Improving the Diagnosis of Dermatitis Herpetiformis Using the Intraepithelial Lymphogram.

Dermatitis herpetiformis is a cutaneous manifestation of celiac disease. Phenotyping of intraepithelial lymphocytes in the small bowel mucosa can strengthen the diagnosis of celiac disease when it is not clear-cut. We aim to evaluate the usefulness of the intraepithelial lymphogram to confirm dermatitis herpetiformis in equivocal cases. We performed a retrospective multicenter study on patients diagnosed with dermatitis herpetiformis and collected data from the intraepithelial lymphogram assessed by flow cytometry. A total of 36 patients were analyzed in relation to the severity of intestinal damage (18 had non-atrophic mucosa) at baseline (N = 28) and/or after the adoption of a gluten-free diet (median follow-up of three years, N = 16). We observed that patients with atrophy more often had positive celiac serology (p = 0.019), celiac clinical symptoms (p = 0.018), and iron-deficiency anemia (p = 0.018), but the severity of skin damage was similar in both groups (p = 0.79). At baseline, increased TCRγδ+ cells were present in 94% of patients with atrophy and 67% with non-atrophic lesions (p = 0.13). After a gluten-free diet, increased TCRγδ+ cells persisted in 100% and 63% of cases, respectively (p = 0.21). We concluded that increased TCRγδ+ cells may be helpful in confirming the diagnosis of dermatitis herpetiformis in equivocal cases, even in patients who were started on a gluten-free diet.

Characterization of Bovine Intraepithelial T Lymphocytes in the Gut.

Intraepithelial T lymphocytes (T-IELs), which constitute over 50% of the total T lymphocytes in the animal, patrol the mucosal epithelial lining to defend against pathogen invasion while maintaining gut homeostasis. In addition to expressing T cell markers such as CD4 and CD8, T-IELs display T cell receptors (TCR), including either TCRαß or TCRγδ. Both humans and mice share similar T-IEL subsets: TCRγδ+, TCRαß+CD8αα+, TCRαß+CD4+, and TCRαß+CD8αß+. Among these subsets, human T-IELs are predominantly TCRαß+ (over 80%), whereas those in mice are mostly TCRγδ+ (~60%). Of note, the majority of the TCRγδ+ subset expresses CD8αα in both species. Although T-IELs have been extensively studied in humans and mice, their profiles in cattle have not been well examined. Our study is the first to characterize bovine T-IELs using flow cytometry, where we identified several distinct features. The percentage of TCRγδ+ was comparable to that of TCRαß+ T-IELs (both ~50% of CD3+), and the majority of bovine TCRγδ+ T-IELs did not express CD8 (CD8-) (above 60%). Furthermore, about 20% of TCRαß+ T-IELs were CD4+CD8αß+, and the remaining TCRαß+ T-IELs were evenly distributed between CD4+ and CD8αß+ (~40% of TCRαß+ T-IELs each) with no TCRαß+CD8αα+ identified. Despite these unique properties, bovine T-IELs, similar to those in humans and mice, expressed a high level of CD69, an activation and tissue-retention marker, and a low level of CD62L, a lymphoid adhesion marker. Moreover, bovine T-IELs produced low levels of inflammatory cytokines such as IFNγ and IL17A, and secreted small amounts of the immune regulatory cytokine TGFß1. Hence, bovine T-IELs' composition largely differs from that of human and mouse, with the dominance of the CD8- population among TCRγδ+ T-IELs, the substantial presence of TCRαß+CD4+CD8αß+ cells, and the absence of TCRαß+CD8αα+ T-IELs. These results provide the groundwork for conducting future studies to examine how bovine T-IELs respond to intestinal pathogens and maintain the integrity of the gut epithelial barrier in animals.

Development and function of natural TCR+ CD8αα+ intraepithelial lymphocytes.

The complexity of intestinal homeostasis results from the ability of the intestinal epithelium to absorb nutrients, harbor multiple external and internal antigens, and accommodate diverse immune cells. Intestinal intraepithelial lymphocytes (IELs) are a unique cell population embedded within the intestinal epithelial layer, contributing to the formation of the mucosal epithelial barrier and serving as a first-line defense against microbial invasion. TCRαß+ CD4- CD8αα+ CD8αß- and TCRγδ+ CD4- CD8αα+ CD8αß- IELs are the two predominant subsets of natural IELs. These cells play an essential role in various intestinal diseases, such as infections and inflammatory diseases, and act as immune regulators in the gut. However, their developmental and functional patterns are extremely distinct, and the mechanisms underlying their development and migration to the intestine are not fully understood. One example is that Bcl-2 promotes the survival of thymic precursors of IELs. Mature TCRαß+ CD4- CD8αα+ CD8αß- IELs seem to be involved in immune regulation, while TCRγδ+ CD4- CD8αα+ CD8αß- IELs might be involved in immune surveillance by promoting homeostasis of host microbiota, protecting and restoring the integrity of mucosal epithelium, inhibiting microbiota invasion, and limiting excessive inflammation. In this review, we elucidated and organized effectively the functions and development of these cells to guide future studies in this field. We also discussed key scientific questions that need to be addressed in this area.

