In vivo evidence that secretion of HLA-G by immunogenic tumor cells allows their evasion from immunosurveillance
Laure Loumagne1,2, Jeremy Baudhuin1,2, Benoit Favier1,2, Florent Montespan1,2, Edgardo D. Carosella1,2, Nathalie Rouas-Freiss1,2
1
CEA, IMETI, Service de Recherches en Hemato-Immunologie (SRHI), Paris, France.
2
UMR_E5, Universite Paris-Diderot-Paris-7, Institut Universitaire d’Hematologie (IUH),
Hopital Saint-Louis, Paris, France.
Short title: Soluble HLA-G mediates tumor escape in vivo
Corresponding author: Nathalie Rouas-Freiss. [email protected] CEA, SRHI, Hopital Saint-Louis, 1, av. Claude Vellefaux, 75010 Paris. Fax: +33157276780.
Disclosure of Potential Conflicts of Interest: There are no potential conflicts of interest to disclose.
Keywords: HLA-G; immunotolerance; myeloid-derived suppressor cells; tumor escape.
Abbreviations: Ab: antibody BM: bone marrow DC: dendritic cell FFPE: formalin fixed, paraffin-embedded
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ijc.28845
International Journal of Cancer
Page 2 of 29
hβ2m: human β2-microglubulin HLA-G: human leukocyte antigen-G IHC: immunohistochemistry ILT: immunoglobulin-like transcript mAb: monoclonal antibody MDSC: myeloid-derived suppressor cells NK: natural killer P1, 2 or 3: phase 1, 2 or 3 PB: peripheral blood PI: post-injection PIR: paired immunoglobulin-like receptor ROS: reactive oxygen species Treg: regulatory T cells TB: tumor bearing TDLN: tumor-draining lymph node
Article category: Tumor Immunology
Novelty impact statement: This is the first in vivo study demonstrating that tolerogenic soluble HLA-G enables immunogenic tumors to escape from immunosurveillance. Impairment of antibody response, defect in T and B cell proportions, function and tumor infiltration as well as MDSC expansion constitute the main mechanisms by which soluble HLA-G bypasses tumor rejection. Owing resistance of certain cancers to current drugs, HLA-G should be considered as a promising target to optimize immunotherapy by blocking its expression or function.
2
John Wiley & Sons, Inc.
Page 3 of 29
International Journal of Cancer
ABSTRACT Human Leukocyte Antigen-G (HLA-G) expression by tumors has been evidenced in numerous malignancies in association with poor prognosis and resistance to immunotherapy in humans. Particularly, soluble form of HLA-G was measured at high concentrations in malignant effusions and plasma from cancer patients and inhibits anti-tumor immune cells in vitro through interaction with immunoglobulin-like transcript (ILT) receptors. Nevertheless, in vivo study demonstrating that HLA-G secretion by tumor cells allows their escape from immunosurveillance remained to be established. Despite non-described murine homolog, direct functional interaction of HLA-G with murine paired immunoglobulin-like receptor (PIR)-B, ortholog of human ILT receptors, enables to investigate its role in vivo. Immunocompetent mice were injected either with syngeneic tumor cells co-expressing HLAG5, the main soluble HLA-G isoform, and the conformation stabilizer human β2microglubulin (hβ2m), or with hβ2m+HLA-G5- tumor cells. hβ2m expressed at both tumor cell surface acted as a tumor antigen triggering a specific humoral response. Interestingly, while hβ2m+HLA-G5- tumors were rejected, secreted HLA-G5 provided hβ2m+HLA-G5+ tumors a protection against hβ2m-elicited immune rejection, enabling such immunogenic tumors to grow similarly to a poorly immunogenic tumor. HLA-G5 tumor expression was associated with local and peripheral immunosuppression, characterized by dampened antihβ2m B cell response, quantitative and functional T and B cell defects, accumulation of myeloid-derived suppressor cells able to inhibit T cell proliferation, and reduced T and B cell tumor-infiltrate. Our study provides the first in vivo proof that soluble HLA-G counteracts tumor rejection and reinforces the importance to consider HLA-G as a promising target to optimize current cancer immunotherapies.
