Serum levels of the IL-6 family of cytokines predict prognosis in renal cell carcinoma (RCC).

PURPOSE: An improved understanding of RCC immunology should shed further light on RCC tumor biology. Our objective was to study to what extent serum levels of the IL-6 family of cytokines at diagnosis were relevant to survival. METHODS: A total of 118 consecutively patients with RCC, in which the tumor was surgically removed at Haukeland University Hospital during the period from 2007 to 2010, were included. The patients were followed-up for 10 years. The morning before surgery blood was sampled and serum frozen, with levels of IL-6, IL-27, IL-31, OSM, CNTF, IL-6Rα and gp130 determined. RESULTS: Among patients with the highest quartile of IL-6 (> 8 pg/ml) (n = 29), six of nine who had metastasis at diagnosis had such high IL-6 values. Among presumed radically treated patients, a high IL-6 and IL-27 strongly predicted recurrence. In particular, the predictions among patients with large (diameter > 7 cm) tumors were excellent regarding both IL-6 and IL-27 values. High gp130 serum levels predicted an overall survival (OS) among RCC patients with large tumors. Patients with a high IL-6 exhibited a strong expression of IL-6 in endothelial- and vascular smooth muscle cells. Moreover, the level of intra-tumoral CD3-positive cells predicted survival. CONCLUSIONS: IL-6 and IL-27 seem to play a role in RCC biology. IL-6 enables the pinpointing of metastatic condition at diagnosis, as well as together with IL-27, the predicting of survival and recurrence. Endothelial cells and vascular smooth muscle cells are both suggested as important sources of IL-6.

Blood type change identifies late dominance reversal of chimerism after double umbilical cord blood transplantation with review of the literature.

BACKGROUND: A 30-year-old man underwent double umbilical cord blood transplantation (UCBT) for acute myeloid leukemia (AML) with reduced intensity conditioning. The cords had identical HLA types and were each a 5/6 match to the patient. Following transplantation, cord 2 initially dominated all tested cell populations. At day +306, we observed an unusual reversal of dominance chimerism pattern in which cord 1 instead dominated all tested populations. STUDY DESIGN & METHODS: Polymerase chain reaction (PCR)-based short tandem repeat (STR) assays were performed on the peripheral blood and bone marrow samples. The white blood cell (WBC) populations from the peripheral blood were manipulated for testing to create subpopulations enriched for CD3, CD33, and CD56. RESULTS: Chimerism studies on day +77 showed the following: cord 1: 44%-CD3; 0%-CD33; 16%-CD56; cord 2: 56%-CD3; 100%-CD33; 84%-CD56. Cord 2 initially dominated in all tested cell populations. Chimerism studies performed on post-transplantation day +306 uncovered a reversal of dominance chimerism pattern in which cord 1 now dominated in all cell populations (cord 1: 82%-CD3; >95%-CD33; 67%-CD56; cord 2: 18%-CD3; <5%-CD33; 33%-CD56). Between days +127 and +244, the patient's blood type shifted from B Rh-positive to A Rh-negative. CONCLUSION: The change in the patient's blood type identified a late reversal of dominance chimerism pattern. This is a rare occurrence, previously cited only once, which is inconsistent with published data that early high CD3 counts and unseparated bone marrow chimerism predominance at day +100 predict long-term cord dominance in double UCBT in the vast majority of cases.

Validation of simple prediction algorithms to consistently achieve CD3+ and postselection CD34+ targets with leukapheresis.