The Critical and Diverse Roles of CD4-CD8- Double Negative T Cells in Nonalcoholic Fatty Liver Disease.

BACKGROUND & AIMS: Hepatic inflammation is a hallmark of nonalcoholic fatty liver disease (NAFLD). Double negative T (DNT) cells are a unique subset of T lymphocytes that do not express CD4, CD8, or natural killer cell markers, and studies have suggested that DNT cells play critical and diverse roles in the immune system. However, the role of intrahepatic DNT cells in NAFLD is largely unknown. METHODS: The proportions and RNA transcription profiling of intrahepatic DNT cells were compared between C57BL/6 mice fed with control diet or methionine-choline-deficient diet for 5 weeks. The functions of DNT cells were tested in vitro and in vivo. RESULTS: The proportion of intrahepatic DNT cells was significantly increased in mice with diet-induced NAFLD. In NAFLD mice, the proportion of intrahepatic TCRγδ+ DNT cells was increased along with elevated interleukin (IL) 17A; in contrast, the percentage of TCRαß+ DNT cells was decreased, accompanied by reduced granzyme B (GZMB). TCRγδ+ DNT cell depletion resulted in lowered liver IL17A levels and significantly alleviated NAFLD. Adoptive transfer of intrahepatic TCRαß+ DNT cells from control mice increased intrahepatic CD4 and CD8 T cell apoptosis and inhibited NAFLD progression. Furthermore, we revealed that adrenic acid and arachidonic acid, harmful fatty acids that were enriched in the liver of the mice with NAFLD, could induce apoptosis of TCRαß+ DNT cells and inhibit their immunosuppressive function and nuclear factor kappa B (NF-κB) or AKT signaling pathway activity. However, arachidonic acid facilitated IL17A secretion by TCRγδ+ DNT cells, and the NF-κB signaling pathway was involved. Finally, we also confirmed the variation of intrahepatic TCRαß+ DNT cells and TCRγδ+ DNT cells in humans. CONCLUSIONS: During NAFLD progression, TCRγδ+ DNT cells enhance IL17A secretion and aggravate liver inflammation, whereas TCRαß+ DNT cells decrease GZMB production and lead to weakened immunoregulatory function. Shifting of balance from TCRγδ+ DNT cell response to one that favors TCRαß+ DNT regulation would be beneficial for the prevention and treatment of NAFLD.

Intestinal and blood lymphograms as new diagnostic tests for celiac disease.

Accurate celiac disease (CD) diagnosis is still challenging for some specific patients or circumstances. Thus, much effort has been expended last decades focused on seronegative or low grade enteropathy CD and, especially, on enable early diagnosis of individuals on a gluten-free diet (GFD). We discuss here two diagnostic approaches based on immunophenotyping by flow cytometry that we expect to reduce the persistent low diagnostic rates and the common diagnostic delay. The intraepithelial lymphogram is based on determining the percentage of TCRγδ+ and surface CD3- lymphocytes in the intestinal epithelium. The concomitant increase in TCRγδ+ and decrease in surface CD3- intraepithelial lymphocytes has been termed the celiac lymphogram and has been proved to be discriminative in seronegative, low grade enteropathy and potential CD, as well as in most CD patients on a GFD. A blood lymphogram based on the analysis of activated gut-homing CD8+ T cells combined with a 3-day gluten challenge is also considered, which has shown high sensitivity and specificity to diagnose seropositive Marsh 1 and Marsh 3 CD in individuals following a GFD. In addition, flow cytometry can be extremely useful in cases of refractory CD type II to identify aberrant cells. Those approaches represent highly accurate methods for CD diagnosis, being simple, fast, highly reproducible and of easy implementation in clinical practice.

Intestinal Cellular Biomarkers of Mucosal Lesion Progression in Pediatric Celiac Disease.

