Phenotypic and Functional Studies of Human Treg Cell Subpopulations.

CD4+ regulatory T (Treg) cells play an important role in maintaining immune homeostasis. Although these cells were initially studied as a homogenous cohort, we now know that they have unprecedented underlying heterogeneity. This heterogeneity is reflected in their phenotype and functions. As human Treg subpopulations are very small in numbers, it is necessary to develop novel ways of isolating and manipulating these cell populations. In this chapter, we discuss immunoassays established to this effect.

Generation of Human Alloantigen-Specific Regulatory T Cells Under Good Manufacturing Practice-Compliant Conditions for Cell Therapy.

Natural regulatory T cells (Tregs) may have a great therapeutic potential to induce tolerance in allogeneic cells and organ transplantations. In mice, we showed that alloantigen-specific Tregs (spe-Tregs) were more efficient than polyclonal Tregs (poly-Tregs) in controlling graft-versus-host disease (GVHD). Here we describe a clinical-grade compliant method for generating human spe-Tregs. Tregs were enriched from leukapheresis products with anti-CD25 immunomagnetic beads, primed twice by allogeneic mature monocyte-derived dendritic cells (mDCs), and cultured during 3 weeks in medium containing interleukin 2 (IL-2), IL-15, and rapamycin. After 3 weeks of culture, final cell products were expanded 8.3-fold from the initial CD25(+) purifications. Immunophenotypic analyses of final cells indicate that they were composed of 88 ± 2.6% of CD4(+) T cells, all expressing Treg-specific markers (FOXP3, Helios, GARP, LAP, and CD152). Spe-Tregs were highly suppressive in vitro and also in vivo using a xeno-GVHD model established in immunodeficient mice. The specificity of their suppressive activity was demonstrated on their ability to significantly suppress the proliferation of autologous effector T cells stimulated by the same mDCs compared to third-party mDCs. Our data provide evidence that functional alloantigen Tregs can be generated under clinical-grade compliant conditions. Taking into account that 130 × 10(6) CD25(+) cells can be obtained at large scale from standard leukapheresis, our cell process may give rise to a theoretical final number of 1 × 10(9) spe-Tregs. Thus, using our strategy, we can propose to prepare spe-Tregs for clinical trials designed to control HLA-mismatched GVHD or organ transplantation rejection.

Clinical validation of IFNγ/IL-10 and IFNγ/IL-2 FluoroSpot assays for the detection of Tr1 T cells and influenza vaccine monitoring in humans.

The type of T cell polarization and simultaneous production of multiple cytokines have been correlated with vaccine efficacy. ELISpot is a T cell detection technique optimized for the measurement of a secreted cytokine at the single cell level. The FluoroSpot assay differs from ELISpot by the use of multiple fluorescent-labeled anticytokine detection antibodies, allowing optimal measurement of multiple cytokines. In the present study, we show that an IFNγ/IL-10 FluoroSpot assay is more sensitive than flow cytometry to detect Tr1 regulatory T cells, an immunosuppressive T cell population characterized by the production of IL-10 and IFNγ. As many tolerogenic vaccines are designed to induce these Tr1 cells, this FluoroSpot test could represent a standard method for the detection of these cells in the future. The use of an IFNγ/IL-2 FluoroSpot assay during influenza vaccine monitoring showed that the influenza-specific IL-2-producing T-cell response was the dominant response both before and after vaccine administration. This study therefore questions the rationale of using the single-color IFNγ ELISpot as the standard technique to monitor vaccine-specific T-cell response. Using this same test, a trend was also observed between baseline levels of IFNγ T cell response and T cell vaccine response. In addition, a lower IFNγ+IL-2+ T-cell response after vaccine was observed in the group of patients treated with TNFα inhibitors (P=0.08). This study therefore supports the use of the FluoroSpot assay due to its robustness, versatility and the complementary information that it provides compared with ELISpot or flow cytometry to monitor vaccine-specific T-cell responses.

A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C.

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

Human CD90 identifies Th17/Tc17 T cell subsets that are depleted in HIV-infected patients.