BACKGROUND: Cellular therapies using engineered T cells, haploidentical transplants, and autologous gene therapy are increasing. Specified CD3+ or high CD34+ doses are typically required for subsequent manufacturing, manipulation, or CD34+ selection. Simple, practical, and reliable lymphocyte and hematopoietic progenitor cell (HPC) collection algorithms accounting for subsequent CD34+ selection have not been published. STUDY DESIGN AND METHODS: In this analysis of 15 haploidentical donors undergoing tandem lymphocyte and HPC collections, we validated one-step, practical prediction algorithms (Appendix S1, available as supporting information in the online version of this paper) that use conservative facility-specific collection efficiencies, CD34+ selection efficiency, and donor-specific peripheral counts to reliably achieve the target CD3+ and CD34+ product doses. These algorithms expand on our previously published work regarding predictive HPC collection algorithms. RESULTS: Ninety-three percent of lymphocyte and 93% of CD34+ collections achieved the final target CD3+ and CD34+ product dose when our algorithm-calculated process volumes were used. Linear regression analysis of our algorithms for CD3+, preselection CD34+, and postselection CD34+ showed statistically significant models with R2 of 0.80 (root mean square error [RMSE], 31.3), 0.72 (RMSE, 385.7), and 0.56 (RMSE, 326.0), respectively, all with p values less than 0.001. CONCLUSION: Because achievement of CD3+ or CD34+ dose targets may be critical for safety and efficacy of cell therapies, these simple, practical, and reliable prediction algorithms for lymphocyte and HPC collections should be very useful for collection facilities.

Absence of influence of peripheral blood CD34+ and CD3+ graft cell counts on outcomes after reduced-intensity conditioning transplantation using post-transplant cyclophosphamide.

The influence of peripheral blood stem cell (PBSC) graft cell contents after transplant with post-transplant cyclophosphamide (PTCY) remains unclear. Here, we retrospectively report on a cohort of 77 adults who received a Baltimore-based reduced-intensity conditioning regimen either with fludarabine (n = 40) or clofarabine (n = 37) and PTCY. With a median follow-up of 29.2 months, [2-]year overall (OS), disease-free (DFS), and GVHD/relapse-free survival (GRFS) rates were 62.8%, 51%, and 36.7%, respectively. The incidence of grades [2-]4 acute GVHD was significantly higher in patients transplanted with a haplodonor (n = 56), at 57.1% vs 19% (p = 0.006). PBSC graft cell contents (CD45+, CD34+, and CD3+ cells) had no impact on any outcome. Considering immune reconstitution until 1 year, only monocytes were above the normal range (as early as day + 30) during the first year post-transplant. In multivariate analysis, an older donor (> 45 years) and a high/very high disease risk index were independently associated with lower OS. A higher monocyte count (> median) at day + 90 was also associated with better OS, DFS, and GRFS. Donor/recipient CMV status matching was independently associated with GRFS. In conclusion, our data support the fact that there is no need to manipulate the graft before infusion in the particular context of PBSC/PTCY Baltimore-based allotransplant.

Pharmacokinetic Analysis of a Novel Human EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry Approach.

Bispecific single chain antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressing T cells to recognize and destroy malignant, EGFRvIII-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we developed a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to a high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ∼8 min and a terminal half-life of ∼2.5 h. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these pharmacokinetic parameters will guide drug optimization and dosing regimens in future IND-enabling and phase I studies of EGFRvIII:CD3 bi-scFv.

Elevated levels of IL-37 correlate with T cell activation status in rheumatoid arthritis patients.

OBJECTIVE: To assess the association between serum levels of IL-37 in rheumatoid arthritis patients and percentage of peripheral blood T lymphocytes expressing the activation marker CD26 and investigate their correlation with disease activity. METHODS: The study included 48 rheumatoid arthritis patients and 42 age and sex matched healthy controls. Serum levels of IL-37 were determined using enzyme linked immunosorbent assay while percentage of CD3+CD26+T cells in peripheral blood mononuclear cells was assayed using flowcytometry. RESULTS: Serum levels of IL-37, as well as the percentage of CD3+CD26+T cells, were significantly higher in rheumatoid arthritis patients than in healthy controls. Also, serum IL-37 levels were higher in patients with severe disease activity than patients with moderate and low disease activity. In rheumatoid arthritis patients, both serum levels of IL-37 and percentage of CD3+CD26+T cells correlated with disease activity (DAS28), C-reactive protein levels and erythrocyte sedimentation rate. In addition, serum levels of the anti-inflammatory cytokine IL-37 positively correlated with the percentage of CD3+CD26+T cells in peripheral blood of rheumatoid arthritis patients. CONCLUSION: Our results indicate a strong correlation between serum levels of IL-37 and frequency of activated T cells in peripheral blood of rheumatoid arthritis patients. Our results suggest that in an active disease status, activated T lymphocytes may be a contributing source to the elevated levels of IL-37 trying to down-regulate the active inflammatory process.

