Mechanism Of Immune Privilege Research Articles

Transmigration across a Steady-State Blood-Brain Barrie Induces Activation of Circulating Dendritic Cells Partly Mediated by Actin Cytoskeletal Reorganization.

The central nervous system (CNS) is considered to be an immunologically unique site, in large part given its extensive protection by the blood–brain barrier (BBB). As our knowledge of the complex interaction between the peripheral immune system and the CNS expands, the mechanisms of immune privilege are being refined. Here, we studied the interaction of dendritic cells (DCs) with the BBB in steady–state conditions and observed that transmigrated DCs display an activated phenotype and stronger T cell-stimulatory capacity as compared to non-migrating DCs. Next, we aimed to gain further insights in the processes underlying activation of DCs following transmigration across the BBB. We investigated the interaction of DCs with endothelial cells as well as the involvement of actin cytoskeletal reorganization. Whereas we were not able to demonstrate that DCs engulf membrane fragments from fluorescently labelled endothelial cells during transmigration across the BBB, we found that blocking actin restructuring of DCs by latrunculin-A significantly impaired in vitro migration of DC across the BBB and subsequent T cell-stimulatory capacity, albeit no effect on migration-induced phenotypic activation could be demonstrated. These observations contribute to the current understanding of the interaction between DCs and the BBB, ultimately leading to the design of targeted therapies capable to inhibit autoimmune inflammation of the CNS.

Intracranial tumors lead to sequestration of T cells in the bone marrow

Abstract Glioblastoma (GBM) is the most common primary, malignant brain tumor and is universally fatal. Immunotherapeutic intervention has shown some success in immunogenic solid tumors, but little efficacy in GBM. Tumor-imposed T cell deficits remain a major obstacle. Recently, we demonstrated that GBM patients exhibit local and systemic T cell dysfunction, severe lymphopenia and contracted lymphoid organs. These observations are recapitulated in multiple murine models of GBM. Although T cell numbers are decreased in blood, lymph nodes, and spleen, we find large numbers of T cells sequestered in the bone marrow of both patients and mice with GBM. T cell sequestration occurs not only in the setting of GBM, but with nearly any cancer placed intracranially. Intriguingly, none of the tumors tested, including GBM, induce sequestration of T cells in the bone marrow when implanted subcutaneously. These data suggest that the capacity to sequester T cells resides within the brain and is usurped by tumors of the intracranial compartment. Such capacities may well represent a novel mechanism of immune privilege. Sequestration of T cells follows loss of sphingosine-1 phosphate-receptor 1 (S1P1) from the T cell surface. Understanding the drivers of S1P1 loss will provide crucial insight for how brain tumors facilitate systemic immune alterations and permit the development of more effective immune-based strategies for brain tumor patients.

Resolution of uveitis.

Autoimmune uveitis is a sight-threatening, rare disease, potentially leading to blindness. Uveitis is a synonym for intraocular inflammation, presenting as various clinical phenotypes with different underlying immune responses in patients, whereas different animal models usually represent one certain clinical and immunological type of uveitis due to genetic uniformity and the method of disease induction. T cells recognizing intraocular antigens initiate the disease, recruiting inflammatory cells (granulocytes, monocytes/macrophages) to the eyes, which cause the damage of the tissue. The treatment of uveitis so far aims at downregulation of inflammation to protect the ocular tissues from damage, and at immunosuppression to stop fueling T cell reactivity. Uveitis is usually prevented by specific mechanisms of the ocular immune privilege and the blood-eye-barriers, but once the disease is induced, mechanisms of the immune privilege as well as a variety of novel regulatory features including new Treg cell populations and suppressive cytokines are induced to downregulate the ocular inflammation and T cell responses and to avoid relapses and chronicity. Here we describe mechanisms of regulation observed in experimental animal models as well as detected in studies with peripheral lymphocytes from patients.