Celiac disease (CD) is a chronic intestinal inflammation caused by gluten ingestion in genetically predisposed individuals. Overt-CD and potential-CD are the two main forms of gluten intolerance in pediatric patients with different grades of intestinal mucosa lesion and clinical management. For overt-CD patients the gluten-free diet is mandatory, while for potential-CD the dietary therapy is recommended only for those subjects becoming clinically symptomatic overtime. To date, specific early biomarkers of evolution to villous atrophy in potential-CD are lacking. We recently observed an expansion of TCRγδ+ T cells and a concomitant disappearance of IL4-producing T cells in the intestinal mucosa of overt-CD patients compared to potential-CD children, suggesting the involvement of these two cells subsets in the transition from potential-CD to overt-CD. In this study, we demonstrated that the intestinal densities of IL4+ T cells inversely correlated with TCRγδ+ T cell expansion (p < 0.005) and with the serum levels of anti-tissue transglutaminase antibodies (p < 0.01). The changes of these two cell subsets strongly correlated with mucosal lesions, according to the histological Marsh classification, as the transition from M0 to M3 lesions was associated with a significant reduction of IL4+ T cells (M0 vs. M1 p < 0.04, M0 vs. M3 p < 0.007) and an increase of TCRγδ+ T cells (M0 vs. M1 p < 0.05, M0 vs. M3 p < 0.0006). These findings strongly suggest that the detection of TCRγδ+ and IL4+ T cells could serve as cellular biomarkers of mucosal lesion and targets of novel immunomodulatory therapies for CD.

Characterization of the chicken T cell receptor γ repertoire by high-throughput sequencing.

BACKGROUND: As one of "γδ-high" species, chicken is an excellent model for the study of γδ T cells in non-mammalian animals. However, a comprehensive characterization of the TCRγδ repertoire is still missing in chicken. The objective of this study was to characterize the expressed TCRγ repertoire in chicken thymus using high-throughput sequencing. METHODS: In this study, we first obtained the detailed genomic organization of the TCRγ locus of chicken based on the latest assembly of the red jungle fowl genome sequences (GRCg6a) and then characterized the TCRγ repertoire in the thymus of four chickens by using 5' Rapid Amplification of cDNA Ends (5' RACE) along with high-throughput sequencing (HTS). RESULTS: The chicken TCRγ locus contains a single Cγ gene, three functional Jγ segments and 44 Vγ segments that could be classified into six subgroups, each containing six, nineteen, nine, four, three and three members. Dot-plot analysis of the chicken TCRγ locus against itself showed that almost all the entire zone containing Vγ segments had arisen through tandem duplication events, and the main homology unit, containing 9 or 10 Vγ gene segments, has tandemly duplicated for four times. For the analysis of chicken TCRγ repertoire, more than 100,000 unique Vγ-region nucleotide sequences were obtained from the thymus of each chicken. After alignment to the germline Vγ and Jγ segments identified above, we found that the four chickens had similar repertoire profile of TCRγ. In brief, four Vγ segments (including Vγ3.7, Vγ2.13, Vγ1.6 and Vγ1.3) and six Vγ-Jγ pairs (including Vγ3.7-Jγ3, Vγ2.13-Jγ1, Vγ2.13-Jγ3, Vγ1.6-Jγ3, Vγ3.7-Jγ1 and Vγ1.6-Jγ1) were preferentially utilized by all four individuals, and vast majority of the unique CDR3γ sequences encoded 4 to 22 amino acids with mean 12.90 amino acids, which exhibits a wider length distribution and/or a longer mean length than CDR3γ of human, mice and other animal species. CONCLUSIONS: In this study, we present the first in-depth characterization of the TCRγ repertoire in chicken thymus. We believe that these data will facilitate the studies of adaptive immunology in birds.

Coeliac Disease in Elderly Patients: Value of Coeliac Lymphogram for Diagnosis.

(1) Background: Although a meta-analysis reported that the sensitivity of CD3+ TCRγδ+ cells for coeliac disease diagnosis was >93%, a recent study has suggested that sensitivity decreased to 65% in elderly patients. (2) Aim: To evaluate whether the sensitivity of intraepithelial lymphocyte cytometric patterns for coeliac disease diagnosis changes with advanced age. (3) Methods: We performed a multicentre study including 127 coeliac disease patients ≥ 50 years: 87 with baseline cytometry (45 aged 50-59 years; 23 aged 60-69 years; 19 aged ≥ 70 years), 16 also with a follow-up cytometry (on a gluten-free diet); and 40 with only follow-up cytometry. (4) Results: In Marsh 3 patients, a sensitivity of 94.7%, 88.9% and 86.7% was observed for each age group using a cut-off value of TCRγδ+ >10% (p = 0.27); and a sensitivity of 84.2%, 83.4% and 53.3% for a cut-off value >14% (p = 0.02; 50-69 vs. ≥70 years), with difference between applying a cut-off of 10% or 14% (p = 0.008). The TCRγδ+ count in the ≥70 years group was lower than in the other groups (p = 0.014). (5) Conclusion: In coeliac patients ≥ 70 years, the TCRγδ+ count decreases and the cut-off point of >10% is more accurate than >14%.

