Peripheral Lymphocyte Populations Research Articles

Peripheral lymphocyte populations in ovarian cancer patients and correlations with clinicopathological features

BackgroundTo investigate the alterations of peripheral lymphocyte subpopulations in ovarian cancer patients compared to benign or borderline counterparts. The possible clinicopathological implications were also evaluated.MethodsWe enrolled 112 treatment-naive ovarian cancer patients, 14 borderline tumor patients and 44 benign tumor patients between 09/2016 and 01/2019. Flow cytometry was used to measure the peripheral lymphocyte subsets consisting of T cells (CD3+, CD3+CD4+, CD3+CD8+ and CD8+CD28+), regulatory T cells (Tregs, CD4+CD25+CD127−), natural killer cells (NK cells, CD3−CD56+) and B cells (CD19+).ResultsMost ovarian cancer patients were high-grade serous carcinoma (84.8%), followed by clear cell carcinoma (8.03%). Late-stage tumor (FIGO III + IV) accounted for 82.1%. The study showed that the proportions of peripheral lymphocyte subsets underwent apparent changes in ovarian cancer patients. We observed elevated levels of Treg cells in patients with both ovarian borderline and malignant tumor compared to those with benign tumors, which achieved statistic significance. In contrast, CD3+CD8+ T and CD8+CD28+ T cells were significantly lower in ovarian cancer patients. Interestingly, low level of B cells was correlated to clear cell carcinoma (P = 0.024), advanced tumor (P = 0.028) and platinum-resistant recurrence (P = 0.014). Regarding the changes of lymphocyte subsets after surgery, CD8+CD28+ T cells had a significant decreasing tendency (P = 0.007) while B cells were the opposite (P < 0.001).ConclusionsOvarian cancer patients have altered circulating lymphocyte profile (elevated Treg cell, depressed CD3+CD8+ T and CD8+CD28+ T cells). Low level of B cells might be related to disease aggressiveness, and it recovered after the removal of tumor, which merits further study.

Features of CD-­­phenotype of peripheral blood lymphocytes in adult patients with atopic dermatitis during the exacerbation

Objective — to study the dynamics of peripheral blood lymphocytic populations in adult patients with atopic dermatitis (AD) with the onset of the disease in childhood, depending on the level of IgE secretion and the method of treatment.&#x0D; Materials and methods. We examined 67 adult patients with AD, which were divided into 4 groups depending on the level of total serum IgE and the basic treatment or treatment in combination with Glycine and Ketotifen. The severity of AD was determined by the SCORAD index. The content of peripheral blood lymphocytes according to the phenotype CD3+, CD4+, CD8+, CD19+, CD65+, HLADR+ and CD95+ was assessed during hospitalization of patients, at the end of the inpatient stage of treatment and after 1 month of outpatient follow-up. The obtained data were compared with the indices of the control group and between the groups of examined patients with AD in the dynamics of their treatment and observation. The results were processed statistically using the methods of parametric and nonparametric statistics.&#x0D; Results and discussion. The indices of the number of cells of peripheral lymphocytic populations of different CD pheno­type in the groups in the dynamics of observation were determined, their relationship with the severity of the course of AD was established, and differences were found depending on the pathogenetic variant of AD. Against the background of an exacerbation of AD, a significant increase in the number of cells in most of the defined populations was revealed, with its gradual decrease as the clinical manifestations of AD subsided. It was established that 1 month after achievement of clinical/subclinical remission, a part of the peripheral blood lymphocytic populations was characterized by higher values compared to the norm. In patients with an IgE-dependent AD variant, aggravation is accompanied by high levels of peripheral lymphocytes with CD3+, CD4+, CD8+, CD19+ and HLA-DR phenotypes, which more often than in the case of an IgE-independent variant of AD, remain above the norm after 1 month of outpatient monitoring. Introduction of glycine and ketotifen to the treatment complex for patients with AD is accompanied by a faster return of peripheral lymphocyte cells to normal values, which is more evident in patients with an IgE-dependent variant of AD.&#x0D; Conclusions. In adult AD patients, the dynamics of the number of peripheral lymphocyte population cells depends on the severity of the disease, its pathogenetic variant and the treatment received by the patient. Against the background of the use of glycine and ketotifen, the normalization of indicators of peripheral lymphocytic populations occurs significantly faster than with only standard basic therapy.

