Pyostomatitis Vegetans: Cellular Immune Profile and Expression of IL-6, IL-8 and TNF-α

The aim of this study was to investigate the cellular immune profile and the expression of IL-6, IL-8 and TNF-α in tissue biopsies of pyostomatitis vegetans (PV). Working hypothesis was that knowledge of the cellular immune profile and role of mediators such as IL-6, IL-8 AND TNF-alpha may contribute to a better understanding of the pathogenesis of this rare entity. Archival tissues from three patients with clinically and histologically confirmed PV were studied. Analysis of the immune profile of the cellular infiltrate and expression of IL-6 and IL-8 were evaluated by immunohistochemistry. ISH was performed to evaluate the expression of TNF-α. Biopsy tissues from erythema multiforme, recurrent aphthous stomatitis, lichen planus and normal buccal mucosa were analyzed as controls. All patients were affected by multiple mucosal ulcerations and yellow pustules mainly located in the vestibular, gingival and palatal mucosa. Histopathologically, all specimens showed ulcerated epithelium with characteristic intraepithelial and/or subepithelial microabscesses containing abundant eosinophils plus a mixed infiltrate composed of lymphocytes and neutrophils. Cellular immune profile of the inflammatory infiltrate revealed a predominance of T-lymphocytes, mainly of cytotoxic (CD3+/CD8+) phenotype, over B-cells. CD20+ B-lymphocytes were also identified to a lesser degree among the lymphoid cells present in the lamina propria. Overexpression of IL-6 and TNF-α was found in both epithelial and inflammatory mononuclear cells. IL-8 expression was shown in the mononuclear cells scattered among the inflammatory infiltrate. Similar findings of overexpression of IL-6, IL-8 and TNF-α were, however, found in control tissues. In PV lesions, the inflammatory infiltrate shows a predominance of cytotoxic lymphocytes. Expression of IL-6, IL-8 and TNF-α, although not specific to PV, appears up-regulated thus these cytokines would represent a suitable therapeutic target. However, the complexity of the cytokine network and their numerous functions require further studies in order to confirm our findings.     

Helper T-cell type 1 or type 2 function of xeno-MHC-restricted T-cell clones in a direct xenoantigen recognition

There have been several reports that xeno-MHC-restricted T-cells have a cytotoxic function through a direct xenoantigen recognition, but yet no report that they have a helper function. Previously we showed that both xeno-MHC-restricted CD4(+) and CD8(+) T-cells recognized xenoantigens directly in a mouse anti-rat combination. In this study, we investigated whether or not xeno-MHC-restricted T-cells had a helper function. Mouse T-cell clones recognizing rat antigens directly were derived from T-cell lines using the limiting dilution method. Phenotype, cytotoxic activity and cytokine production of these clones were analyzed by flow cytometry, 51Cr release assay and ELISA, respectively. Rat-MHC class I-restricted mouse CD8(+) T-cell clones showed a specific cytotoxic activity against rat antigens. One CD4(+) clone produced IL-4 and IL-10, and the other CD4(+) clone produced not T-helper (Th) 2 cytokine but TNF-alpha. Our results suggested that xeno-MHC class I-restricted CD8(+) T-cells should have a cytotoxic function, and xeno-MHC class II-restricted CD4(+) T-cells should have either Th1 or Th2 function.     

Alterations in T-Cell Subset Frequency in Peripheral Blood in Obesity

Background: Obesity affects the regulation of immune and inflammatory responses. This study characterizes differences in peripheral blood lymphocyte phenotype in obese humans. Methods: Frequencies of lymphocyte subsets among peripheral blood mononuclear cells were compared between 10 obese (BMI ≥35) and 10 lean subjects, as determined by antibodies directed against cluster differentiation (CD) markers. Results: Obese patients demonstrated an increased frequency of CD3+CD4+ T-cells (mean difference 12%, P=0.004), a decreased frequency of CD3+CD8+ T-cells (mean difference 9.4%, P=0.016) and an increased frequency of CD3+CD8+CD95+ T-cells (mean difference 13.3%, P=0.032). No other differences among T-cell or monocyte subsets were noted. Conclusions: Obesity is associated with alterations in frequencies of peripheral CD4+ and CD8+ T-cells and aberrations in the expression of CD95 among CD8+ T-cells. These data suggest both CD4+ and CD8+ T-cell compartments, as well as the regulation of CD95 expression on CD8+ T-cells, as targets for further study into obesity's effects on the immune system.     

