Replenish the source within: Rescuing tumor-infiltrating lymphocytes by double checkpoint blockade.

We have recently reported that the PD-1 and CTLA4 signaling pathways are active in both effector and regulatory T cells, causing profound immune dysfunctions in the tumor microenvironment. In line with this notion, the dual blockade of PD-1- and CTLA4-conveyed signals may exert robust therapeutic effects. Here, we discuss the mechanisms possibly underlying such a synergic interaction.

Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma.

BACKGROUND: Immune checkpoint inhibitors targeting programmed cell death 1 (PD1) or its ligand (PD-L1) showed activity in several cancer types. METHODS: We performed immunohistochemistry for CD3, CD8, CD20, HLA-DR, phosphatase and tensin homolog (PTEN), PD-1, and PD-L1 and pyrosequencing for assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status in 135 glioblastoma specimens (117 initial resection, 18 first local recurrence). PD-L1 gene expression was analyzed in 446 cases from The Cancer Genome Atlas. RESULTS: Diffuse/fibrillary PD-L1 expression of variable extent, with or without interspersed epithelioid tumor cells with membranous PD-L1 expression, was observed in 103 of 117 (88.0%) newly diagnosed and 13 of 18 (72.2%) recurrent glioblastoma specimens. Sparse-to-moderate density of tumor-infiltrating lymphocytes (TILs) was found in 85 of 117 (72.6%) specimens (CD3+ 78/117, 66.7%; CD8+ 52/117, 44.4%; CD20+ 27/117, 23.1%; PD1+ 34/117, 29.1%). PD1+ TIL density correlated positively with CD3+ (P < .001), CD8+ (P < .001), CD20+ TIL density (P < .001), and PTEN expression (P = .035). Enrichment of specimens with low PD-L1 gene expression levels was observed in the proneural and G-CIMP glioblastoma subtypes and in specimens with high PD-L1 gene expression in the mesenchymal subtype (P = 5.966e-10). No significant differences in PD-L1 expression or TIL density between initial and recurrent glioblastoma specimens or correlation of PD-L1 expression or TIL density with patient age or outcome were evident. CONCLUSION: TILs and PD-L1 expression are detectable in the majority of glioblastoma samples but are not related to outcome. Because the target is present, a clinical study with specific immune checkpoint inhibitors seems to be warranted in glioblastoma.

Correlation of tumor-associated antigens MAGE, NY-ESO-1 and P53 expression with clinical and pathological relationships of patients with oral squamous cell carcinoma

Despite advances in the diagnosisand treatment of head and neck cancer,survival rates have not improvedover recent years. New therapeuticstrategies, including immunotherapy,are the subject of extensive research.In several types of tumors, the presenceof tumor infiltrating lymphocytes(TILs), notably CD8+ T cellsand dendritic cells, has been correlatedwith improved prognosis. Moreover,some T cells among TILs havebeen shown to kill tumor cells in vitroupon recognition of tumor-associatedantigens. Tumor associated antigensare expressed in a significant proportionof squamous cell carcinoma ofthe head and neck and apparently mayplay a role in the regulation of cancercell growth notably by inhibition ofp53 protein function in some cancers.The MAGE family CT antigens couldtherefore potentially be used as definedtargets for immunotherapy andtheir study bring new insight in tumorgrowth regulation mechanisms. Between1995 - 2005 54 patients weretreated surgically in our institution forsquamous cell carcinoma of the oralcavity. Patient and clinical data wasobtained from patient files and collectedinto a computerized database.For each patient, paraffin embeddedtumor specimens were retrieved andexpression of MAGE CT antigens,p53, NY-OESO-1 were analyzed byimmunohistochemistry. Results werethen correlated with histopathologicalparameter such as tumor depth,front invasion according to Bryne andboth, local control and disease freesurvival. MAGE-A was expressed in52% of patients. NY-ESO-1 and p53expression was found in 7% and 52%cases respectively. A higher tumordepth was significantly correlatedwith expression of MAGE-Aproteins(p = 0.03). No significant correlationcould be made between the expressionof both p53 andNY-OESO-1 andhistopathological parameters. Expressionof tumor-associated antigendid not seem to impact significantlyon patient prognosis. As does thedemonstration of p53 function inhibitionby CT antigens of MAGE family,our results suggest, that tumor associatedantigens may be implicated in tumorprogression mechanisms. Thishypothesis need further investigationto clarify the relationship betweenhost immune response and local tumorbiology.

Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Prognostic value of arginase-II expression and regulatory T-cell infiltration in head and neck squamous cell carcinoma.

Tumor-infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3(+) T cells, and relatively high levels of BDCA2(+) and FOXP3(+) cells in stromal (peripheral) regions of the tumors. Tumor-infiltrating (intraepithelial) FOXP3(+) T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase-II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase-2 by 43%, and B-cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c(+) myeloid dendritic cells, and high numbers of FOXP3(+) T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease-free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.

Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer.

