Intolerance to cereals is not specific for coeliac disease

BACKGROUND: Abdominal complaints after ingestion of cereals are not uncommon. We assessed how reliable such a history is as a marker for the presence of overt coeliac disease, and whether we should also take into account latent coeliac disease and cereal allergy. METHODS: The study group comprised 93 consecutive adults from health centres spontaneously reporting abdominal symptoms after consumption of cereals. Small bowel mucosal morphology, CD3+, alphabeta+ and gammadelta+ intraepithelial lymphocytes (IELs), HLA DQ alleles and serum IgA-class endomysial (EmA), tissue transglutaminase (tTg) and gliadin (AGA) antibodies were determined. Skin prick and patch tests and serum radioallergosorbent tests for cereals were carried out. Thirty non-coeliac adults served as biopsy controls. RESULTS: Eight (9%) patients had coeliac disease and one mild partial villous atrophy. Altogether 17 had an increased density of gamma delta+ IELs without atrophy. However, only seven (8%) showed evidence of latent coeliac disease, i.e. both an increase in gammadelta+ IELs and the presence of coeliac disease-type HLA. One or more of the allergy tests for cereals was positive in 19; 9 adopted a gluten-free diet and abdominal symptoms were alleviated in all. In non-coeliac patients, serum EmA and tTg tests were negative in all, whereas AGA was seen in 40%. CONCLUSIONS: Intolerance to cereals is not a specific sign of overt or latent coeliac disease. All experimental dietary interventions before proper diagnosis of coeliac disease are therefore to be discouraged. Allergy to cereals, on the other hand, should be considered even in adults.     

Intraepithelial gammadelta+ lymphocytes: a comparative study between celiac disease, small intestinal bacterial overgrowth, and irritable bowel syndrome

BACKGROUND: Intraepithelial lymphocytes (IELs) phenotyping has emerged as a useful test in intestinal pathology. In celiac disease (CD), a permanent and marked increase of gammadelta+ IELs has been described. However, there is a lack of knowledge about this peculiar IELs population in other intestinal pathologies. AIM: To analyze the percentage of IELs, specifically gammadelta+ IELs subset, present in duodenal mucosa biopsies from patients with CD and compare it with those obtained from patients with small intestinal bacterial overgrowth (SIBO) or irritable bowel syndrome (IBS). METHODS: Twelve patients with untreated CD, 8 patients with SIBO, and 10 patients with diarrhea-predominant IBS were evaluated. All subjects underwent upper endoscopy for mucosal biopsy and jejunal aspirate. From 2 small bowel biopsies, intraepithelial cells were isolated and labeled with the following monoclonal antibodies CD103-PE (phycoerythrin), CD3-FITC (fluoresecein isothio-cynate), CD-7R-PE, CD45RO-APC (allophycocyanin), and TcR gammadelta-FITC. Flow cytometry analysis was performed on a standard FACScan. Total and IELs subset counts were expressed as percentage. RESULTS: Mean total IELs percentage was 16.7+/-6% in IBS, 25.4+/-17% in SIBO, and 26+/-13% in CD patients (P=0.2). CD and SIBO patients, had significantly higher percentages of gammadelta+ IELs (15.7+/-13% and 14.6+/-8%) than IBS subjects (4.1+/-2.5%, P<0.05). There was no difference between CD and SIBO (P=0.6). CONCLUSIONS: An increased density of gammadelta+ IELs is typical, but not specific for CD. A similar increase was observed in subjects with SIBO. Our findings suggest that this unique T-cell population might have a key role against intestinal bacterial infections.     