Inflammatory infiltrate in prostatic hyperplasia--evidence of a host response to intraprostatic spermatozoa?

The inflammatory infiltrate was characterised in 20 samples of prostatic hyperplasia using cell surface specific monoclonal antibodies. The infiltrate was composed predominantly of CD-3+ T-lymphocytes. The distribution of the T-lymphocyte subsets (CD-4 and CD-8) between the epithelial and stromal components of the gland was significantly different, with most of the intra-epithelial infiltrate being CD8+, T cytotoxic/suppressor cells. This may be a necessary immunological barrier, as in the epididymis, to prevent the development of autoimmunisation to sperm antigens. Large numbers of CD4+ T helper/inducer cells and significant numbers of CD-11c+ macrophages were demonstrated in the extra-glandular stromal tissues, suggesting a delayed type hypersensitivity reaction. Intraprostatic spermatozoa have been demonstrated recently and we have confirmed this finding on post mortem specimens. It is postulated that the presence of an intra-epithelial cytotoxic barrier and the marked stromal infiltrate may be due, in part, to the intermittent presence of spermatozoa within the prostate.     

Characterisation of the cellular infiltrate in the foreign body granuloma of textile meshes with its impact on collagen deposition

Purpose

As part of the foreign body reaction, mesh filaments are surrounded by an infiltrate of inflammatory cells. Though macrophages are considered as being predominant, little is known about the origin of other cells.

Methods

On 55 meshes explanted from humans, we characterised the cells in the inflammatory infiltrate of the granuloma by immunohistochemistry using 10 cellular markers: CD3+ lymphocytes, CD4+ T helper cells, CD8+ cytotoxic T cells, CD20+ B lymphocytes, CD34+ stem cells, CD45R0+ leucocytes, CD68+ macrophages, Mib1 for proliferation, Vimentin for mesenchymal origin, and Desmin for myocytes. Collagen deposits were analysed after staining with Sirius Red.

Results

More than 80 % of the cells in the infiltrate showed a positive expression of CD68, CD8, CD45R0 and Vimentin. CD4 and Desmin were seen in 30–80 % of the cells, unaffected by material or time. A score summarising the expression of all markers positively correlated significantly with an increased percentage of collagen type III (green) in the mesh wound. The analysis of collagen deposits was only affected to a small degree by size of area for investigation.

Conclusions

At the vicinity of the mesh filaments, the accumulated inflammatory cells represent a mixture of cells of various origins. The high expression of at least four markers requires co-expression of different surface markers and thus confirms the existence of multiple transition forms instead of dominance of just macrophages. This offers new options for interventions to attenuate the inflammatory reaction of mesh implants.     

CD8+ T-cell maturation following lung transplantation: the differential impact of CMV and acute rejection

Studies on persistent viral infections demonstrate that CD8(+) T-cells differentiate along distinct pathways following chronic antigen exposure; however the effect of stimulation with non-viral chronic antigens is poorly described. We assessed the contributions that the presence of an allograft or cytomegalovirus (CMV) has on the post-thymic differentiation of CD8(+) T-cells in both the blood and lung allograft in patients undergoing lung transplantation. CD28 expression on blood CD8(+) T-cells was reduced in CMV seropositive patients, and was further reduced following acute episodes of CMV reactivation. These viral-associated changes in phenotype were not seen in CD8(+) T-cells isolated from the lung allograft where a different pattern of CD28 expression was observed. Following transplantation there was a progressive reduction in CD28 expression on BAL CD8(+) T-cells. In contrast to what was observed in peripheral blood, reduced CD28 expression on BAL CD8(+) T-cells was associated with a reduced gamma-IFN production. Furthermore, a high proportion of CD28-CD8(+) T-cells in the BAL was associated with fewer episodes of acute allograft rejection. An expanded CD28-CD8(+) T-cell subset, with reduced function, within the lung allograft may have important prognostic implications in lung transplant recipients.     