Intraepithelial Lymphocyte Cytometric Pattern Is a Useful Diagnostic Tool for Coeliac Disease Diagnosis Irrespective of Degree of Mucosal Damage and Age-A Validation Cohort.

INTRODUCTION: The study of intraepithelial lymphocytes (IEL) by flow cytometry is a useful tool in the diagnosis of coeliac disease (CD). Previous data showed that an increase in %TCRγδ+ and decrease of %CD3- IEL constitute a typical CD cytometric pattern with a specificity of 100%. However, there are no data regarding whether there are differences in the %TCRγδ+ related to sex, age, titers of serology, and degree of histological lesion. STUDY AIMS: To confirm the high diagnostic accuracy of the coeliac cytometric patterns. To determine if there are differences between sex, age, serology titers, and histological lesion grade. RESULTS: We selected all patients who fulfilled "4 of 5" rule for CD diagnosis (n = 169). There were no differences in %TCRγδ+ between sexes (p = 0.909), age groups (p = 0.986), serology titers (p = 0.53) and histological lesion grades (p = 0.41). The diagnostic accuracy of complete CD cytometric pattern was: specificity 100%, sensitivity 82%, PPV 100%, NPV 47%. CONCLUSION: We confirmed, in a validation cohort, the high diagnostic accuracy of complete CD pattern irrespective of sex, age, serology titers, and grade of mucosal lesion.

Granulocyte Colony-Stimulating Factor Effectively Mobilizes TCR γδ and NK Cells Providing an Allograft Potentially Enhanced for the Graft-Versus-Leukemia Effect for Allogeneic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with hematological malignancies but substantial risks of recurrence of the malignant disease remain. TCR γδ and NK cells are perceived as potent innate effector cells in HSCT and have been associated with post-transplant protection from relapse in clinical studies. Immunocompetent cells from the donor are crucial for patient outcomes and peripheral blood stem cells (PBSC) are being increasingly applied as graft source. G-CSF is the preferential mobilizing agent in healthy donors for PBSC grafts, yet effects of G-CSF on TCR γδ and NK cells are scarcely uncovered and could influence the graft composition and potency of these cells. Therefore, we analyzed T and NK cell subsets and activation markers in peripheral blood samples of 49 donors before and after G-CSF mobilization and-for a subset of donors-also in the corresponding graft samples using multicolor flowcytometry with staining for CD3, CD4, CD8, TCRαß, TCRγδ, Vδ1, Vδ2, HLA-DR, CD45RA, CD197, CD45RO, HLA-DR, CD16, CD56, and CD314. We found that TCR γδ cells were mobilized and harvested with an efficiency corresponding that of TCR αß cells. For TCR γδ as well as for TCR αß cells, G-CSF preferentially mobilized naïve and terminally differentiated effector (TEMRA) cells over memory cells. In the TCR γδ cell compartment, G-CSF preferentially mobilized cells of the nonVδ2 types and increased the fraction of HLA-DR positive TCR γδ cells. For NK cells, mobilization by G-CSF was increased compared to that of T cells, yet NK cells appeared to be less efficiently harvested than T cells. In the NK cell compartment, G-CSF-stimulation preserved the proportion of CD56dim NK effector cells which have been associated with relapse protection. The expression of the activating receptor NKG2D implied in anti-leukemic responses, was significantly increased in both CD56dim and CD56bright NK cells after G-CSF stimulation. These results indicate differentiated mobilization and altering properties of G-CSF which could improve the effects of donor TCR γδ and NK cells in the processes of graft-versus-leukemia for relapse prevention after HSCT.

Nodal EBV-positive polymorphic B cell lymphoproliferative disorder with plasma cell differentiation: clinicopathological analysis of five cases.