Levels of peripheral blood polymorphonuclear myeloid-derived suppressor cells and selected cytokines are potentially prognostic of disease progression for patients with non-small cell lung cancer.

Polymorphonuclear-MDSC (PMN-MDSC) have emerged as an independent prognostic factor for survival in NSCLC. Similarly, cytokine profiles have been used to identify subgroups of NSCLC patients with different clinical outcomes. This prospective study investigated whether the percentage of circulating PMN-MDSC, in conjunction with the levels of plasma cytokines, was more informative of disease progression than the analysis of either factor alone. We analyzed the phenotypic and functional profile of peripheral blood T-cell subsets (CD3+, CD3+CD4+ and CD3+CD8+), neutrophils (CD66b+) and polymorphonuclear-MDSC (PMN-MDSC; CD66b+CD11b+CD15+CD14-) as well as the concentration of 14 plasma cytokines (IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p70, IL-17A, IL-27, IL-29, IL-31, and IL-33, TNF-α, IFN-γ) in 90 treatment-naïve NSCLC patients and 25 healthy donors (HD). In contrast to HD, NSCLC patients had a higher percentage of PMN-MDSC and neutrophils (P < 0.0001) but a lower percentage of CD3+, CD3+CD4+ and CD3+CD8+ cells. PMN-MDSC% negatively correlated with the levels of IL1-ß, IL-2, IL-27 and IL-29. Two groups of patients were identified according to the percentage of circulating PMN-MDSC. Patients with low PMN-MDSC (≤ 8%) had a better OS (22.1 months [95% CI 4.3-739.7]) than patients with high PMN-MDSC (9.3 months [95% CI 0-18.8]). OS was significantly different among groups of patients stratified by both PMN-MDSC% and cytokine levels. In sum, our findings provide evidence suggesting that PMN-MDSC% in conjunction with the levels IL-1ß, IL-27, and IL-29 could be a useful strategy to identify groups of patients with potentially unfavorable prognoses.

Lymphocyte apheresis for chimeric antigen receptor T-cell manufacturing in children and young adults with leukemia and neuroblastoma.

BACKGROUND: The first step in the production of chimeric antigen receptor T cells is the collection of autologous T cells using apheresis technology. The procedure is technically challenging, because patients often have low leukocyte counts and are heavily pretreated with multiple lines of chemotherapy, marrow transplantation, and/or radiotherapy. Here, we report our experience of collecting T lymphocytes for chimeric antigen receptor T-cell manufacturing in pediatric and young adult patients with leukemia, non-Hodgkin lymphoma, or neuroblastoma. STUDY DESIGN AND METHODS: Apheresis procedures were performed on a COBE Spectra machine using the mononuclear cell program, with a collection target of 1 × 109 total mononuclear cells per kilogram. Data were collected regarding preapheresis and postapheresis blood counts, apheresis parameters, products, and adverse events. RESULTS: Ninety-nine patients (ages 1.3-25.7 years) and 102 apheresis events were available for analysis. Patients underwent apheresis at a variety of absolute lymphocyte cell counts, with a median absolute lymphocyte count of 944 cells/µL (range, 142-6944 cells/µL). Twenty-two patients (21.6%) had absolute lymphocyte counts less than 500 cells/µL. The mononuclear cell target was obtained in 100% of all apheresis harvests, and chimeric antigen receptor T-cell production was possible from the majority of collections (94%). Mononuclear cell collection efficiency was 65.4%, and T-lymphocyte collection efficiency was 83.4%. Ten patients (9.8%) presented with minor adverse events during the 102 apheresis procedures, with one exception of a severe allergy. CONCLUSIONS: Mononuclear cell apheresis for chimeric antigen receptor T-cell therapy is well tolerated and safe, and it is possible to obtain an adequate quantity of CD3+ lymphocytes for chimeric antigen receptor T-cell manufacturing in heavily pretreated patients who have low lymphocyte counts.