The immunophenotype and activation status of the lymphocytic infiltrate in human breast cancers, the role of the major histocompatibility complex in cell-mediated immune mechanisms, and their association with prognostic indicators

BACKGROUND: The aims of this study were to characterize, phenotypically, the immune infiltrate in human breast cancers, to assess the activation status of tumor-infiltrating lymphocytes (TIL), and to define the association of these findings with established prognostic indicators. METHODS: Immunohistochemistry was performed on frozen sections of 60 primary breast cancers by use of monoclonal antibodies to T lymphocytes (CD3), T-helper cells (CD4), cytotoxic T-cells (CD8), natural killer cells (CD56), interleukin-2 receptors (IL-2R), and major histocompatibility (MHC) class I antigen (HLA-ABC) and MHC class II antigen (HLA-DR). RESULTS: All tumors stained positive for CD3, CD4 and CD8, but with marked variation in the intensity of the infiltrate. In tumors with a moderate infiltrate of TIL, there was a trend toward a greater representation of T-helper cells. However, as the intensity of TIL increased, there was a decline in the proportion of T-helper cells and a concomitant rise in the relative proportion of cytotoxic T cells. There was a relative paucity of natural killer cells. A significant association was found between the intensity of TIL and the number of positive nodes (P=.02) and the intensity of the infiltrate of both T-helper cells and cytotoxic T cells with ER expression (P=.03 and.05, respectively). Most tumors stained positive for IL-2R. The expression of IL-2R was associated with the intensity of TIL (P<.0001), T-helper cells (P<.002), cytotoxic T cells (P=.01) and natural killer cells (P=0.04), and also with the degree of lymph node positivity (P=.02) and histologic tumor grade (P=.05). MHC class II expression was variable, and a large proportion of the tumors showed limited expression in individual cancer cells. There was an association between the expression of HLA-DR in tumor cells and the activation status of TIL (P=.03). CONCLUSION: An immune infiltrate is an invariable finding in breast cancers, and the intensity of the infiltrate is greater in node positive tumors. Additionally, TIL may well be activated, albeit partially, in most tumors, suggesting that cell-mediated immune mechanisms are functionally intact.     

Immunopathology of graft-versus-host disease in the upper gastrointestinal tract

Class I and class II (HLA-DR, DP and DQ) MHC antigen expression and the phenotypic nature of the inflammatory infiltrate in gastric and duodenal biopsies in bone marrow transplantation patients with and without graft-versus-host disease were investigated. Increased expression of class I (P less than 0.016) and class II (HLA-DR, DP) antigens (P less than 0.002) was associated with GVHD. The epithelium in two GVHD-positive biopsies was HLA-DP-positive and HLA-DR-negative. None of the tissues expressed HLA-DQ. Association between MHC antigen expression and phenotype of infiltrating cell was then examined. The majority of GVHD biopsies showed an infiltrate composed of CD4+ cells and CD8+ cells. However, the two DP+, DR- biopsies were associated exclusively with CD8+ intraepithelial cells, suggesting sequential events in GVHD, with CD8+ cells infiltrating tissue first associated with HLA-DP expressions, followed by accumulation of CD4+ as well as CD8+ cells in association with expression of HLA-DR.     

