BackgroundAdoptive cell therapy (ACT) utilizing tumor infiltrating lymphocytes (TIL) has demonstrated durable responses in patients with metastatic melanoma and offers potential for other solid ...tumors. Preclinical experience with expanded TIL from soft tissue sarcoma (STS) demonstrates less frequent tumor-specific reactivity compared to melanoma samples, limiting the potential for efficacy.1 We hypothesized that CD69+ TIL have increased tumor-specific reactivity, which can be manipulated in culture, thereby offering an opportunity to enhance the antitumor effect of this cellular immunotherapy product.MethodsPatients were enrolled on an IRB-approved protocol and TIL were expanded from fresh surgical specimens. After enzymatic digestion, tumor single cell suspensions were cultured in media containing 10% human serum and IL-2 (6000IU/mL). Expanded TIL were then enriched for CD8+ using magnetic bead isolation and CD69+ by flow cytometry cell sorting (FACS). After co-culture with autologous tumor digest, functional capacity was compared between bulk TIL and enriched TIL by evaluation of IFN-gamma (IFNg) and Granzyme B (GzB) secretion. Capacity for direct tumor cytotoxicity was assessed by Cr51 assay after co-culture of autologous immortalized cell lines with expanded TIL subpopulations after enrichment.ResultsFollowing co-culture with autologous tumor digest, CD69+ TIL demonstrated increased IFNg secretion compared to CD69- TIL in 6 samples (1.4–4.2x, p<0.05). CD8+ enriched TIL (75% compared to bulk) had higher relative IFNg secretion in both CD69+ and CD69- subsets (4.2 and 5.8x, respectively, p<0.001). Maximal IFNg secretion was seen from TIL that were both CD69+ sorted and CD8+ enriched, demonstrating an synergistic effect (16.3x vs Bulk CD69-, 4.2x vs Bulk CD69+, 2.8x vs CD8 enriched CD69- ; p<0.001). Functional capacity was also assessed by GzB secretion with similar results. CD69+ TIL had increased relative secretion (1.8–2.2x) compared to CD69- TIL (p< 0.01). CD8+ enriched TIL had increased relative GzB secretion in both CD69- and CD69+ sorted fractions (1.4x, 1.2x, respectively, p<.05). CD69+ sorted and CD8+ enriched TIL demonstrated an additive effect (2.6x vs Bulk CD69-, p<0.01; 1.2x vs Bulk CD69+, p<0.05; 1.8x vs CD8 enriched CD69-, p<0.01). CD8+ enriched CD69+ sorted TIL had greater relative cytotoxicity (3x, p<0.05) at 40:1 E:T ratio against autologous tumor cell lines compared to bulk expanded TIL (figure 1).Abstract 179 Figure 1Functional capacity of CD69+ TIL is demonstrated by increased secretion of GzB (A) and IFNg (B) after co-culture with autologous tumor digest. CD69+ TIL have greater cytotoxicity against autologous immortalized cell lines compared to bulk TIL at 40:1 E:T ratio (C).ConclusionsTIL expanded from STS demonstrate greater tumor-specific functional capacity and cytotoxicity after CD8 enrichment and CD69+ FACS compared to bulk expanded TIL. These data validate the strategy to enhance CD8+CD69+ TIL during culture to yield a more efficacious cellular immunotherapy product.AcknowledgementsThis work was funded by NIH K08CA252642Trial Registration n/aReference1. Mullinax JE, Hall M, Beatty M, Weber AM, Sannasardo Z, Svrdlin T, Hensel J, Bui M, Richards A, Gonzalez RJ, Cox CA, Kelley L, Mulé JJ, Sarnaik AA, Pilon-Thomas S. Expanded Tumor-infiltrating Lymphocytes From Soft Tissue Sarcoma Have Tumor-specific Function. J Immunother 2021 Feb-Mar 01;44(2):63–70.Ethics ApprovalAbstract cites IRB-approved protocol in methods section.Consent n/a