Change of apheresis device decreased the incidence of severe acute graft-versus-host disease among patients after allogeneic stem cell transplantation with sibling donors.

BACKGROUND: The composition of the graft used for allogeneic hematopoietic stem cell transplantation (HSCT) is important for the treatment outcome. Different apheresis devices may yield significant differences in peripheral blood stem cell graft cellular composition. We compared stem cell grafts produced by Cobe Spectra (Cobe) and Spectra Optia (Optia) with use of the mononuclear cell (MNC) protocol, and evaluated clinical outcome parameters such as graft-versus-host disease (GvHD), transplant-related mortality (TRM), relapse, and overall survival. STUDY DESIGN AND METHODS: During 5 years, 31 Cobe Spectra and 40 Spectra Optia grafts were analyzed for CD34, CD3, CD4, CD8, CD19, and CD56 cell content. Clinical outcome parameters were correlated and compared between the two patient groups using different apheresis devices. RESULTS: Optia grafts contained fewer lymphocytes compared to Cobe (p < 0.001). Optia grafts had a significantly lower incidence of acute GvHD Grades II through IV (Cobe 45% vs. Optia 23%; p = 0.039) and TRM (16% vs. 2.5%; p < 0.05) but higher chronic GvHD (32% vs. 67%; p = 0.005) compared to Cobe grafts. Finally, the multivariate analysis showed a significant correlation among the different apheresis devices and both acute GvHD II through IV and severe chronic GvHD. The multivariate analysis also showed a significant correlation between the CD3+ cell dose and the incidence of severe acute GvHD. CONCLUSION: Optia-obtained grafts yielded a lower acute GvHD Grades II-IV and TRM risk, but had no impact on relapse or overall survival in this study. Understanding and further improvement of peripheral blood stem cell (PBSC) apheresis techniques may be used in the future to personalize HSCT by, for example, fine-tuning the GvHD incidence.

Low frequency of CD3+CD4+CD161+ T cells correlates with the occurrence of infections in refractory/relapsed multiple myeloma patients receiving lenalidomide plus low-dose dexamethasone treatment.

The aim of this study was to explore the predictive implications of the composition of immune cell populations prior to lenalidomide plus high-dose dexamethasone (Len-Dex) initiation for the occurrence of infections. We prospectively examined immune cell populations in peripheral blood taken at baseline of lenalidomide plus low-dose dexamethasone (Len-dex) therapy and reviewed clinical and microbiology records in 90 patients with refractory/relapsed multiple myeloma (RRMM). Risk factors for infection were analyzed using logistic regression. During a median of 11 cycles of Len-dex treatment, 52 (57.8%) patients experienced at least 1 infection episode. Of a total of 92 episodes of infection, 58 (63%) episodes were clinically defined, 29 (31.5%) episodes were microbiologically defined, and 5 (5.4%) episodes were fever of unknown origin. Severe episodes were more frequently observed during the first 3 cycles. After adjusting for risk factors for infection based on univariate analyses, multivariate analyses showed that lower Hb (< 10 g/dL) was a clinically independent factor associated with occurrence of infections. Lower frequency (P = 0.044) and absolute count (P = 0.014) of circulating CD3+CD4+CD161+ cells prior to Len-dex treatment were also associated with the occurrence of infection, especially during the first 3 cycles of Len-dex therapy. In addition to several clinical predictive factors, we found that CD3+CD4+CD161+ cells may provide additional information for predicting the occurrence of infection in the early period of Len-dex therapy.