3
John Wiley & Sons, Inc.
International Journal of Cancer
Page 4 of 29
INTRODUCTION
It is now well accepted that both innate and adaptive immune systems protect the host against the development of cancer 1. However, tumor cells circumvent such immunosurveillance through the outgrowth of poorly immunogenic variants and subversion of the immune system by multiple immunosuppressive mechanisms such as inducible expression of the tolerogenic HLA-G molecule 2-5. HLA-G is a non-classical MHC-I molecule which can be expressed as seven isoforms owing to alternative splicing of the primary transcript 3. Among them, the most studied are membrane-bound HLA-G1 and soluble HLA-G5, both associated to β2m, necessary for their stable conformation 6. In healthy individuals, HLA-G tissue distribution is restricted but can be induced under pathological conditions such as organ transplantation, malignant transformation, viral infection, inflammatory and autoimmune diseases 3. HLA-G expression has been described in more than one thousand malignant lesions from various origins using antibodies recognizing both membrane-bound and soluble forms 3. In addition, increased concentrations of soluble HLA-G isoforms have been detected in plasma
7
and malignant
effusions 8, 9 from cancer patients. Its expression was also associated with poor prognosis and resistance to immunotherapy in cancer patients 3, 7. In vitro studies demonstrated that soluble HLA-G inhibits anti-tumor activity of natural killer (NK) 10, αβ 11, 12 and γδ T cells 13 as well as the maturation, differentiation and function of dendritic (DC)
14, 15
and B cells
16, 17
, by
interacting with ILT2 and ILT4 inhibitory receptors on their surface 3. Furthermore, immunosuppressive properties of soluble HLA-G purified from plasma of cancer patients have been demonstrated in vitro
18, 19
. Nevertheless, the contribution of soluble HLA-G
secretion to tumor evasion in vivo remained to be established, especially because systemic effects are expected 12, 18, 19.
4
John Wiley & Sons, Inc.
Page 5 of 29
International Journal of Cancer
Despite non-described murine homolog of HLA-G, investigating its role in vivo is made possible through direct functional interaction with murine PIR-B, ortholog of human ILT
20,
21
. PIR-B is expressed on DC, B lymphocytes, mast cells, macrophages and granulocytes and
binds MHC-I molecules
21
. Its engagement through cross-linking, or by using HLA-G
tetramers or HLA-G transgenic mice resulted in the (i) impairment of DC maturation and function, (ii) inhibition of NK and T cell cytotoxicity, and (iii) prolongation of allogeneic graft survival 20, 22, 23. This HLA-G/PIR-B interaction mediated comparable effects than those described in vitro with human cell lines 14, 15 or in clinical investigations 3, thereby supporting the relevance of HLA-G studies in mouse. To date only three in vivo studies support the pro-tumoral role of HLA-G1 membrane isoform
expressed
by
human
xenotumors
injected
in
immunocompetent
24
or
immunodeficient mice 25, 26. Employing a syngeneic tumor model in immunocompetent mice, we here demonstrate that the HLA-G5 secretion by immunogenic tumor cells enables them to escape from immune response and to grow similarly to a poorly immunogenic tumor through inhibition of B and T cells and expansion of myeloid derived suppressor cells (MDSC).
5
John Wiley & Sons, Inc.
International Journal of Cancer
Page 6 of 29
MATERIAL AND METHODS
Detailed material and methods are provided in Supplementary material.
Lentiviral construction HLA-G5 cDNA was cloned into pWPXL vector after PCR amplification using specific forward
(5’-AAAACGCGTACCATGGTGGTCATGGCGCCC-3’)
and
reverse
(5’-
AAAACTAGTTTAAAGGTCTTCAGAGAGGC-3’) primers. hβ2m-containing pWPXL vector and lentiviral particles were generated as previously described 13.