741 A complex clinical mosaic of severe autoimmune calcific constrictive pericarditis with striking haemodynamic response to immunosuppressive therapy

Abstract Autoimmune constrictive pericarditis constitutes a conundrum to modern cardiology with much uncertainty surrounding both pathophysiology and optimal treatment strategies. We hereby describe the case of a 35-year-old woman of Nigerian origin with severe right heart failure secondary to calcific constrictive pericarditis. Her past medical history included coagulation factor XI deficiency, leukopenia, 2nd trimester miscarriage and premature labour due to placenta previa with fibrin deposition. Further investigations revealed atrial fibrillation, severe biatrial enlargement, moderate tricuspid and mitral regurgitation, pericardial thickening, post-capillary pulmonary hypertension and right ventricular dip-and-plateau pattern, compatible with severe constrictive pericarditis. Extensive screening for infectious and autoimmune causes only revealed borderline positive ANA (1:80). Thereafter, the patient underwent complete surgical pericardiectomy with pericardial biopsies revealing fibrous thickening, diffuse calcification and lymphocyte/macrophage infiltrates, in the absence of giant multinucleated cells or granulomas. The patient was later discharged but soon experienced relapse of exertional dyspnoea presenting with right-sided haemo-pneumothorax (requiring pleural drainage), diffuse alveolar haemorrhage, large right-sided basal and infrascissural pleural effusion, and ascites. She was treated with high dose iv furosemide, oral ibuprofen and colchicine, suspension of rate control medications, achieving initial reduction in pulmonary oedema and ascites, relapsing however after attempts to switch to oral diuretic therapy. Due to the finding of persistent lymphopenia, further immunological tests were conducted, revealing raised IgG1 levels as well as altered peripheral lymphocyte populations (raised CD4+/CD8+ ratio and CD8+ central memory, reduced CD8 effector memory). This finding in conjunction with the history of factor XI deficiency, 2nd trimester miscarriage and placental fibrin deposition as well as the observation of painful cutaneous nodules at sites of venepuncture, suggestive of Koebner’s phenomenon, veered the diagnostic focus to a potential autoimmune aetiology and in particular to systemic lupus erythematosus (&amp;gt;10 ACR-EULAR score points with case reports describing all the above as potential disease manifestations). Furthermore, revision of thoracic CT scans, demonstrated bilateral migratory peribronchovascular nodules with ground-glass halo. CT- guided biopsies thereof were performed revealing focal alveolar damage with capillaritis and alveolar haemorrhage, further corroborating the clinical suspicion of autoimmune disease and justifying the introduction of high-dose oral corticosteroid therapy. In liaison with our tertiary rheumatology centre, the patient was later switched to mycophenolate with gradual weaning from corticosteroid. Concurrent cardiological follow-up revealed persistence of good haemodynamic status (NYHA class II, absence of pulmonary oedema and ascites) with oral diuretic therapy, regression of cutaneous symptoms and echocardiography demonstrating consistent reduction in both mitral and tricuspid regurgitation. This constitutes to our knowledge the first report of autoimmune calcific constrictive pericarditis with significant haemodynamic response to immunosuppressive therapy. Despite the relative rarity of this disease entity, early recognition and instatement of immunosuppressive treatment may prove fundamental to halt and potentially reverse the haemodynamic progression of this highly morbid condition.

Changes in peripheral lymphocyte populations in patients with advanced/recurrent ovarian cancer undergoing splenectomy during cytoreductive surgery

