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1.
EMBO J ; 34(15): 2042-58, 2015 Aug 04.
Article in English | MEDLINE | ID: mdl-26139534

ABSTRACT

T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFß and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFß/IL-6-mediated induction of Maf.


Subject(s)
CD8-Positive T-Lymphocytes/cytology , Cell Differentiation/immunology , Melanoma/metabolism , Proto-Oncogene Proteins c-maf/metabolism , Tumor Microenvironment/physiology , Animals , CD8-Positive T-Lymphocytes/metabolism , DNA Primers/genetics , Flow Cytometry , Gene Expression Profiling , Homeodomain Proteins/genetics , Interleukin-6/metabolism , Luciferases , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-maf/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/metabolism
2.
Immunology ; 150(2): 199-212, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27716898

ABSTRACT

Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A35-43 peptide associated with H-2Ld , although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2Ld . The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+ ]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.


Subject(s)
Antigens, Neoplasm/metabolism , Exocytosis , Mastocytoma/immunology , Melanoma/immunology , Peptide Fragments/metabolism , Secretory Vesicles/metabolism , T-Lymphocytes, Cytotoxic/immunology , Animals , Antigens, Neoplasm/genetics , Calcium Signaling , Cell Line, Tumor , Cytotoxicity, Immunologic , Histocompatibility Antigen H-2D/metabolism , Humans , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Activation , Lymphocyte Function-Associated Antigen-1/metabolism , Mice , T-Cell Antigen Receptor Specificity
3.
Proc Natl Acad Sci U S A ; 111(30): 11115-20, 2014 Jul 29.
Article in English | MEDLINE | ID: mdl-25024217

ABSTRACT

The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cysteine Endopeptidases/immunology , Immunity, Cellular , Intracellular Signaling Peptides and Proteins/immunology , Melanoma/immunology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/pathology , Cysteine Endopeptidases/genetics , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-2/genetics , Interleukin-2/immunology , Intracellular Signaling Peptides and Proteins/genetics , Melanoma/genetics , Melanoma/pathology , Mice , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/immunology , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology
4.
Eur J Immunol ; 45(1): 44-8, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25471823

ABSTRACT

Several recent studies have implicated myeloid cells in providing a microenvironment that promotes tumor cell survival and metastasis, therefore preparing a "premetastatic niche" for cancer progression. In this issue of the European Journal of Immunology, Zhang et al. [Eur. J. Immunol. 2015. 45: 71-81] address the regulation of immune cells in premetastatic lymph nodes in experimental mouse models. The authors show that signal transducer and activator of transcription 3 (STAT3) ablation in murine myeloid cells, which renders the premetastatic niche less receptive to metastasis by B16 melanoma cells, also leads to local activation in the niche of CD8(+) T cells with increased expression of IFN-γ and granzyme B. Data further suggest that STAT3 activation in the myeloid population leads to poor tumor antigen presenting capacity as well as resistance to CD8(+) T-cell killing. Based on these studies in mice and observations in human cancer patients, the authors propose treatments designed to regulate STAT3 activation, which are correlated with increased cytolytic activity of CD8(+) T cells in mouse models.


Subject(s)
Gene Expression Regulation, Neoplastic/immunology , Immunologic Surveillance , Macrophages, Peritoneal/immunology , Melanoma, Experimental/immunology , Skin Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Animals , Humans
5.
Immunology ; 145(1): 24-33, 2015 May.
Article in English | MEDLINE | ID: mdl-25367158

