François M. Lemoine

PD-1–Expressing Tumor-Infiltrating T Cells Are a Favorable Prognostic Biomarker in HPV-Associated Head and Neck Cancer

Head and neck cancers positive for human papillomavirus (HPV) have a more favorable clinical outcome than HPV-negative cancers, but it is unknown why this is the case. We hypothesized that prognosis was affected by intrinsic features of HPV-infected tumor cells or differences in host immune response. In this study, we focused on a comparison of regulatory Foxp3(+) T cells and programmed death-1 (PD-1)(+) T cells in the microenvironment of tumors that were positive or negative for HPV, in two groups that were matched for various clinical and biologic parameters. HPV-positive head and neck cancers were more heavily infiltrated by regulatory T cells and PD-1(+) T cells and the levels of PD-1(+) cells were positively correlated with a favorable clinical outcome. In explaining this paradoxical result, we showed that these PD-1(+) T cells expressed activation markers and were functional after blockade of the PD-1-PD-L1 axis in vitro. Approximately 50% of PD-1(+) tumor-infiltrating T cells lacked Tim-3 expression and may indeed represent activated T cells. In mice, administration of a cancer vaccine increased PD-1 on T cells with concomitant tumor regression. In this setting, PD-1 blockade synergized with vaccine in eliciting antitumor efficacy. Our findings prompt a need to revisit the significance of PD-1-infiltrating T cells in cancer, where we suggest that PD-1 detection may reflect a previous immune response against tumors that might be reactivated by PD-1/PD-L1 blockade.

Novel mode of action of c-kit tyrosine kinase inhibitors leading to NK cell–dependent antitumor effects

Mutant isoforms of the KIT or PDGF receptors expressed by gastrointestinal stromal tumors (GISTs) are considered the therapeutic targets for STI571 (imatinib mesylate; Gleevec), a specific inhibitor of these tyrosine kinase receptors. Case reports of clinical efficacy of Gleevec in GISTs lacking the typical receptor mutations prompted a search for an alternate mode of action. Here we show that Gleevec can act on host DCs to promote NK cell activation. DC-mediated NK cell activation was triggered in vitro and in vivo by treatment of DCs with Gleevec as well as by a loss-of-function mutation of KIT. Therefore, tumors that are refractory to the antiproliferative effects of Gleevec in vitro responded to Gleevec in vivo in an NK cell-dependent manner. Longitudinal studies of Gleevec-treated GIST patients revealed a therapy-induced increase in IFN-gamma production by NK cells, correlating with an enhanced antitumor response. These data point to a novel mode of antitumor action for Gleevec.

Identification of CD8+CD25+Foxp3+ suppressive T cells in colorectal cancer tissue

Background: The antitumoral immune response is one determinant of colorectal cancer (CRC) outcome. Recent work suggests that Foxp3+CD25+CD4+ regulatory T cells (T4reg) might hamper effective immunosurveillance of emerging cancer cells and impede effective immune responses to established tumours. In this descriptive study, we analysed blood and tissue regulatory T cell populations in patients with CRC. Methods: Blood and tissue regulatory Foxp3+ T cells from 40 patients with CRC were compared to regulatory Foxp3+ T cells from normal colonic tissue and from blood of 26 healthy volunteers. Flow cytometry was used to quantify and phenotype all Foxp3+ T cell populations. Correlations were sought with the tumour stage and with micro-invasive status. The suppressive capacity of regulatory Foxp3+ T cells was assessed by their effect on CD4+CD25− T cell proliferation in vitro and by their capacity to inhibit cytokine production by conventional T cells. Results: We found a significant increase of CD8+CD25+Foxp3+ cells (T8reg) in blood and CRC tissue; their phenotype was close to that of T4reg. T8reg cells infiltrating CRC were activated, as suggested by increased cytoxic T lymphocyte-associated antigen-4, glucocorticoid-induced tumour necrosis factor-related protein, and transforming growth factor (TGF)β1 expression compared to T8reg from normal autologous colonic tissue. Moreover, T8reg were able to suppress CD4+CD25− T cell proliferation and Th1 cytokine production ex vivo, demonstrating that tumour-infiltrating T8reg have strong suppressive capacities. T8reg numbers correlated with the tumour stage and with micro-invasive status. Finally, interleukin 6 and TGFβ1 synergistically induced the generation of CD8+CD25+Foxp3+ T cells ex vivo. Conclusions: We have identified a new regulatory T cell population (CD8+Foxp3+) in colorectal tumours. After isolation from cancer tissue these CD8+Foxp3+ cells demonstrated strong immunosuppressive properties in vitro. These data suggest that these cells may contribute to tumoral immune escape and disease progression.