BackgroundTo investigate changes in peripheral lymphocyte subsets after splenectomy during cytoreductive surgery for advanced or recurrent ovarian cancers.MethodsWe enrolled 83 patients with advanced or recurrent ovarian cancer who underwent cytoreductive surgery. Twenty patients who also underwent splenectomy were assigned to the splenectomy cohort and the rest were assigned to the non-splenectomy cohort. Flow cytometry was used to measure peripheral lymphocyte subsets consisting of T cells, regulatory T cells, natural killer cells, B cells, and activation antigens before and after surgery.ResultsThere was no difference in the number and distribution of peripheral lymphocyte subsets between the two cohorts before surgery. After surgery, we observed elevated levels of T cells (CD3+, CD3+CD8+) in the splenectomy cohort compared to those in the non-splenectomy cohort, and the difference was statistically significant. CD8+CD28+ T cells had a significant decreasing tendency (P = 0.011) while CD3+/HLA-DR+ T cells showed the opposite trend (P = 0.001) in the splenectomy cohort. The proportion of Tregs (P = 0.005) and B cells (P < 0.001) including CD3−/HLA-DR+ B cells (P = 0.007) increased after surgery, and the absolute number of T cells and NK cells decreased to different extents (P < 0.001) in the non-splenectomy cohort. The post-operative percentage of CD8+CD28+ T cells was less than the pre-operative percentage (P = 0.022), which was similar to the splenectomy cohort. There was no significant difference in progression-free survival or overall survival between the groups after a median follow-up time of 41 months.ConclusionsThe changes in peripheral lymphocyte populations were different between patients with and those without splenectomy during cytoreductive surgery for ovarian cancers. T cells were increased and activated in the splenectomy cohort, whereas, B cells were increased and activated in the non-splenectomy cohort.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant Using a Human Antibody Drug Conjugate Targeting C-KIT

For patients undergoing hematopoietic stem cell transplantation (HSCT), conditioning remains a primary source of transplant related morbidity and mortality and limits the broader application of this life-saving therapy. Current conditioning regimens, including total body irradiation and high dose chemotherapy, cause acute toxicity, cytokine storm, increased graft-versus-host disease, and organ damage. In addition, these regimens are also associated with the development of secondary malignancies, autoimmunity, and infertility.To address this significant unmet medical need, we have developed a conditioning regimen using an antibody drug conjugate (ADC) targeting C-KIT (CD117) to specifically deplete host hematopoietic stem cells. CD117 is highly expressed on hematopoietic stem and progenitor cells (HSPCs) and binding to its ligand, stem cell factor, promotes hematopoiesis, self-renewal, and rapid internalization of the receptor. The rapid internalization and selective expression of CD117 on HSPCs make it an ideal target for an ADC-based approach to depleting HSPCs, while limiting systemic toxicity. In addition, CD117 is frequently overexpressed on malignant cells in patients with acute myelogenous leukemia. Thus, an ADC targeting CD117 may provide an opportunity for disease control prior to or concomitant with an HSCT. Previous studies have demonstrated that an antagonistic antibody against CD117 lead to high levels of HSPC depletion and donor engraftment in immunocompromised mice, but failed to facilitate this effect in immunocompetent hosts. To overcome the engraftment barrier, additional studies were conducted with an anti-CD117 antibody conjugated to the ribosome inactivating toxin, saporin, which allowed for robust HSPC depletion and high levels of congenic donor chimerism in immunocompetent mice (Czechowicz et.al., Blood 2016, 128:493).To advance this technology toward the clinic, we have developed a fully human anti-CD117 antibody conjugated to a small molecule toxin capable of depleting proliferating and quiescent cells (CD117-ADC), that demonstrated greater than 90% killing of the leukemia cell line Kasumi-1, and was equally effective at killing primary human CD34+ bone marrow cells during in vitro culture. Humanized NSG mice treated with CD117-ADC had greater than 90% depletion of human HSPCs in the bone marrow, 21 days following a single administration of the ADC. The specificity of CD117-ADC for HSPCs was confirmed by the presence of stable peripheral human lymphocyte populations, and absence of human myeloid cells; indicating a lack of stem and progenitor cells capable of replenishing these short-lived cells. In addition to the ability of CD117-ADC to deplete HSPCs in humanized mice, preliminary data suggests that CD117-ADC is also effective at reducing tumor burden in mice challenged with CD117 expressing tumor cells.Eliminating HSPCs from the stem cell niche with a targeted ADC has the promise to enable the engraftment of donor HSCs, while preventing the systemic toxicity associated with irradiation and chemotherapy. We have demonstrated that an ADC targeting CD117 is capable of depleting human HSPCs in vitro and in vivo and has the potential to reduce tumor burden in mice. Ultimately, this novel approach for safer conditioning prior to HSCT could greatly increase the number of patients eligible for transplant and significantly reduce the damaging side effects associated with current protocols and may provide effective non-genotoxic conditioning for all patients suffering from malignant or non-malignant diseases who require a HSCT. [Display omitted] DisclosuresHartigan:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Pearse:Magenta Therapeutics: Employment, Equity Ownership. McDonough:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. McShea:Magenta Therapeutics: Employment, Equity Ownership. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Panwar:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Goncalves:Magenta Therapeutics: Employment, Equity Ownership. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