CD4+CD25+ T-cell populations expressing CD134 and GITR are associated with disease activity in patients with Wegener's granulomatosis

Background. An increased CD4⁺ CD25⁺ T-cell population is observedin Wegener's granulomatosis (WG). This T-cell population isnot well characterized yet and their contribution to the diseasepathogenesis remains obscure. Methods. Thirty patients with WG and 18 healthy controls (HC)were included in this study. The disease activity and extensionwere measured by the Birmingham Vasculitis Activity Score (BVAS)and the Disease Extent Index (DEI). Lymphocytes from peripheralblood were analysed by FACS for the expression of CD4, CD25,CD134 and GITR. Cytokine expression in these subsets was assessedtoo. Nasal, lung and renal tissues from WG patients were immunohistochemicallystained for CD3 and CD134. Results. The percentage of CD134⁺ as well as GITR⁺ expressingCD4⁺CD25⁺ lymphocytes was increased in patients as comparedto HC (37 ± 12% versus 27 ± 8%, P = 0.005; 18± 9% versus 11 ± 6%, P = 0.003). The expressionof CD134 and GITR showed a significant correlation with diseaseactivity (r = 0.5, P = 0.009; r = 0.55, P = 0.001). Most ofthese displayed the phenotype of effector memory T-cells (94± 4% and 91 ± 6%). CD134 T-cells were found intissues affected by WG. Conclusions. CD4⁺CD25⁺ effector memory T-cells expressing CD134and GITR seem to play a role in disease mechanisms, as suggestedby their close association with disease activity and their participationin inflammatory process.     

Immunopathology of primary hypophysitis: implications for pathogenesis

The etiology of primary hypophysitis is still not fully elucidated. Histologically, primary hypophysitis includes three different main subtypes: lymphocytic (LYH), granulomatous (GRH), and xanthomatous (XH) hypophysitis. Clinical and laboratory findings suggest an autoimmune basis in primary hypophysitis. Controversy still exists about the composition of the inflammatory infiltrate and the relevant immunopathogenic effector mechanisms. Therefore, 21 cases of primary hypophysitis of different subtypes were analyzed with respect to the expression of lymphocyte and macrophage antigens as well as MHC class I and II molecules of the inflammatory infiltrate and the resident pituitary acinar cells. Lymphocyte infiltration in LYH (n = 15), but also in GRH (n = 4) and XH (n = 2), mainly consisted of T cells, while B cells were rare. Independent from the histopathologic subtype, T cell subsets showed equal ratios of CD4+ to CD8+ T cells. Highest numbers of activated CD8+ T cells were observed in LYH presenting during pregnancy, surrounding or even infiltrating preserved pituitary acinar cells. Moreover, an increased rate of activated CD8+ T cells correlated with a shorter duration of clinical symptoms. In LYH, aberrant expression of MHC class II antigens as well as overexpression of MHC class I molecules on pituitary cells were observed. Independent of the histologic subtype, macrophages mostly expressed markers of chronic activation and showed MHC class II positivity. LYH, GRH, and XH, although heterogeneous in their histologic appearance and in age distribution, exhibit a similar if not identical immunohistologic profile. It is highly likely that direct T cell-mediated cytotoxicity through CD8+ T cells, with the initial help of CD4+ T cells, is pivotal in the pathogenesis of primary hypophysitis, implicating a target autoantigen expressed by pituitary cells.     

Increased nitric oxide production by T- and B-cells in idiopathic nephrotic syndrome

