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.

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.

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.

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.

Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)

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.

Increases in T cell telomere length in HIV infection after antiretroviral combination therapy for HIV-1 infection implicate distinct population dynamics in CD4+ and CD8+ T cells.

Changes in mean telomeric terminal restriction fragment (TRF) length were examined as a marker for cellular replicative history in HIV-1-infected individuals after institution of anti-retroviral therapy (ART). Increases in mean T cell TRF lengths were observed in most patients following therapy; however, the contribution of individual T cell subsets was complex. An elongation of CD8+ T cell TRF was nearly uniformly observed while changes in mean TRF length in CD4+ T cells were heterogeneous as, despite potent suppression of viral replication, CD4 cell telomeres recovered in some patients, yet continued to decline in others. Increases in CD8 cell TRF correlated with decreased memory cells, suggesting a negative selection in the periphery for CD8 cells with extensive replicative history. In contrast, increases in CD4+ T cell TRF length correlated with increases in naive cell subsets, suggesting that the CD4+ T cell TRF increase may reflect a thymic contribution in some patients. These are the first increases in somatic cell telomere length in a population of cells observed in vivo, and the findings are compatible with therapy-induced reconstitution of the lymphoid compartment with cells having a more extensive replicative potential. These findings further distinguish lymphocytes from other somatic cell populations where only decreases in TRF over time have been noted. Thus, institution of ART in persons with moderately advanced HIV-1 disease reveals distinct population dynamics of CD4 and CD8 T cell subsets and also shows that the lymphocyte replicative history is dynamic.

T cells coactivated with immobilized anti-CD3 and anti-CD28 as potential immunotherapy for cancer.

This report describes the generation of T cells with characteristics that may prove useful for the immunotherapy of cancer. Peripheral blood mononuclear cells obtained from healthy donors were cultured in the presence of anti-CD3/anti-CD28 mAb-coated beads (3/28 beads) at a 3:1 bead to cell ratio. The 3/28 beads were removed after 14 days of culture. Optimal growth conditions for CD3/CD28 coactivated T cells (COACTS) were determined to be X-VIVO 15 containing 5% human AB serum and 100 IU/ml of interleukin-2. The median fold expansion after 14 days was 84-fold. Flow cytometric analyses demonstrated that all cultures were > 90% CD3+ with an increase in the proportion of CD8+ cells. CD28 expression was maintained at very high levels on CD4+ cells and augmented on CD8+ cells. COACTS were induced to secrete high levels of Th1-type cytokines (IFN-gamma and TNF-alpha) after a 24-h restimulation with fresh 3/28 beads and displayed nonmajor histocompatibility complex-restricted lytic activity against a variety of human tumor cell lines in standard 51Cr-release assays. Bead removal from COACT cultures before day 14 greatly enhanced the cell growth and cytokine production without significantly affecting the lytic potential. In summary, large numbers of T cells can be generated by coactivation with anti-CD3/anti-CD28-coated beads for 14 days. This method may provide an advantage over current forms of cellular immunotherapy for cancer because of the ability of COACTS to secrete tumoricidal cytokines and generate antitumor cytotoxicity.

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.

Large-scale production of CD4+ T cells from HIV-1-infected donors after CD3/CD28 costimulation.

We describe a procedure for large-scale enrichment, growth, and harvesting CD4+ T cells. This method may be effective for HIV-1 immunotherapy, as the mode of stimulation, with anti-CD3 plus anti-CD28 coated beads (CD3/CD28 beads) induces a potent antiviral effect. PBMC were obtained by density gradient centrifugation of an apheresis product. Monocytes/macrophages were removed by incubating PBMC with beads coated with IgG. The cells were then magnetically depleted of B cells and CD8+ cells with mouse anti-CD20 and anti-CD8 MAbs and sheep antimouse coated beads. The remaining cells were >80% CD4+ and were transferred to gas-permeable bags containing CD3/CD28 beads and cultured in a closed system. After 14 days, the cell number increased an average of 37-fold, and cells were nearly 100% CD4+. Viral load, assessed by DNA PCR for HIV-1 gag, decreased >10-fold during culture in the absence of antiretroviral agents. Removal of CD3/CD28 beads from the cell suspension was accomplished by passing cells plus beads (3-30 x 10(9) cells in 2-12 L) over a MaxSep magnetic separator using gravity-driven flow. The cells were then concentrated to 300 ml in an automated centrifuge. This process allows safe and efficient growth of large numbers of CD4+ T cells from HIV-1+ donors.

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.

The role of co-stimulation in regulation of chemokine receptor expression and HIV-1 infection in primary T lymphocytes.

Fusion and entry of the human immunodeficiency virus (HIV) into CD4(+) T lymphocytes requires expression of CD4 and a coreceptor. At least eight chemokine receptors can serve as coreceptors for HIV. Accumulating evidence indicates that multiple factors, including the state of cellular differentia- tion and activation, regulate the expression of alpha- and beta-chemokine receptors on lymphocytes. For example, binding of antibodies to the CD28 coreceptor can downregulate expression of beta-chemokine receptors, and this appears to have important consequences on the susceptibility of CD4(+) T lymphocytes to infection by HIV-1. In contrast, binding of the natural CD28 ligand B7 or antibodies to the CD28 homologue CTLA-4 can upregulate CCR5 expression, sug- gesting a reciprocal interaction between CD28 and CTLA-4 and the regulation of beta-chemokine receptor expression. Thus, the CD28/CTLA-4/B7 co-stimulation pathway is identi- fied as a potential novel target for the control of susceptibility to some strains of HIV-1 infection.

CTLA-4 ligation delivers a unique signal to resting human CD4 T cells that inhibits interleukin-2 secretion but allows Bcl-X(L) induction.

We have assessed the functional effects of a panel of CTLA-4 mAbs on resting human CD4+ T cells. Our results demonstrate that some CTLA-4 mAbs can inhibit proliferative responses of resting CD4+ cells and cell cycle transition from G0 to G1. The inhibitory effects of CTLA-4 were evident within 4 h, at a time when cell surface CTLA-4 expression remained undetectable. Other CTLA-4 mAbs had no detectable inhibitory effects, indicating that binding of Ab to CTLA-4 alone is not sufficient to mediate down-regulation of T cell responses. Interestingly, while IL-2 production was shut off, inhibitory anti-CTLA-4 mAbs permitted induction and expression of the cell survival gene bcl-X(L). Consistent with this observation, cells remained viable and apoptosis was not detected after CTLA-4 ligation.

Phorbol esters induce differentiation of human CD34+ hemopoietic progenitors to dendritic cells: evidence for protein kinase C-mediated signaling.

The intracellular signals that mediate the differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have found that the phorbol ester PMA by itself induced 47% +/- 8.7% of input human CD34+ hemopoietic progenitors to differentiate into cells with morphology and surface Ag phenotype characteristic of DC by day 7 of culture. Functionally, PMA-generated DC processed and presented whole soluble Ag and also induced resting T cell proliferation and Ag-specific CTL effector function. Unlike cytokine-driven DC differentiation, PMA suppressed proliferation and induced cell death (in part via apoptosis) in cells that did not differentiate to DC. The effects of PMA were blocked by inhibitors of protein kinase C activation, suggesting a central role for this signaling molecule. PMA-mediated signaling also induced expression of the RelB transcription factor, an NF-kappaB family member implicated in DC differentiation. These findings suggest that phorbol esters activate protein kinase C, which then initiates the terminal component of an intracellular signaling pathway(s) involved in the DC differentiation of CD34+ hemopoietic progenitors.

Intrinsic resistance to T cell infection with HIV type 1 induced by CD28 costimulation.

When HIV-infected leukocytes are activated by the CD28 costimulatory receptor, HIV-1 is rapidly cleared from cultures, suggesting that costimulation can render T cells resistant to HIV-1 infection. In this study we tested the hypothesis that enhanced secretion of cytokines or chemokines could account for CD28-induced antiviral effects. In an acute infection system, resistance to infection with macrophage-tropic strains of HIV-1 was shown to be comprised of both soluble and cell-associated components. Induction of HIV-1 resistance was specific for CD28 costimulation, in that a variety of other accessory receptors, such as CD2, CD4, CD5, and MHC class I, failed to confer the antiviral resistance. The soluble component was secreted by both CD4 and CD8 T cells, was not unique to CD28 costimulation, and could be neutralized by removal of C-C chemokines (RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1alpha and -1beta) from the culture supernatants of costimulated CD4 T cells. In contrast, CD28 stimulation of CD4 cells resulted in the specific induction of a pronounced intrinsic resistance to HIV-1 infection by macrophage tropic isolates of HIV-1.

Impaired induction of the apoptosis-protective protein Bcl-xL in activated PBMC from asymptomatic HIV-infected individuals.

Progression to AIDS in asymptomatic HIV-infected individuals is characterized by a gradual but progressive loss of CD4+ T cells. While the mechanisms underlying this decline are currently unknown, recent evidence suggests that these cells are abnormally sensitive to apoptosis in response to activation signals. Recent work has implicated downregulation of Bcl-2 with the increased spontaneous apoptosis in lymphocytes from HIV-infected patients. We have evaluated the roles of the apoptosis-protective proteins Bcl-2 and Bcl-x in stimulated PBMC from asymptomatic HIV-infected and HIV-uninfected individuals. We found that Bcl-2 was constitutively expressed in PBMC from both HIV-infected and uninfected samples. However, Bcl-x induction was delayed and responses were decreased in stimulated HIV-infected samples. Additionally, single-cell intracellular staining of Bcl-x revealed a significant inverse correlation between PWM-induced Bcl-x expression and apoptosis (r = -0.695, P = 0.05). This was confirmed at the single-cell level in direct experiments when stimulated cells were sorted based on Bcl-x induction and then measured for apoptosis. Furthermore, low Bcl-x expression was not due to reduced lymphocyte activation following PWM stimulation. Our data indicate that the induction of Bcl-x is markedly impaired in asymptomatic HIV-infected patients and that stimuli which induce inadequate expression of Bcl-x are associated with increased levels of apoptosis in these cells.

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.