ABSTRACT

CD8 T cells contribute to long-term protection against Listeria monocytogenes infection by differentiating into memory T cells. These rapidly respond to antigen or inflammation upon secondary infection. In this study we used CD8 T cells from OT1 mice and CD4 T cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing L. monocytogenes (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as 2 days after infection. FACS analysis showed GZMB-Tom expression in undivided CD8 T cells, with its level increasing over one to four divisions. OT1 T cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII-positive cells. Twenty hours later, most OT1 T cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom. Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T cells responding either to Lm-OVA or L. monocytogenes. Differences in the activation phenotype and in the splenic distribution of OT1 T cells were observed, depending on the challenge. Notably, OTI T cells with polarized granules were only observed after challenge with cognate antigen. This work showed that the GZMB-Tom knock-in mice in which GZMB-Tom faithfully reproduced GZMB expression, provide useful tools to dissect mechanisms leading to the development of anti-bacterial effector and memory CD8 T cells and reactivation of the memory response to cognate antigen or inflammatory signals.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Gene Expression Regulation, Enzymologic/immunology , Granzymes/immunology , Listeria monocytogenes/immunology , Listeriosis/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/pathology , Gene Expression Regulation, Enzymologic/genetics , Granzymes/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Listeriosis/genetics , Listeriosis/pathology , Mice , Mice, Transgenic , Signal Transduction/genetics , Signal Transduction/immunology , Time Factors
6.
Immunology ; 145(4): 543-57, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25882552

ABSTRACT

CD8 T cells used in adoptive immunotherapy may be manipulated to optimize their effector functions, tissue-migratory properties and long-term replicative potential. We reported that antigen-stimulated CD8 T cells transduced to express an active form of the transcription factor signal transducer and activator of transcription 5 (STAT5CA) maintained these properties upon adoptive transfer. We now report on the requirements of STAT5CA-expressing CD8 T cells for cell survival and proliferation in vivo. We show that STAT5CA expression allows for greater expansion of T cells in vivo, while preserving dependency on T-cell-receptor-mediated tonic stimulation for their in vivo maintenance and return to a quiescent stage. STAT5CA expression promotes the formation of a large pool of effector memory T cells that respond upon re-exposure to antigen and present an increased sensitivity to γc receptor cytokine engagement for STAT5 phosphorylation. In addition, STAT5CA expression prolongs the survival of what would otherwise be short-lived terminally differentiated KLRG1-positive effector cells with up-regulated expression of the senescence-associated p16(INK) (4A) transcripts. However, development of a KLRG1-positive CD8 T cell population was independent of either p16(INK) (4A) or p19(ARF) expression (as shown using T cells from CDKN2A(-/-) mice) but was associated with expression of transcripts encoding p15(INK) (4B) , another protein involved in senescence induction. We conclude that T-cell-receptor- and cytokine-dependent regulation of effector T cell homeostasis, as well as mechanisms leading to senescent features of a population of CD8 T cells are maintained in STAT5CA-expressing CD8 T cells, even for cells that are genetically deficient in expression of the tumour suppressors p16(INK) (4A) and p19(ARF) .


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p15/immunology , Cyclin-Dependent Kinase Inhibitor p16/immunology , STAT5 Transcription Factor/immunology , Animals , Cell Differentiation/genetics , Cellular Senescence/genetics , Cellular Senescence/immunology , Cyclin-Dependent Kinase Inhibitor p15/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Gene Expression Regulation/immunology , Lectins, C-Type , Mice , Mice, Knockout , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , STAT5 Transcription Factor/genetics
7.
J Immunol ; 191(7): 3712-24, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-24006458

ABSTRACT

In adoptive therapy, CD8 T cells expressing active STAT5 (STAT5CA) transcription factors were found to be superior to unmanipulated counterparts in long-term persistence, capacity to infiltrate autochthonous mouse melanomas, thrive in their microenvironment, and induce their regression. However, the molecular mechanisms sustaining these properties were undefined. In this study, we report that STAT5CA induced sustained expression of genes controlling tissue homing, cytolytic granule composition, type 1 CD8 cytotoxic T cell-associated effector molecules granzyme B(+), IFN-γ(+), TNF-α(+), and CCL3(+), but not IL-2, and transcription factors T-bet and eomesodermin (Eomes). Chromatin immunoprecipitation sequencing analyses identified the genes possessing regulatory regions to which STAT5 bound in long-term in vivo maintained STAT5CA-expressing CD8 T cells. This analysis identified 34% of the genes differentially expressed between STAT5CA-expressing and nonexpressing effector T cells as direct STAT5CA target genes, including those encoding T-bet, Eomes, and granzyme B. Additionally, genes encoding the IL-6R and TGFbRII subunits were stably repressed, resulting in dampened IL-17-producing CD8 T cell polarization in response to IL-6 and TGF-ß1. The absence of T-bet did not affect STAT5CA-driven accumulation of the T cells in tissue or their granzyme B expression but restored IL-2 secretion and IL-6R and TGFbRII expression and signaling, as illustrated by IL-17 induction. Therefore, concerted STAT5/T-bet/Eomes regulation controls homing, long-term maintenance, recall responses, and resistance to polarization towards IL-17-producing CD8 T cells while maintaining expression of an efficient type 1 CD8 cytotoxic T cell program (granzyme B(+), IFN-γ(+)).