Wheat starch-containing gluten-free flour products in the treatment of coeliac disease and dermatitis herpetiformis. A long-term follow-up study

BACKGROUND: We investigated whether wheat starch-based gluten-free products are safe in the treatment of gluten intolerance. METHODS: The study involved 41 children and adults with coeliac disease and 11 adults with dermatitis herpetiformis adhering to a gluten-free diet for 8 years on average. Thirty-five newly diagnosed coeliac patients at diagnosis and 6 to 24 months after the start of a gluten-free diet and 27 non-coeliac patients with dyspepsia were investigated for comparison. Daily dietary gluten and wheat starch intake were calculated. Small-bowel mucosal villous architecture, CD3+, alphabeta+, and gammadelta+ intraepithelial lymphocytes, mucosal HLA-DR expression, and serum endomysial, reticulin, and gliadin antibodies were investigated. RESULTS: Forty of 52 long-term-treated patients adhered to a strict wheat starch-based diet and 6 to a strict naturally gluten-free diet; 6 patients had dietary lapses. In the 46 patients on a strict diet the villous architecture, enterocyte height, and density of alphabeta+ intraepithelial lymphocytes were similar to those in non-coeliac subjects and better than in short-term-treated coeliac patients. The density of gammadelta(+)cells was higher, but they seemed to decrease over time with the gluten-free diet. Wheat starch-based gluten-free flour products did not cause aberrant upregulation of mucosal HLA-DR. The mucosal integrity was not dependent on the daily intake of wheat starch in all patients on a strict diet, whereas two of the six patients with dietary lapses had villous atrophy and positive serology. CONCLUSION: Wheat starch-based gluten-free flour products were not harmful in the treatment of coeliac disease and dermatitis herpetiformis.     

Villous tip intraepithelial lymphocytes as markers of early-stage coeliac disease

BACKGROUND: An investigation was conducted to determine whether the density of small-intestinal villous tip intraepithelial lymphocytes would be of value in clinical practice in uncovering early-stage coeliac disease. METHODS: Villous tip, CD3+ and gammadelta+ intraepithelial lymphocytes were counted in patients with definite early-stage coeliac disease without villous atrophy, in classic coeliac disease with manifest mucosal lesion and in non-coeliac controls with normal mucosal structure. Villous tip analysis was made of haematoxylin-eosin specimens and CD3+ and gammadelta+ of immunohistochemical stainings from frozen samples. RESULTS: The villous tip intraepithelial lymphocyte count was statistically significantly higher in patients with early-stage coeliac disease than in non-coeliac controls. The sensitivity of this method to detect untreated coeliac disease with normal villous architecture was 0.84; the specificity was 0.88. This method proved superior to CD3+ analysis and was at least as good as gammadelta+ analysis in detecting early-stage coeliac disease. In detecting classic coeliac disease, villous tip analysis also reached a higher sensitivity than CD3+ and gammadelta+ cells. CONCLUSIONS: Villous tip analysis seems to distinguish early coeliac from non-specific changes, thus providing a valuable tool in routine practice, especially when borderline findings are involved. Its value appears to be similar to counting of gammadelta+ cells, which, however, requires frozen biopsy samples.     

Diagnostic methods beyond conventional histology in coeliac disease diagnosis

BackgroundCoeliac disease diagnostic criteria currently require the detection of small bowel mucosal villous atrophy and crypt hyperplasia.AimsTo compare conventional histological examination to the determination of small bowel mucosal intraepithelial lymphocytes (IELs) and to serum and intestinal coeliac autoantibodies in untreated coeliac disease with villous atrophy and in mild enteropathy coeliac disease.Patients and methodsStudy comprised consecutive adult patients with coeliac disease suspicion; villous height–crypt depth ratio (Vh/CrD), the densities of CD3+, γδ+ and villous tip IELs and serum and intestinal transglutaminase 2 (TG2)-targeted autoantibodies were studied. Coeliac disease was diagnosed in 223 and excluded in 608 patients. Further, 66 patients were considered to suffer from mild enteropathy coeliac disease. Control group consisted of 138 patients.ResultsVh/CrD determination detected 77% of untreated coeliac disease patients. Serum coeliac autoantibodies had 84% sensitivity for untreated coeliac disease with villous atrophy and 70% sensitivity for mild enteropathy coeliac disease; the specificity was 100%. Intestinal TG2-targeted autoantibodies had sensitivities of 100% and 93%, and 100% specificity, respectively. γδ+ and villous tip IELs proved more reliable than CD3+ IELs.ConclusionsConventional histological examination as the golden standard in coeliac disease diagnosis is questionable. Serum and especially intestinal TG2-targeted autoantibodies seem promising in future coeliac disease diagnostics.     

Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after gammadelta T cell depletion and no effect of alphabeta T cell depletion

BACKGROUND AND AIM: Indirect evidence suggests that CD4+ T cells have a pathogenic while gammadelta T cells have a protective role in the initiation and perpetuation of inflammatory bowel disease. To define the role of T cell subsets in a rat colitis model (2,4,6-trinitrobenzene sulphonic acid (TNBS)) we analysed colitis severity after effective depletion of T helper cells, alphabeta T cells, or gammadelta T cells. METHODS: T helper cells, alphabeta T cells, or gammadelta T cells were depleted using previously described monoclonal antibodies directed at the CD4 molecule (OX38), the CD2 molecule (OX34, both depleting CD4+ T cells), the alphabeta T cell receptor (R73), and the gammadelta T cell receptor (V65). Depletion was verified by flow cytometry and/or immunohistology. Colitis was induced using intracolonic application of TNBS. RESULTS: Surprisingly, depletion of T helper cells or alphabeta T cells had no influence on survival, macroscopic or microscopic scores, or myeloperoxidase activity following colitis induction. In contrast, depletion of gammadelta T cells resulted in significantly increased mortality (V65: 73%, n=15) compared with controls (30%, n=13; p<0.03). In addition, colitis was histologically more severe in the gammadelta T cell depleted group compared with controls (p<0.05). CONCLUSIONS: T helper cells or alphabeta T cells did not influence the initiation or perpetuation of rat TNBS colitis. In contrast, gammadelta T cells had a protective role in rat TNBS colitis as depletion caused increased mortality.     

In vitro and in vivo effects of 2'-deoxycoformycin (Pentostatin) on tumour cells from human gammadelta+ T-cell malignancies

Hepatosplenic gammadelta+ T-cell lymphoma represents a rare neoplasm of post-thymic phenotype, characterized by an aggressive clinical course and a poor response to conventional chemotherapy. In the present study, we have examined the cytotoxic effects of the purine analogue 2'-deoxycoformycin (dCF) on cultured mononuclear cells and purified gammadelta+ tumour cells from bone marrow or peripheral blood of four patients with hepatosplenic gammadelta+ T-cell lymphoma. At a concentration of 10 microM, dCF, in the presence of 2'-deoxyadenosine (dAdo), displayed an early and selective cytotoxic effect on gammadelta+ tumour T cells. After 48 h of in vitro exposure to dCF, the absolute number of viable CD3+/gammadelta+ tumour T cells was reduced by more than 90% in all samples with respect to control cultures, with absolute counts of viable CD3+/alphabeta+ lymphocytes being reduced only by 6-40% of the initial cell input. Analysis of cultures after 5 d of exposure to dCF plus dAdo revealed the persistence of normal CD3+/alphabeta+ T cells, which accounted, however, for only 20-25% of the initial cell input. Accordingly, the combination of dCF (10-100 microM) plus dAdo was able to induce a dose-dependent inhibition of clonogenic growth and [3H]-thymidine incorporation in purified CD3+/CD4-/CD8- gammadelta+ tumour cells. We also report that one patient with hepatosplenic gammadelta+ T-cell lymphoma in terminal leukaemic phase showed a striking haematological response to single-agent dCF given as fourth-line treatment. In particular, the selective clearance of gammadelta+ tumour T cells in peripheral blood and bone marrow was observed starting after the second course of treatment. Our results suggest that dCF may represent a potentially active drug for the management of this aggressive form of T-cell lymphoma.     

