma secretion. Nonresponders stimulated with alphaCD3/alphaCD28 beads reversed immune unresponsiveness and induced a type 1 cytokine response. Bead-generated ATCs from patient 11 in the responder group lysed autologous and allogeneic HNSCC in vitro, suggesting a possible effective immunotherapeutic modality in the treatment of HNSCC.

Regulation of telomerase RNA template expression in human T lymphocyte development and activation.

Telomeres are unique DNA-protein complexes at the terminals of chromosomes that appear to play a critical role in protecting chromosomal integrity and in maintaining cellular replicative potential. Telomerase is a ribonuclear protein that is capable of elongating telomeres by the addition of telomeric hexanucleotide repeats and therefore contributing to the capacity for cell replication. Telomerase activity is expressed in human germline cells and malignant cells, and it has recently been demonstrated that telomerase activity is highly regulated in normal lymphocytes at specific stages of development and activation. However, these studies have not elucidated whether telomerase activity is regulated at the level of specific gene expression or whether the regulation of telomerase RNA template (hTR) and/or protein components contributes to the regulation of telomerase activity in normal somatic cells. To characterize at a molecular level the regulation of telomerase expression in human T lymphocytes, we analyzed the expression of hTR during lineage development and after in vitro activation. It was found that hTR is expressed in subsets of thymocytes with strong telomerase activity at levels that are consistently higher (1.5 times; p < 0.01) than those found in peripheral blood resting T cells. In addition, hTR is up-regulated two- to fivefold in peripheral blood naive and memory CD4+ T cells after in vitro activation with anti-CD3 plus anti-CD28. These results establish that hTR expression is regulated in normal human T cells during lineage development and after activation, and indicate that regulation of hTR expression may contribute to the regulation of telomerase activity in normal lymphoid cells.

Response of Jurkat T cells to phorbol ester and bryostatin. Development of sublines with distinct functional responses and changes in protein kinase C activity.

T lymphocyte activation is initiated as a result of the interaction between the TCR complex and Ag as seen in the framework of a membrane-bound MHC molecule. Receptor stimulation results in a rise in free intracellular Ca2+ and the activation of protein kinase C (PKC). Bryostatin (Bryo) and phorbol esters (e.g., 12-O-tetradecanoylphorbol 13-acetate (TPA] are PKC activators with somewhat different immunologic effects. We compared the effect of Bryo and TPA on the T cell tumor line Jurkat and derivatives of Jurkat cells grown in media supplemented with 100 nM Bryo ("BR100" cells) or 100 nM TPA ("TP100" cells). In untreated Jurkat cells, there is a dose- and time-dependent decrease in proliferation, compared to media controls, after the administration of as little as 10 nM TPA. This can be reversed in a dose- and time-dependent manner by Bryo. Interestingly, the expression of the transferrin receptor parallelled this effect on proliferation. Furthermore, Jurkat cells grown continuously in 100 nM TPA regained full proliferative capacity after several weeks in culture and transferrin receptor expression returned to near the level seen in untreated Jurkat cells. The chromatographic separation of PKC activity in these three cell lines showed that total PKC activity was dramatically decreased in both the TP100 and BR100 cells when compared to untreated Jurkat cells. However, in the TP100 cells there exists a peak of activity that is activated by Bryo, but not TPA. Western blots of whole cell lysates of the three cell lines showed that PKC-alpha and PKC-beta II were both down-regulated in BR100 and TP100 cells compared to untreated Jurkat cells. PKC-gamma was not detected in any of the cell lines. Therefore, the Bryo-specific peak seen in TP100 cells may be PKC-delta, -epsilon, -zeta, -eta, or a novel PKC isoform. This could provide the basis for a molecular characterization of the differences in PKC activation between phorbol esters and Bryo.

Human naive and memory T lymphocytes differ in telomeric length and replicative potential.

