Increased Expression Of MHC Research Articles

Intermittent MEK Inhibition with GITR Costimulation Rescues T-cell Function for Increased Efficacy with CTLA-4 Blockade in Solid Tumor Models.

MEK inhibitors (MEKi) have shown limited success as a treatment for MAPK/ERK pathway-dependent cancers due to various resistance mechanisms tumor cells can employ. CH5126766 (CKI27) is an inhibitor that binds to MEK and prevents release of RAF, reducing the relief of negative feedback commonly observed with other MEKis. We observed that CKI27 increased MHC expression in tumor cells and improved T cell-mediated killing. Yet, CKI27 also decreased T-cell proliferation, activation, and cytolytic activity by inhibiting the MAPK/ERK pathway that is activated downstream of T-cell receptor signaling. Therefore, we aimed to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. Intermittent administration of CKI27 allowed T cells to partially recover and costimulation via GITR and OX-40 agonist antibodies completely alleviated inhibition of function. In Kras mutant lung and colon tumor mouse models, intermittent CKI27 and anti-GITR significantly decreased tumor growth and prolonged survival when further combined with CTLA-4 immune checkpoint blockade. Moreover, this triple combination increased CD8+ and CD4+ T-cell proliferation, activation, and effector/memory subsets in the tumor-draining lymph nodes and tumors and led to intratumoral regulatory T-cell destabilization. These data, collectively, will allow for more informed decisions when optimizing combination regimens by overcoming resistance, reducing toxicity, and generating long-term immune responses.

CD4 +Treg expansion arrests disease progression in a pre-clinical model of alopecia areata

Abstract Alopecia Areata (AA) is a prevalent autoimmune disease that results in nonscarring hair loss. Disease is associated with the collapse of an immune privileged state of the hair follicle, characterized by increased MHC expression, immune cell infiltration and the presence of pro-inflammatory cytokines in the microenvironment. CD8 T cells have been identified as the main contributors to disease and the activating receptor NKG2D can identify this pathogenic population. There is currently one FDA-approved drug for AA, a JAK1/2 inhibitor, which is thought to act by dampening inflammatory mediators during disease. However, the potential of adverse effects and refractory patients necessitates the development of alternative therapeutics. Emerging data from our lab suggests that the observed expansion of CD4 T regulatory cells (Tregs), relative to effector CD8 T cells during AA, is inadequate to prevent disease onset. We hypothesized that pharmacologic enhancement of Treg numbers may restrain development of AA. To address this, we investigated the protective potential of Treg expansion using a murine model of AA. In vivo expansion of CD4 +CD25 +FoxP3 +Tregs was achieved using an IL-2 cytokine/antibody complex (IL-2C), which is known to selectively expand and enhance Tregs. We found that 6 weeks of treatment with IL-2C halted the progression of hair loss, while control mice exhibited robust hair loss. Additionally, IL-2C treatment led to a reduction in NKG2D +CD8 T cells and IFNγ-producing CD8 T cells found infiltrating the skin, suggesting that increasing Tregs in the system is an effective strategy for restraining disease. Our findings provide translational insight for the use of Treg enrichment as a therapeutic strategy for patients with AA. Supported by grants from NIH (R01 AR077194) and Dept. of VA (I01 BX004907)

Abstract 4175: Combining a novel MEK inhibitor with immunomodulation to promote an anti-tumor response