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Interleukin-6/metabolism , STAT5 Transcription Factor/metabolism , T-Box Domain Proteins/genetics , Transforming Growth Factor beta1/metabolism , Animals , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Line, Tumor , Chromatin Immunoprecipitation , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Interleukin-17/metabolism , Mice , Mice, Transgenic , Receptors, Cytokine/genetics , Receptors, Cytokine/metabolism , STAT5 Transcription Factor/genetics , Signal Transduction , T-Box Domain Proteins/deficiency , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Transcription, Genetic
8.
J Immunol ; 188(1): 111-21, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-22140254

ABSTRACT

Central tolerance toward tissue-restricted Ags is considered to rely on ectopic expression in the thymus, which was also observed for tumor Ags encoded by cancer-germline genes. It is unknown whether endogenous expression shapes the T cell repertoire against the latter Ags and explains their weak immunogenicity. We addressed this question using mouse cancer-germline gene P1A, which encodes antigenic peptide P1A(35-43) presented by H-2L(d). We made P1A-knockout (P1A-KO) mice and asked whether their anti-P1A(35-43) immune responses were stronger than those of wild-type mice and whether P1A-KO mice responded to other P1A epitopes, against which wild-type mice were tolerized. We observed that both types of mice mounted similar P1A(35-43)-specific CD8 T cell responses, although the frequency of P1A(35-43)-specific CD8 T cells generated in response to P1A-expressing tumors was slightly higher in P1A-KO mice. This higher reactivity allowed naive P1A-KO mice to reject spontaneously P1A-expressing tumors, which progressed in wild-type mice. TCR-Vß usage of P1A(35-43)-specific CD8 cells was slightly modified in P1A-KO mice. Peptide P1A(35-43) remained the only P1A epitope recognized by CD8 T cells in both types of mice, which also displayed similar thymic selection of a transgenic TCR recognizing P1A(35-43). These results indicate the existence of a minimal tolerance to an Ag encoded by a cancer-germline gene and suggest that its endogenous expression only slightly affects diversification of the T cell repertoire against this Ag.


Subject(s)
Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , Immune Tolerance , Neoplasms/immunology , Peptides/immunology , Animals , Antigens, Neoplasm/genetics , Cell Line, Tumor , Epitopes/genetics , Mice , Mice, Knockout , Neoplasms/genetics , Peptides/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology
9.
Eur J Immunol ; 41(8): 2414-23, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21590766

ABSTRACT

Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo-rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H-2K(b) (K(b) )-associated peptides and critically required to induce full activation of H-2(k) monoclonal CD8(+) TLs expressing the cognate TCR-BM3.3. Here, we asked whether a pBM1/K(b) -specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K(b) -binding CD8(+) TLs increased from <0.04% in naïve mice to 3% of activated CD44(+) CD8(+) TLs in H-2(k) mice rejecting K(b) -expressing cells. Among these, TCR-Vß2 usage was greatly enriched, and 75% of them shared a TCR-Vß2 CDR3ß motif with the prototype TCR-BM3.3. Fewer than 5% of K(b) -reactive CD44(+) CD8(+) TLs not binding pBM1/K(b) displayed this CDR3ß motif. We found that the recurrent CDR3ß motif of pBM1/K(b) -binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR-peptide/MHC fit. Thus, a CDR3ß motif generated by a process akin to "convergent recombination" accounts for a sizable fraction of the alloreactive anti-K(b) TCR repertoire.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Complementarity Determining Regions/immunology , Peptides/immunology , Receptors, Antigen, T-Cell/immunology , Amino Acid Motifs/immunology , Amino Acid Sequence , Animals , CD8-Positive T-Lymphocytes/metabolism , Complementarity Determining Regions/genetics , Complementarity Determining Regions/metabolism , Flow Cytometry , H-2 Antigens/immunology , H-2 Antigens/metabolism , Hyaluronan Receptors/immunology , Hyaluronan Receptors/metabolism , Immunization , Mice , Mice, Inbred CBA , Peptides/metabolism , Protein Binding/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Recombination, Genetic/immunology , Sequence Analysis, DNA , VDJ Exons/genetics , VDJ Exons/immunology
10.
Immunology ; 129(1): 41-54, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20050329