Plasma cell differentiation (PCD) is frequently observed in some entities of non-Hodgkin B cell lymphoma, including both low-grade and high-grade lymphomas. However, except for plasmablastic lymphoma and primary effusion lymphoma, EBV+ B cell lymphoproliferative disorder (LPD) with PCD has not been well addressed due to its rarity. We clinicopathologically examined five cases of nodal EBV+ polymorphic B cell LPD with PCD (PBLPD-PCD) initially diagnosed as polymorphic EBV+ diffuse large B cell lymphoma, not otherwise specified (DLBCL-NOS) with PCD (n = 3) and methotrexate-associated B cell LPD (MTX-associated B-LPD) (n = 2). One case had a concomitant brain lesion which was clinically diagnosed as EBV-related encephalitis. This patient received therapy with vidarabine, and both the brain lesion and the nodal EBV+ PBLPD-PCD lesions disappeared. Another case was characterized by Mott cell differentiation. This case was the first reported case of EBV+ B cell lymphoma or LPD with Mott cell differentiation. The two cases of MTX-associated B cell LPD which arose in patients with rheumatoid arthritis spontaneously regressed after MTX cessation. TCRγ and IGH PCR analysis was performed in four cases. Two cases had TCRγ rearrangements, but no IGH rearrangements. The other two cases had no rearrangements in these genes. We concluded that nodal EBV+ PBLPD-PCD is rare, with heterogeneous characteristics. PCR analysis revealed that nodal EBV+ PBLPD-PCD may have only TCR clonality and no IGH clonality. Considering the partial or complete loss of CD20 expression on the tumor cells, this result may be confusing for accurate diagnosis of EBV+ PBLPD-PCD, and pathologists need to be aware of this phenomenon to avoid misdiagnosis.

Intestinal intraepithelial lymphocytes: Maintainers of intestinal immune tolerance and regulators of intestinal immunity.

Intestinal immune tolerance is essential for the immune system, as it prevents abnormal immune responses to large quantities of antigens from the intestinal lumen, such as antigens from commensal microorganisms, and avoids self-injury. Intestinal intraepithelial lymphocytes (IELs), a special group of mucosal T lymphocytes, play a significant role in intestinal immune tolerance. To accomplish this, IELs exhibit a high threshold of activation and low reactivity to most antigens from the intestinal lumen. In particular, CD8αα+ TCRαß+ IELs, TCRγδ+ IELs, and CD4+ CD8αα+ IELs show great potential for maintaining intestinal immune tolerance and regulating intestinal immunity. However, if the intestinal microenvironment becomes abnormal or intestinal tolerance is broken, IELs may be activated abnormally and become pathogenic.

Clonality test by PCR - PARR in real time of canine lymphomas

Lymphoma is a neoplasm of hematopoietic origin that affects canines. The proper establishment of prognosis and rapid institution of treatment are essential for a better quality of life, and immunophenotyping is one of the tools used for this purpose. The objective of this study was to perform a clonality test for immunophenotypic characterization of canine lymphomas using the polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) technique in real-time from samples fixed in formalin and embedded in paraffin. The 23 analyzed samples were fixed in formalin and embedded in paraffin canine lymphoma from the collection Laboratory of Histopathology of the Animal Pathology Area of the Departament of Veterinary Medicine - Federal Rural University of Pernambuco (UFRPE). Samples were processed, their DNA was extracted, quantified, diluted, and standardized at a concentration of 50 ng/µL. After extraction, all samples were subjected to conventional PCR for endogenous control (detection of the IgM target region), in which the extracted DNA was amplified in a final volume of 25 µL. The 128 bp amplified product was detected by 1.5% agarose gel electrophoresis. Of the 23 samples analyzed for the detection of the conserved region referring to the endogenous gene, 91.30% (21/23) amplified the conserved region Cµ by conventional PCR, and two samples 8.70% (2/23) were negative. Endogenous control positive samples were subjected to real-time PCR-PARR for detection of IgH Major and IgH Minor for B lymphocytes (LB), and TCRy for lymphocytes T (LT) target regions. All reactions were performed in duplicate to reduce the risk of false-positive or false-negative results due to technical errors. Samples previously confirmed by immunohistochemistry were used as positive controls for T cell and B cell lymphoma, and MilliQ water was used as a negative reaction control. After amplification, the melting curve gradually increased the temperature by 1o C/5 s to 95o C during continuous fluorescence monitoring. Of the 21 samples analyzed, 100.00% (21/21) demonstrated clonal amplification. Of these, 57.15% (12/21) were positive for phenotype B, and 42.85% (9/21) were positive for phenotype T. Due to the importance of researching and confirming samples from files fixed and embedded in paraffin samples in laboratories, PCR-PARR is a good tool for this purpose. In the present study, real-time PCR analysis demonstrated greater sensitivity in the characterization of the immunophenotype of lymphomas from old samples fixed in formalin and embedded in paraffin. The temperature of melting curve analysis may vary depending on the amount of DNA and its quality. In the present study, it was found that the average melting temperature in the samples varied between ± 3o C when compared to that in the control sample for LB and LT, 83.5o C and 80o C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4o C. Real-time PCR-PARR was satisfactory in the characterization of the immunophenotype of canine lymphomas from formalin-fixed and paraffin-embedded samples; therefore, its use is recommended for both retrospective studies. The use of PCR-PARR associated with histopathological and/or cytopathological examination in cases of canine lymphomas strongly helps pathologists, provide a safe establishment of the immunophenotype, minimize errors, and optimize the diagnosis, thus directly contributing to the establishment of the prognosis.(AU)

Immune Modulation Properties of Zoledronic Acid on TcRγδ T-Lymphocytes After TcRαß/CD19-Depleted Haploidentical Stem Cell Transplantation: An analysis on 46 Pediatric Patients Affected by Acute Leukemia.

TcRαß/CD19-cell depleted HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) represents a promising new platform for children affected by acute leukemia in need of an allograft and lacking a matched donor, disease recurrence being the main cause of treatment failure. The use of zoledronic acid to enhance TcRγδ+ lymphocyte function after TcRαß/CD19-cell depleted haplo-HSCT was tested in an open-label, feasibility, proof-of-principle study. Forty-six children affected by high-risk acute leukemia underwent haplo-HSCT after removal of TcRαß+ and CD19+ B lymphocytes. No post-transplant pharmacological graft-versus-host disease (GvHD) prophylaxis was given. Zoledronic acid was administered monthly at a dose of 0.05 mg/kg/dose (maximum dose 4 mg), starting from day +20 after transplantation. A total of 139 infusions were administered, with a mean of 3 infusions per patient. No severe adverse event was observed. Common side effects were represented by asymptomatic hypocalcemia and acute phase reactions (including fever, chills, malaise, and/or arthralgia) within 24-48 h from zoledronic acid infusion. The cumulative incidence of acute and chronic GvHD was 17.3% (all grade I-II) and 4.8% (all limited), respectively. Patients given 3 or more infusions of zoledronic acid had a lower incidence of both acute GvHD (8.8 vs. 41.6%, p = 0.015) and chronic GvHD (0 vs. 22.2%, p = 0.006). Transplant-related mortality (TRM) and relapse incidence at 3 years were 4.3 and 30.4%, respectively. Patients receiving repeated infusions of zoledronic acid had a lower TRM as compared to those receiving 1 or 2 administration of the drug (0 vs. 16.7%, p = 0.01). Five-year overall survival (OS) and disease-free survival (DFS) for the whole cohort were 67.2 and 65.2%, respectively, with a trend toward a better OS for patients receiving 3 or more infusions (73.1 vs. 50.0%, p = 0.05). The probability of GvHD/relapse-free survival was significantly worse in patients receiving 1-2 infusions of zoledonic acid than in those given ≥3 infusions (33.3 vs. 70.6%, respectively, p = 0.006). Multivariable analysis showed an independent positive effect on outcome given by repeated infusions of zoledronic acid (HR 0.27, p = 0.03). These data indicate that the use of zoledronic acid after TcRαß/CD19-cell depleted haploHSCT is safe and may result in a lower incidence of acute GvHD, chronic GvHD, and TRM.

Immunophenotype and function define TCRγδ + T-ALL as a distinct subgroup from TCRαß + T-ALL patients.

We recently demonstrated TCRγδ + T-ALL as a distinct subgroup from TCRαß + T-ALL at genomic level. TCRγδ + T-ALL subgroup possess significant survival advantage compared to TCRαß + T-ALL. In the present study, functional level differences in these two subgroups of T-ALL were studied to understand the immune scenario contributing to survival benefit of TCRγδ + T-ALL subgroup. TCRγδ clonal T-ALL patients showed significantly high levels of γδ T cells compared to TCRαß clonal T-ALL patients. TCRγδ + T-ALL patients expressed significantly high central memory and terminally differentiated (TemRA) Vδ1 and Vδ2 T cells. TCR γδ clonal leukemic blasts stimulated increased number of Vδ2 T cells from healthy lymphocytes. TCR γδ clonal leukemic blasts were able to form efficient immune synapse with effector γδ T cells. The differences in immunophenotype, cytotoxicity and immune synapse formations corroborate TCRγδ + T-ALL as a distinct subgroup from TCRαß + T-ALL patients and explain the survival benefit of TCRγδ + T-ALL patients.

Distinct Features of Canine Non-conventional CD4-CD8α- Double-Negative TCRαß+ vs. TCRγδ+ T Cells.

The role of conventional TCRαß+CD4+ or TCRαß+CD8α+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, non-conventional T cells expressing TCRαß or TCRγδ but lacking CD4 and CD8α expression [i.e., CD4-CD8α- double-negative (dn) T cells] are thought to play a role at the interface between the innate and adaptive immune system. Dn T cells are frequent in swine, cattle or sheep and predominantly express TCRγδ. In contrast, TCRγδ+ T cells are rare in dogs. In this study, we identified a high proportion of canine dn T cells in the TCRαß+ T cell population of PBMC, lymphatic and non-lymphatic organs. In PBMC, the frequency of this T cell subpopulation made up one third of the frequency of TCRαß+CD4+ sp, and almost half of the frequency of TCRαß+CD8α+ sp T cells (i.e., ~15% of all TCRαß+ T cells). Among TCRαß+CD4-CD8α- dn T cells of PBMC and tissues, FoxP3+ cells were identified indicating regulatory potential of this T cell subset. 80% of peripheral blood FoxP3+TCRαß+CD4-CD8α- dn T cells co-expressed CD25, and, interestingly, also the FoxP3-negative TCRαß+CD4-CD8α- dn T cells comprised ~34% CD25+ cells. Some of the FoxP3-positive TCRαß+CD4-CD8α- dn T cells co-expressed GATA-3 suggesting stable function of regulatory T cells. The frequency of GATA-3 expression by FoxP3-TCRαß+CD4-CD8α- dn T cells was even higher as compared with TCRαß+CD4+ sp T cells (20.6% vs. 11.9%). Albeit lacking FoxP3 and CD25 expression, TCRγδ+CD4-CD8α- dn T cells also expressed substantial proportions of GATA-3. In addition, TCRαß+CD4-CD8α- dn T cells produced IFN-γ and IL-17A upon stimulation. T-bet and granzyme B were only weakly expressed by both dn T cell subsets. In conclusion, this study identifies two dn T cell subsets in the dog: (i) a large (~7.5% in Peyer's patches, ~15% in lung) population of TCRαß+CD4-CD8α- dn T cells with subpopulations thereof showing an activated phenotype, high expression of FoxP3 or GATA-3 as well as production of IFN-γ or IL-17A and (ii) a small TCRγδ+CD4-CD8α- dn T cell subset also expressing GATA-3 without production of IFN-γ or IL-17A. It will be exciting to unravel the function of each subset during immune homeostasis and diseases of dogs.

The intestinal expansion of TCRγδ+ and disappearance of IL4+ T cells suggest their involvement in the evolution from potential to overt celiac disease.

Celiac disease (CD) is characterized by a spectrum of intestinal inflammatory lesions. Most patients have villous atrophy (overt-CD), while others have a morphologically normal mucosa, despite the presence of CD-specific autoantibodies (potential-CD). As the mechanism responsible for villous atrophy is not completely elucidated, we investigated biomarkers specific for the different celiac lesions. Phenotype and cytokine production of intestinal mucosa cells were analyzed by flow cytometry in gut biopsies of children with overt- or potential-CD and in healthy controls. Density of TCRγδ+ T cells was found markedly enhanced in intestinal mucosa of children with overt-CD compared to potential-CD or controls. By contrast, very few IL4+ T cells infiltrated the mucosa with villous atrophy compared to morphologically normal mucosa. IL4+ T cells were classical CD4+ T-helper cells (CD161- ), producing or not IFN-γ, and negative for IL17A. Our study demonstrated that the transition to villous atrophy in CD patients is characterized by increased density of TCRγδ+ T cells, and concomitant disappearance of IL4+ cells. These findings suggest that immunomodulatory mechanisms are active in potential-CD to counteract the inflammatory cascade responsible of villous atrophy. Further studies are required to validate the use of IL4+ and TCRγδ+ T cells as biomarkers of the different CD forms.

Human γδ T-cell receptor repertoire is shaped by influenza viruses, age and tissue compartmentalisation.

BACKGROUND: Although γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T-cell receptor (TCR) clonal expansions. We dissected anti-viral functions of human γδ T cells towards influenza viruses and defined influenza-reactive γδ TCRs in the context of γδ-TCRs across the human lifespan. METHODS: We performed 51Cr-killing assay and single-cell time-lapse live video microscopy to define mechanisms underlying γδ T-cell-mediated killing of influenza-infected targets. We assessed cytotoxic profiles of γδ T cells in influenza-infected patients and IFN-γ production towards influenza-infected lung epithelial cells. Using single-cell RT-PCR, we characterised paired TCRγδ clonotypes for influenza-reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues. RESULTS: We provide the first visual evidence of γδ T-cell-mediated killing of influenza-infected targets and show distinct features to those reported for CD8+ T cells. γδ T cells displayed poly-cytotoxic profiles in influenza-infected patients and produced IFN-γ towards influenza-infected cells. These IFN-γ-producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9-TCRγ, capable of pairing with numerous TCR-δ chains, suggesting their significant role in γδ T-cell immunity. Neonatal γδ T cells displayed extensive non-overlapping TCRγδ repertoires, while adults had enriched γ9δ2-pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ-pairings characterised by large clonal expansions, a profile also prominent in adult tissues. CONCLUSION: Human TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge.

Systematic Review and Meta-Analysis: Accuracy of Both Gamma Delta+ Intraepithelial Lymphocytes and Coeliac Lymphogram Evaluated by Flow Cytometry for Coeliac Disease Diagnosis.

It has been suggested that in doubtful cases of coeliac disease, a high CD3+ T-cell receptor gamma delta+ (TCRγδ+) intraepithelial lymphocyte count increases the likelihood of coeliac disease. AIM: To evaluate the diagnostic accuracy of both an isolated increase of TCRγδ+ cells and a coeliac lymphogram (increase of TCRγδ+ plus decrease of CD3- intraepithelial lymphocytes) evaluated by flow cytometry in the diagnosis of coeliac disease. METHODS: The literature search was conducted in MEDLINE and EMBASE. The inclusion criteria were: an article that allows for the construction of a 2 × 2 table of true and false positive and true and false negative values. A diagnostic accuracy test meta-analysis was performed. RESULTS: The search provided 49 relevant citations, of which 6 were selected for the analysis, which represented 519 patients and 440 controls. Coeliac lymphogram: The pooled S and Sp were 93% and 98%, without heterogeneity. The area under the SROC curve (AUC) was 0.98 (95% CI, 0.97-0.99). TCRγδ+: Pooled S and Sp were both 95%, with significant heterogeneity. The AUC was 0.97 (95% CI, 0.95-0.98). Conclusions: Both TCRγδ+ count and coeliac lymphogram assessed by flow cytometry in duodenal mucosal samples are associated with a high level of diagnostic accuracy for and against coeliac disease.

Molecular characterization and expression analysis of T cell receptor (TCR) γ and δ genes in dojo loach (Misgurnus anguillicaudatus) in response to bacterial, parasitic and fungal challenge.

In mammalian, T-cell receptors (TCRs) play a key role in recognizing the presented antigen from external to protect organisms against environmental pathogens. To understand the potential roles of TCRγ and TCRδ in dojo loach (Misgurnus anguillicaudatus), Ma-TCRγ and Ma-TCRδ cDNAs were cloned and their gene expression profiles were investigated after bacterial, parasitic and fungal challenge. The open reading frame (ORF) of Ma-TCRγ and Ma-TCRδ cDNAs contained 948 and 867 bp, encoding 316 and 288 amino acid residues, respectively. Structurally, Ma-TCRγ and Ma-TCRδ were consisted of a signal peptide, a variable region, a constant region (IgC), a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic domain (CYT), which were similar to those of other vertebrates. Multiple sequence alignment and phylogenetic analysis showed Ma-TCRγ and Ma-TCRδ were closely related to fish of Cyprinidae family. Ma-TCRγ and Ma-TCRδ were widely expressed in all tested organs/tissues, as the highest expressions of Ma-TCRγ and Ma-TCRδ were detected in kidney and gill, respectively. In addition, three infection models of dojo loach with bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia sp.) were constructed. The morphological changes of gills and skin after challenged with F. columnare G4 and Ichthyophthirius multifiliis were investigated. Compared to F. columnare G4 infection, mRNA expression of both TCRγ and TCRδ showed higher sensitivity in classical immune organs (kidney and spleen) and mucosal tissues (skin and gill) after challenge with Ichthyophthirius multifiliis and Saprolegnia sp. Our results first indicated that TCRγ and TCRδ of dojo loach might function differently in response to challenge with different pathogens.