Bone Marrow T-Cell Sequestration and Lymphopenia Accompanying Ischemic Stroke are Mediated by T-Cell S1P1 Loss

Abstract INTRODUCTION Sequestration of T-cells in bone marrow is a phenomenon recently characterized by our group in the setting of intracranial tumors. Our findings suggest that it is the intracranial location rather than tumor histology that elicits this phenotype. Sequestration is accompanied by lymphopenia and lymphoid organ contraction and is mediated by loss of the S1P1 receptor from the T-cell surface. We now reveal that this phenomenon is not only unique to brain tumors, but accompanies additional intracranial pathologies, most notably ischemic stroke. METHODS Blood, bone marrow, and spleens were collected from mice at day 2, 5, 7, or 14 following stroke via middle cerebral artery occlusion or sham surgery and analyzed by flow cytometry. T-cell S1P1 levels were assessed, along with T-cell counts in each compartment. S1P1 receptor stabilization was achieved with a knock-in model that inhibited receptor internalization. RESULTS Following stroke induction, T-cells accumulated in the bone marrow of injured mice. T-cell numbers peaked at day 7 poststroke before returning to normal levels by day 14. Bone marrow accumulation was accompanied by transient T-cell lymphopenia and splenic contraction following stroke. T-cells in the bone marrow yielded decreased levels of S1P1 on their surface. Conversely, mice with genetically stabilized T-cell S1P1 were protected against sequestration, lymphopenia, and splenic contraction following stroke. CONCLUSION Bone marrow T-cell sequestration occurs transiently following stroke and is mediated by the S1P-S1P1 axis. This may prove to be an adaptive mechanism to limit intracranial inflammation following an initial insult. Better understanding of this phenomenon may uncover a novel mechanism of immune privilege and allow for therapeutic modulation in the setting of stroke, brain tumor, and other types of intracranial injury.

Lymphopenia and bone marrow T cell sequestration accompanying stroke are mediated by T cell S1P1 loss

Abstract Sequestration of T cells in bone marrow is a phenomenon recently characterized by our group in the setting of intracranial tumors, regardless of histology. It is accompanied by lymphopenia and lymphoid organ regression and is mediated by loss of S1P1 receptor from the T cell surface. We now reveal that this phenomenon is not unique to tumors, but accompanies additional intracranial pathologies, most notably stroke. In this study, blood, bone marrow, and spleens were collected from mice at day 2, 5, 7 or 14 following stroke via middle cerebral artery occlusion (MCAO) or sham surgery and analyzed by flow cytometry. T cell S1P1 levels were assessed, as were T cell counts in each compartment. S1P1 receptor stabilization was achieved with a knock-in model in which receptor internalization is inhibited. Following stroke, T cells accumulated in the bone marrow of injured mice. T cell numbers peaked at day 7 post-stroke before returning to normal by day 14. Bone marrow accumulation was accompanied by transient lymphopenia and splenic involution following stroke. T cells in the bone marrow yielded decreased levels of S1P1 on their surface. Conversely, mice with genetically stabilized T cell S1P1 protected against sequestration, lymphopenia, and splenic regression following stroke. In conclusion, bone marrow T cell sequestration occurs transiently following stroke and is mediated by the S1P-S1P1 axis. This may be prove an adaptive mechanism to limit intracranial inflammation following an initial insult. Better understanding of this phenomenon may uncover a novel mechanism of immune privilege and allow for therapeutic modulation in the setting of stroke, brain tumor, and other types of intracranial injury.

Human dendritic cell sequestration onto the Necator americanus larval sheath during ex-sheathing: a possible mechanism for immune privilege.

Despite the profound health implications of Necator americanus infection in humans, many aspects of its interaction with the host immune system are poorly understood. Here we investigated the early events at the interface of N. americanus larvae (L3) and human dendritic cells (DCs). Our data show that co-culturing DCs and the larvae trigger ex-sheathing of hookworms rapidly where a majority of DCs are sequestered onto the larval sheath allowing the ex-sheathed larvae to migrate away unchallenged. Intriguingly, DCs show negligible interaction with the ex-sheathed larvae, alluding to differences between the surface chemistry of the larva and its sheath. Furthermore, blocking of two key C-type lectin receptors on DC surface (i.e. DC-SIGN and mannose receptor) resulted in inhibition of ex-sheathing process and DC sequestration, highlighting the importance of C-type lectins on DCs in the induction of the ex-sheathing. Analyses of DC phenotype and cytokine profile after co-culture with the N. americanus larvae showed an immature phenotype as evidenced by the low expression of the maturation markers and cytokines. These data provide new insights into early events at the interface of human DCs and N. americanus larvae and could explain how L3 evade immune recognition upon initial interaction with DCs.