Abstract To enhance the therapeutic scope of MEK inhibitors (MEKis), we aim to develop new strategies to extend their usage to MEKi resistant RAS mutant cancers, which represent an unmet clinical need. CH5126766 (CKI27) binds the allosteric site of MEK to inhibit its kinase activity but is novel due to its interaction with MEK S218 and 228, which blocks their phosphorylation by RAF. CKI27 bound MEK binds to RAF and cannot be released by phosphorylation, thus becoming a dominant negative inhibitor of RAF activation. This prevents the induction of MEK phosphorylation observed with other MEKis. Although this results in more potent tumor control, CKI27 is also capable of inhibiting T cell function because the MAPK/ERK pathway is activated downstream of T cell receptor signaling. We aim to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. We observed that CKI27 increased MHC expression on tumor cells and improved T cell mediated killing. Yet, CKI27 also decreased T cell proliferation, activation, and cytolytic activity. Intermittent administration of CKI27 allowed T cells to recover and partially relieved these inhibitory effects. Further combination with agonist antibodies anti-OX40 and GITR completely alleviated T cell inhibition and increased combination efficacy with immune checkpoint blockade antibody anti-CTLA-4. We also observed an increase in proliferation and T cell activation markers in LLC tumor bearing mice treated with the combination of CKI27, anti-GITR, and anti-CTLA-4. Understanding the immunomodulatory effects of combining CKI27 with immunotherapy will elucidate the mechanism behind this increased efficacy. This will allow us to make more informed decisions in dosing regimens, overcoming resistance, and generating long-term immune responses in future clinical trials treating patients with RAS mutant cancers. Citation Format: Lauren Dong, Hyejin Choi, Sadna Budhu, Isabell Schulze, Svena Verma, Nezar Mehanna, Neal Rosen, Taha Merghoub, Jedd Wolchok. Combining a novel MEK inhibitor with immunomodulation to promote an anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4175.

Dual Targeting of EZH2 and HDAC with Tazemetostat and Belinostat Promotes Immunogenicity in GC-DLBCL

Epigenetic remodeling is essential for the proper differentiation and function of germinal center (GC) B cells. Competing actions of histone acetyltransferases (HATs), such as CREBBP and EP300, and inhibitory histone deacetylases (HDACs) and methyltransferases (HMTs), such as EZH2, modulate the epigenomic program of GC B cells and control key activating and immunogenic processes, such as BCR and CD40 signaling, antigen processing and presentation, and cell cycle regulation. In line with the critical importance of epigenetic regulation of GC B cell function, inactivating mutations in CREBBPor EP300and gain-of-function mutations in EZH2 have been identified in 39% and 21% of GC B-cell like diffuse large B-cell lymphoma (GC-DLBCL), respectively. Strategies to target epigenetic dysfunction in GC-DLBCL have largely focused on indirectly restoring the activity of HATs through the use of histone deacetylase (HDAC) inhibitors, such as belinostat, and by directly inhibiting EZH2 with tazemetostat. Despite showing great promise in preclinical studies, single agent therapies with HDAC or EZH2 inhibitors have shown only modest efficacy in the clinic. Recently, our group demonstrated synergistic effects of dual therapy with EZH2 and HDAC inhibitors in inducing lethality of GC-DLBCL lines. As combination therapies begin to be tested in clinical trials, whether dual inhibition of EZH2 and HDAC is able to restore immunogenic features of GC-DLBCL and lead to enhanced T cell-mediated killing in vivo remains unknown. Here, we utilize transcriptomic and flow cytometric methods to assess whether epigenetic remodeling with tazemetostat (EZH2 inhibitor) and belinostat (HDAC inhibitor) can alter the immunogenicity of GC-DLBCL. We find that combination treatment with both tazemetostat and belinostat promotes an antigen processing and presentation program in GC-DLBCL lines and results in significant increases in MHC-I and MHC-II surface expression. This effect was only modestly appreciated with single agent treatment and required at least 4-7 days post-treatment to become apparent. Importantly, the increased MHC expression in response to combination therapy was not dependent on EZH2 or CREBBPmutation status, suggesting broad applications of dual therapy in patients with diverse mutation burdens. Furthermore, we found that combination treatment also altered surface expression of costimulatory molecules, such as CD80 and CD86, suggesting that tazemetostat and belinostat can modulate interactions with T cells in a multifaceted manner. These findings thus uncover that dual targeting of EZH2 and HDAC with tazemetostat and belinostat promotes antigen presentation pathways in GC-DLBCL and suggests that dual therapies could restore immunogenicity in GC-DLBCL and enhance immune-mediated killing of tumor cells.This work was funded, in part, by the ASH HONORS Award Summer Program. [Display omitted] DisclosuresAmengual: Seagen: Consultancy; Appia Pharmaceuticals: Research Funding; Daiichi Sankyo, Inc: Consultancy; Epizyme, Inc.: Speakers Bureau.