Cell lines Murine mammary 4T1 cell line was obtained from ATCC. hβ2m-deficient melanoma cell line FON- was previously described 4. Cell lines expressing hβ2m (i.e., 4T1-hβ2m and FON-hβ2m cells) or hβ2m plus HLA-G5 (i.e., 4T1-hβ2m-HLA-G5 cells) were obtained by lentiviral transduction as previously described 13. All cell lines were grown in complete RPMI (see Supplementary material).
Flow cytometry analysis Leukocyte and tumor cell stainings were carried out as previously described
8, 10, 24
. For
intracellular leukocyte staining, acquisition informations and antibodies (Abs) used in flow cytometry refer to Supplementary material.
Immunoprecipitation and Western blotting The HLA-G5 protein was detected following immunoprecipitation and SDS-PAGE analysis, as described in Supplementary material.
6
John Wiley & Sons, Inc.
Page 7 of 29
International Journal of Cancer
ELISA 0.5x106 tumor cells were cultured in 6-well plates in 2 ml of complete RPMI for 4 days. Supernatants were collected, centrifuged and stored at -20°C. Levels of hβ2m-associatedsoluble HLA-G were determined by MEM-G/9 ELISA as previously described 17.
Subcutaneous tumor model Female Balb/c mice (6-10 weeks old) were obtained from Janvier. All experimental protocols were approved by the ethics review committee for animal experimentation of Hopital SaintLouis (Paris, France) and followed the guiding principles for the care and use of animals approved by our local committee. At day 0, 5x106 early passaged tumor cells resuspended in 200 µl of PBS 1X were injected subcutaneously in mice flank (3-5 mice per group). Tumor volume was monitored at indicated time points as previously described 24.
Plasma Ig detection Plasma were diluted (1/10) in PBS 1X, 0.1% BSA and incubated with target cells for 30 min at 4°C. Mouse IgG2a was used as control. Ig binding was then detected using goat antimouse (GAM)-PE Ab followed by flow cytometry analysis.
Preparation of leukocyte suspensions Leukocytes from spleen, tumor-draining lymph node (TDLN), peripheral blood (PB) and bone marrow (BM) were obtained as described in Supplementary material.
7
John Wiley & Sons, Inc.
International Journal of Cancer
Page 8 of 29
Proliferation assays Refer to Supplementary material for details concerning splenocyte proliferation under antiCD3/CD28 stimulation and MDSC immunosuppression assay.
T cells sorting, stimulation and cytotoxicity assay Splenic T cells were sorted using the EasySep mouse T cell enrichment kit following the fully automated protocol for Robosep (Stem Cell), stimulated for 3 days in anti-CD3/CD28coated wells and evaluated for their cytotoxic potential towards 4T1-hβ2m target cells using flow cytometry. Refer to Supplementary material for further details.
Ly6G+ cell sorting In mice, MDSC are a heterogeneous population of immature myeloid cells broadly defined as Gr-1 (Ly6G/Ly6C)+ CD11b+ cells 27. These cells are divided into 2 subsets, granulocytic (GMDSC, CD11b+ Ly6G+) and monocytic (M-MDSC, CD11b+ Ly6G- Ly6Chigh) MDSC. 21 days post-injection (PI), we isolated G-MDSC from splenocytes by magnetic bead sorting using Ly6G Ab and MS columns (Miltenyi Biotec) according to manufacturer’s instructions.
Immunohistochemistry Formaldehyde-fixed, paraffin-embedded (FFPE) tumor sections were stained with anti-Gr-1, -B220, -CD3 and -CD31 Abs by immunohistochemistry (IHC). Further information is provided in Supplementary material.