The pathogenesis of idiopathic nephrotic syndrome (INS) remains unclear. To study the role of nitric oxide (NO) in INS, we measured intracellular NO produced by T- and B-cells using a novel fluorescent indicator. Twelve children with INS (mean age 7.3 years; group A-1: in relapse, group A-2: in remission) were enrolled in the study together with 16 children with other renal diseases (9.5 years; group B) and 42 healthy control children (7.7 years; group C). The amount of NO produced by CD3+ cells (T-cells) and CD19+ cells (B-cells) and of plasma NO_x was measured by flow cytometry and colorimetry, respectively. The average amount of NO produced by CD3+ and CD19+ cells in group A-1 subjects was significantly higher than that produced by these cells in group A-2 and B patients and the healthy controls (group C), respectively (P < 0.01), and it decreased after the patients achieved remission (P < 0.01). Plasma NO_x levels in group A-1 patients was also highest among the different groups (P < 0.01). There were no significant differences in intracellular NO and plasma NO_x among group A-2, B, and C subjects (P > 0.05). A significant correlation between plasma NO_x and urinary protein excretion was found only in group A patients and not in group B patients. We conclude that an aberrant immune system may exist not only in T-cells but also in B-cells, and NO may play some role in INS.     

Granzyme B-mediated death of pancreatic beta-cells requires the proapoptotic BH3-only molecule bid

Perforin-deficient NOD mice are protected from diabetes, suggesting that cytotoxic granule contents of CD8(+) T-cells have a significant role in killing beta-cells. Despite this, cytotoxic granule effects on human or mouse pancreatic islets have not been reported. We tested the susceptibility of human and mouse islet cells to purified recombinant perforin and granzyme B and measured apoptotic death using a number of assays. Perforin and granzyme B impaired insulin secretion from islet cells, and this was accompanied by cytochrome c release, caspase activation, and DNA fragmentation. Granzyme B-mediated apoptotic changes only occurred in the presence of perforin. When compared with hemopoietic cells, traditionally used as targets to measure cytotoxic T-cell function in vitro, islet cells were relatively resistant to perforin and granzyme B. Inhibition of caspases prevented DNA fragmentation but not cytochrome c release, indicating that mitochondrial disruption due to granzyme B is independent of caspase activation. Consistent with this, islet cells from mice deficient in the BH3-only protein Bid were resistant to cytochrome c release and were protected from apoptosis after exposure to perforin/granzyme B. Our data suggest that Bid cleavage by granzyme B precedes mitochondrial disruption and apoptosis in pancreatic islets.     

The nature of the myenteric infiltrate in achalasia: an immunohistochemical analysis

Achalasia is an esophageal motor disorder characterized by abnormal relaxation of the lower esophageal sphincter and absence of progressive peristalsis in the esophageal body. Previous studies evaluating esophagomyotomy and esophageal resection specimens have shown the presence of myenteric inflammation to be a consistent and early pathologic change in patients with achalasia. Thus, the goal of this study was to determine the immunohistochemical characteristics of the inflammatory infiltrate within the myenteric plexus in patients with clinically early and end-stage achalasia. Using formalin-fixed tissue, we analyzed the immunohistochemical features of the myenteric lymphocytes using antibodies that recognize B cells (CD20), T cells (CD3), T cell subsets (CD8), and the activation state of T cell subpopulations (TIA-1 and granzyme B) in nine patients with clinically early achalasia who underwent esophagomyotomy and 13 patients with clinically endstage achalasia who underwent esophageal resection. The myenteric infiltrate in all nine esophagomyotomy specimens was composed predominantly of T cells (CD3-positive), the majority of which also stained for CD8. In five of nine specimens, the majority of CD8-positive cells stained for TIA-1. In the esophageal resection specimens, the myenteric infiltrate was composed predominantly of CD3-positive T cells in seven of 13 cases. In three cases, there was a predominance of CD20-positive B cells, and in the remaining three cases there were relatively equal numbers of T and B cells. In eight of 13 cases, the majority of T cells stained for CD8. TIA-1 immunoreactivity was found in the majority of CD8-positive cells in nine of 13 cases. In all esophagomyotomy and esophageal resection specimens studied, rare granzyme B-positive cells were detected. In conclusion, the majority of myenteric inflammatory cells in patients with achalasia are CD3-positive T cells, most of which are also CD8-positive, although the relative percentage of such cells appears to decrease with disease progression. Furthermore, many of the CD3-positive/CD8-positive myenteric lymphocytes also express TIA-1, suggesting they are resting or activated cytotoxic T cells. The immunohistochemical demonstration of granzyme B in a subpopulation of these cells supports the contention that achalasia is an immune-mediated disease, although the inciting antigen remains an enigma.     