Lack or loss of tumor antigenicity represents one of the key mechanisms of immune escape and resistance to T cell-based immunotherapies. Evidence suggests that activation of stimulator of interferon ...genes (STING) signaling in tumor cells can augment their antigenicity by triggering a type I IFN-mediated sequence of autocrine and paracrine events. Although suppression of this pathway in melanoma and other tumor types has been consistently reported, the mechanistic basis remains unclear. In this study, we asked whether this suppression is, in part, epigenetically regulated and whether it is indeed a driver of melanoma resistance to T cell-based immunotherapies. Using genome-wide DNA methylation profiling, we show that promoter hypermethylation of
and
genes mediates their coordinated transcriptional silencing and contributes to the widespread impairment of the STING signaling function in clinically-relevant human melanomas and melanoma cell lines. This suppression is reversible through pharmacologic inhibition of DNA methylation, which can reinstate functional STING signaling in at least half of the examined cell lines. Using a series of T cell recognition assays with HLA-matched human melanoma tumor-infiltrating lymphocytes (TIL), we further show that demethylation-mediated restoration of STING signaling in STING-defective melanoma cell lines can improve their antigenicity through the up-regulation of MHC class I molecules and thereby enhance their recognition and killing by cytotoxic T cells. These findings not only elucidate the contribution of epigenetic processes and specifically DNA methylation in melanoma-intrinsic STING signaling impairment but also highlight their functional significance in mediating tumor-immune evasion and resistance to T cell-based immunotherapies.
•HDAC6 interacts with HDAC11 in antigen presenting cells.•HDAC6 is a positive regulator of IL10 production.•Nuclear role of HDAC6 as transcriptional regulator.•HDACs as modulators of the APC ...plasticity to trigger T-cell activation.
The anti-inflammatory cytokine IL-10 is a key modulator of immune responses. A better understanding of the regulation of this cytokine offers the possibility of tipping the balance of the immune response toward either tolerance, or enhanced immune responses. Histone deacetylases (HDACs) have been widely described as negative regulators of transcriptional regulation, and in this context, the primarily nuclear protein HDAC11 was shown to repress il-10 gene transcriptional activity in antigen-presenting cells (APCs). Here we report that another HDAC, HDAC6, primarily a cytoplasmic protein, associates with HDAC11 and modulates the expression of IL-10 as a transcriptional activator. To our knowledge, this is the first demonstration of two different HDACs being recruited to the same gene promoter to dictate divergent transcriptional responses. This dynamic interaction results in dynamic changes in the expression of IL-10 and might help to explain the intrinsic plasticity of the APC to determine T-cell activation versus T-cell tolerance.
STING (stimulator of IFN genes) signaling is an innate immune pathway for induction of a spontaneous antitumor T-cell response against certain immunogenic tumors. Although antigen-presenting cells ...are known to be involved in this process, insight into the participation of tumor cell-intrinsic STING signaling remains weak. In this study, we find diversity in the regulation of STING signaling across a panel of human melanoma cell lines. We show that intact activation of STING signaling in a subset of human melanoma cell lines enhances both their antigenicity and susceptibility to lysis by human melanoma tumor-infiltrating lymphocytes (TIL) through the augmentation of MHC class I expression. Conversely, defects in the STING signaling pathway protect melanoma cells from increased immune recognition by TILs and limit their sensitivity to TIL lysis. Based on these findings, we propose that defects in tumor cell-intrinsic STING signaling can mediate not only tumor immune evasion but also resistance to TIL-based immunotherapies.