Statistical analyses Analyses were performed using Graph-Pad Prism (version 5.01). Except when specified, twotailed Mann-Whitney test was performed, p values
Laure Loumagne1,2, Jeremy Baudhuin1,2, Benoit Favier1,2, Florent Montespan1,2, Edgardo D. Carosella1,2, Nathalie Rouas-Freiss1,2
1
CEA, IMETI, Service de Recherches en Hemato-Immunologie (SRHI), Paris, France.
2
UMR_E5, Universite Paris-Diderot-Paris-7, Institut Universitaire d’Hematologie (IUH),
Hopital Saint-Louis, Paris, France.
Short title: Soluble HLA-G mediates tumor escape in vivo
Corresponding author: Nathalie Rouas-Freiss. [email protected] CEA, SRHI, Hopital Saint-Louis, 1, av. Claude Vellefaux, 75010 Paris. Fax: +33157276780.
Disclosure of Potential Conflicts of Interest: There are no potential conflicts of interest to disclose.
Keywords: HLA-G; immunotolerance; myeloid-derived suppressor cells; tumor escape.
Abbreviations: Ab: antibody BM: bone marrow DC: dendritic cell FFPE: formalin fixed, paraffin-embedded
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ijc.28845
International Journal of Cancer
Page 2 of 29
hβ2m: human β2-microglubulin HLA-G: human leukocyte antigen-G IHC: immunohistochemistry ILT: immunoglobulin-like transcript mAb: monoclonal antibody MDSC: myeloid-derived suppressor cells NK: natural killer P1, 2 or 3: phase 1, 2 or 3 PB: peripheral blood PI: post-injection PIR: paired immunoglobulin-like receptor ROS: reactive oxygen species Treg: regulatory T cells TB: tumor bearing TDLN: tumor-draining lymph node
Article category: Tumor Immunology
Novelty impact statement: This is the first in vivo study demonstrating that tolerogenic soluble HLA-G enables immunogenic tumors to escape from immunosurveillance. Impairment of antibody response, defect in T and B cell proportions, function and tumor infiltration as well as MDSC expansion constitute the main mechanisms by which soluble HLA-G bypasses tumor rejection. Owing resistance of certain cancers to current drugs, HLA-G should be considered as a promising target to optimize immunotherapy by blocking its expression or function.
2
John Wiley & Sons, Inc.
Page 3 of 29
International Journal of Cancer
ABSTRACT Human Leukocyte Antigen-G (HLA-G) expression by tumors has been evidenced in numerous malignancies in association with poor prognosis and resistance to immunotherapy in humans. Particularly, soluble form of HLA-G was measured at high concentrations in malignant effusions and plasma from cancer patients and inhibits anti-tumor immune cells in vitro through interaction with immunoglobulin-like transcript (ILT) receptors. Nevertheless, in vivo study demonstrating that HLA-G secretion by tumor cells allows their escape from immunosurveillance remained to be established. Despite non-described murine homolog, direct functional interaction of HLA-G with murine paired immunoglobulin-like receptor (PIR)-B, ortholog of human ILT receptors, enables to investigate its role in vivo. Immunocompetent mice were injected either with syngeneic tumor cells co-expressing HLAG5, the main soluble HLA-G isoform, and the conformation stabilizer human β2microglubulin (hβ2m), or with hβ2m+HLA-G5- tumor cells. hβ2m expressed at both tumor cell surface acted as a tumor antigen triggering a specific humoral response. Interestingly, while hβ2m+HLA-G5- tumors were rejected, secreted HLA-G5 provided hβ2m+HLA-G5+ tumors a protection against hβ2m-elicited immune rejection, enabling such immunogenic tumors to grow similarly to a poorly immunogenic tumor. HLA-G5 tumor expression was associated with local and peripheral immunosuppression, characterized by dampened antihβ2m B cell response, quantitative and functional T and B cell defects, accumulation of myeloid-derived suppressor cells able to inhibit T cell proliferation, and reduced T and B cell tumor-infiltrate. Our study provides the first in vivo proof that soluble HLA-G counteracts tumor rejection and reinforces the importance to consider HLA-G as a promising target to optimize current cancer immunotherapies.