Immunization with donor splenocytes in testis of recipient mice promotes induction of long-term survival of islet allografts transplanted in a conventional site (TRAN1P.920)

Abstract Immune responses are subdued in immune privileged sites, which provide a unique opportunity to prolong cellular allograft survival. We and others have previously shown that inducing tolerance of islet allografts transplanted in murine testes is easier than in conventional sites. However, it is not practical to transplant islets in human testes. Here we found that immunization of B6 recipients with irradiated Balb/C splenocytes through testicular injection, 7 and 14 days before islet transplantation under the kidney capsule, promotes long-term allograft survival induced by low doses of anti-CD40L (MR1) or CTLA4-Ig (0.1mg/day, days 0,2,4,6), with 75% islet allografts achieving long-term survival (>120 days). Same Ab treatment without the immunization failed to do so. Immunization without MR1 or CTLA4-Ig did not prolong allograft survival. We also found that islet transplantation under renal capsule of previously immunized recipients generates much less CD44+CD8+ memory T cells but induces more CD4+CD25+ Tregs than under renal capsule of unimmunized recipients. These Tregs exhibited donor-specific suppression of alloimmune responses in vitro. These findings reveal new mechanisms of immune privilege and may provide novel approaches to inducing long-term allograft survival or transplant tolerance in both immunologically privileged and unprivileged sites.

Mechanisms of Immune Privilege in the Posterior Eye

Immune privilege protects vital organs and their functions from the destructive interference of inflammation. Because the eye is easily accessible for surgical manipulation and for assessing and imaging the outcomes, the eye has been a major tissue for the study of immune privilege. Here, we focus on the immune regulatory mechanisms in the posterior eye, in part, because loss of immune privilege may contribute to development of certain retinal diseases in the aging population. We begin with a background in immune privilege and then focus on the select regulatory mechanisms that have been studied in the posterior eye. The review includes a description of the immunosuppressive environment, regulatory surface molecules expressed by cells in the eye, types of cells that participate in immune regulation and finally, discusses animal models of retinal laser injury in the context of mechanisms that overcome immune privilege.

FasL Expression on Human Nucleus Pulposus Cells Contributes to the Immune Privilege of Intervertebral Disc by Interacting with Immunocytes

The mechanisms of immune privilege in human nucleus pulposus (NP) remain unclear. Accumulating evidence indicates that Fas ligand (FasL) might play an important role in the immune privilege of the disc. We aimed for addressing the role of FasL expression in human intervertebral disc degeneration (IDD) and immune privilege in terms of the interaction between NP cells and immunocytes via the FasL-Fas machinery. We collected NP specimens from 20 patients with IDD as degenerative group and 8 normal cadaveric donors as control. FasL expression was detected by qRT-PCR, western blotting and flow cytometry (FCM). We also collected macrophages and CD8+ T cells from the peripheral blood of patients with IDD for co-cultures with NP cells. And macrophages and CD8+ T cells were harvested for apoptosis analysis by FCM after 2 days of co-cultures. We found that FasL expression in mRNA, protein and cellular resolutions demonstrated a significant decrease in degenerative group compared with normal control (p<0.05). FCM analysis found that human NP cells with increased FasL expression resulted in significantly increased apoptosis ratio of macrophages and CD8+ T cells. Our study demonstrated that FasL expression tends to decrease in degenerated discs and FasL plays an important role in human disc immune privilege, which might provide a novel target for the treatment strategies for IDD.

Ocular immune privilege

It has been over 60 years since the phrase immune privilege was used by Sir Peter Medawar to describe the lack of an immune response against allografts placed into the ocular microenvironment. Since then, we have come to understand that the mechanisms of ocular immune privilege include unique anatomical features of a blood barrier and a lack of direct lymphatic drainage. Also, we know that the ocular microenvironment is rich with immunosuppressive molecules that influence the activity of immune cells. Moreover, the placement of foreign antigen into the ocular microenvironment can induce a systemic form of tolerance to the foreign antigen called anterior chamber-associated immune deviation (ACAID). Many soluble immunomodulators are found in aqueous humour, and are a mixture of growth factors, cytokines, neuropeptides, and soluble receptors. This is a continuously growing list. The mechanisms of ocular immune privilege induce apoptosis, promote the production of anti-inflammatory cytokines, and mediate the activation of antigen-specific regulatory immunity. These mechanisms of immune privilege also attempt to impose themselves upon immunity within the uveitic eye. The adaptation of several anatomical and biochemical mechanisms to establish an immune privileged microenvironment within the eye makes the eye immunologically unique. It is a tissue site where we may learn how immunity is regulated in inflammation and at rest. Success in translating the lessons of ocular immune privilege to other tissues has the potential to drastically change the therapy and clinical outcomes of autoimmune diseases and allograft survival.

