The novel roles of DNA damage repair proteins ATM and DNA-PK in the regulation of the ER stress response and IL-23 expression in dendritic cells

Dendritic cells (DC) are the principal, cross-priming antigen-presenting cell responsible for naïve T cell activation and differentiation. Specific programmes of cytokine secretion by DC are particularly important for the polarisation of CD4+ T-cells to discrete effector populations i.e. Th1, Th17, Tr1 etc. Our knowledge of control for some of these cytokine signals is currently limited and modulating the polarisation of defined Th-subsets remains a challenge. Improved control of Th-differentiation has considerable potential in the fields of immune disorders and infectious disease. We describe a novel immune-regulatory function of two DNA-repair proteins signal to modulate IL-23 secretion, a key signal for Th17 differentiation. ATM and DNA-PK, are two protein kinases classically regarded for their paramount importance in repair of DNA double strand breaks. We characterised an alternate immune-regulatory function for ATM and DNA-PK for IL-23 production by DC. Therefore, constitutive expression of activated ATM and DNA-PK in DC, but not in CD14+ monocytes, related not only to their ability to repair damaged DNA, but to secrete IL-23. Increased IL-23 secretion was observed in activated human monocyte derived-DC (moDC), mouse bone marrow derived-DC and macrophages following inhibition of ATM and DNA-PK using the highly selective antagonists KU55399 and NU7441, respectively. In contrast, inhibiting ATR, a closely related single-strand DNA repair kinase, had no effect on IL-23. Interestingly, whilst inhibition of ATM only activated IL-23 secretion, inhibition of DNA-PK also led to increased IL-6, IL-1β and IL-10. IL-23 regulation through ATM/DNA-PK only occurred following toll-like receptor 4 (TLR4) activation and not with TLR1/2, 3, 5, 7/8, Dectin-1 and Dectin-1/TLR2 agonists. In support of a proposed immune-regulatory role for DNA-repair proteins, ionising radiation activated ATM and DNA-PK and consequently repressed IL-23. Radiation-dependent ATM/DNA-PK phosphorylation was prevented by KU55399 and NU7441, however only the ATM inhibitor restored IL-23 levels. To determine the mechanism of action we tested the hypotheses that increased IL-23 resulted from a) Altered activation of the Unfolded Protein Response (UPR) pathways, b) Increased IL-23 was IL-1β dependent, or c) Differential activation of NF-κB. These were not supported by experimental evidence as a) whilst inhibition of ATM/DNA-PK activated the UPR pathways they could not be shown to regulate IL-23 b) blocking IL-1β receptor with IL-1RA did not attenuate ATM/DNA-PK regulated IL-23. c) Evidence for prolonged activation of NF-κB following DNA-PK inhibition was obtained, however its role remained unclear. A role for the CREB transcription factor was established for ATM-dependent IL-23 regulation. Activated ATM phosphorylated CREB at Ser121, a residue associated with negative CREB activity and consequently ATM inhibition enhanced CREB activity in a luciferase reporter assay. The addition of an inhibitor of the interaction of CREB with its transcriptional co-activator, CBP, abolished ATM-enhanced IL-23 response. Control of IL-23 dependent immune responses is increasingly important in autoimmunity and cancer. This study identifies a novel immune-regulatory role for DNA-repair systems in DC resulting in the regulation of IL-23 and other Th17 associated cytokines (IL-1, IL-6). Further studies could explore the potential of either small molecule inhibitors of ATM/DNA-PK to enhance, or stress signals (e.g. radiation) to repress IL-23 responses

T cell repertoire to citrullinated self-peptides in healthy humans is not confined to the HLA-DR SE alleles; targeting of citrullinated self-peptides presented by HLA-DP4 for tumour therapy

Post-translational modifications are induced in stressed cells which cause them to be recognised by the immune system. One such modification is citrullination where the positive charged arginine is modified to a neutral citrulline. We demonstrate most healthy donors show an oligoclonal CD4 response in vitro to at least one citrullinated vimentin or enolase peptide. Unlike rheumatoid arthritis patients, these T cell responses were not restricted by HLA-DRB1 shared epitope (SE) alleles, suggesting they could be presented by other MHC-II alleles. As HLA-DP is less polymorphic than HLA-DR, we investigated whether the common allele, HLA-DP4 could present citrullinated epitopes. The modification of arginine to citrulline enhanced binding of the peptides to HLA-DP4 and induced high frequency CD4 responses in HLA-DP4 transgenic mouse models. Our previous studies have shown that tumours present citrullinated peptides restricted through HLA-DR4 which are good targets for anti-tumour immunity. In this study we show that citrullinated vimentin and enolase peptides also induced strong anti-tumour immunity (100% survival,

Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy

IntroductionPost translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy.MethodsA citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model.ResultsWe show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide.ConclusionWe propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach

Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation

Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-0/IL-113 from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk over¬comes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radia¬tion (1–6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-10, -6, -10, and -27 or TNF-a and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Impor¬tantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-0/IL-10 and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would other¬wise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity. J. Leukoc. Biol. 100: 000–000; 2016

The novel roles of DNA damage repair proteins ATM and DNA-PK in the regulation of the ER stress response and IL-23 expression in dendritic cells

Dendritic cells (DC) are the principal, cross-priming antigen-presenting cell responsible for naïve T cell activation and differentiation. Specific programmes of cytokine secretion by DC are particularly important for the polarisation of CD4+ T-cells to discrete effector populations i.e. Th1, Th17, Tr1 etc. Our knowledge of control for some of these cytokine signals is currently limited and modulating the polarisation of defined Th-subsets remains a challenge. Improved control of Th-differentiation has considerable potential in the fields of immune disorders and infectious disease. We describe a novel immune-regulatory function of two DNA-repair proteins signal to modulate IL-23 secretion, a key signal for Th17 differentiation. ATM and DNA-PK, are two protein kinases classically regarded for their paramount importance in repair of DNA double strand breaks. We characterised an alternate immune-regulatory function for ATM and DNA-PK for IL-23 production by DC. Therefore, constitutive expression of activated ATM and DNA-PK in DC, but not in CD14+ monocytes, related not only to their ability to repair damaged DNA, but to secrete IL-23. Increased IL-23 secretion was observed in activated human monocyte derived-DC (moDC), mouse bone marrow derived-DC and macrophages following inhibition of ATM and DNA-PK using the highly selective antagonists KU55399 and NU7441, respectively. In contrast, inhibiting ATR, a closely related single-strand DNA repair kinase, had no effect on IL-23. Interestingly, whilst inhibition of ATM only activated IL-23 secretion, inhibition of DNA-PK also led to increased IL-6, IL-1β and IL-10. IL-23 regulation through ATM/DNA-PK only occurred following toll-like receptor 4 (TLR4) activation and not with TLR1/2, 3, 5, 7/8, Dectin-1 and Dectin-1/TLR2 agonists. In support of a proposed immune-regulatory role for DNA-repair proteins, ionising radiation activated ATM and DNA-PK and consequently repressed IL-23. Radiation-dependent ATM/DNA-PK phosphorylation was prevented by KU55399 and NU7441, however only the ATM inhibitor restored IL-23 levels. To determine the mechanism of action we tested the hypotheses that increased IL-23 resulted from a) Altered activation of the Unfolded Protein Response (UPR) pathways, b) Increased IL-23 was IL-1β dependent, or c) Differential activation of NF-κB. These were not supported by experimental evidence as a) whilst inhibition of ATM/DNA-PK activated the UPR pathways they could not be shown to regulate IL-23 b) blocking IL-1β receptor with IL-1RA did not attenuate ATM/DNA-PK regulated IL-23. c) Evidence for prolonged activation of NF-κB following DNA-PK inhibition was obtained, however its role remained unclear. A role for the CREB transcription factor was established for ATM-dependent IL-23 regulation. Activated ATM phosphorylated CREB at Ser121, a residue associated with negative CREB activity and consequently ATM inhibition enhanced CREB activity in a luciferase reporter assay. The addition of an inhibitor of the interaction of CREB with its transcriptional co-activator, CBP, abolished ATM-enhanced IL-23 response. Control of IL-23 dependent immune responses is increasingly important in autoimmunity and cancer. This study identifies a novel immune-regulatory role for DNA-repair systems in DC resulting in the regulation of IL-23 and other Th17 associated cytokines (IL-1, IL-6). Further studies could explore the potential of either small molecule inhibitors of ATM/DNA-PK to enhance, or stress signals (e.g. radiation) to repress IL-23 responses

DataSheet_1_Citrullinated glucose-regulated protein 78 is a candidate target for melanoma immunotherapy.docx

IntroductionPost translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy.MethodsA citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model.ResultsWe show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (pConclusionWe propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.</p