The physiological role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, remained largely unknown until the discovery of its regulatory function in immune cells. Among them, ...the regulation of cytokine production by antigen-presenting cells and the modulation of the suppressive ability of myeloid-derived suppressor cells (MDSCs) (Sahakian et al. Mol Immunol 63: 579-585, 2015; Wang et al. J Immunol 186: 3986-3996, 2011; Villagra et al. Nat Immunol 10: 92-100, 2009). Our earlier data has demonstrated that HDAC11, by interacting at the chromatin level with the IL-10 promoter, downregulates il-10 transcription in both murine and human APCs in vitro and ex vivo models (Villagra et al. Nat Immunol 10: 92-100, 2009). However the role of HDAC11 in other cell types still remains unknown. Here we present several methods that can potentially be used to identify the functional role of HDAC11, assigning special attention to the evaluation of immunological parameters.
Histone deacetylase 10 (HDAC10) belongs to the class IIb HDAC family and its biological role remains mostly unidentified. A decreased HDAC10 expression has been reported in patients with aggressive ...solid tumors (Osada et al. Int J Cancer 112: 26-32, 2004; Jin et al. Int J Clin Exp Pathol 7: 5872-5879, 2014), suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Consequently, results from our lab suggests that overexpression of HDAC10 in aggressive mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) Z138c and MEC1 cells, respectively, resulted in a rapid induction of cell death in vitro with only 5 % of cells being alive at 48 h, cell cycle arrest, and up-regulation of co-stimulatory molecules. Here we present several standard methods to study the function of HDAC10 in B cell malignancies.
Cutaneous T-cell lymphoma (CTCL) includes a spectrum of non-Hodgkin’s T-cell lymphomas. While most patients with CTCL experience non-life threatening skin symptoms, those who do progress to later ...stages of the disease may develop serious complications. For patients with tumor stage or lymph node involvement there is a significant decline in treatment response & few treatment options are available. Current available therapeutic options include the use of histone deacetylase (HDAC) inhibitors as Romidepsin, Panobinostat and Vorinostat. However, their mechanism of action is still unknown. The role of histone deacetylases (HDACs) in cell biology, initially limited to their effects upon histones, now encompasses more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, pathophysiological conditions and stage of cellular differentiation. Although major advances have been made in understanding the role of specific HDACs in cell proliferation and the survival of cancer cells, their individual participation in specific intracellular pathways is not completely understood. Here we present data demonstrating the changes in several immune-related pathways in two CTCL cell lines after exposure to both pan-HDAC inhibitors and selective HDAC6 inhibitors.
We compared the pharmacological effects of the pan-HDAC inhibitor LBH589 (Panbinostat), the class I selective Romidepsin, and the selective HDAC6 inhibitors such as TubastatinA and NexturstatB, on two CTCL cell lines (HuT78, HuT102). Our findings thus far demonstrate the following: First, we observed a marked inhibition of proliferation capacity of both cell lines when treated with either pan-HDACi or the more selective HDAC6 inhibitors. However, the selective HDAC6 inhibitors showed less cytotoxicity. Second, we observed important changes in the expression of the co-inhibitory molecules, such as PD-L1. Given our results, we conclude that the selective targeting of HDAC6 could recapitulate the anti-tumor effects of pan-HDAC inhibitors without having the non-target cytotoxic effects often encountered when using pan-HDAC inhibitors.
No relevant conflicts of interest to declare.
Histone deacetylases (HDACs), originally discovered as histone modifiers are now proposed as important regulators of non-chromatin related processes, including the regulation of cellular pathways ...involved in the production of anti- and pro-inflammatory cytokines and the subsequent function of antigen-presenting cells (APCs). We have recently identified HDAC6 as a positive regulatory factor in the production of IL-10. However, the participation of this HDAC in other immune related cellular processes remains unknown. In this work we present evidence of the important role of HDAC6 in the modulation of the JAK/STAT pathway through the IL-6 regulation.