By revisiting CD90, a GPI-anchored glycoprotein, we show that CD90 is expressed by a subset of CD4(+) and CD8(+) human T cells. CD4(+)CD90(+) cells share similarities with Th17 cells because they express the Th17-specific transcription factor RORC2 and produce IL-17A. CD4(+)CD90(+) cells are activated memory T cells that express the gut mucosal markers CCR6, CD161, and the α(4) and ß(7) integrins. Compared with CD90-depleted CCR6(+) memory Th17 cells, CD4(+)CD90(+) cells express higher levels of IL-22 and proinflammatory cytokines (IL-6, TNF-α and GM-CSF), but they produce lower levels of IL-21 and no IL-9. Analyses of CD8(+)CD90(+) cells reveal that they express RORC2 and are able to produce higher levels of IL-17A, IL-22, and CCL20 compared with CD90-depleted CD8(+) cells. These data show that CD90 identifies Th17 and Tc17 cells with a peculiar cytokine profile. Studies of circulating CD90(+) cells in HIV patients show that CD90(+) cells are decreased with an imbalance of the CD4(+)CD90(+)/regulatory T cell ratio in nontreated patients compared with treated patients and healthy donors. Overall, human CD90 identifies a subset of Th17 and Tc17 cells within CD4(+) and CD8(+) T cells, respectively, which are depleted during HIV infection.

CD4+CD25+ regulatory T cell depletion improves the graft-versus-tumor effect of donor lymphocytes after allogeneic hematopoietic stem cell transplantation.

Donor T cells play a pivotal role in the graft-versus-tumor effect after allogeneic hematopoietic stem cell transplantation. Regulatory T cells (T(reg)s) may reduce alloreactivity, the major component of the graft-versus-tumor effect. In the setting of donor lymphocyte infusion after hematopoietic stem cell transplantation, we postulated that T(reg) depletion could improve alloreactivity and likewise the graft-versus-tumor effect of donor T cells. The safety and efficacy of T(reg)-depleted donor lymphocyte infusion was studied in 17 adult patients with malignancy relapse after hematopoietic stem cell transplantation. All but one had previously failed to respond to at least one standard donor lymphocyte infusion, and none had experienced graft-versus-host disease. Two of the 17 patients developed graft-versus-host disease after their first T(reg)-depleted donor lymphocyte infusion and experienced a long-term remission of their malignancy. Four of the 15 patients who did not respond after a first T(reg)-depleted donor lymphocyte infusion received a second T(reg)-depleted donor lymphocyte infusion combined with lymphodepleting chemotherapy aimed to also eliminate recipient T(reg)s. All four developed acute-like graft-versus-host disease that was associated with a partial or complete and durable remission. In the whole cohort, graft-versus-host disease induction through T(reg) depletion was associated with improved survival. These results suggest that T(reg)-depleted donor lymphocyte infusion is a safe, feasible approach that induces graft-versus-host or graft-versus-tumor effects in alloreactivity-resistant patients. In patients not responding to this approach, the combination of chemotherapy-induced lymphodepletion of the recipient synergizes with the effect of T(reg)-depleted donor lymphocyte infusion. These findings offer a rational therapeutic approach for cancer cellular immunotherapy.

Massive expansion of regulatory T-cells following interleukin 2 treatment during a phase I-II dendritic cell-based immunotherapy of metastatic renal cancer.

Cytotoxic chemotherapy is ineffective in metastatic renal cancer. However, systemic administration of interleukin 2 (IL-2) or infusion of dendritic cells (DCs) loaded with tumor extracts can lead to some response rates with concomitant survival improvements. We report the results of a phase I-II pilot study combining DCs and IL-2 where six patients were included. DCs were derived from bone marrow CD34+ cells and loaded with autologous tumor extracts. CD34-DC vaccines were infused subcutaneously at day 45, 52, 59, 90 and 120 following surgery in combination with IL-2, that was subsequently administrated after the 3rd and 4th DC vaccinations. Preparation of tumor extracts and CD34-DCs were satisfactory in all patients but one. Due to rapid tumor progression, one patient was excluded before vaccination. In the 4 remaining patients, two received 3 vaccinations, while the 2 others received 5 vaccinations and the full IL-2 treatment. No adverse effect due to the vaccinations was observed. A specific immune response against autologous tumor cells was observed in the 2 patients who completed the treatment. Interestingly, these 2 patients had a more prolonged survival than the patients receiving 3 vaccinations. Importantly, a transient and massive increase of circulating natural regulatory T-cells (nTregs) was evidenced in 3 patients following IL-2 administration. Overall, the use of CD34-DC vaccines is feasible, safe and non-toxic. A specific anti-tumor immune response can be detected. However, our data highlights that IL-2 is a potent inducer of nTregs in vivo and as such may have a negative impact on cancer immunotherapy.