The present study has assessed the replicative history and the residual replicative potential of human naive and memory T cells. Telomeres are unique terminal chromosomal structures whose length has been shown to decrease with cell division in vitro and with increased age in vivo for human somatic cells. We therefore assessed telomere length as a measure of the in vivo replicative history of naive and memory human T cells. Telomeric terminal restriction fragments were found to be 1.4 +/- 0.1 kb longer in CD4+ naive T cells than in memory cells from the same donors, a relationship that remained constant over a wide range of donor age. These findings suggest that the differentiation of memory cells from naive precursors occurs with substantial clonal expansion and that the magnitude of this expansion is, on average, similar over a wide range of age. In addition, when replicative potential was assessed in vitro, it was found that the capacity of naive cells for cell division was 128-fold greater as measured in mean population doublings than the capacity of memory cells from the same individuals. Human CD4+ naive and memory cells thus differ in in vivo replicative history, as reflected in telomeric length, and in their residual replicative capacity.

Signal transduction properties of the T cell activation monoclonal antibody panel: ubiquitous increase in cellular substrate tyrosine phosphorylation in the absence of detectable calcium mobilization.

A panel of coded monoclonal antibodies (mAbs) submitted to the T cell section of the Vth International Workshop and Conference on Human Leukocyte Differentiation Antigens was examined for the ability to induce cellular activation. Intracellular calcium levels were examined by loading resting T cells with the Ca(+2)-specific dye Indo-1, incubating the cells with Ab, cross-linking with goat anti-mouse Ab and analyzing by flow cytometry. Only 11 out of 68 Abs induced a detectable rise in Ca+2; two of these Abs induced a substantial rise in Ca+2. In resting T cells, 63 of 68 Abs induced increased tyrosine phosphorylation of substrates compared to negative control. Approximately half of the Abs that induced tyrosine phosphorylation induced substrates different from those seen following treatment of cells with anti-CD3 Ab. All mAbs that induced a rise in Ca+2 also induced increased tyrosine phosphorylation. These experiments show a distinct difference in the ability of cross-linked Ab to induce changes in tyrosine phosphorylation and intracellular free Ca+. Furthermore, these results indicate that transient increases in cellular substrate phosphorylation may have questionable biologic significance in T cells.

Efficient priming of protein antigen-specific human CD4(+) T cells by monocyte-derived dendritic cells.

Dendritic cells (DCs) have the unique ability to initiate an immune response in vivo by capturing antigens (Ags) in peripheral tissues and migrating to secondary lymphoid organs, where they sensitize naive CD4(+) T cells. To mimic this process in vitro, previous studies have shown that DCs directly isolated from peripheral blood can be used to elicit primary responses to neoantigens (neoAgs). In other studies, when monocyte-derived DCs have been utilized to sensitize total CD4(+) T cells in vitro, only secondary proliferation to neoAgs could be elicited. In the present study, the relative abilities of CD40 ligation, protein kinase C activation, and culture in tumor necrosis factor alpha (TNF-alpha) to induce functional and phenotypic maturation of human DCs from monocyte precursors were compared. Optimal TNF-alpha-induced maturation of DCs required a prolonged 4-day culture. It was then found that loading immature DCs with the neoAgs keyhole limpet hemocyanin or human immunodeficiency virus-1 p24 gag prior to TNF-alpha-induced maturation, rather than after maturation, was crucial to sensitize CD4(+) T cells to new Ags. This primary proliferation to neoAgs was initiated from the CD4(+) CD45RA(+) naive T-cell population. Finally, it was found that monocyte-derived DCs acquired the ability to secrete interleukin-12 p70, after contact with Ag-specific T cells. The ability to prime and expand Ag-specific CD4(+) T cells ex vivo to neoAgs in serum-free conditions has potential application for cellular vaccination and adoptive immunotherapy.

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro.

The interaction of CD28 and its ligands is critical for antigen-induced T cell activation. Recent studies have demonstrated the existence of at least two members of the B7 receptor family. In this report, the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2) were compared to CD28 ligation by mAb. We demonstrate that the kinetics of induction of T cell proliferation after anti-CD3 stimulation was similar regardless of the form of co-stimulation. Similarly, B7-1 and B7-2 could both maintain long-term expansion of CD4 cells. The co-stimulatory effects of both B7-1 and B7-2 were dependent on CD28 cross-linking, based on complete inhibition of proliferation by CD28 antibody Fab fragments. Co-stimulation with B7-1 and B7-2 induced high levels of cytokine secretion by resting T cells, and the effects of B7-1 and B7-2 could not be distinguished. This conclusion is based on analysis of the initial activation of CD28+ T cells, as well as T cell subpopulations consisting of CD4+ and CD8+ T cells. Both B7-1 and B7-2 could elicit IL-4 secretion from CD4+ T cells while anti-CD28 antibody induced substantially less IL-4 secretion. Furthermore, both B7-1 and B7-2 could stimulate high levels of IFN-gamma and IL-4 from CD4+CD45RO+ cells, while neither B7 receptor could co-stimulate IFN-gamma and IL-4 secretion from CD4+CD45RA+ T cells. B7-1 and B7-2 could, however, co-stimulate CD4+CD45RA+ T cells to secrete IL-2. By contrast, when previously activated T cells were tested, re-stimulation of CD4+ T cell blasts with B7-1 or B7-2 resulted in higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulation with anti-CD28 antibody maintained a higher level of secretion of IL-2 and IFN-gamma than B7-1 or B7-2. These observations may have important implications because they suggest that the manner of CD28 ligation can be a critical determinant in the development of cytokine secretion that corresponds to Th1- and Th2-like patterns of differentiation. Together these observations suggest that there are no intrinsic differences between B7-1 and B7-2 in their ability to co-stimulate the populations of cells that we have tested.

Tales of tails: regulation of telomere length and telomerase activity during lymphocyte development, differentiation, activation, and aging.

Telomerase activity and the regulation of telomere length are factors which have been implicated in the control of cellular replication. These variables have been examined during human lymphocyte development, differentiation, activation, and aging. It was found that telomere length of peripheral blood CD4+ T cells decreases with age as well as with differentiation from naive to memory cells in vivo, and decreases with cell division in vitro. These results provide evidence that telomere length correlates with lymphocyte replicative history and residual replicative potential. In contrast, telomere length appears to increase during tonsil B-cell differentiation and germinal center (GC) formation in vivo. It was also found that telomerase activity is highly regulated during T-cell development and B-cell differentiation in vivo, with high levels of telomerase activity expressed in thymocytes and GC B cells, and low levels of telomerase activity in resting mature peripheral blood lymphocytes. Finally, resting lymphocytes retain the ability to upregulate telomerase activity upon activation, and this capacity does not appear to decline with age. Although the precise role of telomerase in lymphocyte function remains to be elucidated, telomerase may contribute to protection from telomere shortening in T and B lymphocytes, and may thus play a critical role in lymphocyte development, differentiation and activation. The future study of telomerase and its regulation of telomere length may enhance our understanding of how the replicative lifespan is regulated in lymphocytes.

Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells.