Local challenge on oral mucosa with an alpha-gliadin related synthetic peptide in patients with celiac disease

OBJECTIVE: Gluten-derived peptides (e.g., amino-acids 31-49 of alpha-gliadin) have been shown to cause changes typical of celiac disease in the gut. Gluten-derived peptides have mostly been used in in vitro studies. The easiest access to the gastrointestinal system may be the mouth. In the present study we were interested to see whether a synthetic peptide corresponding to amino-acids 31-49 of alpha-gliadin could induce inflammatory changes in the oral mucosa after a local challenge in celiac disease patients. METHODS: The challenge was made by injecting the peptide solution at a concentration of 10 microg/ml submucosally into the oral mucosa of 10 celiac disease patients after a gluten-free diet (GFD) and 12 healthy control subjects. B and CD45RO+ T cells, mast cells, CD3+, CD4+, CD8+ lymphocytes, and alphabeta and gammadelta T-cell receptor-bearing (TcR alphabeta, TcR gammadelta) lymphocytes were counted and HLA DR expression was determined. The expression of CD25 and Ki-67 antigen was also examined. RESULTS: The peptide significantly increased the total number of T cells in the lamina propria of the celiac disease patients. The expression of T-cell activation marker CD25 (IL-2 receptor), but not that of cell proliferation marker Ki-67, was also significantly increased in the lamina propria after peptide challenge. Such a reaction was not observed in the controls. The numbers of CD3+ and CD4+ T cells in the lamina propria were also increased in celiac disease patients after the challenge. The count of TcR gammadelta+ cells was very small in the oral mucosa in celiac disease and showed no increase when the oral mucosa was challenged with the peptide. The expression of HLA DR staining was enhanced after the submucosal peptide challenge in celiac disease; however, the difference was not statistically significant. CONCLUSIONS: The results show that in the celiac disease patients after the peptide challenge the oral mucosal lamina propria responds with a nonproliferative increase of lymphocytes. Thus, submucosal challenge with the peptide 31-49 can be used as an aid in the diagnosis of celiac disease. However, further studies with optimized methodology, including various concentrations of the peptide, adjuvants, other peptides, etc., are warranted, especially because the oral mucosa provides the easiest access to an in vivo peptide challenge in celiac disease.     

HIV感染者/AIDS患者外周血T细胞抗原受体γδT细胞的检测及其临床意义

目的　探讨外周血T细胞抗原受体 (TCR)γδT细胞在艾滋病病毒 (HIV)感染者 /艾滋病 (AIDS)患者疾病进程中的作用。方法　应用流式细胞仪采用三色荧光抗体染色技术分别检测 18例HIV感染者、2 9例AIDS患者、2 5例伴有机会性感染的AIDS患者、2 0例高效抗逆转录病毒联合疗法 (HAART)治疗后的AIDS患者及 2 0例正常对照者外周血的TCRγδT细胞与TCRαβT细胞。 结果　HIV感染者组和AIDS患者组的外周血TCRγδT细胞的百分率比正常对照组明显地增高 (P <0 0 1) ,而TCRαβT细胞的百分率明显地低于正常对照组 (P <0 0 1) ,AIDS患者组的CD+ 3 细胞的百分率明显地低于HIV感染者组和正常对照组 (P <0 0 1)。AIDS患者伴机会性感染组的外周血TCRγδT细胞的百分率明显地高于HAART治疗后的AIDS患者组 (P <0 0 1) ,该组的TCRαβT细胞的百分率及CD+ 3 T细胞的百分率则明显地低于经HAART治疗后的AIDS患者组 (P <0 0 1)。结论　TCRγδT细胞数量增多是机体受到病毒感染时的一种早期非特异性免疫反应 ,在AIDS的疾病进程中及机会性感染时起一定的免疫防御作用。     

Nonhepatosplenic gamma delta T-cell lymphoma with initial testicular compromise

We report here a case of nonhepatosplenic gammadelta T-cell lymphoma with undescribed initial localization in testis, without hepatosplenomegaly or adenopathies, and subsequent development in the maxillary sinus. The maxillar mass biopsy revealed a T-cell infiltration, and its immunologic characterization by flow cytometry showed a gammadelta T-cell phenotype (CD45+, CD3+, CD2+, TCR gammadelta+), without expression of CD7, CD5, CD1a, TdT, CD4, CD8, TCR alphabeta, or NK antigens (CD16, CD56, and CD57). Clonal gamma-chain gene rearrangement by polymerase chain reaction (PCR) was detected in testicular and maxillar biopsies. Epstein-Barr virus type 1 (EBV) sequences were detected by molecular biology in the biopsy material, suggesting that this oncogenic virus may play a role in the genesis of the clonal expansion of gammadelta T-cells. The patient was initially treated with standard chemotherapeutic protocols, with poor response and aggressive course.     