Abstract 1892: Proteomic interrogation of the metabolic control of MHC class I antigen presentation in metastatic melanoma

Abstract Since 2014, immune checkpoint inhibitors (ICIs) have led the charge against metastatic melanoma as the most commonly utilized first-line treatment. While ICIs have dramatically increased the 5-year overall survival rate, only half of patients respond to treatment. Recently, a proteomic study correlated high levels of tumor mitochondrial metabolic proteins and MHC-related proteins to responsiveness to anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitor treatment. In a study of 91 melanoma cell lines in the European Searchable Tumor Cell and Databank, 67% of the cell lines exhibited HLA class antigen alterations, most commonly being down-regulation of HLA-B. Loss of MHC expression is a mechanism proposed to aid in evading the immune system by decreasing T-cell recognition and killing. Approaches to reverse tumor MHC loss is needed as it is a known mechanism utilized by melanoma and increased MHC expression has been linked to ICI responders. The connection between metabolism and MHC correlation has not been fully explored, and this study sought to further explore that relationship through a proteomic analysis of a metabolically conditioned B16F10 murine melanoma model. To expand our understanding of the metabolic control of MHC Class I antigen presentation, we cultured B16F10 melanoma cells in galactose media (B16.Gal), forcing the cells into an oxidative phosphorylative state relative to B16F10 melanoma cells cultured in glucose media (B16.Glu) which exhibit a glycolytic metabolic profile. We show that enforcing mitochondrial respiration restores the lost basal MHC expression and enhances the response to interferon gamma. Further, B16.Gal cells show higher rates of T-cell killing and increased PD-L1 expression. This phenotype proved reversible as shown by moving B16.Gal cells back into glucose media (B16.GalGlu), supporting the formation of a metabolically conditioned cell model rather than a favorable selection of compatible cells. We used proteomics to identify the driving forces behind the restoration of MHC expression. The identification of these forces could provide novel therapeutic targets for enhancing immunotherapy of melanoma. Citation Format: Alyson McKinnon, Nathan Avaritt, Alan Tackett, Brian Koss. Proteomic interrogation of the metabolic control of MHC class I antigen presentation in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1892.

Abstract S02-02: Covid-19 treatment candidate hydroxychloroquine impairs tumor response to anti-PD1

Abstract Background and Aims: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a worldwide health crisis. As of now, only one treatment has been established for the coronavirus infection disease 2019 (Covid-19), which has disproportionally affected patients with comorbidities such as cancer. Hydroxychloroquine (HCQ) has been studied as a potential treatment for Covid-19. Here we studied whether HCQ treatment could influence the effect of immunotherapy against colorectal and liver cancer. Methods: In vitro studies were conducted to assess the effect of HCQ on lymphocyte activation and proliferation as well as cell growth and antigen presentation. Mouse cell lines used included MC38, CT26, RIL-175, SB1, and LD1 while human tumor cell lines used were TFK-1 and EGI-1. Mice bearing subcutaneous MC38, CT26, and RIL-175 tumors were treated with HCQ, anti-PD1, or both. Tumor volume was measured over 19 days. Systemic and tumor-infiltrating immune cells were analyzed by flow cytometry. Results: HCQ treatment impairs T-cell production of TNF-α and IFNγ in vitro and in vivo. Whereas cell growth was decreased in all tumor cell lines, in vitro treatment of tumor cells with HCQ revealed an upregulation of MHC-1 in CT26, SB1, LD1, TFK-1, and EGI-1 but not in MC38 and RIL-175. HCQ treatment impaired response to anti-PD1 treatment in all subcutaneous models. Compared to control, HCQ treatment led to increased tumor growth in MC38 tumors. Flow cytometry analysis revealed an impaired activation of CD4+ T cells as well as tumor-infiltrating cytotoxic antigen-specific CD8+ T cells. Conclusion: This study shows that HCQ treatment can result in immunotherapy failure due to a systematic immunosuppressive effect that overcomes the increased MHC expression on tumor cells. Citation Format: Simon Wabitsch, Jack C McVey, Chi Ma, Benjamin Ruf, Laurence Diggs, Bernd Heinrich, Tim F Greten. Covid-19 treatment candidate hydroxychloroquine impairs tumor response to anti-PD1 [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S02-02.