Su.98. Mechanisms of Immune Privilege in High Grade Cervical Dysplasia

Su.98. Mechanisms of Immune Privilege in High Grade Cervical Dysplasia

Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells

Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8(+) T cells but induces more Ag-specific CD4(+)CD25(+) regulatory T cells than in a conventional site. These CD4(+)CD25(+) cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8(+) T cells or deletion of CD25(+) T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.

Inducible immune regulation following autoimmune disease in the immune-privileged eye

The immune-privileged eye has the potential to induce regulatory immunity along with local mechanisms of immunosuppression. Rodent models of human autoimmune uveoretinitis [experimental autoimmune uveoretinitis (EAU)] recover without spontaneous recurrence of uveitis, which differs from uveitis in some humans. This raises the possibility that the mechanism of immune privilege in the rodent eye can reimpose itself during autoimmune uveoretinitis and re-establish tolerance to autoantigen. To investigate this possibility, we examined the spleens of EAU-recovered mice for regulatory immunity. We detected regulatory immunity when we adoptively transferred post-EAU spleen cells into other mice immunized for EAU. We could not detect this regulatory immunity in enucleated mice nor in naive mice. Moreover, unlike the mechanisms of anterior chamber-associated immune deviation, the suppression was only mediated by post-EAU CD4+ T cells, which required activation with autoantigen presented by post-EAU spleen antigen-presenting cells (APC). Our results demonstrate that when the immune-privileged ocular microenvironment recovers from an autoimmune response, it has influenced systemic immunity to retinal autoantigen by affecting APC and mediating induction of potential regulatory CD4+ T cells laying in wait in the post-EAU spleen for restimulation.

Mechanisms of immune privilege in the eye and hair follicle.

It has been recognized for over a century that the eye is endowed with remarkable properties that permit the long-term survival of foreign tumor and tissue grafts that are normally rejected at extraocular sites. This ocular immune privilege was originally attributed to a putative sequestration of antigens in the eye as a result of the conspicuous absence of intraocular lymphatic drainage channels. In the last 30 years, a sizeable body of information indicates that ocular immune privilege is a product of multiple anatomical, physiological, and immunoregulatory processes. Ocular tissues and fluids express a wide variety of anti-inflammatory and immunosuppressive molecules, including CD95L (FasL), transforming growth factor-beta, macrophage migration inhibitory factor, alpha-melanocyte-stimulating hormone, calcitonin gene-related peptide, somatostatin, and complement regulatory proteins. Moreover, antigens entering the anterior chamber of the eye evoke a unique form of immune deviation that culminates in the antigen-specific suppression of TH1 immune responses. Finally, the intraocular milieu contains both cell membrane and soluble factors that inhibit both the adaptive and innate immune systems. The hair follicle is also recognized for its immune privilege. Like the anterior chamber of the eye, it produces anti-inflammatory and immunosuppressive cytokines, such as transforming growth factor-beta and adrenocorticotrophic hormone. The cells of the hair follicle display limited expression of class Ia MHC molecules and, like cells that line the anterior chamber of the eye, are protected against CD8+ cytotoxic T lymphocyte attack. Gaining a better understanding of the immune privilege of the hair follicle may provide insights into the regulation and pathogenesis of immune-mediated diseases of the skin.

Regulated Expression and Ultrastructural Localization of Galectin-1, a Proapoptotic β-Galactoside-Binding Lectin, During Spermatogenesis in Rat Testis1