3
John Wiley & Sons, Inc.
International Journal of Cancer
Page 4 of 29
INTRODUCTION
It is now well accepted that both innate and adaptive immune systems protect the host against the development of cancer 1. However, tumor cells circumvent such immunosurveillance through the outgrowth of poorly immunogenic variants and subversion of the immune system by multiple immunosuppressive mechanisms such as inducible expression of the tolerogenic HLA-G molecule 2-5. HLA-G is a non-classical MHC-I molecule which can be expressed as seven isoforms owing to alternative splicing of the primary transcript 3. Among them, the most studied are membrane-bound HLA-G1 and soluble HLA-G5, both associated to β2m, necessary for their stable conformation 6. In healthy individuals, HLA-G tissue distribution is restricted but can be induced under pathological conditions such as organ transplantation, malignant transformation, viral infection, inflammatory and autoimmune diseases 3. HLA-G expression has been described in more than one thousand malignant lesions from various origins using antibodies recognizing both membrane-bound and soluble forms 3. In addition, increased concentrations of soluble HLA-G isoforms have been detected in plasma
7
and malignant
effusions 8, 9 from cancer patients. Its expression was also associated with poor prognosis and resistance to immunotherapy in cancer patients 3, 7. In vitro studies demonstrated that soluble HLA-G inhibits anti-tumor activity of natural killer (NK) 10, αβ 11, 12 and γδ T cells 13 as well as the maturation, differentiation and function of dendritic (DC)
14, 15
and B cells
16, 17
, by
interacting with ILT2 and ILT4 inhibitory receptors on their surface 3. Furthermore, immunosuppressive properties of soluble HLA-G purified from plasma of cancer patients have been demonstrated in vitro
18, 19
. Nevertheless, the contribution of soluble HLA-G
secretion to tumor evasion in vivo remained to be established, especially because systemic effects are expected 12, 18, 19.
4
John Wiley & Sons, Inc.
Page 5 of 29
International Journal of Cancer
Despite non-described murine homolog of HLA-G, investigating its role in vivo is made possible through direct functional interaction with murine PIR-B, ortholog of human ILT
20,
21
. PIR-B is expressed on DC, B lymphocytes, mast cells, macrophages and granulocytes and
binds MHC-I molecules
21
. Its engagement through cross-linking, or by using HLA-G
tetramers or HLA-G transgenic mice resulted in the (i) impairment of DC maturation and function, (ii) inhibition of NK and T cell cytotoxicity, and (iii) prolongation of allogeneic graft survival 20, 22, 23. This HLA-G/PIR-B interaction mediated comparable effects than those described in vitro with human cell lines 14, 15 or in clinical investigations 3, thereby supporting the relevance of HLA-G studies in mouse. To date only three in vivo studies support the pro-tumoral role of HLA-G1 membrane isoform
expressed
by
human
xenotumors
injected
in
immunocompetent
24
or
immunodeficient mice 25, 26. Employing a syngeneic tumor model in immunocompetent mice, we here demonstrate that the HLA-G5 secretion by immunogenic tumor cells enables them to escape from immune response and to grow similarly to a poorly immunogenic tumor through inhibition of B and T cells and expansion of myeloid derived suppressor cells (MDSC).
5
John Wiley & Sons, Inc.
International Journal of Cancer
Page 6 of 29
MATERIAL AND METHODS
Detailed material and methods are provided in Supplementary material.
Lentiviral construction HLA-G5 cDNA was cloned into pWPXL vector after PCR amplification using specific forward
(5’-AAAACGCGTACCATGGTGGTCATGGCGCCC-3’)
and
reverse
(5’-
AAAACTAGTTTAAAGGTCTTCAGAGAGGC-3’) primers. hβ2m-containing pWPXL vector and lentiviral particles were generated as previously described 13.