ABSTRACT

In mice expressing a transgenic T-cell receptor (TCR; TCRP1A) of DBA/2 origin with reactivity towards a cancer-germline antigen P1A, the number of TCRP1A CD8+ T cells in lymphoid organs is lower in DBA/2 than in B10.D2 or B10.D2(x DBA/2)F1 mice. This reduction results from haemopoietic cell autonomous differences in the differentiation of the major histocompatibility complex class I-restricted TCRP1A thymocytes controlled by DBA/2 versus B10.D2-encoded genes. We report here that the lower number of TCRP1A CD8+ T cells in DBA/2 mice correlated with their poor resistance to P1A-expressing mastocytoma solid tumours. Functional potency of CD8+ cytolytic T lymphocytes (CTL) from the above strains was not compromised, but their number after expansion appeared to be influenced by their genetic background. Intriguingly, non-transgenic DBA/ 2 mice resisted P1A+ tumours more efficiently despite poor representation of P1A-specific CTL. This was partly the result of their more heterogeneous TCR repertoire, including reactivity to non-P1A tumour antigens because mice that had rejected a P1A+ tumour became resistant to a P1A) variant of the tumour. Such 'cross-resistance' did not develop in the TCRP1A transgenic mice. Nonetheless, reconstitution of RAGo/o mice with TCRP1A CD8+ T cells, with or without CD4+ T cells, or exclusive representation of TCRP1A CD8+ T cells in RAGo/o TCRP1A transgenic mice efficiently resisted the growth of P1A-expressing tumours. Natural killer cells present at a higher number in RAGo/o mice also contributed to tumour resistance, in part through an NKG2D-dependent mechanism. Hence, in the absence of a polyclonal T-cell repertoire, precursor frequencies of natural killer cells and tumour-specific CTL affect tumour resistance.


Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Cell Count , Killer Cells, Natural/metabolism , Mastocytoma/immunology , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Adoptive Transfer , Animals , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Growth Processes , Clonal Deletion/genetics , Immunity, Cellular , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Lymphocyte Cooperation , Mastocytoma/pathology , Mice , Mice, Inbred DBA , Mice, Transgenic , NK Cell Lectin-Like Receptor Subfamily K/immunology , Neoplasm Transplantation , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , T-Cell Antigen Receptor Specificity/genetics
11.
Cancers (Basel) ; 11(12)2019 Nov 21.
Article in English | MEDLINE | ID: mdl-31766350

ABSTRACT

Oncogene-induced STAT3-activation is central to tumor progression by promoting cancer cell expression of pro-angiogenic and immunosuppressive factors. STAT3 is also activated in infiltrating immune cells including tumor-associated macrophages (TAM) amplifying immune suppression. Consequently, STAT3 is considered as a target for cancer therapy. However, its interplay with other STAT-family members or transcription factors such as NF-κB has to be considered in light of their concerted regulation of immune-related genes. Here, we discuss new attempts at re-educating immune suppressive tumor-associated macrophages towards a CD8 T cell supporting profile, with an emphasis on the role of STAT transcription factors on TAM functional programs. Recent clinical trials using JAK/STAT inhibitors highlighted the negative effects of these molecules on the maintenance and function of effector/memory T cells. Concerted regulation of STAT3 and STAT5 activation in CD8 T effector and memory cells has been shown to impact their tumor-specific responses including intra-tumor accumulation, long-term survival, cytotoxic activity and resistance toward tumor-derived immune suppression. Interestingly, as an escape mechanism, melanoma cells were reported to impede STAT5 nuclear translocation in both CD8 T cells and NK cells. Ours and others results will be discussed in the perspective of new developments in engineered T cell-based adoptive therapies to treat cancer patients.