CD4+CD25+ Regulatory T Cell Depletion Improves the Graft-Versus-Tumor Effect of Donor Lymphocytes After Allogeneic Hematopoietic Stem Cell Transplantation

Immunological effects of donor lymphocyte infusion for treatment of recurrent malignancy after allogeneic hematopoietic cell transplantation can be enhanced by depleting T regulatory cells in the infused cells and in the recipient. Regulatory T Cells Interfere with Graft-Versus-Tumor Effects Patients search for organ donors who are close genetic matches to avoid immune reactions. But sometimes a little immune activation is a good thing. Hematopoietic stem cells in the form of bone marrow are often used to treat blood cancers, and the donated cells not only engraft in bone to provide a source of healthy blood cells but also contain T cells that attack and destroy any remaining cancerous cells. This graft-versus-tumor effect is also harnessed when such patients suffer a relapse and donor T cells are infused into the patient. These infusions often fail to quell the malignancy, however. The reason for failure, Maury et al. have now shown, is that the donor cells can often include regulatory T cells (Tregs), a class of T cells that dampens the immune response. Removing these cells before the infusion markedly improved the graft-versus-tumor effect and the patients’ survival. The beneficial graft-versus-tumor effect of transplantation can be accompanied by the not-so-desirable graft-versus-host disease. Like the transplanted T cells that perceive cancer cells as foreign, T cells can also attack the host’s skin, liver, intestinal lining, and other internal organs—a condition that is serious but can be treated. The authors of this study used the presence of graft-versus-host disease as a sign that there were active, functioning T cells that also provided graft-versus-tumor effects. They treated 17 patients with relapsed blood cancer who had previously received an infusion of lymphocytes and had neither clinical manifestations of graft-versus-host disease nor control of their malignancy. After receiving a new infusion of lymphocytes from which the Tregs had been removed, two of the patients developed graft-versus-host disease for the first time in their transplant history. Hypothesizing that this low rate of response was a result of Treg cells present in the recipient, they treated four of the patients who needed more infusions with the same Treg-depleted cells but now infused them immediately after recipient Tregs were eliminated with lymphopdepletive chemotherapy. These four patients, all of whom had Hodgkin’s lymphoma, reacted to the infused cells by developing graft-versus-host disease, a sign that the infused cells were likely attacking the tumor cells as well. When the whole group was assessed 1 year after treatment, the patients who had experienced a graft-versus-host reaction after cell infusion were found to have survived longer, likely a result of successful immune control of the cancer cells by the infused Treg-depleted lymphocytes. This preliminary study shows that depleting donor lymphocytes of inhibitory regulatory T cells can be a safe and effective way to free active T cells from inhibition so that they can fight cancer cells in the recipient. Further studies are needed, but this seemingly inappropriate encouragement of immune reactions in transplant recipients may prove a boon to patients with blood cancers. Donor T cells play a pivotal role in the graft-versus-tumor effect after allogeneic hematopoietic stem cell transplantation. Regulatory T cells (Tregs) 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 Treg depletion could improve alloreactivity and likewise the graft-versus-tumor effect of donor T cells. The safety and efficacy of Treg-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 Treg-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 Treg-depleted donor lymphocyte infusion received a second Treg-depleted donor lymphocyte infusion combined with lymphodepleting chemotherapy aimed to also eliminate recipient Tregs. 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 Treg depletion was associated with improved survival. These results suggest that Treg-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 Treg-depleted donor lymphocyte infusion. These findings offer a rational therapeutic approach for cancer cellular immunotherapy.

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.

Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy.

Dendritic cells (DC) are professional antigen‐presenting cells that can be used as immune adjuvant for anti‐tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) alone or combined with interleukin‐4 (IL‐4) or Flt3 ligand (Flt3‐L) on the number, immunophenotype and functions of bone marrow‐derived DC. In GM‐CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC‐IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC‐205+ F4/80+), and MHC‐IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC‐205−; F4/80+). The addition of Flt3‐L to GM‐CSF induced a twofold increase in MHC‐IIhi DC number; besides, the MHC‐IIlo cells lost all DC markers. In contrast, after addition of IL‐4 to GM‐CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1− DEC‐205+ F4/80−), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty‐four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM‐CSF+Flt3‐L. Finally, we showed that MHC‐IIhi were more efficient than MHC‐IIlo cells in an anti‐tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro‐generated DC before use in immunotherapy.

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

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.

High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells.

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.

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 α4 and β7 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.

IL-3-INDUCED COEXPRESSION OF HISTIDINE DECARBOXYLASE, IL-4 AND IL-6 MRNA BY MURINE BASOPHIL PRECURSORS

Murine low-density bone marrow cells sorted from the blast cell window on the basis of high rhodamine-123 retention (Rh-bright), are highly enriched in histamine-, IL-4-, and IL-6-producing cells. We established by in situ hybridization that up to 50% of this population (around 0.25% of the whole bone marrow) coexpressed the transcripts for these molecules upon stimulation with 1L-3. Rh-bright cells were also positive for mRNA encoding the alpha, beta, and gamma chains of the Fc(epsilon)RI which was functional since aggregated IgE induced the same percentage of cells hybridizing with the HDC probe as IL-3. Clonogenic progenitors and histamine- and cytokine-producing cells copurified in the Rh-bright population, but could be distinguished by their c-kit expression, CFU-C being more frequent in the c-kit(high) fraction, while histamine and IL-6 producers were enriched in the kit(low) counterpart. Ultrastructural analysis of Rh-bright cells revealed essentially two subsets, namely undifferentiated blast cells and basophil precursors. No other lineage-committed population was enriched by this sorting procedure, and it can therefore be concluded that coexpression of HDC, IL-6, and IL-4 transcripts in response to IL-3 or aggregated IgE takes place mainly in hematopoietic precursors belonging to the basophil lineage.