Galectin-1, a highly conserved beta-galactoside-binding protein, induces apoptosis of activated T cells and suppresses the development of autoimmunity and chronic inflammation. To gain insight regarding the potential role of galectin-1 as a novel mechanism of immune privilege, we investigated expression and ultrastructural localization of galectin-1 in rat testis. Galectin-1 expression was assessed by Western blot analysis and immunocytochemical localization in testes obtained from rats aged from 9 to 60 days. Expression of this carbohydrate-binding protein was developmentally regulated, and its immunolabeling exhibited a stage-specific pattern throughout the spermatogenic process. Immunogold staining using the anti-galectin-1 antibody revealed the typical Sertoli cell profile in the seminiferous epithelium, mainly at stages X-II. During spermiation (stages VI-VIII), a strong labeling was observed at the luminal pole of seminiferous epithelium, localized on apical stalks of Sertoli cells, on heads of mature spermatids, and on bodies of residual cytoplasm. Moreover, spermatozoa released into the lumen showed a strong immunostaining. Following spermiation (stage VIII), galectin-1 expression was restored at the basal portion of Sertoli cells and progressively spread out through the whole cells as differentiation of germinal cells proceeded. Immunoelectron microscopy confirmed distribution of galectin-1 in nuclei and cytoplasmic projections of Sertoli cells and on heads and tails of late spermatids and residual bodies. Surface localization of galectin-1 was evidenced in spermatozoa from caput epididymis. Thus, the regulated expression of galectin-1 during the spermatogenic cycle suggests a novel role for this immunosuppressive lectin in reproductive biology.

Apoptotic depletion of infiltrating mucosal lymphocytes associated with Fas ligand expression by Helicobacter pylori-infected gastric mucosal epithelium: human glandular stomach as a site of immune privilege.

H. pylori infection almost invariably results in chronic gastritis, but only a proportion of patients develops severe destruction of epithelial glandular structure or peptic ulcer. To confirm the recent data obtained in testis and eye, showing that Fas ligand is involved in the phenomenon of "immune privilege," expression of Fas receptor and its ligand of the stomach was investigated in a panel of gastric biopsies obtained from patients H. pylori-positive (N = 42) and with H. pylori-negative (N = 18) by two-color flow cytometry. The results show that membrane-bound Fas ligand protein is constitutively expressed on freshly isolated human gastric mucosal epithelium coupled with infiltrating lymphocytes. There was significant overexpression of Fas receptor and its ligand, and a higher frequency of apoptotic cell death detected by TUNEL in epithelium and infiltrating lymphocytes in H. pylori-infected patients. These findings suggest that involvement of Fas receptor and its ligand system contributes to some extent to mucosal damage in H. pylori-associated gastritis. However, the more specific findings are apoptotic depletion of invading mucosal lymphocytes associated with Fas ligand expression by gastric epithelium. These provide the first direct quantitative evidence to support Fas receptor counterattack and/or paracrine fratricide as a mechanism of immune privilege in vivo in the H. pylori-infected glandular stomach.

Experimental ocular toxoplasmosis induced in naive and preinfected mice by intracameral inoculation

We have developed a murine model to investigate the pathogenesis of acquired ocular toxoplasmosis. Tachyzoites of PLK strain of Toxoplasma gondii were intracamerally inoculated under anesthesia into the right eyes of naive or perorally preinfected C57BL/6 and MRL-MpJ mice. Clinical and histopathological observations of responses to intraocular infection were analyzed. Ocular inflammation from Toxoplasma gondii is dose-dependent in both strains of mice. After inoculation of fifty parasites, no evidence of inflammation was observed in the eyes of naive mice. The eyes of naive mice that received 500 or 5,000 parasites developed inflammatory changes by day 6 post challenge. By day 8, the changes progressed to moderate to severe intraocular inflammation. Histologic analysis of the ocular lesions demonstrated mononuclear cell infiltration and necrosis predominantly in the anterior segment of the eyes of the naive mice. Inoculation of 50,000 tachyzoites induced a destructive ocular inflammatory response and was uniformly lethal to the mice by approximately one week after challenge. In contrast, eyes from mice previously orally infected with Toxoplasma gondii and that received a 50 or 500 parasite intracameral challenge revealed no inflammation, but the eyes receiving 5,000 parasites demonstrated necrotic focal retinochoroiditis with vitreitis by day 8 after challenge. The murine model reproduces some features of ocular toxoplasmosis in humans and may be suitable for large-scale controlled studies of the pathogenesis and therapeutics of acquired ocular toxoplasmosis as well as for study of the mechanisms of immune privilege in the eye.

Acute anterior uveitis and spondyloarthropathies.