Clinical-grade preparation of human natural regulatory T-cells encoding the thymidine kinase suicide gene as a safety gene.

BACKGROUND: Human CD4+CD25+FOXP3+ natural regulatory T-cells (nTreg) have a great therapeutic potential for the induction of tolerance in allo-transplanted patients or for the control of severe auto-immune diseases. However, clinical-grade production of nTreg remains difficult to achieve because of the absence of a truly specific surface marker and of their low frequency that implies a need for their ex vivo expansion. Furthermore, safety issues should be taken into consideration due to the risk of either uncontrolled nTreg-induced immunosuppression or uncontrolled proliferation of autoreactive contaminating T-cells particularly in an auto-immune context. METHODS: We compared different clinical-grade conditions for immuno-magnetic selection and ex vivo expansion of nTreg. For safety, expanded cells were genetically modified with retroviral vectors co-expressing human CD90 and HSV1 thymidine kinase. The CD90 surface marker and thymidine kinase allow for selection and elimination of transduced cells by ganciclovir, respectively. RESULTS: We showed that (i) nTreg could be enriched in a one step using CD25 microbeads, were functionally suppressive and mainly FOXP3+; (ii) using anti-CD28- and anti-CD3-coated beads, interleukin-2 and rapamycin, nTreg were expanded 150-200-fold after 3 weeks. Under these clinical-grade conditions, they remained suppressive, and no major alteration of the TCR repertoire was observed; (iii) after efficient retroviral transduction and CD90 selection, nTreg maintained their suppressive activity; (iv) transduced nTreg could be eliminated by ganciclovir upon activation. CONCLUSIONS: The efficient procedure reported here for the preparation of nTreg, whose safety has been ensured, is now applicable for further clinical trials.

Initial depletion of regulatory T cells: the missing solution to preserve the immune functions of T lymphocytes designed for cell therapy.

We investigated the causes of the altered functionality of T cells cultured under conditions designed for cell and gene therapy and the strategies to prevent their defects. We first showed that human T cells cultured for 6 days with anti-CD3 +/- anti-CD28 antibodies and interleukin-2 presented a 50% decrease of their proliferative responses to allogeneic or recall antigens. Similarly, day-6 cultured murine T cells completely lost their capacity to reject allogeneic skin grafts and to provoke graft-versus-host disease (GVHD) when infused into irradiated semi-allogeneic mice. Interestingly, injection of higher amounts of cultured T cells restored GVHD induction. Moreover, depletion of CD25+ cells prior to T-cell cultures can prevent these deficiencies both in mice and humans. Therefore, we demonstrated that culture conditions used for T-cell therapy preferentially activated and expanded regulatory T cells (Treg's). Thus, we showed that dividing cells sorted from T-cell cultures strongly suppressed the proliferation of autologous T cells in response to allogeneic stimulation. An increased detection of Foxp3 at mRNA and protein levels in the cultures confirmed the Treg expansion. Overall, we demonstrate that T-cell cultures promote Treg expansion over effector T cells, leading to deleterious immune functions, and that this imbalance can be prevented by an initial depletion of CD25+ cells.

Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters.

BACKGROUND: Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes. METHODS: We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration. RESULTS: For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels. CONCLUSIONS: Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications.

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.

BACKGROUND: T-cells expressing the HSV1-TK suicide gene can be used for the control of graft-versus-host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel 'split genome' human packaging cell line (1704 cells). METHODS: To minimize the risk of generating replication-competent retroviruses, pol was truncated to remove sequences overlapping with env. To improve retroviral infection and selection of transduced T-cells, high titers of GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector coding for the CD90 GPI-anchored membrane molecule were produced by 1704 cells. RESULTS: Using 1704 cell supernatant and an optimized transduction protocol, approximately 50% of primary T-cells were transduced and could then be purified (approximately 95%) using clinical-grade immunomagnetic beads directed against CD90. Over 96% of these OKT3/IL-2-activated CD90(+)-selected T-cells were killed by ganciclovir. Cell proliferation and cytokine production of transduced T-cells and HLA-restricted cytotoxicity of transduced T-cell clones were identical to those of their non-transduced counterparts cultured under the same conditions. CONCLUSIONS: GALV-pseudotyped retroviral particles harboring a bicistronic Thy1-IRES-TK vector allow efficient transduction and rapid selection of human T-cells under conditions applicable for clinical trials using the new human 1704 packaging cell line.