NKG2D Enhances Double-Negative T Cell Regulation of B Cells.

Numerous studies reported a small subpopulation of TCRαβ+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.

Adenosine monophosphate deaminase 3 null mutation causes reduction of naive T cells in mouse peripheral blood

AbstractAdenosine monophosphate deaminase 3 (Ampd3) encodes the erythrocyte isoform of the adenosine monophosphate (AMP) deaminase gene family. Mutations in this gene have been reported in humans, leading to autosomal-recessive erythrocyte AMP deaminase deficiency. However, the mutation is considered clinically asymptomatic. Using N-ethyl-N-nitrosourea mutagenesis to find mutations that affect peripheral lymphocyte populations, we identified 5 Ampd3 mutations (Ampd3guangdong, Ampd3carson, Ampd3penasco, Ampd3taos, and Ampd3commanche) that strongly correlated with a reduction in naive CD4+ T and naive CD8+ T-cell populations. Causation was confirmed by targeted ablation of Ampd3. Knockout mice had reduced frequencies of CD62LhiCD44lo CD4+ naive and CD8+ naive T cells. Interestingly, these phenotypes were restricted to T cells circulating in peripheral blood and were not seen in T cells from secondary lymphoid organs (lymph nodes and spleen). We found that reduction of naive T cells in the peripheral blood of Ampd3−/− mice was caused by T-cell–extrinsic factor(s), which we hypothesize to be elevated levels of adenosine triphosphate released by Ampd3-deficient erythrocytes. These findings provide an example in which disruption of an erythrocyte-specific protein can affect the physiological status of lymphocytes in peripheral blood.

Non-therapeutic administration of antimicrobial growth promoters to interrogate suppression mechanisms of innate and adaptive immune responses using an ovine model of stress

Abstract Antimicrobial growth promoters (AGPs) have been administered in non-therapeutic concentrations in livestock feed to increase weight gain per unit of feed consumed. Daily low-dose antibiotic administration is capable of modulating immunity by suppressing inflammation, potentially increasing metabolic resources that can be allocated to muscle development. It is unknown how the AGPs tylosin (TYL) and chlortetracycline (CTC) influence acquired immunity. The objective of this study was to measure acquired immune responses, assess impacts on innate immunity and to elucidate mechanisms of immunosuppression by immunizing 10-week old lambs (male Suffolk cross) with keyhole limpet hemocyanin (KLH) during AGP administration (TYL, CTC or Control diets) while enduring intermittent physical stress. Over a 4-week period, 18 lambs (n=3 per diet/stress group) were monitored. Between the third-fourth experimental week, stressed lambs gained less weight (1.50 ± 0.49 kg) than non-stressed lambs (3.06 ± 0.37 kg, P &amp;lt; 0.03). The weight of lambs fed AGPs did not surpass those on the control diet. Diet and stress did not impact serum concentrations of total IgG and IgM, or anti-KLH IgG and IgM specific titres. Endpoint hematological analysis revealed that stressed lambs had decreased red cell distribution width (RDW%, 29% vs. 31%, P&amp;lt;0.01) and peripheral lymphocyte populations (5.72 × 103/μL vs. 4.69 × 103/μL, P&amp;lt;0.02). A diet × stress interaction was observed for peripheral granulocyte population (P=0.02). The impending restriction of AGP usage in North America has generated interest in identifying AGP alternatives, and elucidation of the mechanisms of AGP function is needed to develop effective non-antibiotic alternatives that mimic their action.

Immunological Responses to the Relapsing Fever Spirochete Borrelia turicatae in Infected Rhesus Macaques: Implications for Pathogenesis and Diagnosis.