Acute anterior uveitis (AAU) is characterized by sudden-onset, mostly unilateral exacerbations of an inflammation of the iris and ciliary body. The duration of illness is short if the patient is treated with corticosteroids. Half of all patients with any type of anterior uveitis are HLA-B27-positive, and more than half of the B27-positive patients have spondyloarthropathy. Ophthalmologists should therefore refer all patients with AAU who are HLA-B27-positive to a rheumatologist. Because attacks of AAU are extremely painful and frightening, most spondyloarthropathy patients with AAU will seek out an ophthalmologist on their own. The anterior chamber of the eye and the joints are mesenchymal cavities that are cleaned by macrophages. Anterior chamber-associated immune deviation is the mechanism by which specific regulatory T cells normally produce sufficient transforming growth factor-beta to impair inflammatory reactions that might hamper vision. Another mechanism of immune privilege is Fas-ligand induced apoptosis. Because the cells of the anterior eye express Fas-ligand, infiltrating cells are apoptotically killed. Comparable mechanisms may occur at a lower level in joints. The cause of AAU and spondyloarthropathy is unknown. B27 is probably only responsible for one quarter of the pathogenesis, other non-B27 genetic factors for another quarter, and unknown exogenous factors for the remaining half. It is possible that Gram-negative bacteria such as Klebsiella or Yersinia are involved in the pathogenesis in a yet unknown way.

The Fas Counterattack In Vivo: Apoptotic Depletion of Tumor-Infiltrating Lymphocytes Associated with Fas Ligand Expression by Human Esophageal Carcinoma

Various cancer cell lines express Fas ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression has been demonstrated in several human malignancies in vivo. We sought to determine whether human esophageal carcinomas express FasL, and whether FasL expression is associated with increased apoptosis of tumor-infiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Using in situ hybridization and immunohistochemistry, respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n = 6; adenocarcinoma, n = 2). The Extent of FasL expression was variable, with both FasL-positive and FasL-negative neoplastic regions occurring within tumors. TIL were detected by immunohistochemical staining for the leukocyte common Ag, CD45. FasL expression was associated with a mean fourfold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6- to 12-fold, n = 6,p < 0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean twofold increase in detectable cell death among TIL in FasL-positive areas compared with FasL-negative areas (range 1.6- to 2.4-fold, n = 6, p < 0.05). In conclusion, we demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest Fas-mediated apoptotic depletion of TIL in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer.

Evidence for immune evasion by the ‘Fas counterattack’ in vivo in esophageal cancer

Background Various cancer cell lines express ligand (FasL) and can kill lymphoid cells by Fas-mediated apoptosis in vitro. FasL expression was first demonstrated by us in colonic cancer and confirmed by others in extraintestinal human malignancies in vivo. Relative resistance of cancer cells to Fas-mediated apoptosis coupled with the ability to make a pre-emptive strike against infiltrating lymphocytes (by expressing FasL and triggering apoptosis in lymphocytes) theoretically confers a privileged status on a cancer and has been termed the counterattack. The Aims of this study were to determine: (i) whether human esophageal carcinomas express FasL; and (ii) whether FasL expression is associated with increased apoptosis of tumorinfiltrating lymphocytes (TIL) in vivo, thereby contributing to the immune privilege of the tumor. Methods and Results Using in situ hybridization and immunohistochemistry respectively, FasL mRNA and protein were colocalized to neoplastic esophageal epithelial cells in all esophageal carcinomas (squamous, n=6; adenocarcinoma, n=2). Extent of FasL expression was variable, with both FasL-positive and -negative neoplastic regions occurring within individual tumors. TIL were detected by immunohistochemical staining for the leukocyte common antigen, CD45. FasL expression was associated with a mean four-fold depletion of TIL when compared with FasL-negative areas within the same tumors (range 1.6-12.0fold, n=6, p<0.05). Cell death of TIL was detected by dual staining of CD45 (immunohistochemistry) and DNA strand breaks (TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). There was a mean two-fold increase in detectable cell death among TIL in FasL-positive areas compared to FasL-negative areas (range 1.6-2.4-fold, n=6, p<0.05). In conclusion We demonstrate a statistically significant, quantitative reduction of TIL concomitant with significantly increased TIL apoptosis within FasL-expressing areas of esophageal tumors. Our findings suggest that Fas-mediated apoptotic depletion of TIL occurs in response to FasL expression by esophageal cancers, and provide the first direct, quantitative evidence to support the Fas counterattack as a mechanism of immune privilege in vivo in human cancer. Supported by a grant from the Health Research Board of Ireland.