The tumor microenvironment mediates induction of the immunosuppressive programmed cell death-1 (PD-1) pathway, and targeted interventions against this pathway can help restore antitumor immunity. To gain insight into these responses, we studied the interaction between PD-1 expressed on T cells and its ligands (PD-1:PD-L1, PD-1:PD-L2, and PD-L1:B7.1), expressed on other cells in the tumor microenvironment, using a syngeneic orthotopic mouse model of epithelial ovarian cancer (ID8). Exhaustion of tumor-infiltrating lymphocytes (TIL) correlated with expression of PD-1 ligands by tumor cells and tumor-derived myeloid cells, including tumor-associated macrophages (TAM), dendritic cells, and myeloid-derived suppressor cells (MDSC). When combined with GVAX or FVAX vaccination (consisting of irradiated ID8 cells expressing granulocyte macrophage colony-stimulating factor or FLT3 ligand) and costimulation by agonistic α-4-1BB or TLR 9 ligand, antibody-mediated blockade of PD-1 or PD-L1 triggered rejection of ID8 tumors in 75% of tumor-bearing mice. This therapeutic effect was associated with increased proliferation and function of tumor antigen-specific effector CD8(+) T cells, inhibition of suppressive regulatory T cells (Treg) and MDSC, upregulation of effector T-cell signaling molecules, and generation of T memory precursor cells. Overall, PD-1/PD-L1 blockade enhanced the amplitude of tumor immunity by reprogramming suppressive and stimulatory signals that yielded more powerful cancer control.

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

Dual Blockade of PD-1 and CTLA-4 Combined with Tumor Vaccine Effectively Restores T-Cell Rejection Function in Tumors.

Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression. Cancer Res; 73(12); 3591-603. ©2013 AACR.

Interactions between cancer stem cells and their niche govern metastatic colonization.

Metastatic growth in distant organs is the major cause of cancer mortality. The development of metastasis is a multistage process with several rate-limiting steps. Although dissemination of tumour cells seems to be an early and frequent event, the successful initiation of metastatic growth, a process termed 'metastatic colonization', is inefficient for many cancer types and is accomplished only by a minority of cancer cells that reach distant sites. Prevalent target sites are characteristic of many tumour entities, suggesting that inadequate support by distant tissues contributes to the inefficiency of the metastatic process. Here we show that a small population of cancer stem cells is critical for metastatic colonization, that is, the initial expansion of cancer cells at the secondary site, and that stromal niche signals are crucial to this expansion process. We find that periostin (POSTN), a component of the extracellular matrix, is expressed by fibroblasts in the normal tissue and in the stroma of the primary tumour. Infiltrating tumour cells need to induce stromal POSTN expression in the secondary target organ (in this case lung) to initiate colonization. POSTN is required to allow cancer stem cell maintenance, and blocking its function prevents metastasis. POSTN recruits Wnt ligands and thereby increases Wnt signalling in cancer stem cells. We suggest that the education of stromal cells by infiltrating tumour cells is an important step in metastatic colonization and that preventing de novo niche formation may be a novel strategy for the treatment of metastatic disease.

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity.

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.

Selecting highly affine and well-expressed TCRs for gene therapy of melanoma.

A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

An infrared spectral signature of human lymphocyte subpopulations from peripheral blood.

Metastatic melanomas are frequently refractory to most adjuvant therapies such as chemotherapies and radiotherapies. Recently, immunotherapies have shown good results in the treatment of some metastatic melanomas. Immune cell infiltration in the tumor has been associated with successful immunotherapy. More generally, tumor infiltrating lymphocytes (TILs) in the primary tumor and in metastases of melanoma patients have been demonstrated to correlate positively with favorable clinical outcomes. Altogether, these findings suggest the importance of being able to identify, quantify and characterize immune infiltration at the tumor site for a better diagnostic and treatment choice. In this paper, we used Fourier Transform Infrared (FTIR) imaging to identify and quantify different subpopulations of T cells: the cytotoxic T cells (CD8+), the helper T cells (CD4+) and the regulatory T cells (T reg). As a proof of concept, we investigated pure populations isolated from human peripheral blood from 6 healthy donors. These subpopulations were isolated from blood samples by magnetic labeling and purities were assessed by Fluorescence Activated Cell Sorting (FACS). The results presented here show that Fourier Transform Infrared (FTIR) imaging followed by supervised Partial Least Square Discriminant Analysis (PLS-DA) allows an accurate identification of CD4+ T cells and CD8+ T cells (>86%). We then developed a PLS regression allowing the quantification of T reg in a different mix of immune cells (e.g. Peripheral Blood Mononuclear Cells (PBMCs)). Altogether, these results demonstrate the sensitivity of infrared imaging to detect the low biological variability observed in T cell subpopulations.

A role for T-bet-mediated tumour immune surveillance in anti-IL-17A treatment of lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. The cytokine interleukin-17A supports tumour vascularization and growth, however, its role in lung cancer is unknown. Here we show, in the lungs of patients with lung adenocarcinoma, an increase in interleukin-17A that is inversely correlated with the expression of T-bet and correlated with the T regulatory cell transcription factor Foxp3. Local targeting of interleukin-17A in experimental lung adenocarcinoma results in a reduction in tumour load, local expansion of interferon-γ-producing CD4(+) T cells and a reduction in lung CD4(+)CD25(+)Foxp3(+) regulatory T cells. T-bet((-/-)) mice have a significantly higher tumour load compared with wild-type mice. This is associated with the local upregulation of interleukin-23 and induction of interleukin-17A/interleukin-17R-expressing T cells infiltrating the tumour. Local anti-interleukin-17A antibody treatment partially improves the survival of T-bet((-/-)) mice. These results suggest that local anti-interleukin-17A antibody therapy could be considered for the treatment of lung tumours.