How do anxiety affect CD34 and CD3 cells in allogeneic peripheral blood stem cell transplantation?

Allogeneic peripheral blood stem cells (PBHSCs) transplantation using apheresis is a curative method for malignant and non-malignant hematologic diseases. The aim of this study was to assess the possible effects of anxiety as well as other variables on PBHSCs apheresis success. In this cross-sectional study, different demographic and clinical data such as granulocyte-colony stimulating factor (G-CSF) dosage, CD 34+ cells count (before apheresis), CD 34+, CD3+ cells count in apheresis product and also complete blood count were assessed. Furthermore, for evaluation of anxiety level in donors, the Beck's anxiety inventory (BAI) was administered. In this study, 111 donors were randomly enrolled after meeting inclusion and exclusion criteria. Results of BAI showed the mean score of 22.85 ±â€¯15.43 (mild to moderate anxiety) for the participants. According to the bootstrapped quantile regression analysis, a statistically significant linear association was found between CD34+ cell count and BAI score (P-value < 0.001) after adjusting for confounding variables. Moreover, the BAI score had a statistically significant effect on CD3+ cells count (P-value = 0.021) after adjusting for confounding variables. Taken together, results showed that anxiety affects both CD34+ and CD3+ cells count. Thus, the authors suggest that anxiety levels would be evaluated as well as other variables in donors in order to run a proper intervention by professionals.

Transplantation outcomes for severe combined immunodeficiency, 2000-2009.

BACKGROUND: The Primary Immune Deficiency Treatment Consortium was formed to analyze the results of hematopoietic-cell transplantation in children with severe combined immunodeficiency (SCID) and other primary immunodeficiencies. Factors associated with a good transplantation outcome need to be identified in order to design safer and more effective curative therapy, particularly for children with SCID diagnosed at birth. METHODS: We collected data retrospectively from 240 infants with SCID who had received transplants at 25 centers during a 10-year period (2000 through 2009). RESULTS: Survival at 5 years, freedom from immunoglobulin substitution, and CD3+ T-cell and IgA recovery were more likely among recipients of grafts from matched sibling donors than among recipients of grafts from alternative donors. However, the survival rate was high regardless of donor type among infants who received transplants at 3.5 months of age or younger (94%) and among older infants without prior infection (90%) or with infection that had resolved (82%). Among actively infected infants without a matched sibling donor, survival was best among recipients of haploidentical T-cell-depleted transplants in the absence of any pretransplantation conditioning. Among survivors, reduced-intensity or myeloablative pretransplantation conditioning was associated with an increased likelihood of a CD3+ T-cell count of more than 1000 per cubic millimeter, freedom from immunoglobulin substitution, and IgA recovery but did not significantly affect CD4+ T-cell recovery or recovery of phytohemagglutinin-induced T-cell proliferation. The genetic subtype of SCID affected the quality of CD3+ T-cell recovery but not survival. CONCLUSIONS: Transplants from donors other than matched siblings were associated with excellent survival among infants with SCID identified before the onset of infection. All available graft sources are expected to lead to excellent survival among asymptomatic infants. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Exacerbation of oxygen-glucose deprivation-induced blood-brain barrier disruption: potential pathogenic role of interleukin-9 in ischemic stroke.