Studies on autoimmunity for T-cell-mediated beta-cell destruction. Distinct difference in beta-cell destruction between CD4+ and CD8+ T-cell clones derived from lymphocytes infiltrating the islets of NOD mice.

Six CD4+ and three CD8+ islet-reactive T-cell clones were established from lymphocytes infiltrating the pancreatic islets of NOD mice. Two of six CD4+ T-cell clones responded to NOD islet cells only, not to spleen cells. The remaining four clones responded to both islet cells and spleen cells from NOD mice, but not to cells from other strains of mice, including SJL, C3H, C57BL/6, and DBA/2 mice. None of the CD4+ T-cell clones had a cytotoxic effect on the cultured islet cells. On the other hand, all of the CD8+ T-cell clones showed both a proliferative response and a cytotoxic effect on the islet cells, with the restriction of MHC class I H-2Db. Electron microscopic studies revealed that islet-specific CD4+ T-cells attached closely to islet cells but did not destroy them. In contrast, CD8+ T-cell clones showed pseudopodialike protrusions into beta-cells, but not alpha- or delta-cells, leading to selective destruction of beta-cells. CD8+ CTLs could not be isolated from islets of NOD mice less than 10 wk of age, even if the islets showed lymphocytic infiltration, whereas CD4+ T-cells could be isolated from islets of these younger NOD mice. On the basis of these observations, we concluded that CD4+ and CD8+ T-cells interact differently with beta-cells at different stages in T-cell--mediated beta-cell destruction. CD4+ T-cells may secrete cytokines, which in turn activate effector cell populations, whereas CD8+ T-cells may act as a final effector directly involved in beta-cell destruction.     

Wegener's granulomatosis: inflammatory cells and markers of repair and fibrosis in renal biopsies--a clinicopathological study.

OBJECTIVE: This study aimed to quantitate inflammatory cells in renal biopsies from patients with Wegener's granulomatosis (WG) and to identify cells participating in early fibrogenesis. The goal was to determine whether these cells correlated with the severity of renal disease and whether their presence had a bearing on renal prognosis. MATERIAL AND METHODS: Sixty-one patients with WG who had a renal biopsy taken at the time of diagnosis were included in the study. Immunostaining with monoclonal antibodies towards macrophages (CD68), T- and B-lymphocytes, alpha-smooth muscle actin (alpha-SMA) and vimentin was done. RESULTS: The dominating intraglomerular leucocytes were macrophages (29.9 +/- 15 cells/glomerular cross-section) and to a lesser extent T-cells (2.57 +/- 1.8 cells/glomerular cross-section). No B-lymphocytes were detected in the glomeruli. More than two-thirds of the T-cells were CD8+ (cytotoxic) cells. Macrophages and T-lymphocytes were distributed equally in the renal interstitium and were numerous around crescentic glomeruli. Glomerular and interstitial macrophages and interstitial T-cells correlated significantly with serum (S-) creatinine at the time of biopsy but not after 1 year. S-creatinine at the time of biopsy and after 1 year differed significantly among the three levels of interstitial alpha-SMA staining. S-creatinine at biopsy was highest when tubular vimentin staining was strongest, and tubular vimentin staining was strongest in patients with acute tubular damage. CONCLUSIONS: Evidence was found for a cellular type IV immune response in WG, with CD8+ T-lymphocytes and macrophages dominating the cellular infiltrate. The detection of interstitial alpha-SMA, probably staining myofibroblasts implicated in renal fibrogenesis, indicated a low glomerular filtration rate 1 year after renal biopsy.     