13.
Cancer Res ; 66(6): 3278-86, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16540681

ABSTRACT

Cancer immunotherapy based on vaccination with defined tumor antigens has not yet shown strong clinical efficacy, despite promising results in preclinical models. This discrepancy might result from the fact that available preclinical models rely on transplantable tumors, which do not recapitulate the long-term host-tumor interplay that occurs in patients during progressive tumor development and results in tumor tolerance. To create a faithful preclinical model for cancer immunotherapy, we generated a transgenic mouse strain developing autologous melanomas expressing a defined tumor antigen recognized by T cells. We chose the antigen encoded by P1A, a well-characterized murine cancer germ line gene. To transform melanocytes, we aimed at simultaneously activating the Ras pathway and inactivating tumor suppressor Ink4a/Arf, thereby reproducing two genetic events frequently observed in human melanoma. The melanomas are induced by s.c. injection of 4-OH-tamoxifen (OHT). By activating a CreER recombinase expressed from a melanocyte-specific promoter, this treatment induces the loss of the conditional Ink4a/Arf gene in melanocytes. Because the CreER gene itself is also flanked by loxP sites, the activation of CreER also induces the deletion of its own coding sequence and thereby allows melanocyte-specific expression of genes H-ras and P1A, which are located downstream on the same transgene. All melanomas induced in those mice with OHT show activation of the Ras pathway and deletion of gene Ink4a/Arf. In addition, these melanomas express P1A and are recognized by P1A-specific T lymphocytes. This model will allow to characterize the interactions between the immune system and naturally occurring tumors and thereby to optimize immunotherapy approaches targeting a defined tumor antigen.


Subject(s)
Antigens, Neoplasm/biosynthesis , Melanoma, Experimental/immunology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Cyclin-Dependent Kinase Inhibitor p16/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p16/genetics , Disease Models, Animal , Melanoma, Experimental/genetics , Mice , Mice, Transgenic , Recombination, Genetic , Tamoxifen/analogs & derivatives , Tumor Suppressor Protein p14ARF/antagonists & inhibitors , Tumor Suppressor Protein p14ARF/biosynthesis , Tumor Suppressor Protein p14ARF/genetics , ras Proteins/genetics
14.
Nat Commun ; 8(1): 1404, 2017 11 10.
Article in English | MEDLINE | ID: mdl-29123081

ABSTRACT

Despite impressive clinical success, cancer immunotherapy based on immune checkpoint blockade remains ineffective in many patients due to tumoral resistance. Here we use the autochthonous TiRP melanoma model, which recapitulates the tumoral resistance signature observed in human melanomas. TiRP tumors resist immunotherapy based on checkpoint blockade, cancer vaccines or adoptive T-cell therapy. TiRP tumors recruit and activate tumor-specific CD8+ T cells, but these cells then undergo apoptosis. This does not occur with isogenic transplanted tumors, which are rejected after adoptive T-cell therapy. Apoptosis of tumor-infiltrating lymphocytes can be prevented by interrupting the Fas/Fas-ligand axis, and is triggered by polymorphonuclear-myeloid-derived suppressor cells, which express high levels of Fas-ligand and are enriched in TiRP tumors. Blocking Fas-ligand increases the anti-tumor efficacy of adoptive T-cell therapy in TiRP tumors, and increases the efficacy of checkpoint blockade in transplanted tumors. Therefore, tumor-infiltrating lymphocytes apoptosis is a relevant mechanism of immunotherapy resistance, which could be blocked by interfering with the Fas/Fas-ligand pathway.