Combining a novel dual RAF/MEK inhibitor with immunomodulation to promote an anti-tumor response

Abstract The therapeutic scope of MEK inhibitors (MEKis) is currently limited to use in BRAF mutant melanoma. Therefore, we aim to develop new strategies to extend their usage to MEKi resistant RAS mutant cancers, which represent an unmet clinical need. A strategy we investigated is to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. In Ras mutant murine lung cancers, CH5126766 (CKI27) is novel due to its ability to inhibit both RAF and MEK, preventing the rebound of ERK that normally results from the relief of negative feedback in the MAPK pathway. We observed that CKI27 increased MHC expression on tumor cells and T cell mediated killing. Yet, CKI27 also decreased T cell proliferation, activation, and cytolytic activity. Implementing a break for T cells to recover with intermittent dosing of CKI27 partially relieved these inhibitory effects. Further combination with co-stimulatory agonist antibodies targeting OX40 and GITR completely alleviated these T cell toxicities and increased combination efficacy with checkpoint blockade antibody anti-CTLA-4. Understanding the immunomodulatory effects of combining CKI27 with immunotherapy will elucidate the mechanism behind their increased efficacy. This will allow us to make more informed decisions in dosing regimens, overcoming resistance, and generating long-term immune responses in current and future clinical trials treating patients with RAS mutant cancers.

Abstract MS1-1: Immunotherapy in breast cancer: enhancing response to checkpoint blockade

Abstract Historically, breast tumors have been categorized as non-immunogenic in part due to a paucity of tumor infiltrating lymphocytes (TIL) when compared to other histologies. Multiple studies have now demonstrated an endogenous immune response against breast cancer with tumors having TIL present in the tumor and surrounding stroma. The extent of TIL infiltrate is dictated by the subtype of disease being highest in triple negative breast cancer (TNBC) and lowest in hormone receptor positive, HER2 negative (HR+/HER2-) disease. Recent studies have shown that immunotherapy, in the form of immune checkpoint blockade, can be effective in treating breast cancer patients. Specifically, the IMpassion 130 trial, a phase III study that enrolled over 900 patients with metastatic TNBC and randomized them to the immune checkpoint blockade agent atezolizumab plus nab-paclitaxel versus placebo plus nab-paclitaxel, demonstrated benefit to the addition of immunotherapy with respect to progression free survival as well as overall survival; a difference that was greater in patients with PD-L1 positive disease. Based on these data, the combination of atezolizumab plus nab-paclitaxel was approved for use in patients with PD-L1+ metastatic TNBC. More recently, data from a large phase III trial evaluating the use of the immune checkpoint blockade agent pembrolizumab in the neoadjuvant setting for TNBC patients showed a significant improvement in the rates of pathologic complete response. Based in part on these successes, there is significant enthusiasm for using immunotherapy to treat breast cancer patients. It must be recognized however, that a minority of patients respond. Additional work must therefore be done to expand the number of patients with TNBC who respond as well as to identify strategies for successfully employing immunotherapy for HER2+ and HR+ positive breast cancer. Development of these strategies requires understanding of the cancer immunity cycle and how it can be altered to enhance endogenous immune responses to breast cancer as well as improved understanding of the immune aspects of the tumor microenvironment and how standard of care therapies alter that microenvironment. As an example, there is emerging data showing that PARP inhibition leads to cytoplasmic DNA which activates the STING pathway leading to increased expression of release of type 1 interferons, increased MHC expression and antigen presentation and increased infiltration of effector T cells. Ongoing trials are therefore evaluating those agents in combination with immunotherapy. Our lab has shown that PARP inhibition further modifies the tumor microenvironment suggesting additional therapeutic targets. In addition, there is mounting evidence regarding potential mechanisms behind why some subtypes of breast cancer mount a robust immune response while others remain immunologically “cold”. Differences in antigen processing and presentation likely contribute and understanding how HR+ tumors “hide” from immune recognition could contribute to developing strategies to make these tumors susceptible to immunotherapy. Finally, our group has recently published data demonstrating a difference in susceptibility to immune checkpoint blockade based on age suggesting that a patient’s systemic immune status could predict therapeutic response and potentially be manipulated to enhance that response. Citation Format: E Mittendorf. Immunotherapy in breast cancer: enhancing response to checkpoint blockade [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr MS1-1.

