Jenny H. Pan

Ionic immune suppression within the tumour microenvironment limits T cell effector function.

Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt–mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt–mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.

BACH2 regulates CD8+ T cell differentiation by controlling access of AP-1 factors to enhancers

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8+ T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

Collapse of the Tumor Stroma is Triggered by IL-12 Induction of Fas

Engineering CD8⁺ T cells to deliver interleukin 12 (IL-12) to the tumor site can lead to striking improvements in the ability of adoptively transferred T cells to induce the regression of established murine cancers. We have recently shown that IL-12 triggers an acute inflammatory environment that reverses dysfunctional antigen presentation by myeloid-derived cells within tumors and leads to an increase in the infiltration of adoptively transferred antigen-specific CD8⁺ T cells. Here, we find that local delivery of IL-12 increased the expression of Fas within tumor-infiltrating macrophages, dendritic cells, and myeloid-derived suppressor cells (MDSC), and that these changes were abrogated in mice deficient in IL-12-receptor signaling. Importantly, upregulation of Fas in host mice played a critical role in the proliferation and antitumor activity of adoptively transferred IL-12-modified CD8⁺ T cells. We also observed higher percentages of myeloid-derived cell populations within tumors in Fas-deficient mice, indicating that tumor stromal destruction was dependent on the Fas death receptor. Taken together, these results describe the likely requirement for costimulatory reverse signaling through Fasl on T cells that successfully infiltrate tumors, a mechanism triggered by the induction of Fas expression on myeloid-derived cells by IL-12 and the subsequent collapse of the tumor stroma.

The transcription factor BACH2 promotes tumor immunosuppression

The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.

A complicated case of vascular Pythium insidiosum infection treated with limb-sparing surgery

INTRODUCTION Pythiosis is a serious life- and limb-threatening infection endemic to Thailand, but rarely seen in the Western hemisphere. Here, we present a unique case of vascular pythiosis initially managed with limb-sparing vascular bypass grafts complicated by a pseudoaneurysm in our repair. PRESENTATION OF CASE The patient is a 17 year-old Jamaican male with severe aplastic anemia. He sustained a minor injury to his left leg while fishing in Jamaica, which evolved to become an exquisitely tender inguinal swelling. His physical exam and imaging were significant for arteriovenous fistula with limb ischemia. Pathology obtained during surgery for an extra-anatomic vascular bypass showed extensive invasion by Pythium insidiosum. He later developed a pseudoaneurysm at the site of proximal anastomosis and required urgent intervention. DISCUSSION This patient presented with a rare, but classic case of vascular pythiosis, which was unrecognized at the time of presentation. A variety of therapeutic modalities have been used to treat this disease, including antibiotics, antifungals, and immunotherapy, but the ultimate management of vascular pythiosis is surgical source control. CONCLUSION A high index of suspicion in susceptible patients is needed for timely diagnosis of vascular pythiosis to achieve optimal source control.

Abstract 1463: Ionic immune suppression within the tumor microenvironment limits T cell effector function

Tumors progress in immunocompetent hosts despite the local infiltration of tumor-specific effector T cells. Recent advances have identified ion transport as important for T cell activation and function. We report that the concentration of potassium is markedly elevated within murine and human tumors in comparison to other tissues while other ions are minimally perturbed. Additionally, ex vivo stimulation of effector T cells in hyperkalemic conditions led to profound suppression of T cell activation, cytokine production, and glycolytic metabolism without affecting viability - suggesting a role for potassium in tumor immune evasion. Characterization of this phenomenon with whole-transcriptome RNA-Sequencing revealed that elevated potassium specifically repressed TCR induced transcripts with the effector cytokine interferon gamma (IFNγ) being among the most depressed. Moreover, hyperkalemia limited TCR induced glucose uptake and consumption, metabolic changes required for full effector function. Prior investigations into the role of potassium transport in cellular physiology have centered on the presumption that the ion9s sole function is to maintain cellular membrane potential and calcium influx. However, we find that elevated potassium had no effect on TCR induced calcium signaling. Although hyperkalemia increased the cytoplasmic membrane potential of effector T cells as expected, other methods to similarly depolarize the cell without the addition of extracellular potassium did not produce the inhibitory phenomenon. Therefore, we conclude that the immunosuppressive effect induced by hyperkalemia is independent of potassium9s previously understood role in the regulation of cytoplasmic membrane potential and calcium signaling. Causatively, elevated potassium led to blunted serine/threonine phosphorylation within the Akt/mTOR signaling cascade following TCR ligation, while pharmacologic or genetic means to restore Akt signaling rescued effector function. Mechanistically, the suppressive effect of elevated extracellular potassium was directly related to an increase in intracellular levels, and depletion of intracellular potassium restored full T cell activation and cytokine production. T cells genetically reprogrammed to express the potassium efflux channel Kcna3 were resistant to potassium mediated suppression - displaying lower intracellular potassium levels and increased cytokine production. Finally, tumor-specific Kcna3 expressing cells exhibited augmented in vivo intratumoral IFNγ production, Akt/mTOR signaling, and enhanced anti-tumor activity against established melanoma in tumor bearing mice. These results uncover a novel axis of tumor-induced immune suppression via an ‘ionic’ checkpoint. The manipulation of cellular ion transport represents an entirely new approach for cancer immunotherapy. Citation Format: Robert L. Eil, Rahul Roychoudhuri, Madhu Sukumar, David Clever, Jenny H. Pan, Shashank Patel, Douglas C. Palmer, Nicholas P. Restifo. Ionic immune suppression within the tumor microenvironment limits T cell effector function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1463.