The global public health impact of relapsing fever (RF) spirochetosis is significant, since the pathogens exist on five of seven continents. The hallmark sign of infection is episodic fever and the greatest threat is to the unborn. With the goal of better understanding the specificity of B-cell responses and the role of immune responses in pathogenicity, we infected rhesus macaques with Borrelia turicatae (a new world RF spirochete species) by tick bite and monitored the immune responses generated in response to the pathogen. Specifically, we evaluated inflammatory mediator induction by the pathogen, host antibody responses to specific antigens, and peripheral lymphocyte population dynamics. Our results indicate that B. turicatae elicits from peripheral blood cells key inflammatory response mediators (interleukin-1β and tumor necrosis factor alpha), which are associated with preterm abortion. Moreover, a global decline in peripheral B-cell populations was observed in all animals at 14 days postinfection. Serological responses were also evaluated to assess the antigenicity of three surface proteins: BipA, BrpA, and Bta112. Interestingly, a distinction was observed between antibodies generated in nonhuman primates and mice. Our results provide support for the nonhuman primate model not only in studies of prenatal pathogenesis but also for diagnostic and vaccine antigen identification and testing.

T cell population expansion in response to allogeneic cancer vaccine alone (DPV-001) or with granulocyte-macrophage colony-stimulating factor (GM-CSF) or imiquimod (I) for definitively-treated stage III NSCLC patients (pts).

e14639 Background: The DPV-001 DRibble is a dendritic cell-targeted microvesicle (proteasome blocked autophagosome) vaccine derived from an adenocarcinoma and a mixed histology cell line. It contains multiple TLR agonists and &gt; 130 potential NSCLC antigens, many as prospective altered-peptide ligands. In preclinical studies, DRibble immunotherapy provided significant anti-cancer effects in a dozen models. We hypothesize that DRibble’ vaccination efficacy can be attributed to their capacity to present tumor-derived short-lived proteins (SLiPs) and defective ribosomal products (DRiPs) that are typically not processed and presented by professional antigen presenting cells and against which the host may be less tolerant. Methods: Pts received induction cyclophosphamide, 7 vaccines every 3-weeks, then every 6 weeks x 4 more doses. Pts were randomized to receive DRibble alone (A), or with I (B) or GM-CSF (C). PBMCs /serum were collected at baseline and at each vaccination to assess changes in antibodies (Ab) (ProtoArray and microsphere affinity proteomics), peripheral lymphocyte populations (flow cytometry) and T cell receptor (TCR) repertoires (Adaptive immunoSEQ). Results: 13 pts were enrolled (Arm A: 5; B: 4; C: 4). We previously reported that vaccination induced or increased IgG Ab responses against targets over-expressed by NSCLC. Patients receiving DPV-001 had a significant (p &lt; 0.04) increase in total (CD4 + CD8) TCRs that increased 10 fold over baseline compared to normal controls (independent from trial, n = 3) and the increase in CD4 clones was similar to that seen following ipilimumab (melanoma pts, independent from trial, n = 9). Analysis of a resected metastasis (progressing on treatment), identified brisk infiltration of T cells and tumor that was strongly PD-L1+. Conclusions: Vaccination with DPV-001 expanded populations of T cells over that observed in controls and the increase in CD4 T cells was similar to that observed in patients receiving ipilimumab and may represent vaccine-reactive T cells. Clinical Trial Identifier: NCT01909752, Support: R44 CA121612 Clinical trial information: NCT01909752.

Характер изменений в популяционном и субпопуляционном составе лимфоцитов периферической крови и экспрессии Toll-рецепторов на клетках у больных хроническим генерализованным пародонтитом I-II степени тяжести в сочетании с паразитарными инвазиями

Характер изменений в популяционном и субпопуляционном составе лимфоцитов периферической крови и экспрессии Toll-рецепторов на клетках у больных хроническим генерализованным пародонтитом I-II степени тяжести в сочетании с паразитарными инвазиями

