10 research outputs found
Intratumoral Immunotherapy with XCL1 and sFlt3L Encoded in Recombinant Semliki Forest Virus-Derived Vectors Fosters Dendritic Cell-Mediated T-cell Cross-Priming
: Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38- and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8β T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNAR-deficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltrating CD8 T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. SIGNIFICANCE: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates cross-priming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion.We are in debt to Eneko Elizalde for excellent animal facility care. Critical reading and suggestions by Drs. Sandra Hervas, Carmen Ochoa, Jose L. Perez-Gracia, Ana Rouzaut, and Juan Jose Lasarte are also much appreciated. This work was supported by grants MINECO SAF 2014-52361-R and SAF 2017-83267-C2-1R and Cancer Research Institute (CRI) CLIP Grant 2017 and the Horizon 2020 Programme from the European Comission (PROCROP project, ref. #635122) to I. Melero; FIS (PI14/01442 and PI17/01859 (to C. Smerdou); and Fundacion Echebano fellowship (to M.C. Ballesteros-Briones).S
Deubiquitinases A20 and CYLD modulate costimulatory signaling via CD137 (4-1BB)
TRAF2 dependent K63-polyubiquitinations have been recently shown to connect CD137 (4–1BB) stimulation to NF-κB activation. In a search of deubiquitinase enzymes (DUBs) that could regulate such a signaling route, A20 and CYLD were found to coimmunoprecipitate with CD137 and TRAF2 complexes. Indeed, overexpression of A20 or CYLD downregulated CD137-elicited ubiquitination of TRAF2 and TAK1 upon stimulation with agonist monoclonal antibodies. Moreover, overexpression of A20 or CYLD downregulated CD137-induced NF-κB activation in cultured cells and in gene-transferred hepatocytes in vivo, while silencing these deubiquitinases enhanced CD137 costimulation of primary human CD8 T cells. Therefore A20 and CYLD directly downregulate the signaling from a T and NK-cell costimulatory receptor under exploitation for cancer immunotherapy in clinical trials.MICINN (SAF2011-22831 and SAF2014-52361-R) (I.M), Departamento de Salud del Gobierno de Navarra, Redes temáticas de investigación cooperativa RETICC, European Commission VII Framework and Horizon 2020 programs (AICR and PROCROP), Fundación de la Asociación Española Contra el Cáncer (AECC), Fundación BBVA. MER-R receives support for a Rio Ortega Contract.Peer reviewe
Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death
Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a
fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role
of TNF in AICD of CD8 T lymphocytes.
Methods: Human peripheral mononuclear cells were isolated from healthy donors
and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were
activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinicalgrade TNF blocking agents.
Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially
prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also
observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The
mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals
which damage DNA.
Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and
the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in
human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in
immunotherapy strategies
Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death
Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a
fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role
of TNF in AICD of CD8 T lymphocytes.
Methods: Human peripheral mononuclear cells were isolated from healthy donors
and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were
activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinicalgrade TNF blocking agents.
Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially
prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also
observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The
mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals
which damage DNA.
Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and
the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in
human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in
immunotherapy strategies
Repurposing the yellow fever vaccine for intratumoral immunotherapy
Live 17D is widely used as a prophylactic vaccine strain for yellow
fever virus that induces potent neutralizing humoral and cellular
immunity against the wild-type pathogen. 17D replicates and kills
mouse and human tumor cell lines but not non-transformed
human cells. Intratumoral injections with viable 17D markedly
delay transplanted tumor progression in a CD8 T-cell-dependent
manner. In mice bearing bilateral tumors in which only one is
intratumorally injected, contralateral therapeutic effects are
observed consistent with more prominent CD8 T-cell infiltrates
and a treatment-related reduction of Tregs. Additive efficacy
effects were observed upon co-treatment with intratumoral 17D
and systemic anti-CD137 and anti-PD-1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized
with 17D, intratumoral 17D treatment achieved better local and
distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8
T-cell infiltration in the treated tumor. The repurposed use of a
GMP-grade vaccine to be given via the intratumoral route in
prevaccinated patients constitutes a clinically feasible and safe
immunotherapy approach
Cellular cytotoxicity is a form of immunogenic cell death
Background The immune response to cancer is often
conceptualized with the cancer immunity cycle. An essential
step in this interpretation is that antigens released by dying
tumors are presented by dendritic cells to naive or memory
T cells in the tumor-draining lymph nodes. Whether tumor
cell death resulting from cytotoxicity, as mediated by T cells
or natural killer (NK) lymphocytes, is actually immunogenic
currently remains unknown.