Changes in the proportions of peripheral blood lymphocytes in patients with worn implants

We compared the peripheral blood and periprosthetic tissues of 53 patients at revision arthroplasty with those of 30 patients at primary arthroplasty to determine whether there is a systemic difference in lymphocytes in patients with worn hip implants. The absolute number and relative proportion of lymphocytes bearing CD2, CD3, CD4, CD8, CD16, CD19, HLA-DR, kappa and lambda antigens were compared with the levels of IL-1beta, IL-6 and PGE2 in the pseudosynovial membrane as well as with a semiquantitative estimate of metal and polyethylene particles, necrosis and chronic inflammation and the total concentration of metals within the periprosthetic tissues. There was a significant increase in the relative proportion of CD2-positive T-cells and CD16-positive natural killer cells in the peripheral blood at revision arthroplasty compared with primary arthroplasty and an increased proportion of CD8-positive T-cells and a decreased ratio of CD4 to CD8 (helper inducer/suppressor cytotoxic cells). Three control patients, who went on to have revision surgery, had values at primary arthroplasty which were similar to those of patients at the time of revision surgery. These differences did not correlate with the local concentration of metal, plastic or cement or inflammatory response or the type of prosthesis. An inverse correlation was noted between the necrosis in the periprosthetic tissue and both the local production of IL-6 and the absolute numbers of T-cells in peripheral blood. We conclude that there may be several cell-mediated systemic immune responses to aseptic loosening, at least one of which may be directly related to events in the periprosthetic tissues. We cannot exclude the possibility that the changes in the proportion of CD8-positive cells reflected a predisposition, rather than a reaction, to loosening of the implant.     

An immunohistopathological characterisation of mixed non-seminomatous germ cell tumors

Summary Immunohistological techniques were used to characterise inflammatory cell infiltrates in mixed germ cell tumours. The distribution of these infiltrates was much more variable than in pure seminomas but could not be related accurately to any particular tumour type. There were approximately equal numbers of B and T cells in these areas and helper/inducer T cells were more common than suppressor/cytotoxic lymphocytes. Within these areas of inflammatory cells, the subtype composition was similar to that seen in pure seminomas.     

The role of systemic inflammatory cells in meningiomas

The aim of this review is to describe the inflammatory systemic cell infiltrate and its role in pathophysiology and prognostic implications of meningiomas. Articles from PubMed describing inflammation and immune cells in meningioma were systematically selected and reviewed. Infiltrating inflammatory cells are common in meningiomas and correlate with tumor behavior and peritumoral edema. The immune cell infiltrate mainly comprised macrophages, CD4?+?T cells of the Th1 and Th2 subtype, CD8?+?cytotoxic T cells, mast cells, and to a lesser degree B cells. The polarization of macrophages to M1 or M2 states, as well as the differentiation of T-helper cells to Th1 or Th2 subsets, is of prognostic value, but whether or not the presence of macrophages is associated with the degree of malignancy of the tumor is controversial. The best documented immunosuppressive and tumor-promoting mechanism is the expression of programmed cell death protein 1 (PD-1/PD-1L) which is found on both tumor cells and tumor-infiltrating immune cells. The immune cell infiltration varies between different meningiomas. It contributes to a microenvironment with potential contradictory effects on tumor growth and edema. The immune mechanisms are potential therapeutic targets provided that their effects can be comprehensively understood.

     

Simultaneous Detection of Circulating Autoreactive CD8+ T-Cells Specific for Different Islet Cell�CAssociated Epitopes Using Combinatorial MHC Multimers

OBJECTIVE

Type 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8⁺ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8⁺ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive.

RESEARCH DESIGN AND METHODS

Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B_10–18, prepro-insulin (PPI)_15–24, islet antigen (IA)-2_797–805, GAD65_114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)_265–273, and prepro islet amyloid polypeptide (ppIAPP)_5–13–specific CD8⁺ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients.

RESULTS

Using this kit, islet autoreactive CD8⁺ T-cells recognizing insulin B_10–18, IA-2_797–805, and IGRP_265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI_15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome.