Immune monitoring of immunosuppression withdrawal of liver transplant recipients

Several studies have shown that some liver transplant recipients may tolerate immunosuppression withdrawal. Mechanisms and biomarkers of tolerance are not well known. MethodsTwenty-four LT patients with immunosuppression side-effects underwent progressive immunosuppression withdrawal. Peripheral lymphocyte populations and secretion of cytokines were analyzed at baseline and during withdrawal until tolerance (n=15) or rejection (n=9), as well as 3months after tolerance achievement or rejection resolution (as follow-up). Immunological markers were compared among groups. ResultsThe percentages of CD3+CD4+ cells progressively decreased in both groups. CD3+CD8+ cells gradually increased in tolerant patients. B lymphocytes gradually decreased in tolerant and initially in non-tolerant patients, reverting at rejection. Regulatory T cells progressively increased until rejection in non-tolerants, decreasing to basal levels after renewing immunosuppression; no significant changes were found in tolerant patients. The percentages and absolute counts of natural killer cells significantly increased in both groups, being more evident in tolerant patients. The secretion of several cytokines was higher in non-tolerant patients when rejection was diagnosed. ConclusionsThe greater increase of natural killer cells in tolerant patients suggests their potential role in the tolerance phenomenon.

Distribution of Peripheral Lymphocyte Populations in Primary Sjögren's Syndrome Patients.

Purpose of this study was to evaluate the lymphocyte populations' distribution changes in peripheral blood of patients with primary Sjögren's syndrome (pSS). Lymphocyte populations' distribution changes in peripheral blood of pSS patients were investigated in 52 patients with pSS and in 28 healthy controls by flow cytometry. We found decreased absolute count of CD3+ T cell population in pSS patients. Analysis of CD4+ T cell population showed significant proportion and absolute count differences in pSS patient's blood with SSA/SSB antibodies (Abs) in comparison to controls. No significant differences were observed analyzing CD4+ and CD8+ Treg subpopulation. Proportion and absolute counts of Th17 cells were significantly lower in pSS patient's blood. Absolute counts of CD8+ T cells were significantly lower in pSS patients in comparison to controls and also impaired proportion and absolute counts of CD8+ subpopulations according to CD27+ and CD57+ were observed. Absolute counts of NKT and NK cells were decreased in pSS with Abs. B cells proportion was increased only in blood of pSS with Abs. Lymphocyte distribution impairment can be due to genetically determined lymphopenia or lymphocyte migration from periphery to inflammatory sites or/and increased susceptibility to apoptosis.

Accentuated hypo- and hyper-NK lymphocyte CD8 expression is a marker of NK subsets’ misbalance and is predictive for reproductive failures

IntroductionNK cells play critical yet poorly defined role in reproductive processes. Role of CD8αα expression on a part of peripheral blood NK lymphocyte population is not clear. Patients and methodsDuring isolated double-blinded clinical investigation we studied blood samples from 153 women with multiple reproductive failures undergoing IVF which were drawn 5–12d before embryo transfer procedure. 90 women had IVF failures (IVFf), 24 became pregnant with subsequent pregnancy failure (Pf), 39 became pregnant with subsequent successful pregnancy.During retrospective study we analyzed our laboratory data of 1045 infertile women which underwent routine immunology investigation after IVF failures. Lymphocyte phenotype and NK cytotoxicity was studied by FACScan flow cytometer using BD monoclonal abs. ResultsWe showed that NK-CD8 expression being increased↑ (>60%) was predictive for IVFf (OR 3.523, p=0.0193, n=23) while being decreased↓ (<40%) was significantly predictive for subsequent Pf (OR 4.571, p=0.0418, n=37). Balanced “conditionally normal” NKCD8 expression (40–60%, n=93) was very significantly predictive for whole reproductive success after IVF (OR 3.972, p=0.0021). Analysing retrospective data, we found decreased CD3− CD56++ and T lymphocyte frequency in both “accentuated” groups compared to patients with “conditionally normal” NK-CD8 expression (n=562). NK-CD8 expression↓ (n=341) was associated with elevated HLADR expression on NK, CD3+, CD3+ CD8+, CD3+ CD4+ cells. Meanwhile, NK-CD8 expression ↑ (n=142) was associated with elevated NK frequency, NK cytotoxicity levels and CD158a expression on NK cells, simultaneously with decreased CD3CD8 and CD3+ CD56+ numbers. ConclusionBoth hypo- and hyper-NK lymphocyte CD8 expression “accentuations” associated with NK subsets’ misbalance (NK and T lymphocyte phenotype and cytotoxicity). Both hypo- and hyper-NK lymphocyte CD8 expression “accentuations” associated with poor IVF outcome, possibly through their association with other unfavorable accentuated parameters that result into unfavorable combination of “accentuated phenotype”.