Cell lines Murine mammary 4T1 cell line was obtained from ATCC. hβ2m-deficient melanoma cell line FON- was previously described 4. Cell lines expressing hβ2m (i.e., 4T1-hβ2m and FON-hβ2m cells) or hβ2m plus HLA-G5 (i.e., 4T1-hβ2m-HLA-G5 cells) were obtained by lentiviral transduction as previously described 13. All cell lines were grown in complete RPMI (see Supplementary material).
Flow cytometry analysis Leukocyte and tumor cell stainings were carried out as previously described
8, 10, 24
. For
intracellular leukocyte staining, acquisition informations and antibodies (Abs) used in flow cytometry refer to Supplementary material.
Immunoprecipitation and Western blotting The HLA-G5 protein was detected following immunoprecipitation and SDS-PAGE analysis, as described in Supplementary material.
6
John Wiley & Sons, Inc.
Page 7 of 29
International Journal of Cancer
ELISA 0.5x106 tumor cells were cultured in 6-well plates in 2 ml of complete RPMI for 4 days. Supernatants were collected, centrifuged and stored at -20°C. Levels of hβ2m-associatedsoluble HLA-G were determined by MEM-G/9 ELISA as previously described 17.
Subcutaneous tumor model Female Balb/c mice (6-10 weeks old) were obtained from Janvier. All experimental protocols were approved by the ethics review committee for animal experimentation of Hopital SaintLouis (Paris, France) and followed the guiding principles for the care and use of animals approved by our local committee. At day 0, 5x106 early passaged tumor cells resuspended in 200 µl of PBS 1X were injected subcutaneously in mice flank (3-5 mice per group). Tumor volume was monitored at indicated time points as previously described 24.
Plasma Ig detection Plasma were diluted (1/10) in PBS 1X, 0.1% BSA and incubated with target cells for 30 min at 4°C. Mouse IgG2a was used as control. Ig binding was then detected using goat antimouse (GAM)-PE Ab followed by flow cytometry analysis.
Preparation of leukocyte suspensions Leukocytes from spleen, tumor-draining lymph node (TDLN), peripheral blood (PB) and bone marrow (BM) were obtained as described in Supplementary material.
7
John Wiley & Sons, Inc.
International Journal of Cancer
Page 8 of 29
Proliferation assays Refer to Supplementary material for details concerning splenocyte proliferation under antiCD3/CD28 stimulation and MDSC immunosuppression assay.
T cells sorting, stimulation and cytotoxicity assay Splenic T cells were sorted using the EasySep mouse T cell enrichment kit following the fully automated protocol for Robosep (Stem Cell), stimulated for 3 days in anti-CD3/CD28coated wells and evaluated for their cytotoxic potential towards 4T1-hβ2m target cells using flow cytometry. Refer to Supplementary material for further details.
Ly6G+ cell sorting In mice, MDSC are a heterogeneous population of immature myeloid cells broadly defined as Gr-1 (Ly6G/Ly6C)+ CD11b+ cells 27. These cells are divided into 2 subsets, granulocytic (GMDSC, CD11b+ Ly6G+) and monocytic (M-MDSC, CD11b+ Ly6G- Ly6Chigh) MDSC. 21 days post-injection (PI), we isolated G-MDSC from splenocytes by magnetic bead sorting using Ly6G Ab and MS columns (Miltenyi Biotec) according to manufacturer’s instructions.
Immunohistochemistry Formaldehyde-fixed, paraffin-embedded (FFPE) tumor sections were stained with anti-Gr-1, -B220, -CD3 and -CD31 Abs by immunohistochemistry (IHC). Further information is provided in Supplementary material.
Statistical analyses Analyses were performed using Graph-Pad Prism (version 5.01). Except when specified, twotailed Mann-Whitney test was performed, p values