Subject(s)
Immunotherapy , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma, Experimental/immunology , Melanoma, Experimental/therapy , Animals , Apoptosis/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Fas Ligand Protein/antagonists & inhibitors , Fas Ligand Protein/genetics , Fas Ligand Protein/immunology , Female , Humans , Immunotherapy/methods , Immunotherapy, Adoptive , Lymphocytes, Tumor-Infiltrating/pathology , Male , Melanoma, Experimental/pathology , Mice , Mice, Transgenic , Tumor Microenvironment/immunology , fas Receptor/antagonists & inhibitors , fas Receptor/immunology
15.
Oncoimmunology ; 4(2): e974959, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25949872

ABSTRACT

Tumors with reduced expression of MHC class I (MHC-I) molecules may be unrecognized by tumor antigen-specific CD8+ T cells and thus constitute a challenge for cancer immunotherapy. Here we monitored development of autochthonous melanomas in TiRP mice that develop tumors expressing a known tumor antigen as well as a red fluorescent protein (RFP) reporter knock in gene. The latter permits non-invasive monitoring of tumor growth by biofluorescence. One developing melanoma was deficient in cell surface expression of MHC-I, but MHC-I expression could be rescued by exposure of these cells to IFNγ. We show that CD8+ T cells specific for tumor antigen/MHC-I were efficient at inducing regression of the MHC-I-deficient melanoma, provided that the T cells were endowed with properties permitting their migration into the tumor and their efficient production of IFNγ. This was the case for CD8+ T cells transfected to express an active form of STAT5 (STAT5CA). The amount of IFNγ produced ex vivo from T cells present in tumors after adoptive transfer of the CD8+ T cells was correlated with an increase in surface expression of MHC-I molecules by the tumor cells. We also show that these CD8+ T cells expressed PD-1 and upregulated its ligand PDL-1 on melanoma cells within the tumor. Despite upregulation of this immunosuppressive pathway, efficient IFNγ production in the melanoma microenvironment was found associated with resistance of STAT5CA-expressing CD8+ T cells to inhibition both by PD-1/PDL-1 engagement and by TGFß1, two main immune regulatory mechanisms hampering the efficiency of immunotherapy in patients.

16.
Ann N Y Acad Sci ; 975: 68-76, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12538155

ABSTRACT

When initial antigen encounter involves optimal antigenic and costimulatory stimuli, naïve CD8 T cells undergo a developmental program that leads to their activation, expansion and acquisition of effector functions (including production of IL-2, IFNgamma and expression of cytolytic effector molecules). A subset of the activated CD8 T cells thrives as long-lived memory cells. Encounter of tissue-associated, and in particular tumor-associated antigen, may often be suboptimal in terms of antigenicity and costimulation, however. We previously developed a model of naïve CD8 T cells from transgenic mice expressing an alloreactive TCR for which a mutant alloantigen behaved as a partial agonist, inducing only some of the effector functions induced by the native alloantigen. To ascertain the molecular bases for the establishment of divergent fates within the same naïve CD8 T cells, we have used cDNA microarrays to monitor sequential gene expression patterns in conditions of full or partial response of these naïve CD8 T cells. Of the 5000 different genes monitored on the array, 18% showed changes in expression in activated versus naïve CD8 T cells, independent of whether stimulation was with full or partial agonist. These included antigen-induced upregulated as well as downregulated genes. Clusters of genes that were differentially expressed were also identified, being either (i) weakly versus strongly, or (ii) transiently versus stably expressed in response to partial and full agonist, respectively. They included (i) genes encoding costimulatory molecules and (ii) genes controlling cytolytic function, cytokine production, and chemokines. Therefore, the cDNA microarray approach was a sensitive tool to provide an exhaustive picture of T cell activation as it could discriminate quantitative, qualitative and dynamic differences in mRNA expression profiles between fully or partially activated T cells.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Lymphocyte Activation/genetics , Animals , CD8-Positive T-Lymphocytes/metabolism , Cells, Cultured , Gene Expression Profiling , Isoantigens/genetics , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis/methods , Receptors, Antigen, T-Cell/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
17.
Oncoimmunology ; 3(10): e958951, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25941582

ABSTRACT

Mechanisms controlling immune reactivity prevent excessive inflammation and autoimmunity, but generally dampen antitumor activity. We recently showed that adoptively transferred antitumor CD8+ T cells harboring a deletion of A20/Tnfaip3, a molecule controlling NF-κB activation, possessed heightened antitumor activity in vivo. The boosted immunity of A20-deleted CD8+ T cells correlated with a heightened capacity to produce IFNγ and TNFα while expressing reduced levels of the immune checkpoint molecule PD-1.

18.
PLoS One ; 8(6): e67239, 2013.
Article in English | MEDLINE | ID: mdl-23840635

ABSTRACT

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells' plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo.