CONCLUSIONS

A kit was developed that allows simultaneous detection of CD8⁺ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.Type 1 diabetes results from a selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. It is becoming increasingly clear that islet epitope–specific CD8⁺ T-cells play a pivotal role in the destruction process and constitute a significant portion of insulitis (1,2). In accordance, nonobese diabetic mice lacking the expression of major histocompatibility complex (MHC) class I are resistant to autoimmune diabetes (3,4), whereas HLA-A2 transgenic nonobese diabetic mice develop accelerated disease (5). Additionally, transfer of CD8⁺ T-cell clones resulted in transfer of type 1 diabetes (6,7). Thus, detection and monitoring of specific CD8⁺ T-cells may provide a valuable tool to assess the disease activity.Islet cell transplantation has considerable potential as a cure for type 1 diabetes (8). Several groups have reported short-term success, using different islet isolation and immunosuppressive regimens (9–12), but long-term insulin independence is rare (13). The rationale behind transplantation of islet cells is replenishment of destructed cells. Yet, as the insulin-producing cells were destructed by an autoimmune response, islet cell transplantation could also result in reactivation of the autoimmune response. Recently, we have shown that proliferation of CD4⁺ T-cells specific for GAD and IA-2 in patients who underwent islet cell transplantation is associated with clinical outcome (14). Yet, ultimately, the destruction of β-cells is likely to be caused by CD8 T-cells.The epitopes recognized by the diabetes-specific human autoreactive CD8⁺ T-cells are primarily derived from β-cell antigens, most importantly (pre-)(pro-)insulin. Previously, we showed that the presence of CD8⁺ T-cells reactive to the naturally processed insulin–peptide B_10–18 in HLA-A2 correlated with islet cell destruction (15). Recently, another important epitope that was uncovered as the signal peptide of pro-insulin was shown to contain a glucose-regulated CD8⁺ T-cell epitope (prepro-insulin [PPI]_15–24) (16), but many other epitopes derived from insulin and a range of other β-cell–derived antigens, such as GAD65 (17), islet antigen (IA)-2 (18), islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) (19,20), and prepro islet amyloid polypeptide (ppIAPP) (21), have been reported (rev. in 22). Ideally, monitoring for the presence of CD8⁺ T-cells reactive to all of the above-mentioned epitopes simultaneously would be desired, posing considerable constraints on blood volumes accessible for monitoring of islet autoimmunity with conventional immune assays.Currently, monitoring of CD8⁺ T-cells reactive to β-cell–derived antigens requires staining of a large number of, usually fresh, cells with HLA tetramers loaded with a single peptide, or in vitro culture for functional immune assays (proliferation, cytokine production [ELISPOT]). Monitoring multiple epitope-specific CD8⁺ T-cell populations by conventional tetramer technology is generally impossible because of the scarcity of material. Furthermore, detection of islet autoreactive T-cells is hampered by their low precursor frequencies in circulation (23,24), low T-cell receptor (TCR) avidity (15), potentially low binding affinity of peptide epitopes to HLA (25), a wide range of candidate islet epitopes (22), and the existence of regulatory T-cells (26,27).Therefore, we used the recently described combinatorial quantum dot (Qdot) technique (28) to simultaneously detect CD8⁺ T-cells specific for six different β-cell–derived antigens, a naturally occurring HLA-A2 derived peptide, and a mix of viral epitopes in HLA-A2 multimers. Using peripheral blood cells from recent-onset type 1 diabetic patients, their siblings, and control subjects, we validated this technique and established the specificity of these stainings. Subsequently, we monitored the presence of reactive CD8⁺ T-cells before and at several time points after clinical islet cell transplantation. Altogether, we developed a high-throughput and relatively sensitive and specific Diab-Q-kit, allowing simultaneous detection of autoreactive CD8⁺ T-cells to multiple islet epitopes, which is applicable to small volumes of stored blood samples to allow screening in multicenter immune intervention trials.