Late Epstein-Barr virus infection of a hepatosplenic gamma delta T-cell lymphoma arising in a kidney transplant recipient

BACKGROUND AND OBJECTIVE: gd T-cell lymphomas are only exceptionally observed in transplanted patients. Aim of this study was the detailed characterization of one such case. DESIGN AND METHODS: The patient developed spontaneous splenic rupture six years after kidney transplantation. The splenic red pulp was infiltrated by medium-sized and large lymphoid cells with two or more nucleoli. At autopsy, similar lymphoid cells infiltrated the hepatic sinusoids. Histologic, immunologic and molecular studies were carried out. RESULTS: By immunohistochemistry, the atypical lymphoid cells were found to express CD3, CD45 and CD43, indicating their T-lineage origin. Approximately 99% of spleen mononuclear cells (MNC) were CD3(+), gammadelta TcR+, CD4-, CD8-, alphabeta TcR-. A clonal gammadelta TcR rearrangement (Vgamma1-Jgamma1.3/2.3-Cgamma2; Vdelta1-Ddelta2-Jdelta1) was detected. The final diagnosis was peripheral T-cell lymphoma, hepato-splenic gammadelta-type. EBV infection of spleen MNC was documented by molecular studies. However, in situ hybridization for EBER-1 (EBV-RNA) showed that only a minority of malignant lymphoid cells (5-7%) were EBV-infected. INTERPRETATION AND CONCLUSIONS: It is concluded that EBV infection was as a late event involving an already transformed gd T-cell clone.     

Flow cytometric analysis of gut mucosal lymphocytes supports an impaired Th1 cytokine profile in spondyloarthropathy

OBJECTIVE: To quantify the fraction of gut mucosal lymphocytes expressing the T helper type 1 (Th1) cytokines, interferon gamma (IFNgamma) and interleukin (IL)2, and the Th2 cytokines, IL4 and IL10, at the single cell level in patients with spondyloarthropathy (SpA) in comparison with healthy controls. METHODS: An improved extraction protocol was used for the enrichment of intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from colonic and ileal biopsy specimens obtained from patients with SpA (n=20) and healthy controls (n=13). After stimulation with phorbol ester/ionomycin, expression of the intracellular cytokines IFNgamma, IL2, IL4, and IL10 was determined in CD3+, CD3+CD8+ and CD3+CD8- T cells by flow cytometry. RESULTS: In colonic LPLs, a significant decrease in IFNgamma-producing CD3+ cells was observed (p=0.02) in patients with SpA. In the CD3+CD8- subset, the proportion of cells producing IFNgamma and IL2 was decreased in patients with SpA (p=0.021 and p=0.027 respectively). In ileal LPLs, the percentage of IL10-producing CD3+CD8- cells was significantly increased (p=0.046). CONCLUSION: An impaired Th1 cytokine profile is observed in gut mucosal lymphocytes from patients with SpA. This adds to the existing evidence that the gut mucosal immune apparatus is involved in the pathogenesis of SpA.     

Duodenal intraepithelial lymphocytes in inflammatory disorders of the esophagus and stomach.

BACKGROUND & AIMS: Duodenal cluster designation 3 positive (CD3+) intraepithelial T lymphocytes (IELs) are increased in gluten-sensitive enteropathy (GSE) and, because of the dispersed nature of the gut immune system, might also be increased in mucosa distant from the duodenum. Conversely, little is known about their frequency in the duodenum during inflammatory conditions of the stomach and esophagus. This study assessed whether CD3+ IELs are increased in duodenal biopsies in patients with esophagitis or gastritis relative to normal control subjects. METHODS: Cases (n=46) with concurrent mucosal biopsies of the duodenum, stomach, and esophagus were divided into 4 groups: I, no inflammation in any site; II, active esophagitis only; III, chronic active gastritis only, with Helicobacter pylori bacteria; IV, chronic gastritis only, without H pylori bacteria. Immunostains against CD3 were performed by using standard techniques, the number of CD3+ cells/100 enterocytes in 3 well-oriented villi was recorded, and the results for the groups were compared statistically. RESULTS: The average number of CD3+ IELs/100 enterocytes for each group was I, 6.7; II, 11.8; III, 7.2; and IV, 9.1. The differences among the groups were not statistically significant. There was no correlation between the number of duodenal IELs and severity of inflammation, patient age or sex, or symptoms. CONCLUSIONS: Duodenal mucosal biopsies from patients with esophagitis and/or gastritis may have a slightly increased number of CD3+ IELs relative to normal control subjects. This finding may reflect an underlying mechanism of diffuse inflammation in the gastrointestinal tract.     