Interleukin (IL)-9 exerts a variety of functions in autoimmune diseases. However, its role in ischemic brain injury remains unknown. The present study explored the biological effects of IL-9 in ischemic stroke (IS). We recruited 42 patients newly diagnosed with IS and 22 age- and sex-matched healthy controls. The expression levels of IL-9 and percentages of IL-9-producing T cells, including CD3+CD4+IL-9+ and CD3+CD8+IL-9+ cells, were determined in peripheral blood mononuclear cells (PBMCs) obtained from patients and control individuals. We also investigated the effects of IL-9 on the blood-brain barrier (BBB) following oxygen-glucose deprivation (OGD) and the potential downstream signaling pathways. We found that patients with IS had higher IL-9 expression levels and increased percentages of IL-9-producing T cells in their PBMCs. The percentages of CD3+CD4+IL-9+ and CD3+CD8+IL-9+ T cells were positively correlated with the severity of illness. In in vitro experiments using bEnd.3 cells, exogenously administered IL-9 exacerbated the loss of tight junction proteins (TJPs) in cells subjected to OGD plus reoxygenation (RO). This effect was mediated via activation of IL-9 receptors, which increased the level of endothelial nitric oxide synthase (eNOS), as well as through up-regulated phosphorylation of signal transducer and activator of transcription 1 and 3 and down-regulated phosphorylated protein kinase B/phosphorylated phosphatidylinositol 3-kinase signaling. These results indicate that IL-9 has a destructive effect on the BBB following OGD, at least in part by inducing eNOS production, and raise the possibility of targetting IL-9 for therapeutic intervention in IS.

Reactive oxygen species mediated T lymphocyte abnormalities in an iron-overloaded mouse model and iron-overloaded patients with myelodysplastic syndromes.

The adverse effects of iron overload have raised more concerns as a growing number of studies reported its association with immune disorders. This study aimed to investigate alterations in the immune system by iron overload in patients with myelodysplastic syndrome (MDS) and an iron-overloaded mouse model. The peripheral blood from patients was harvested to test the effect of iron overload on the subsets of T lymphocytes, and the level of reactive oxygen species (ROS) was also evaluated. The data showed that iron-overloaded patients had a lower percentage of CD3+ T cells and disrupted T cell subsets, concomitant with higher ROS level in lymphocytes. In order to explore the mechanism, male C57Bl/6 mice were intraperitoneally injected with iron dextran at a dose of 250 mg/kg every 3 days for 4 weeks to establish an iron-overloaded mouse model and the blood of each mouse was collected for the analysis of the T lymphocyte subsets and T cell apoptosis. The results showed that iron overload could reduce the percentage of CD3+ T cells and the ratio of Th1/Th2 and Tc1/Tc2 but increase the percentage of regulatory T (Treg) cells and the ratio of CD4/CD8. We also found that iron overload induced the apoptosis of T lymphocytes and increased its ROS level. Furthermore, these effects could be partially recovered after treating with antioxidant N-acetyl-L-cysteine (NAC) or iron chelator deferasirox (DFX). Taken together, these observations indicated that iron overload could selectively affect peripheral T lymphocytes and induce an impaired cellular immunity by increasing ROS level.

Association of Immune Factors with Drug-Resistant Tuberculosis: A Case-Control Study.

BACKGROUND Presently, studies of factors associated with drug-resistant tuberculosis (TB) focus on patients' socio-demographic characteristics and living habits, to the exclusion of biochemical indicators, especially immune factors. This study was carried out to determine whether immune factors are associated with drug-resistant TB. MATERIAL AND METHODS A total of 227 drug-resistant pulmonary TB patients and 225 drug-susceptible pulmonary TB patients were enrolled in this study. Information on socio-demographic characteristics and biochemical indicators were obtained through their clinical records. Non-conditional logistic regression was used to analyze the association of these indicators with drug-resistant TB. RESULTS There were significant differences in re-treatment, marital status, alanine aminotransferase (ALT), blood uric acid (BUA), carcino-embryonic antigen (CEA), T-spot, and CD3 and CD4 counts between the 2 groups. In multivariable analysis, re-treatment [Odds Ratio (OR)=5.290, 95% Confidence Interval [CI]=2.652-10.551); CD3 (OR=1.034, 95% CI=1.001-1.068); CD4 (OR=1.035, 95% CI =1.001-1.070) and IgM (OR=1.845, 95% CI=1.153-2.952) were associated with drug-resistant TB. CONCLUSIONS These results suggest the need for greater attention to re-treatment cases and immune function when treating drug-resistant TB.