Subject(s)
Cell Degranulation , Granzymes/biosynthesis , Secretory Vesicles/metabolism , T-Lymphocytes, Cytotoxic/physiology , Animals , Cell Differentiation , Cells, Cultured , Gene Expression , Gene Knock-In Techniques , Granzymes/genetics , Killer Cells, Natural/metabolism , Kinetics , Listeriosis/immunology , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Lymph Nodes/immunology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Microscopy, Fluorescence , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Secretory Vesicles/enzymology , Single-Cell Analysis , Red Fluorescent Protein
19.
Pigment Cell Melanoma Res ; 26(2): 167-75, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23217139

ABSTRACT

Discovery of tumor antigen (TA) recognized by autologous T cells (TCs) in patients with melanoma has led to clinical protocols using either vaccination or adoptive transfer of TA-specific TCs. However, efficacy of these treatments has been hampered by inhibitory effects exerted on tumor-infiltrating TCs by tumor-intrinsic mediators or by recruitment of immunosuppressive cells. A mouse model of autochthonous melanoma recapitulates some aspects of inflammatory melanoma development in patients. These include a systemic Th2-/Th17-oriented chronic inflammation, recruitment of immunosuppressive myeloid cells and acquisition by tumor-infiltrating TCs of an 'exhausted' phenotype characterized by expression of multiple inhibitory receptors including programmed death-1, also expressed on patients' melanoma-infiltrating TCs. Rather than using extracellular blocking reagents to inhibitory surface molecules on TCs, we sought to dampen negative signaling exerted on them. Adoptively transferred TCs presenting increased cytokine receptor signaling due to expression of an active Stat5 transcription factor were efficient at inducing melanoma regression in the preclinical melanoma model. These transferred TCs thrived and retained expression of effector molecules in the melanoma microenvironment, defining a protocol endowing TCs with the ability to resist melanoma-induced immunosuppression.


Subject(s)
Adoptive Transfer , Immunosuppression Therapy , Inflammation/pathology , Melanoma/immunology , Melanoma/therapy , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Adaptive Immunity/immunology , Animals , Antigens, Neoplasm/immunology , Humans , Immunotherapy, Adoptive , Lymphocytes, Tumor-Infiltrating/metabolism , Melanoma/pathology , Mice , Skin Neoplasms/immunology , Skin Neoplasms/pathology , Skin Neoplasms/therapy
20.
Cancer Res ; 72(1): 76-87, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-22065720

ABSTRACT

Immunotherapy based on adoptive transfer of tumor antigen-specific CD8(+) T cell (TC) is generally limited by poor in vivo expansion and tumor infiltration. In this study, we report that activated STAT5 transcription factors (STAT5CA) confer high efficiency on CD8(+) effector T cells (eTC) for host colonization after adoptive transfer. Engineered expression of STAT5CA in antigen-experienced TCs with poor replicative potential was also sufficient to convert them into long-lived antigen-responsive eTCs. In transplanted mastocytoma- or melanoma-bearing hosts, STAT5CA greatly enhanced the ability of eTCs to accumulate in tumors, become activated by tumor antigens, and to express the cytolytic factor granzyme B. Taken together, these properties contributed to an increase in tumor regression by STAT5CA-transduced, as compared with untransduced, TCs including when the latter control cells were combined with infusion of interleukin (IL)-2/anti-IL-2 complexes. In tumors arising in the autochthonous TiRP transgenic model of melanoma associated with systemic chronic inflammation, endogenous CD8(+) TCs were nonfunctional. In this setting, adoptive transfer of STAT5CA-transduced TCs produced superior antitumor effects compared with nontransduced TCs. Our findings imply that STAT5CA expression can render TCs resistant to the immunosuppressive environment of melanoma tumors, enhancing their ability to home to tumors and to maintain high granzyme B expression, as well as their capacity to stimulate granzyme B expression in endogenous TCs.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Melanoma/physiopathology , STAT5 Transcription Factor/physiology , Adoptive Transfer , Animals , Flow Cytometry , Melanoma/pathology , Melanoma/therapy , Mice , Transplantation, Heterologous
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