Abnormal proliferation of CD4- CD8+ gammadelta+ T cells with chromosome 6 anomaly: role of Fas ligand expression in spontaneous regression of the cells

We report a case of granular lymphocyte proliferative disorder accompanied with hemolytic anemia and neutropenia. Phenotypes of the cells were T cell receptor gammadelta+ CD3+ CD4- CD8+ CD16+ CD56- CD57-. Southern blot analysis of T cell receptor beta and gamma chains demonstrated rearranged bands in both. Chromosomal analysis after IL-2 stimulation showed deletion of chromosome 6. Sorted gammadelta+ T cells showed an increase in Fas ligand expression compared with the levels in sorted alphabeta+ T cells. The expression of Fas ligand on these gammadelta+ T cells increased after IL-2 stimulation. The patient's anemia improved along with a decrease in granular lymphocyte count and disappearance of the abnormal karyotype without treatment. The expression of Fas ligand may be involved in spontaneous regression of granular lymphocyte proliferation with hemolytic anemia.     

Adoptive transfer of immunity with intraepithelial lymphocytes in Cryptosporidium parvum-infected severe combined immunodeficient mice

BACKGROUND: Intestinal infections with the protozoan parasite Cryptosporidium parvum are prevalent in both immunocompetent and immunocompromised hosts. Although C parvum is an important cause of outbreaks and opportunistic infections worldwide, little is known about protective mucosal immune responses. This is in part because animal models of infection are limited to those with genetic or induced immunodeficiencies. METHOD: In this report, we isolated immune (primed) or nonimmune (unprimed) intraepithelial lymphocytes (IEL) from BALB/cJ mouse intestines, adoptively transferred them into C parvum-infected severe combined immunodeficient (SCID) mice, and evaluated infection and cell phenotype responses. RESULTS: Control SCID mice that received no IEL shed large numbers of oocysts throughout the experimental period (day 18 to day 72). Transfer of primed IEL significantly reduced fecal oocyst shedding in recipient SCID mice compared with SCID mice that received unprimed IEL or no IEL. SCID mice transferred with unprimed IEL shed variable numbers of fecal oocysts that increased and decreased in bursts until day 57 after infection. SCID mice transferred with primed IEL exhibited significantly higher proportions of T-cell receptor (TCR) alphabeta+, CD8+, and CD8alphabeta+ EL compared with inoculated SCID mice that received unprimed or no IEL. CONCLUSION: We conclude that primed IEL from immunocompetent mice may influence protective mucosal response against cryptosporidiosis when transferred into SCID mice. In addition, the increased percentage of TCR alphabeta+, CD8+, CD8alphabeta+ IEL in recipient SCID mice may reflect mucosal cell populations involved in these responses during chronic C parvum infection.     

High- and low-affinity single-peptide/MHC ligands have distinct effects on the development of mucosal CD8αα and CD8αβ T lymphocytes