Canadine from Corydalis turtschaninovii Stimulates Myoblast Differentiation and Protects against Myotube Atrophy.

Cachexia and sarcopenia are the main causes of muscle atrophy. These result in a reduction in the muscle fiber area, myo-protein content, and muscle strength, with various molecular modulators being involved. Although several reports have proposed potential therapeutic agents, no effective treatments have been found for muscle atrophy. We searched for myogenic modulators from medicinal plants to treat muscle diseases. We isolated six alkaloids from Corydalis turtschaninovii and evaluated their myogenic potential by using the MyoD reporter gene assay in C2C12 cells. Among the tested compounds, canadine showed the strongest transactivation of MyoD and increased MHC expression during myogenesis. The activation of p38 MAP kinase and Akt are major mechanisms that contribute to the myogenesis by canadine. Canadine increased the number of multinucleated and cylinder-shaped myotubes during myogenesis of C2C12 myoblasts. To determine the preventive effect of canadine in cancer-induced muscle wasting, differentiated C2C12 myotubes were treated with conditioned media from CT26 colon carcinoma culture (CT26 CM) in the presence of canadine. Canadine ameliorated the muscle protein degradation caused by CT26-CM by down-regulating the muscle specific-E3 ligases, MAFbx/atrogin-1 and MuRF1. In this study, we found that canadine from C. turtschaninovii stimulates myogenesis and also inhibits muscle protein degradation. Therefore, we suggest canadine as a protective agent against muscle atrophy.

Role of MHC class I expression and CD8(+) T cells in the evolution of iodine-induced thyroiditis in NOD-H2(h4) and NOD mice.

Dietary iodine has long been known to influence the development of human autoimmune thyroid disease. In nonobese diabetic (NOD) and NOD-H2(h4) mice elevated dietary iodine has been shown to induce autoimmune thyroid disease. Immune responses to thyroid antigens can be detected in these mouse strains, including T cell responses in the NOD-H2(h4) mouse to thyroid peroxidase. Cell transfer studies and antibody depletion experiments reveal a requirement for both CD4(+) T cells and CD8(+) T cells in the development of thyroid autoimmunity. Histological analyses of the thyroids show that following 1 week of iodine administration MHC class I expression is elevated on thyroid follicular cells and CD4(+) and CD8(+) T cells have begun to infiltrate the gland. Although MHC class II expression on thyroid epithelial cells was also elevated, the tempo of expression was slower and the extent of expression was far less than that for MHC class I. Depletion of CD8(+) T cells at early stages of disease induction inhibited not only thyroid infiltration and autoantibody production but also reduced the levels of MHC expression in the thyroid, suggesting that cytokine production by infiltrating lymphocytes was responsible for the increased MHC expression.

IFN regulatory factor-1 plays a central role in the regulation of the expression of class I and II MHC genes in vivo.