MAJOR AND LYMPHOCYTE POPULATIONS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES AND THEIR REFERENCE VALUES, AS ASSAYED BY MULTI-COLOUR CYTOMETRY

Determination of lymphocyte subpopulations and their phenotypes is an important diagnostic feature, in order to elucidate some disturbances connected with immune system functioning. However, insufficient data are obtained when analyzing only major populations of peripheral lymphocytes. In order to perform clinical diagnostics, the data about minor lymphocytic populations and activated cellular pools seem to be more pertinent. Studies of peripheral blood cell subpopulations of healthy donors performed in different Russian regions allowed to assess quantitative distribution intervals for both major and minor immune cell subpopulations in humans. The results obtained, as compared with data from literature, provide an evidence for similar reference intervals for main immune cell subpopulations in healthy donors, independent on their habitation area. Present work has resulted into development of algorithms for cytometric studies and generation of certain panels of monoclonal antibodies enabling evaluation of all main lymphocyte subpopulations, as well as their minor subsets participating in emerging immune response. The distribution intervals have been estimated for such minor subpopulations, as B1- and B2-lymphocytes, memory B-cells, γδ- and αβT-cells, regulatory and naїve T-cells, cytotoxic and secretory NK-cell polupations. The results of present study, while been performed with peripheral blood of healthy donors, may provide a basis of reference values when studying subpopulation profile of immune cells.

Differential influence of vemurafenib and dabrafenib on patients' lymphocytes despite similar clinical efficacy in melanoma.

Since the majority of melanomas eventually become resistant and progress, combining selective BRAF inhibitors (BRAFi) with immunotherapies has been proposed to achieve more durable treatment responses. Here, we explored the impact of selective BRAFi on the hosts' immune system. Clinical data, whole blood counts (WBC) and serum lactate dehydrogenase (LDH) of 277 vemurafenib- and 65 dabrafenib-treated melanoma patients were evaluated. The frequency and phenotype of lymphocyte subpopulations were determined by flow cytometry while T cell cytokine secretion was measured by multiplex assays. Progression-free survival (PFS) as well as overall survival (OS) were similar in patients treated with either BRAFi. High pretreatment LDH was associated with shorter PFS and OS in both groups. During therapy, peripheral lymphocytes decreased by 24.3% (median, P < 0.0001) in vemurafenib-treated patients but remained unchanged in dabrafenib-treated patients (+1.2%, P = 0.717). Differentiation of peripheral lymphocytes of vemurafenib-treated patients showed a significant decrease in CD4(+) T cells (P < 0.05). Within CD4(+) T cells obtained during treatment, an increase in CCR7(+)CD45RA(+) (naïve) and a decrease in CCR7(+)CD45RA(-) (central memory) populations were found (P < 0.01 for both). Furthermore, secretion of interferon-γ and interleukin-9 by CD4(+) T cells was significantly lower in samples obtained during vemurafenib treatment compared with baseline samples. While both compounds have comparable clinical efficacy, vemurafenib but not dabrafenib decreases patients peripheral lymphocyte counts and alters CD4(+) T cell phenotype and function. Thus, selective BRAFi can significantly affect patients' peripheral lymphocyte populations. Fully understanding these effects could be critical for successfully implementing combinatorial therapies of BRAFi with immunomodulatory agents.