Methods In this study, tumor cells were killed by antigenspecific T-cell receptor (TCR) transgenic CD8 T cells or
activated NK cells. Immunogenic cell death was studied
analyzing the membrane exposure of calreticulin and the
release of high mobility group box 1 (HMGB1) by the dying
tumor cells. Furthermore, the potential immunogenicity of the
tumor cell debris was evaluated in immunocompetent mice
challenged with an unrelated tumor sharing only one tumorassociated antigen and by class I major histocompatibility
complex (MHC)-multimer stainings. Mice deficient in
Batf3, Ifnar1 and Sting1 were used to study mechanistic
requirements.
Results We observe in cocultures of tumor cells and effector
cytotoxic cells, the presence of markers of immunogenic
cell death such as calreticulin exposure and soluble HMGB1
protein. Ovalbumin (OVA)-transfected MC38 colon cancer
cells, exogenously pulsed to present the gp100 epitope are
killed in culture by mouse gp100-specific TCR transgenic
CD8 T cells. Immunization of mice with the resulting
destroyed cells induces epitope spreading as observed by
detection of OVA-specific T cells by MHC multimer staining
and rejection of OVA+
EG7 lymphoma cells. Similar results
were observed in mice immunized with cell debris generated
by NK-cell mediated cytotoxicity. Mice deficient in Batf3-
dependent dendritic cells (conventional dendritic cells type 1,
cDC1) fail to develop an anti-OVA response when immunized
with tumor cells killed by cytotoxic lymphocytes. In line with
this, cultured cDC1 dendritic cells uptake and can readily
cross-present antigen from cytotoxicity-killed tumor cells to
cognate CD8+
T lymphocytes.
Conclusion These results support that an ongoing
cytotoxic antitumor immune response can lead to
immunogenic tumor cell death
Repurposing the yellow fever vaccine for intratumoral immunotherapy
Live 17D is widely used as a prophylactic vaccine strain for yellow
fever virus that induces potent neutralizing humoral and cellular
immunity against the wild-type pathogen. 17D replicates and kills
mouse and human tumor cell lines but not non-transformed
human cells. Intratumoral injections with viable 17D markedly
delay transplanted tumor progression in a CD8 T-cell-dependent
manner. In mice bearing bilateral tumors in which only one is
intratumorally injected, contralateral therapeutic effects are
observed consistent with more prominent CD8 T-cell infiltrates
and a treatment-related reduction of Tregs. Additive efficacy
effects were observed upon co-treatment with intratumoral 17D
and systemic anti-CD137 and anti-PD-1 immunostimulatory monoclonal antibodies. Importantly, when mice were preimmunized
with 17D, intratumoral 17D treatment achieved better local and
distant antitumor immunity. Such beneficial effects of prevaccination are in part explained by the potentiation of CD4 and CD8
T-cell infiltration in the treated tumor. The repurposed use of a
GMP-grade vaccine to be given via the intratumoral route in
prevaccinated patients constitutes a clinically feasible and safe
immunotherapy approach
Nivolumab and urelumab enhance antitumor activity of human T lymphocytes engrafted in Rag2-/-IL2Rγnull immunodeficient mice
A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations
TGFβ Blockade Enhances Radiotherapy Abscopal Efficacy Effects in Combination with Anti-PD1 and Anti-CD137 Immunostimulatory Monoclonal Antibodies
CXCR1 and CXCR2 chemokine receptor agonists produced by tumors induce neutrophil extracellular traps that interfere with immune cytotoxicity
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2
chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular
traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+
T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target
cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the
immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic
cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells