Lymphocyte Immunophenotyping Research Articles

Lung Transplantation and Extracorporeal Photopheresis as Induction Therapy in Cystic Fibrosis Patients: Immune System Profile Changes

<h3>Purpose</h3> Acute rejection (AR), common during the first year after lung transplantation (LuTx), can trigger chronic rejection (CR), the leading cause of late morbidity and mortality of LuTx. Extracorporeal photopheresis (ECP) has emerged as a promising treatment for chronic rejection. Only few studies are focus on ECP as prophylactic therapy of AR and CR. This study aims to verify, in recipients affected by cystic fibrosis (CF), whether the induction therapy with ECP can decrease the rate of AR, in order to impact positively on CR (primary end point: survival, AR). The expected results are the reduction of AR episodes in its clinical and histopathological manifestations. <h3>Methods</h3> This is a pilot clinical trial on 20 CF lung-transplanted patients, randomize, 2 parallel arms: standard immunosuppressive therapy and ECP (ECP) vs standard immunosuppressive therapy alone (CTR). We investigated the effect of ECP by the evaluation of lymphocyte immunophenotype by multiplex essay (CD4 + and CD25 +), the cytokine profile, the leukocytes subsets (by flow cytometry) in blood and BAL at different time points. AR episodes and infections were recorded, as far as ECP-related adverse events. <h3>Results</h3> No differences were detected in terms of AR episodes. Treg cells were significantly increased in the ECP group at 3 weeks post LuTx, and this difference was more evident 1 year post LuTx . Th17 cells were diminished in the ECP group. The anti-inflammatory IL10-producing NKs were significantly increased in the ECP group. Cytokine profile, both in BAL and plasma obtained at defined time points shows that in ECP group pro-inflammatory cytokines were early reduced and anti-inflammatory cytokines were upregulated. <h3>Conclusion</h3> In FC lung transplanted patients ECP was well tolerated without increasing in opportunistic infections. Its tolerogenic effect was not pointed out in terms of AR episodes, but has been confirmed in the immunological setting of ECP patients vs control: it prevents decline in Treg and NK observed with standard immunosuppressive drugs, with higher expression of anti-inflammatory cytokines (Il10, Il1RA) and less pro-inflammatory ones (Il1beta, Il6). Its effect is more evident months after the end of ECP treatment. The schedule of ECP prophylactic treatment has to be tested in an ampler cohort in order to reach its best immunomodulatory effect.

Prescription of Blood Lymphocyte Immunophenotyping in the Diagnosis of Lymphoid Neoplasms in Older Adults.

Lymphoid neoplasms are a heterogeneous group of lymphoid neoplastic diseases with multiple presentations, and varying prognoses. They are especially frequent in older patients (OPs) and the atypism of this frail elderly population can make the diagnostic process even more difficult. Blood lymphocyte immunophenotyping (BLI) is essential in rapid noninvasive diagnosis orientation and guides complementary investigations. To our knowledge, BLI prescription has never been evaluated in OPs. We hypothesized that, when there is a suspicion of lymphoid neoplasm in the geriatric population, a BLI is performed in view of various clinical or biological abnormalities. This study aimed to: (1) describe the characteristics of hospitalized OPs having undergone BLI for suspected lymphoid neoplasm, (2) identify the causes leading to BLI prescription, and (3) identify the most profitable criteria for BLI prescription. This was a descriptive retrospective study on 151 OPs aged ≥75 years who underwent BLI over a 2-year period. Regarding BLI prescriptions, eight had lymphocytosis, constituting the “lymphocytosis group” (LG+), while the 143 others had BLI prescribed for reasons other than lymphocytosis (LG−), mainly general weakness and anemia. In the LG−, we compared OPs with positive and negative BLI results. The criteria found to be profitable for BLI prescription were lymphadenopathy, splenomegaly, lymphocytosis, and thrombocytopenia. BLI identified circulating lymphoid neoplasms (positive BLI) in 21/151 OPs, mainly marginal zone lymphoma and chronic lymphocytic leukemia. In polymorbid OPs, as per our study population, the diagnostic and therapeutic complexity explained in part the sole use of indirect and minimally invasive diagnostic techniques such as BLI.

Main lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in HIV-1 subtypes C and B.

HIV-1 subtype C (HIV-1C) shows reduced Tat protein chemoattractant activity compared with HIV-1B. The impact of HIV-1C Tat on the chemotaxis of the main lymphocyte subpopulations in the cerebrospinal fluid (CSF) and the peripheral blood (PB) is unclear. We hypothesized that there would be a lower frequency of specific lymphocyte subpopulations CD3+ or CD19+ in CSF in HIV-1C than in HIV-1B. The objectives were to detect the differences in the proportions of main lymphocyte subpopulations in CSF and PB, between people with HIV (PWH) and HIV-1-uninfected volunteers (PWoH) and in HIV-1B and HIV-1C. Lymphocyte immunophenotyping was studied in CSF and paired PB samples of PWH (n = 22) and PWoH (n = 14). Lymphocytes were analyzed within the CD45+ gated region. The proportions of CSF CD3+CD4+, CD3+CD8+, and CD3-CD19+ lymphocytes in CSF were comparable in HIV-1B and C. There was an increase in the proportion of CD3+CD8+ cells and a decrease in CD3+CD4+ T cells (ps = 0.016) in the CSF samples of the PWH compared with the PWoH group. In the PWH group, both CD3+CD4+ and CD3+CD8+ lymphocytes were significantly higher in the CSF than in the PB (p = 0.047 and 0.005). The proportion of CD3+CD4+ was lower and that of CD3+CD8+ was higher in the CSF samples of the aviremic group than that of HIV-negative control (p = 0.0008 and < 0.0001, respectively). HIV-1C Tat substitution (C30S) did not interfere with the CNS migration of the main lymphocyte subpopulations. This is the first study to evaluate these lymphocytes in CSF and PB of HIV-1C compared with HIV-1B.

High Frequency of Post-Transfusion Microchimerism Among Multi-Transfused Beta-Thalassemic Patients.

BackgroundTransfusion-associated microchimerism implies the presence of allogeneic hematopoietic cells in an individual, following the transfusion of a blood product. It is a transfusion-related adverse effect/long-term consequence, which has not been well-investigated among regularly transfused patients with thalassemia.Patients and MethodsWe investigated 64 regularly transfused, homozygous β-thalassemic patients and 21 never-transfused healthy volunteer blood donors (controls) for the presence of microchimerism in their sera, using real-time PCR targeting circulating allogeneic, both, Human Leukocyte Antigen-DR isotype (HLA-DR) and non-HLA alleles. The investigation was longitudinally repeated in patient subsets for more than 2 years. Results were correlated with clinical and laboratory parameters, peripheral blood lymphocyte immunophenotype, blood storage time, and donor's gender to identify potential contributing factors for microchimerism generation.ResultsOverall, microchimerism was detected in 52 of the 64 patients (81.2%) and in 6 of the 21 controls (28.5%, p = 0.0001). Forty-four patients (68.7%) exhibited long-term microchimerism (persisted for more than 6 months), confirmed at all time-points investigated. Microchimerism was more frequent among elderly, women, splenectomized and more heavily transfused patients, and among those who exhibit higher serum ferritin levels. In these patients, a distinct descending pattern of CD16dim+CD56dim+ natural killer (NK)-cells (p < 0.001) and an ascending pattern of CD4+CD25brightCD127– regulatory T-cells (p = 0.022) for increasing allelic burden were noticed, suggesting the establishment of recipient immune tolerance against the donor-derived chimeric alleles. Both splenectomized and non-splenectomized thalassemic patients exhibited the same trend. The storage time of transfused blood products and donor/gender mismatch had no impact on the development of microchimerism.Discussion-Conclusive RemarksTransfusion-associated microchimerism appears to be a very common complication among multi-transfused thalassemic patients. The potential clinical consequences of this phenomenon remain as yet unclear. Immune tolerance attributed to disease itself and to repeated transfusions might at least in part explain its appearance.

Immune-Mediated Mechanisms in Patients Testing Positive for SARS-CoV-2: Protocol for a Multianalysis Study.

BackgroundThe novel coronavirus has a high mortality rate (over 1% for patients older than 50 years). This can only be partially ascribed to other comorbidities. A possible explanation is a factor that assures a prompt response to SARS-CoV-2 in younger people, independent from the novelty of the virus itself. A factor is believed to stimulate the immune system and provide immunity against more antigens. The only external stimulation received by healthy people is vaccination (eg, the diphtheria, tetanus, and pertussis [DTP] vaccine). One hypothesis is that vaccination helps develop specific immunity but generates sprouting immunity against antigens in transit. The underlying immunological phenomena are the “bystander effect” and “trained immunity.” The developed immunity gives protection for years until it naturally fades out. After the fifth decade of life, the immune system is almost incompetent when a viral infection occurs, and thus, at this stage, the novel coronavirus can enter the body and cause acute respiratory distress syndrome.ObjectiveThe initial aim is to demonstrate that blood monocytes and natural killer cells show overpowering hyperactivity, while CD4+ and CD8+ T cells experience impediments to their defensive functions in patients with severe SARS-CoV-2 infection. The secondary objectives are to correlate clinical data and vaccination history with laboratory immune patterns in order to identify protective factors. Subsequently, we are also interested in characterizing the phenotypes and state of the degree of activation of peripheral blood mononuclear cells, including monocytes, natural killer cells, and CD4+ and CD8+ T cells, in healthy subjects vaccinated with the Pfizer vaccine.MethodsData will be collected using the following 3 approaches: (1) an experimental analysis to study the innate immune response and to identify genetic profiles; (2) an epidemiological analysis to identify the patients’ vaccination history; and (3) a clinical analysis to detect the immunological profile.ResultsThe protocol was approved by the Ethics Committee on April 16, 2020, and the study started on April 27, 2020. As of February 2021, enrollment has been completed. Immunological analysis is ongoing, and we expect to complete this analysis by December 2022.ConclusionsWe will recognize different populations of patients, each one with a specific immunological pattern in terms of cytokines, soluble factor serum levels, and immune cell activity. Anamnestic data, such as preceding vaccinations and comorbidities, biochemical findings like lymphocyte immunophenotyping, and pre-existing persistent cytomegalovirus infection, allow depicting the risk profile of severe COVID-19. Proof of the roles of these immunological phenomena in the development of COVID-19 can be the basis for the implementation of therapeutic immunomodulatory treatments.Trial RegistrationClinicalTrials.gov NCT04375176; https://clinicaltrials.gov/ct2/show/NCT04375176International Registered Report Identifier (IRRID)DERR1-10.2196/29892

B-cell cytopenia and time to last B-cell-depleting therapy predict response to SARS-COV-2 vaccines in patients with lymphoproliferative disorders

Patients with B-non-Hodgkin lymphoma (NHL) are at increased risk of morbidity and mortality from SARS-CoV-2. We investigated the relationship between B cell cytopenia and the SARS-CoV-2 vaccine response in B-NHL patients. We measured anti-RBD antibodies and the lymphocyte immunophenotype in 19 controls, 22 lymphoma patients on observation (cohort 1) and 55 lymphoma patients receiving their vaccines post B-cell depleting therapy (cohort 2). Half of the lymphoma patients in both cohorts achieved seropositivity compared to 100% of controls. In cohort 2, only 5% achieved an antibody response in the first year post B-cell depleting treatment, vs 88% treated >2 years prior. Also, 28% of patients with <50 B cells/µl achieved a serologic response vs 86% of patients with B-cell >50 B cells/µl. B-cell cytopenia is profound within the first year of exposure to B-cell depleting treatment, therefore an additional dose of vaccine within that timeframe is unlikely to raise antibody levels.

Лейкоцитарний склад та імунофенотип лімфоцитів периферичної крові у ДААт-позитивних і ДААт-негативних щодо вмісту діабетасоційованих автоантитіл дітей та підлітків на доклінічній та ранній клінічній стадіях розвитку цукрового діабету 1-го типу

У статті проаналізовані дані щодо особливостей лейкоцитарного складу та імунофенотипу лімфоцитів периферичної крові (ПК) у 94 ДААт-позитивних і 272 ДААт-негативних щодо вмісту діабетасоційованих автоантитіл (ДААт) дітей та підлітків, 49 ДААт-позитивних пацієнтів із дебютом цукрового діабету 1-го типу (ЦД1) та 84 дітей контрольної групи на доклінічній та ранній клінічній стадіях розвитку ЦД1. Визначення вмісту й аналіз розподілу лімфоцитів різного імунологічного фенотипу здійснювали методом проточної цитометрії, а визначення ДААт — радіоімунологічним методом. Встановлено, що найбільш значні зміни адаптивного та природного імунітету відбуваються на доклінічній стадії розвитку захворювання, що обумовлено найактивнішою фазою перебігу автоімунного процесу. У доклінічний період розвитку ЦД1 у ДААт-позитивних дітей реєструвалося збільшення абсолютної кількості моноцитів та зниження кількості природних клітин-кілерів (CD56+ лімфоцитів/великих грануловмісних лейкоцитів) у ПК, що свідчило про патогенетично значуще ослаблення вродженого імунітету на цьому етапі формування захворювання. Латентний, доклінічний період розвитку ЦД1 у дітей із підвищеними титрами ДААт до острівцевих антигенів порівняно з маркер-негативними пацієнтами щодо наявності ДААт і здоровими дітьми із групи контролю характеризувався порушеннями адаптивного імунітету — зниженням у циркуляції загальної кількості CD3+, CD4+, CD8+ Т-клітин, що є матеріальним субстратом детермінованої автоімунної агресії на доклінічній стадії розвитку ЦД1.

Altered immune response in organic acidemia.

Most patients with organic acidemia suffer from recurrent infections. Although neutropenia has been reported in multiple studies, other components of the immune system have not been evaluated thoroughly. This study was conducted to assess the immune status of patients with organic acidemia (OA). Thirty-three patients with OA who were followed up in Istanbul University-Cerrahpasa, Cerrahpasa School of Medicine, Nutrition and Metabolism Department and a total of 32 age- and sex-matched healthy controls were enrolled to the study. The demographic and clinical data were recorded retrospectively from patient files. Complete blood counts, immunoglobulins, and lymphocyte immunophenotyping were recorded prospectively in a symptom- (infection-) free period. Of the 33 patients enrolled to the study, 21 (88%) were diagnosed with methylmalonic acidemia, 10 (33%) with propionic acidemia, and two (6.6%) with isovaleric acidemia. The mean age of the patients with OA and healthy subjects were 5.89 ± 4.11 years and 5.34 ± 4.36, respectively (P = 0.602). Twenty-nine (88%) of the patients had experienced frequent hospital admission, 13 (39%) were admitted to pediatric intensive care unit, and 18 (55%) suffered from sepsis. Naïve helper T cells and recent thymic emigrants were significantly lower in OAs (P < 0.001). Various defects in humoral immunity have also been documented including memory B cells and immunoglobulins. Patients with OAs may show adaptive immune defects rendering them susceptible to infections. Metabolic reprogramming based on nutritional modifications may be a promising therapeutic option in the future.

ІМУННІ ЦИТОПЕНІЇ У ХВОРИХ НА ХРОНІЧНУ ЛІМФОЦИТАРНУ ЛЕЙКЕМІЮ (ОСОБЛИВОСТІ ПЕРЕБІГУ, ПРОГНОСТИЧНІ МАРКЕРИ)

Background: Immune cytopenia (IC) is one of the major complications in chronic lymphocytic leukemia (CLL). The paper describes the peculiarities of different immune cytopenia in CLL patients and the importance of individual prognostic markers in the course of the disease. Methods: We observed 62 patients with CLL complicated by immune cytopenia. Among these patients 30 had autoimmune hemolytic anemia (AIHA), 18 experienced immune thrombocytopenia (ITP), 10 had Fisher-Evans syndrome (FES), 3 were diagnosed with partial red cell aplasia (PRCA), and immune neutropenia (IN) was revealed in 1 patient. In addition to general examination and laboratory studies, the following examinations were performed: immunophenotyping of peripheral blood lymphocytes, flow cytometry (CD5; CD19; CD20; CD23; CD38; ZAP70), Coombs test, a molecular cytogenetic study of peripheral blood lymphocytes using the FISH method with TP53 and ATM probes, the level of ß2-microglobulin. Results: It was established that the overall survival of CLL patients with IC depends on the form of the latter. The median overall survival in patients with Fisher-Evans syndrome was the shortest (75 months), slightly better survival was observed in patients with AIHA (median 80 months), the best survival was found in patients with ITP (median not reached). Among unfavorable markers of CLL with IC, there is the presence of del 11q22.3. Unfavorable prognostic markers were also the following: a positive Coombs test, high levels of ZAP 70 expression, and high levels of ß2-microglobulin

182P Comprehensive assessment of the immunophenotype of peripheral blood lymphocytes, inflammatory factors, the immunophenotype of lymphocytes infiltrating the tumor and clinical indicators for overall survival and progression-free survival in patients with gastric cancer

182P Comprehensive assessment of the immunophenotype of peripheral blood lymphocytes, inflammatory factors, the immunophenotype of lymphocytes infiltrating the tumor and clinical indicators for overall survival and progression-free survival in patients with gastric cancer

Hematopoietic stem cell transplantation for Inborn Errors of Immunity

Hematopoietic stem cell transplantation for Inborn Errors of Immunity

Immunophenotyping of intraepithelial lymphocytes in canine chronic enteropathy and intestinal T-cell lymphoma using endoscopic samples.

Human enteropathy-associated T-cell lymphoma (EATL) is considered to be derived from intraepithelial lymphocytes (IELs); however, the origin of canine intestinal T-cell lymphoma (ITCL) remains unclear. Histological, immunohistochemical, and clonality examinations were performed using endoscopically collected canine duodenum samples of mucosal lesions of chronic enteropathy (CE; 73 cases) and ITCL without transmural neoplastic mass lesions (64 cases). Histopathological examinations revealed the intraepithelial accumulation of lymphocytes (called "intraepithelial lymphocytosis") in 54/73 CE cases (74%) and the epitheliotropism of neoplastic lymphocytes in 63/64 ITCL cases (98%). Immunohistochemically, IELs in CE with intraepithelial lymphocytosis (IEL+CE) were diffusely immunopositive for CD3, with scattered immunopositivity for CD5, CD8, CD20, and granzyme B (GRB). The percentage of CD8+ in CD3+ IELs was significantly lower in IEL+CE than in CE without intraepithelial lymphocytosis (IEL-CE). Double-labeling immunohistochemistry revealed a high percentage of GRB expression in CD8- IEL among IEL+CE. Among 64 ITCL cases, CD3 was immunopositive in 64 (100%), CD5 in 22 (34%), CD8 in 8 (13%), CD20 in 12 (19%), CD30 in 13 (20%), and GRB in 49 (77%). In CD3+ cells, Ki67 immunopositivity was highest in ITCL, intermediate in IEL+CE, and lower in IEL-CE. A clonal TCR gene rearrangement was detected in 1/19 IEL-CE cases (5%), 15/54 IEL+CE (28%), and 38/58 ITCL (66%). These results indicate that the immunophenotype of canine ITCL (CD8-GRB+) is similar to that of the increased IELs in CE. The high proliferative activity and clonality of T cells in IEL+CE suggest that canine ITCL originates from these IELs, similar to human EATL.

Multisystem Autoimmune Inflammatory Disease, Including Colitis, Due to Inborn Error of Immunity

Our understanding of inflammatory bowel disease is changing as we identify genetic variants associated with immune dysregulation. Inflammatory bowel disease undetermined, even when diagnosed in older children and adolescents, in the setting of multiple inflammatory and infectious diseases should raise the suspicion of complex immune dysregulation with a monogenic basis. We report a case of inflammatory bowel disease undetermined triggered by exposure to a nonsteroidal antiinflammatory drug in a 16-year-old girl with a background history of juvenile idiopathic arthritis, cytopenias, recurrent respiratory tract and middle ear infections, and esophageal candidiasis. Immunologic assessment included measurement of immunoglobulin levels, lymphocyte immunophenotyping, B-cell functional tests, and whole-exome sequencing. Laboratory investigation revealed defects of humoral immunity, including mild persistent hypogammaglobulinemia affecting all 3 isotypes and absent isohemagglutinins. Whole exome sequencing revealed a heterozygous TNFRSF13B (Tumor Necrosis Factor Receptor Superfamily Member 13B, or Transmembrane Activator and Calcium-modulating cyclophilin ligand Interactor, TACI) gene variant, which is associated with common variable immunodeficiency and the development of autoimmune diseases. In conclusion, a clinical history of recurrent infections, atypical histologic features of inflammatory bowel disease, additional autoimmune manifestations, and an inadequate response to conventional therapy should prompt the physician to refer to an immunologist with the query of inborn error of immunity. We report how extensive immune evaluation and genetic diagnosis can individualize care and facilitate a multidisciplinary team approach.

Téloméropathies de recrutement hématologique : étude de 15 cas

IntroductionTelomeres are composed of a repeated sequence of double-stranded nucleotides TTAGGG and numerous proteins including the Shelterin complex. Their main role is to maintain the stability of the genome during cell replication through a mechanism of copying the repeted sequence by the telomerase complexe. All the diseases involving a deregulation of this complex are now grouped together under the term telomeropathies. They are difficult to diagnose and manage. Our objective was to describe the clinico-biological characteristics and treatments used, in patients affected by telomeropathies previously seen by an hematologist followed at the Lille University Hospital Center. MethodsThis is a retrospective, single-center study carried out within the department of internal medicine-clinical immunology, Reference center for rare autoimmune and systemic diseases at Lille University Hospital Center between 2005 and 2020 including all patients followed for telomeropathy. ResultsProbands and relatives were included. Fifteen patients were studied from 10 independant families. Sixty percent had an heterozygous TERC gene mutation. Sixty seven percent had haematological diseases including macrocytosis, anemia and/or thrombocytopenia, 20 % had a fibrotic hepatic disease, 27 % had a fibrotic pulmonary disease. Lymphocyte immunophenotyping showed a double negative T lymphocyte population with γδ TCR expression in 5 (33 %) patients. Forty-seven percent of the patients had not received any treatment. Twenty-seven percent were on androgen therapy. Twenty percent had received cyclosporine and 13 % anti-lymphocyte serum in the context of initial misdiagnosis. ConclusionIt is important to be aware of the complexity of telomeropathies, a differential diagnosis of immune aplastic anemia, in order to optimize management and avoid inappropriate treatments. Allografting of hematopoietic stem cells is the only potentially curative treatment. Our analysis found particularities in immunophenotyping lymphocyte not previously described to our knowledge, whose physiopathological imputability remains to be demonstrated.

Состояние клеточного иммунитета и цитокинового баланса у беременных женщин при внутриутробном инфицировании

The question of studying changes in the immune responses of mother and foetus against the background of infections acquired via various routes remains relevant. This study aimed to assess the state of T-cell immunity and cytokine balance in pregnant women with various pathways of intrauterine infection and the condition of newborns. The study involved 205 pregnant women at high risk for intrauterine infection. In the 1st trimester, bacteriological and DNA analysis of the lower urogenital tract and cytological examination of the cervical canal were performed, blood levels of immunoglobulins M and G for herpes simplex virus type 1 and 2, cytomegalovirus infection, toxoplasmosis, and rubella were determined. In the whole blood of the women, lymphocyte immunophenotyping was performed using CYTO-STAT® triCHROMETM CD8-FITC/CD4-RD1/CD3-FITC monoclonal antibodies; the content of cytokines (IL-6, IL-10) was analysed by means of enzyme-linked immunosorbent assay. After delivery, a pathomorphological examination of the placenta was performed in line with the generally accepted method. According to the results of the study, the following groups of women were identified: 1) without infectious or inflammatory changes (n = 59); 2) with confirmed ascending infection (n = 69); 3) with haematogenous infection (n = 33); 4) with mixed infection (n = 44). The condition of newborns was assessed with the help of laboratory and instrumental methods, using the INTERGROWTH-21st charts and the Apgar score. We found that the functioning of the immune system of pregnant women is affected by viral infections acquired via the haematogenous route, resulting in a relative increase in suppressor T cells and a decrease in helper T cells, as well as ina growing absolute number of lymphocytes in the blood. The identified inhibition of IL-6 and IL-10 production in the groups with signs of placental lesions due to infection at 16–18 weeks can indicate a strain on the immune processes and development of placental insufficiency. Newborns with morphological signs of haematogenous infection are characterized by changes in the cytological parameters of residual cord blood, signs of placental insufficiency, low birth weight, and hypoxic-ischemic damage to the central nervous system.

Tocilizumab and Desensitization in Kidney Transplant Candidates: Personal Experience and Literature Review.

Desensitization (DES) allows kidney transplantation for highly HLA-sensitized subjects. Due to the central role of IL-6 in the immunological response, tocilizumab may improve DES efficacy. Thus, we conducted a PubMed systematic review using the MeSH terms tocilizumab, interleukin-6, kidney transplantation, and desensitization. Tocilizumab (TCZ) was first studied for DES as the second-line treatment after failure of a standard DES protocol (SP) (apheresis, rituximab +/− IVIg). Although TCZ (as a monotherapy) attenuated anti-HLA antibody rates, it did not permit transplantation. However, lymphocyte immuno-phenotyping has shown that TCZ hinders B-cell maturation and thus could improve the long-term efficacy of DES by limiting anti-HLA rebound and so avoid antibody-mediated rejection. This hypothesis is supported by a recent study where clazakizumab, a monoclonal antibody directed against IL-6, was continued after kidney transplantation in association with an SP. Nine out of ten patients were then eligible for transplantation, and there were no donor-specific antibodies at 6 months post-transplantation. In association with an SP, tocilizumab does not seem to significantly improve kidney-allograft access (short-term efficacy) vs. a SP only. However, it could improve the long-term prognosis of HLA-incompatible transplantation by hindering B-cell maturation and, thereby, avoiding donor-specific antibody rebounds post-transplantation.

An Ulcerating Perineal Rash in an 8-month-old Girl.

An Ulcerating Perineal Rash in an 8-month-old Girl.

Prognostic role of NK cell percentages in bronchoalveolar lavage from patients with different fibrotic interstitial lung diseases

BAL cellularity and lymphocyte immunophenotyping offer insights into lung inflammatory status. Natural killer (NK) cells are efficient effector cells, producing pro-inflammatory cytokines. A better understanding of the biology of NK cells in BAL in the lungs is necessary to improve the pathogenesis of fibrotic ILD and develop prospective targeted treatments. Our aim was to analyse NK and NKT-like cell percentages in BAL from 159 patients with different ILD: f-HP, f-NSIP, IPF and CTD-ILD, to evaluate their potential diagnostic/prognostic role. BAL NK cell percentages showed significantly higher values in IPF than in f-HP and f-NSIP, while BAL NKT-like cells showed significantly lower values in the f-NSIP than the f-HP and IPF. A cut-off of 4%NK cells in BAL of IPF showed a significant difference in survival rate. It suggests a possible new marker of survival and raises the possibility of new targeted approach in treatment and management of IPF.

Lymphocyte immunophenotyping in inflammatory myositis: a review.

This is a comprehensive review of the current knowledge on predominant immune cell phenotypes involved in idiopathic inflammatory myopathies (IIM). Major circulating immune cell subpopulations described in IIM encompass the lymphocyte compartment. An unbalance in T cell subsets seems to consistently affect the peripheral and muscle compartment, with a predominance of CD4+ T and B cells in dermatomyositis, CD8+ T cells in polymyositis/inclusion body myositis (IBM) and novel findings highlighting novel proinflammatory T subsets, that is, CD8+Tbet+ and CD28- T cells across different IIM subsets. On the other hand, an impairment in Treg cells number and function has been described especially across polymyositis/dermatomyositis and IBM. Total T follicular helper (Tfh) cells, increased in immune-mediated necrotizing myopathy, skewed toward Tfh2 and Tfh17 in dermatomyositis, polymyositis, and juvenile dermatomyositis. B cell compartment is more rarely described in IIM, yet an unbalance in this pool is as well likely. Evidence of plasma cells increased in polymyositis, dermatomyositis, IBM, and Bregs decreased in dermatomyositis have been reported. Perturbations in the memory and naïve subsets are common in dermatomyositis/polymyositis and antisynthetase syndrome. Protean immune cell abnormalities characterize different IIM subsets, reflecting the complexity of these autoimmune conditions. A deeper understanding of B-cell and T-cell immunophenotyping may promote early diagnosis and identification of new potential therapeutic targets.

Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes

The gut associated lymphoid tissue (GALT) effector sites play a crucial role on the pathogenesis of many immune-mediated gastrointestinal diseases. The lymphocytes at these effector sites are principally T cells which present important morphological, phenotypical and functional differences. Flow cytometry (FC) is one of the most commonly used techniques to characterize intestinal lymphocytes in human and animal models. Published studies with a focus on dogs for intraepithelial lymphocytes (IEL) immunophenotyping exist in very limited numbers. Moreover, no lamina propria lymphocytes (LPL) isolation protocols in the canine species have been described for FC evaluation. In addition to immune intestinal dysregulation, imbalances in the peripheral blood immune system have been described in both human and animal gastrointestinal disorders. The aim of this study was to provide a protocol for canine IEL and LPL isolation for FC immunophenotyping of T cells subsets. Specifically, T helper, T cytotoxic, activated Th and Tc lymphocytes, regulatory, double negative, double positive, IFN-γ and IL-4 producing T cells, and to compare their respective populations between these effector sites and with the blood stream compartment in healthy dogs. The potential relationship of these cells distributions with age, sex and breed was also evaluated. This study included sixteen healthy dogs of different sexes and breeds with a mean age of 4.55 ± 2.93 years old. The selected protocols for the three immune compartments showed proper cell yield, purity, viability, and the absence of phenotypic and functional disturbances. Histologically, an adequate separation of the duodenal epithelium from the lamina propria was also observed. All the proposed T cells subsets were identified in the three immune compartments studied, showing some statistically significant differences in their distributions at these locations that highlight the importance of their individual evaluation. This study provides an adequate method for canine small intestine IEL and LPL isolation for FC immunophenotyping and is key for future studies on the gastrointestinal immune system associated with different canine diseases.