Abstract 4034: Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies

Host immune surveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. Less is known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application of a T cell-dependent selection pressure in the form of a whole tumor vaccine would alter the immunogenic architecture in our transplantable murine melanoma model SB-3123. We generated an escape variant to test this hypothesis. We performed whole-exome and RNA-sequencing of the tumor line SB-3123 and two fresh tumors generated by subcutaneous implantation of SB-3123 to identify mutations and to assess levels of expression. There were 349 mutations shared between the tumor line and fresh tumors and less than 5 mutations were unique to each sample. We then investigated whether tumors that had undergone an additional round of host immunoediting would acquire different mutations. To test this, we generated a re-derived tumor cell line from a fresh SB-3123 tumor. Remarkably, the re-derived tissue culture line retained 333 mutations in common with the prior samples and produced only 2 unique mutations. To test the immunogenicity of SB-3123, we vaccinated mice with irradiated SB-3123 and administered a live tumor challenge two weeks after vaccination. This resulted in a profound vaccination response with mice being completely protected from tumor for over 200 days. However, one vaccinated mouse developed a recrudesced tumor at the site of implantation 40 days after challenge and a tumor line was created (SB-3123-esc). We asked whether SB-3123-esc represented an escape variant by immunizing mice with SB-3123 and then challenging with either SB-3123 or SB-3123-esc. Vaccination with SB-3123 did not protect against SB-3123-esc. We also performed sequencing on SB-3123-esc and discovered that 441 nonsynonymous mutations were common to both tumor lines while 80 were unique to SB-3123 and 40 to SB-3123-esc. Furthermore, we were able to determine that SB-3123-esc lost 2 immunogenic neoantigens but had acquired 5. In addition, we identified a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. Our results demonstrate that the expressed mutations of SB-3123 tumor did not change, regardless of whether we sequenced the tumor line, fresh tumors, or a re-derived tumor cell line. However, the escape tumor had new unique mutations and at least one of these was demonstrably immunogenic; we were able to develop a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. In conclusion, we posit that these observations offer a proof of concept that the process of cancer immune evasion can create new opportunities to treat escape tumors with T cell based therapies. Citation Format: Jenny H. Pan, Suman Vodnala, Robert L. Eil, Zhiya Yu, Jared Gartner, David Clever, Rahul Roychoudhuri, Shashank J. Patel, Christopher A. Klebanoff, Madhu Sukumar, Tori Yamamoto, Nicholas P. Restifo. Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4034.

Elevated potassium levels suppress T cell activation within tumors

Tumors progress in immunocompetent hosts despite the ability of the adaptive immune system to recognize cancer cells. Ion gradients regulate T cell function but their role in intratumoral immune responses is unexplored. We found that the concentration of K+ was strikingly elevated within tumors while the concentration of the divalent cations Ca2+ and Mg2+ was similar to serum levels. High K+ levels significantly blunted cytokine production and suppression TCR stimulation induced gene transcription in CD8+ and CD4+ effector T cells. Moreover, polarization of CD8+ and CD4+ T cells in high K+ suppressed effector differentiation and promoted the formation of CD4+ Foxp3+ Treg cells. Surprisingly, this was not due to an attenuation of TCR induced Ca2+ flux, but rather to reduced activation of the serine/threonine Akt-mTOR pathway and could be partially reversed by overexpression of constitutively active Akt1. This coincided with the finding that okadaic acid, an inhibitor of the serine/threonine phosphatase PP2A, rendered effector cells resistant to the inhibitory effects of high K+ and restored cytokine function within tumors. Additionally, expression of a peptide inhibitor targeting the PP2A complex provided resistance to the inhibitory effect of elevated K+. These findings identify a novel mechanism of ionic regulation of TCR induced signals and immunosuppression within tumors whereby locally high extracellular concentrations of normally intracellular ions suppress immune function to promote tumor growth.

Immunotherapy generates selective pressure for acquisition of immunogenic neoantigens in escape tumors.

Host immunosurveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. It is less known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application of a T cell-dependent selection pressure in the form of a whole tumor vaccine would alter the immunogenic architecture in our transplantable murine melanoma model SB-3123, and generated an escape variant to test this hypothesis. We performed whole-exome and RNA-sequencing of the tumor line SB-3123 and two fresh tumors generated by subcutaneous implantation of SB-3123 to identify mutations and to assess levels of expression. There were 349 mutations shared between the tumor line and fresh tumors, with less than 5 mutations that were unique to each sample. We then determined the stability of these mutations and created a re-derived tissue culture line from a fresh SB-3123 tumor. Remarkably, the re-derived tissue culture line retained 333 mutations in common with the prior samples and produced only 2 unique mutations. To test the immunogenicity of SB-3123, we vaccinated mice with irradiated SB-3123 and administered a live tumor challenge two weeks after vaccination. This resulted in a profound vaccination response and mice were completely protected from tumor for over 200 days. Surprisingly, one vaccinated mouse developed a recrudesced tumor at the site of implantation 40 days after challenge, and a tumor line was created (SB-3123-esc). We asked whether SB-3123-esc represented an antigen escape variant by immunizing mice with SB-3123 and challenging with either SB-3123 or SB-3123-esc. Vaccination with SB-3123 did not protect against SB-3123-esc. We also performed sequencing on SB-3123-esc and determined that no known genes involved in antigen processing and presentation were mutated, and SB-3123-esc upregulated class I molecules at similar levels compared to SB-3123 upon IFN-γ treatment. Our most striking results were the differences in mutational constituents between SB-3123 and SB-3123-esc. Of the nonsynonymous mutations called in the coding region, 441 were common to both tumor lines while 80 were unique to SB-3123 and 40 to SB-3123-esc. Furthermore, we were able to determine that SB-3123-esc lost 2 immunogenic neoantigens but had acquired 5. Future investigations are directed towards identifying the rejection antigen responsible for the escape phenotype of SB-3123-esc.