Abstract Transcription factor interferon regulatory factor-1 (IRF-1) is implicated in regulating class I MHC expression in vitro. We investigated the in vivo relationship between IRF-1 and MHC expression in kidney and other nonlymphoid organs, assessing MHC expression in mice with disrupted IRF-1 genes (IRF-1 KO) compared with mice with intact IRF-1 genes (WT). In kidneys of IRF-1 KO mice, basal class I expression was decreased, particularly on arterial endothelium, but basal class II expression was unchanged. The induction of both class I and class II expression by injected rIFN-gamma was reduced in IRF-1 KOs, compared with WT mice. Similarly, stimuli that induce endogenous IFN-gamma production (LPS or oxazolone) massively increased MHC expression in kidneys of WT mice, with little increase in IRF-1 KO mice. Impaired class II induction by rIFN-gamma in IRF-1 KO mice probably reflects the role of IRF-1 in regulating class II transactivator (CIITA) expression: rIFN-gamma induced CIITA mRNA less in kidneys of IRF-1 KO mice than in WT mice. In organs of WT mice, IRF-1 mRNA was expressed in the basal state, and rIFN-gamma treatment increased IRF-1 mRNA before the induction of class I or CIITA mRNA. Treatment of WT mice with cycloheximide plus rIFN-gamma superinduced IRF-1 mRNA expression, but partially inhibited CIITA mRNA expression, indicating that IRF-1 mRNA induction is not dependent on new protein synthesis, unlike CIITA. Thus, in vivo, IRF-1 plays a major role in basal and induced class I expression and in induction of class II by IFN-gamma, probably via CIITA induction.

The induction of class I and II major histocompatibility complex by allogeneic stimulation is dependent on the transcription factor interferon regulatory factor 1 (IRF-1): observations in IRF-1 knockout mice.

Hosts undergoing allograft rejection show increased MHC expression locally in the graft and systemically in the normal host organs, mediated principally by IFN-gamma. The transcription factor IRF-1 has been implicated in the regulation of MHC expression by IFNs in vitro as well as in the regulation of production of some cytokines. We investigated the role of IRF-1 in vivo in the systemic regulation of MHC expression in hosts undergoing rejection of allogeneic tumors by comparing MHC induction in mice with normal IRF-1 genes (wild type or WT mice) with mice with disrupted IRF-1 genes (IRF-1 knockout or IRF-1 KO mice). We assessed MHC product expression by immunohistology and by radiolabeled antibody binding to tissue homogenates, and MHC mRNA levels by Northern blotting. By immunohistology in mice undergoing allogeneic stimulation by the ascites tumor cells, kidneys of WT mice showed massive class I and II induction, but kidneys from IRF-1 KO mice showed almost no class I and II induction. Allograft rejection also increased class I and II product levels by radiolabeled antibody binding and steady state mRNA levels, but again IRF-1 KO mice showed severe impairment of MHC induction. Similar impaired MHC class I and II induction was seen in heart and spleen, but in liver the IRF-1 mice showed impaired class I induction but unimpaired class II induction. The results indicate that IRF-1 has an essential role in both class I and class II MHC induction in allogeneic responses, but that a component of IRF-1 independent MHC induction is also demonstrable in some tissues. The reduction in MHC induction by allogeneic stimulation probably reflects decreased response to IFN-gamma and other cytokines as well as some reduction in the amount of cytokines produced.

Local T cell responses induce widespread MHC expression. Evidence that IFN-gamma induces its own expression in remote sites.

Local inflammation induces increased expression of MHC and other genes in the affected tissue because of the paracrine effects of cytokines such as IFN-gamma. We previously reported that one such process--local allograft rejection--was accompanied by increased expression of MHC in a remote tissue, namely kidney. To explore how local inflammation affects gene expression in remote tissues, we studied MHC, beta 2-microglobulin, and IFN-gamma expression in mice undergoing either of two T cell-dependent localized inflammatory processes: rejection of an ascites tumor allograft, and skin sensitization by oxazalone. As assessed by binding of radiolabeled mAb and by immunohistology, each stimulus increased MHC expression in many remote tissues, including liver, heart, pancreas, and kidney. This was associated with increases in steady state mRNA for class I, class II, and beta 2-microglobulin. MHC induction was inhibited by the in vivo administration of cyclosporine or anti-IFN-gamma mAb and did not occur in nude mice, confirming the key role of IFN-gamma released from T cells. When we examined tissues of mice with these localized inflammatory lesions for IFN-gamma mRNA levels by polymerase chain reaction, we found that IFN-gamma steady state mRNA levels were increased in the spleen and, more surprisingly, in the kidney, and in uninvolved skin. Moreover, anti-IFN-gamma inhibited the induction of IFN-gamma mRNA in the kidney, suggesting that IFN-gamma expression was induced by IFN-gamma in an autoregulatory fashion. Thus the systemic MHC induction accompanying local T cell-mediated inflammation reflects the release of IFN-gamma from the site of inflammation, but may be amplified by the ability of IFN-gamma to induce its own expression in remote tissues. This self-amplification of IFN-gamma may contribute to the ability of local inflammation to induce extensive systemic effects.