In this study, we compared the influence of two peptides on the selection of CD8alphaalpha and CD8alphabeta intraepithelial lymphocytes (IELs) of the intestine, which develop by a unique and partially thymus-independent process. Mice were used in which all T cells carried one transgenic T cell antigen receptor (TCR) (F5), and in which only well defined transgenic peptides were presented by H-2Db. The first peptide, for which the F5 TCR has a high affinity, derives from the influenza virus nucleoprotein (NP68). The second peptide, NP34, is an antagonistic variant of NP68 and is recognized by the F5 TCR with low affinity. To avoid presentation of endogenous peptides or production of T cells carrying alternative TCRs, F5 TCR transgenic mice were generated that were deficient for Tap-1 and Rag-1. In these mice, no CD3(+)CD8(+) cells were found in lymph nodes, spleen, or intestine. Introduction of transgenes encoding either NP34 or NP68 along with an endoplasmic reticulum signal sequence enabled Tap-1-independent expression of each peptide in these mice. Positive selection of F5TCR+CD8(+) thymocytes was not rescued by these transgenic peptides. However, the high-affinity NP68 peptide induced maturation of CD8alphaalpha IEL, whereas the low-affinity NP34 peptide stimulated development of both CD8alphabeta and CD8alphaalpha IEL, but in smaller numbers. When both peptides were present, CD8alphabeta T cells failed to develop and the number of CD8alphaalpha IELs was lower than in mice carrying the NP68 transgene alone. These data demonstrate that single ligands with a high or low affinity for TCR are capable of inducing or inhibiting the maturation of alternative subsets of IELs.     

Clinical-scale single-step CD4(+) and CD8(+) cell depletion for donor innate lymphocyte infusion (DILI)

The ability to selectively deplete or enrich cells of specific phenotype by immunomagnetic selection to reduce the risk of GVHD holds significant promise for application in adoptive immunotherapy. Current clinical-scale approaches for T-cell depletion (e.g., CD34(+) selection, CD3(+) depletion), usually deplete gammadelta T cells, which may be advantageous in mediating graft-versus-tumor (GVT) effects and augmenting the innate immune response against infections. Here, we present a new method for depletion of T cells with potential GVHD reactivity by using a single-step immunomagnetic protocol, which efficiently depletes CD4(+) and CD8(+) alphabeta T cells under good manufacturing practice (GMP) conditions. Depletion from unstimulated leukapheresis products (n=6) containing up to 2.0 x 10(10) cells showed high efficiency (mean log depletion of CD4(+) cells: 4.12, CD8(+) cells: 3.77). In addition, immunomagnetic CD4/CD8 depletion resulted in passive enrichment of innate lymphocytes (mean recovery of natural killer (NK) cells: 38%, gammadelta T cells: 50%). We demonstrated that gammadelta/NK cells preserved their proliferative and cytotoxic capacity and conclude that simultaneous large-scale depletion of CD4(+)/CD8(+) T cells is feasible and can be performed under GMP conditions with high-depletion efficacy for alphabeta T cells and recovery of functionally intact innate effector lymphocytes for potential use in adoptive immunotherapy studies.     

Selective expansion of intraepithelial lymphocytes expressing the HLA-E-specific natural killer receptor CD94 in celiac disease

BACKGROUND & AIMS: Celiac disease is a gluten-induced enteropathy characterized by the presence of gliadin-specific CD4(+) T cells in the lamina propria and by a prominent intraepithelial T-cell infiltration of unknown mechanism. The aim of this study was to characterize the subset(s) of intraepithelial lymphocytes (IELs) expanding during active celiac disease to provide insights into the mechanisms involved in their expansion. METHODS: Flow-cytometric analysis of isolated IELs and/or immunohistochemical staining of frozen sections were performed in 51 celiac patients and 50 controls with a panel of monoclonal antibodies against T-cell and natural killer (NK) receptors. In addition, in vitro studies were performed to identify candidate stimuli for NK receptor expression. RESULTS: In normal intestine, different proportions of IELs, which were mainly T cells, expressed the NK receptors CD94/NKG2, NKR-P1A, KIR2D/3D, NKp46, Pen5, or CD56. During the active phase of celiac disease, the frequency of CD94(+) IELs, which were mostly alphabeta T cells, was conspicuously increased over controls. In contrast, the expression of other NK markers was not modified. Furthermore, expression of CD94 could be selectively induced in vitro by T-cell receptor activation and/or interleukin 15, a cytokine produced by intestinal epithelial cells. CONCLUSIONS: The gut epithelium favors the development of T cells that express NK receptors. In active celiac disease, there is a specific and selective increase of IELs expressing CD94, the HLA-E-specific NK receptor that may be related to T-cell receptor activation and/or interleukin 15 secretion.