In this report, conditions for prolonged in vitro proliferation of polyclonal adult CD4+ T cells via stimulation with immobilized anti-CD3 plus anti-CD28 have been established. CD4+ cells maintained exponential growth for more than 60 days during which a total 10(9)- to 10(11)-fold expansion occurred. Cell cultures exhibited cyclical changes in cell volume, indicating that, in terms of proliferative rate, cells do not have to rest before restimulation. Indeed, electronic cell size analysis was the most reliable method to determine when to restimulate with additional immobilized mAb. The initial approximately 10(5)-fold expansion was autocrine, occurring in the absence of exogenous cytokines or feeder cells. Addition of recombinant human IL-2 after the initial autocrine expansion resulted in continued exponential proliferation. Phorbol ester plus ionomycin also induced long-term growth when combined with anti-CD28 stimulation. Analysis of the T cell repertoire after prolonged expansion revealed a diverse repertoire as assessed by anti-TCR Vbeta Abs or a PCR-based assay. Cytokines produced were consistent with maintenance of both Th1 and Th2 phenotypes; however, the mode of CD3 and CD28 stimulation could influence the cytokine secretion pattern. When anti-CD3 and anti-CD28 were immobilized on the same surface, ELISAs on culture supernatants revealed a pattern consistent with Th1 secretion. Northern analysis revealed that cytokine gene expression remained inducible. Spontaneous growth or cell transformation was not observed in more than 100 experiments. Together, these observations may have implications for gene therapy and adoptive immunotherapy. Furthermore, these culture conditions establish a model to study the finite lifespan of mature T lymphocytes.

Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes.

Human telomerase consists of two essential components, telomerase RNA template (hTER) and telomerase reverse transcriptase (hTERT), and functions to synthesize telomere repeats that serve to protect the integrity of chromosomes and to prolong the replicative life span of cells. Telomerase activity is expressed selectively in germ-line and malignant tumor cells but not in most normal human somatic cells. As a notable exception, telomerase is expressed in human lymphocytes during development, differentiation, and activation. Recent studies have suggested that regulation of telomerase is determined by transcription of hTERT but not hTER. The highly regulated expression of telomerase in lymphocytes provides an opportunity to analyze the contribution of transcriptional regulation of hTERT and hTER. We report here an analysis of hTERT expression by Northern and in situ hybridization. It was found that hTERT mRNA is expressed at detectable levels in all subsets of human lymphocytes isolated from thymus, tonsil, and peripheral blood, regardless of the status of telomerase activity. hTERT expression is regulated as a function of lineage development, differentiation, and activation. Strikingly, however, telomerase activity in these cells is not correlated strictly with the levels of hTERT and hTER transcripts. The absence of correlation between telomerase activity and hTERT mRNA could not be attributed to the presence of hTERT splice variants or to detectable inhibitors of telomerase activity. Thus, transcriptional regulation of hTERT is not sufficient to account for telomerase activity in human lymphocytes, indicating a likely role of posttranscriptional factors in the control of enzyme function.

Naïve and memory CD4 T cells differ in their susceptibilities to human immunodeficiency virus type 1 infection following CD28 costimulation: implicatip6s for transmission and pathogenesis.

In vitro evidence suggests that memory CD4(+) cells are preferentially infected by human immunodeficiency virus type 1 (HIV-1), yet studies of HIV-1-infected individuals have failed to detect preferential memory cell depletion. To explore this paradox, we stimulated CD45RA+ CD4(+) (naïve) and CD45RO+ CD4(+) (memory) cells with antibodies to CD3 and CD28 and infected them with either CCR5-dependent (R5) or CXCR4-dependent (X4) HIV-1 isolates. Naïve CD4(+) cells supported less X4 HIV replication than their memory counterparts. However, naïve cells were susceptible to R5 viral infection, while memory cells remained resistant to infection and viral replication. As with the unseparated cells, mixing the naïve and memory cells prior to infection resulted in cells resistant to R5 infection and highly susceptible to X4 infection. While both naïve and memory CD4(+) subsets downregulated CCR5 expression in response to CD28 costimulation, only the memory cells produced high levels of the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta upon stimulation. Neutralization of these beta-chemokines rendered memory CD4(+) cells highly sensitive to infection with R5 HIV-1 isolates, indicating that downregulation of CCR5 is not sufficient to mediate complete protection from CCR5 strains of HIV-1. These results indicate that susceptibility to R5 HIV-1 isolates is determined not only by the level of CCR5 expression but also by the balance of CCR5 expression and beta-chemokine production. Furthermore, our results suggest a model of HIV-1 transmission and pathogenesis in which naïve rather than memory CD4(+) T cells serve as the targets for early rounds of HIV-1 replication.