INCREASED MAJOR HISTOCOMPATIBILITY COMPLEX ANTIGEN EXPRESSION IN UNILATERAL ISCHEMIC ACUTE TUBULAR NECROSIS IN THE MOUSE

In many studies of renal transplant recipients, acute tubular necrosis has been shown to predispose to a higher rate of graft loss, apparently due to rejection, but the mechanism of this effect is unknown. One possibility is an increased immunogenicity of the graft. To study this possibility, we examined the expression of major histocompatibility complex antigens in kidneys damaged by ischemia, using a mouse model of ischemic ATN. ATN was produced in the left kidney of male CBA mice by temporary clamping of the vascular pedicle for up to 60 min. Class I and II MHC expression was quantified by the extent of binding of monoclonals in radioimmunoassay, after 1 to 35 days in both kidneys. MHC induction was localized by indirect immunoperoxidase staining. Specific steady state mRNA for beta 2 microglobulin and class II were quantified by northern blotting using 32P-labeled probes. Changes in MHC expression were assessed by comparing the ischemically injured left kidney to the control right kidney. By day 1, ATN was evident by histology but there was no change in MHC expression. By day 3, class I was increased in the left kidney by 3-6-fold over the right. In tissue sections, the class I increase was localized to tubular epithelial cells. Starting on day 7 and persisting to day 35, class II was increased by 1.5 to 3 times for the ischemic kidney over the control, primarily in interstitial cells but also in tubular cells. This increase in class II was associated with the appearance of Thy 1.2-positive cells in the interstitial areas. Increased antigen expression was preceded by increased steady state mRNA. Thus unilateral ischemic ATN causes increased MHC expression in tubular cells and the accumulation of an inflammatory infiltrate, both of which may contribute to the increased rate of rejection and graft loss in ischemically injured kidneys.

Multiple low dose streptozotocin induces systemic MHC expression in mice by triggering T cells to release IFN-gamma.

Multiple low dose streptozotocin (STZ) is believed to induce immunologically mediated islet cell necrosis. We sought to establish whether this agent also increased MHC expression, as has been reported for other diabetes models. STZ (40 mg/kg/day for 5 days) produced increased levels of both class I and II MHC products in kidney, liver, heart, and pancreas. Class I expression was induced in renal tubular cells, Kupffer cells, hepatocytes, occasional cardiac myocytes, and cells within the pancreatic islet. In contrast, class II products were increased in dendritic cells, renal tubules, and cells within the pancreatic islet. Steady state mRNA levels for class I, class II, and beta 2-microglobulin correlate closely with the level of MHC products measured by radiolabeled antibody binding, suggesting that changes in MHC expression reflect changes in gene transcription. The effect is T cell dependent and inhibitable by cyclosporine. IFN-gamma is an essential mediator of the MHC induction; administration of a neutralizing antibody blocks the increase in expression. Furthermore, this antibody attenuates the hyperglycemic response to STZ, demonstrating a pathogenic role for IFN-gamma in mediating beta cell damage. We conclude that the MHC induction observed after low dose STZ is due to immunologic mechanisms, in particular the release of lymphokines such as IFN-gamma from T cells. The release of IFN-gamma and changes in MHC expression may be relevant to the injury seen with this agent.