Cord Blood Units with High CD3(+) Cell Counts Predict Early Lymphocyte Recovery After In Vivo T Cell-Depleted Single Cord Blood Transplantation.

Although high absolute lymphocyte count (ALC) early after transplantation is a simple surrogate for immune reconstitution, few studies to date have established the predictive factors for ALC after umbilical cord blood transplantation (UCBT). We retrospectively studied the factors associated with early lymphocyte recovery and the impact of the ALC on day +42 (ALC42) of ≥300 × 10(6)/L on outcomes in 210 consecutive pediatric and adult patients (112 males; median age, 15 years; range, 0.3 to 60 years; interquartile range, 4 to 36 years) who underwent myeloablative in vivo T cell-depleted single UCBT between 2005 and 2014 for malignant and nonmalignant disorders. In a logistic multivariate regression model, factors favoring a higher ALC42 were higher infused CD3(+) cell dose (odds ratio [OR], 2.7; 95% CI, 1.4 to 5.2; P = .004), lower antithymocyte globulin dose (OR, 2.3; 95% CI, 1.2 to 4.5; P = .01), and better HLA match (OR, 2.1; 95% CI, 1.1 to 4.1; P = .03). In multivariate analysis, lower ALC42 was associated with higher nonrelapse mortality (hazard ratio [HR], 1.76; 95% CI, 1.34 to 2.32; P = .001), whereas a higher ALC42 was associated with better disease-free survival (HR, 2.03; 95% CI, 1.15 to 3.6; P < .001) and overall survival (HR, 2.03; 95% CI, 1.17 to 3.6; P < .001). Our study suggests that the selection of better HLA-matched cord blood units containing higher CD3(+) cell counts and the use of conditioning regimens with lower ATG doses could improve immune reconstitution after UCBT.

CD4+ CD56+ natural killer T-like cells secreting interferon-γ are associated with incident coronary events.

BACKGROUND: CD3(+) CD56(+) natural killer T (NKT)-like cells are a subset of T cells characterized by expression of NK receptors and potent antitumour activity. It has also been suggested that they have a role in autoimmune disease, and levels of NKT-like cells are elevated in patients with coronary disease. OBJECTIVES: To investigate whether high levels of CD3(+) CD56(+) NKT-like cells are associated with an increased incidence of cardiovascular disease and a lower incidence of cancer. METHODS: This was a prospective study including 700 subjects participating in the baseline investigation of the Malmö Diet and Cancer study between 1991 and 1994. Leucocytes obtained at the baseline investigation and stored at -140 °C were thawed and CD3(+) CD56(+) cells analysed by flow cytometry. The incidence rates of cancer and coronary events during a mean follow-up of 15 years were determined through national registers. RESULTS: Subjects in the lowest tertile of interferon (IFN)-γ-expressing CD4(+) CD56(+) cells were found to have an increased risk of incidence of coronary events (log-rank test: P < 0.05). This association remained significant after controlling for age, sex, smoking, body mass index, hypertension, diabetes and the Th1/Th2 and Th1/Treg cell ratios in a Cox proportional hazards regression model (hazard ratio 1.98, 95% confidence interval 1.24-3.16), but not when the LDL/HDL ratio was included in the model. There were no associations between CD3(+) CD56(+) NKT-like cells and incident cancer. CONCLUSIONS: The present results could not confirm the hypothesis that low levels of CD3(+) CD56(+) NKT-like cells are associated with a higher incidence of cancer and a lower incidence of cardiovascular disease. However, we found that low levels of IFN-γ-expressing CD3(+) CD4(+) CD56(+) NKT-like cells were associated with an increased incidence of coronary events and that this association may be dependent on lipoproteins.

Response of steroid-refractory chronic graft-versus-host disease to extracorporeal photopheresis correlates with the dose of CD3+ lymphocytes harvested during early treatment cycles.