We generated knockdown cell lines of HDAC6 (HDAC6KD) and non-target (NT) cells as a control in RAW264.7 murine macrophages using lentiviral shRNA. Two HADC6KD and two NT cell lines were treated with LPS or were left untreated and then analyzed by microarray. In HDAC6KD cells we found 1542 genes were down-regulated and 775 up-regulated in HDAC6KD cells. Their ontology distribution revealed significant changes in immune-related and apoptosis/cell cycle control genes. Importantly, we observed that most STAT3 and SP1 target genes were down regulated in HDAC6KD cells, suggesting the participation of HDAC6 in the regulation of these two transcription factors. Further analysis evidenced that the phosphorylation of STAT3 and the acetylation of Sp1 were diminished in HDAC6KD cells when compared against control cells. Chromatin immuneprecipitacion (CHIP) assays indicate that this particular effect of abrogation of HDAC6 involved histone modifications at the IL-6 promoter level, and more importantly, the recruitment of STAT3 and Sp1 to the IL-6 promoter was abrogated. Then, we analyzed the relevance of these findings by studying the tolerogenic JAK/STAT signaling pathway, which is known to be activated by IL-6 and critical in the final outcome of APCs in response to stimuli. Our observations included a complete abrogation in the phosphorylation of JAK2 and STAT3 proteins in HDAC6KD cells in response to LPS, which was reverted when these cells were treated with exogenous IL-6.
Our final results demonstrate a critical role of HDAC6 in the modulation of IL-6 and the potential role of HDAC6 in the regulation of the JAK/STAT3 pathway. In addition HDAC6 is a regulator of SP1 and STAT3 target genes. These findings provide insight into the molecular mechanisms controlling the immunogenicity of APCs, supporting the use of HDAC6 inhibitors to enhance immune activation, and positioning HDAC6 as a potential therapeutic target.
No relevant conflicts of interest to declare.
Abstract 3724
MCL is an aggressive and incurable subtype of B-cell Non-Hodgkin's lymphomas. Although patients often respond to first-line chemotherapy plus monoclonal antibodies, relapse and decrease ...response to further lines of treatment eventually occurs. Manipulation of the immune system to unleash its specificity and long-lasting protective effect might provide a unique opportunity to induce more durable responses or prevent relapse in MCL.
Previous studies by our group have shown that HDAC6 is required for production of the immunosuppressive cytokine, IL-10 in antigen-presenting cells (APCs) and that genetic or pharmacologic disruption of HDAC6 in these cells triggers potent antigen-specific T-cell responses. Given the role of IL-10 in suppressing the immunogenicity of B-cells, we asked therefore whether inhibition of HDAC6 with the isotype-selective inhibitor, Tubastatin-A (Tub-A) could reverse the tolerogenic properties of malignant B-cells in a murine model of MCL1. First, in vitro treatment of FC-muMCL1 cells with Tub-A resulted in increased acetylation of a-tubulin, a known HDAC6 target. Treated B-cells also displayed an enhanced expression of MHC class II and the co-stimulatory molecules B7.1, B7.2 and CD40 relative to untreated cells. Such changes resulted in more immunogenic MCL cells able to effectively activate naïve antigen-specific CD4+ T-cells and more importantly, capable of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Second, in vivo treatment of FC-muMCL1-bearing C57BL/6 mice with Tub-A resulted in lymphoma rejection. This antitumor effect was not observed in immunodeficient (SCID) C57BL/6 mice treated with Tub-A, pointing to the immunological effects triggered by HDAC6 inhibition in B-cells as playing a dominant role in Tub-A induced anti-MCL activity. Third, mechanistically we have found that HDAC6 physically interacts with STAT3 and it is required for STAT3 phosphorylation and recruitment to the IL-10 gene promoter. Furthermore, Tub-A treated MCL cells displayed a diminished STAT3 phosphorylation and abrogation of IL-10 gene transcriptional activity.
Taken together, a concerted regulatory mechanism involving HDAC6 and STAT3 influence the immunogenicity of MCL cells. Targeting HDAC6 with specific inhibitors to disrupt the tolerogenic STAT3/IL-10 axis represents a novel strategy to trigger effective anti-MCL immunity.
Martin:Millennium Pharmaceuticals, Inc.: Speakers Bureau.
PDF