Clinical safety of induced CTL infusion through recombinant adeno-associated virus-transfected dendritic cell vaccination in Chinese cancer patients

The aim of the study is to explore the safety of cytotoxic T lymphocytes (CTLs) infusion by transfected dendritic cells (DCs) with recombinant adeno-associated virus vector (rAAV) carrying CEA cDNA among advanced cancer patients. A total of 27 cancer patients with tumor tissue expression positivity and/or sera-elevated level of CEA were subsequently divided into cohort A and B resulted from the ex vivo expansion number of CTLs generated from co-culture of specific transfected DCs with autologous T lymphocytes. Based on the variations of infused number of specific CTL derived from different yields of individualized patients who had experienced various anti-cancer treatments, we compared the patients of low number of CTL cells (2-8 × 10(8) infused, cohort A, 6 cases) with those of higher number (above 8 × 10(8) infused, cohort B, 21 cases) to testify the possible adverse reactions caused by amount of infused CTLs. This study resembled a phase I study aiming for setting up clinical trial of adoptive cellular therapy that conceptually comes from conventional cytotoxic drugs. The results showed that one case from the each cohort had experienced moderate fever, and four cases with fatigue were seen in cohort B. The symptoms were transient without serious adverse events. For the consideration of clinical response 2 partial remission (8.0 %, 2/25), 1 minor remission, and 9 stable disease (40 %, 10/25) were observed in 25 patients eligible for evaluation. Sera levels of CEA assay were lowered in six patients. During a median follow-up of 8.1 months, we could not observe severe or chronic adverse reactions related to rAAV-DC infusions. Meanwhile, the variation of number of CTLs infused in this setting did not alter the status of peripheral lymphocyte population. These preliminary data suggest that the rAAV-DC immunotherapy is well-tolerated and showed no severe adverse reactions in cancer patients.

Rapid and Massive Virus-Specific Plasmablast Responses during Acute Dengue Virus Infection in Humans

Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.

Loss of Immunological Tolerance in Gimap5-Deficient Mice Is Associated with Loss of Foxo in CD4+ T Cells

Previously, we reported the abrogation of quiescence and reduced survival in lymphocytes from Gimap5(sph/sph) mice, an ENU germline mutant with a missense mutation in the GTPase of immunity-associated protein 5 (Gimap5). These mice showed a progressive loss of peripheral lymphocyte populations and developed spontaneous colitis, resulting in early mortality. In this study, we identify the molecular pathways that contribute to the onset of colitis in Gimap5(sph/sph) mice. We show that CD4(+) T cells become Th1/Th17 polarized and are critically important for the development of colitis. Concomitantly, regulatory T cells become reduced in frequency in the peripheral tissues, and their immunosuppressive capacity becomes impaired. Most importantly, these progressive changes in CD4(+) T cells are associated with the loss of Forkheadbox group O (Foxo)1, Foxo3, and Foxo4 expression. Our data establish a novel link between Gimap5 and Foxo expression and provide evidence for a regulatory mechanism that controls Foxo protein expression and may help to maintain immunological tolerance.

IL-6 and IL-10 in post-transplant lymphoproliferative disorders development and maintenance: a longitudinal study of cytokine plasma levels and T-cell subsets in 38 patients undergoing treatment

IL-6 and IL-10 have previously been implicated in the pathogenesis of post-transplant lymphoproliferative disorders (PTLD) and, like peripheral lymphocyte populations, are markers of immune status that are amenable to study in vivo. Thus, we analyzed cytokine plasma levels as well as lymphocyte subsets in a longitudinal analysis of 38 adult transplant recipients undergoing treatment for PTLD. Pretherapeutically, we found significantly elevated IL-6 (13.8 pg/ml) and IL-10 plasma levels (54.7 pg/ml) - in the case of IL-10, even higher in treatment nonresponders than in responders (116 vs. 14 pg/ml). Over time, however, IL-10 levels did not correlate with the course of disease, whereas those of IL-6 did, falling in responders and rising in nonresponders. These findings were independent of histological EBV-status, treatment type, and total peripheral T-cell counts, which were significantly reduced in patients with PTLD. Our observations support the idea that although IL-10 is important for creating a permissive environment for post-transplant lymphoma development, IL-6 is associated with PTLD proliferation. The analysis of lymphocyte subsets further identified HLA-DR+ CD8+ lymphocyte numbers as significantly different in non-PTLD controls (33%), treatment responders (44%) and nonresponders (70%). Although the specificity of these cells is unclear, their increase might correlate with the impaired tumor-specific cytotoxic-T-lymphocyte (CTL)-response in PTLD.