BACKGROUND: Extracorporeal photopheresis (ECP) is a recognized second-line treatment for steroid-refractory chronic graft-versus-host disease (cGVHD). Treatment course is usually long, expensive, and demanding for patients, so predictors for response are needed. We carried out a retrospective study on cGVHD patients treated at our institution with the aim to identify a possible correlation between apheretic yields composition and probability of response. STUDY DESIGN: Patients treated for at least 6 months were eligible for the study. Flow cytometry data, including absolute counts of lymphocytes and their subpopulations in ECP products from cGVHD patients, were collected. For each cell population 1) the median dose per procedure harvested during the first 3 months of treatment and 2) the cumulative dose collected in the same period were compared with clinical response. RESULTS: A total of 726 ECP procedures were performed in 15 patients. Overall response, defined as either a complete response (CR) or a partial response according to National Institutes of Health criteria, was obtained in 10 of 15 patients (66.7%), and CR, in eight of 15 (53.3%). According to Cox regression analysis, the probability of achieving an overall response is significantly correlated with the median number of CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes collected during the early treatment phase (first 3 months). CONCLUSION: Our data suggest that CD3+ cell evaluation in ECP during the early phase of treatment course could predict response and help identify patients who deserve further treatment.

Chemotherapy-induced B-cell depletion in hepatoblastoma patients undergoing ABO-incompatible living donor liver transplantation.

LT from ABO-I donors requires preconditioning regimens to prevent postoperative catastrophic AMR. NAC for HBL is known to cause myelosuppression leading to a reduction in the number and function of lymphocytes. We investigated this chemotherapy-induced myelosuppression in HBL patients listed for LT from ABO-I donors with reference to the kinetics of B, T cells, and anti-ABO blood type isoagglutinin titers. Between 2005 and 2015, of the 319 patients who underwent LDLT at our institute, 12 were indicated for unresectable HBL. Three patients with unresectable HBL who underwent LDLT from ABO-I donors are included in this study. Immunosuppression consisted of a standard regime of tacrolimus and low-dose steroids as in ABO compatible/identical LDLT. No additional preoperative therapies for B-cell depletion were used. Absolute lymphocyte counts, lymphocyte subsets (including CD20+ B cells, CD3+CD4+ T cells and CD3+CD8+ T cells), and anti-ABO blood type isoagglutinin titers were measured before LDLT and postoperatively. The median age at diagnosis was 19 months (range, 3-31 months). The median follow-up was seven months (range, 6-15 months). The median interval from the last NAC to LDLT was 33 days (range, 25-52 days). The median interval from LDLT to adjuvant chemotherapy was 28 days (range, 22-36 days). The counts of CD20+ B cells before LDLT were depleted to median 5 cells/mm(3) (range, 0-6 cells/mm(3)). There was a transient rebound in the CD20+ B cell counts on day seven (maximum of 82 cells/mm(3)) followed by a decline starting at 14 days after LDLT that was sustained for the duration of adjuvant chemotherapy. Anti-ABO blood type isoagglutinin titers were lowered to between 1:1 and 1:16 before LDLT and remained low for the duration of follow-up in this study. All of the three patients remained in good health without either acute cellular or AMR after LDLT. The B-cell depletion that occurs after cisplatin-based chemotherapy for HBL may help accomplish safe ABO-I LDLT in children without the use of additional conditioning regimens for prevention of AMR.

Comparison of Simultaneous CD34+ and CD3+ Quantification with a Modified Stem Cell Enumeration Kit on Two Different Flow Cytometers.

BACKGROUND: Quantification of CD34+ cells in peripheral blood stem cell apheresis is normally performed by single platform flow cytometric measurements according to the ISHAGE protocol. Peripheral blood stem cell concentrates (PBSC) produced by apheresis normally contain many T cells. Those T cells can be used for production of donor lymphocyte infusion doses, if abundant amounts of CD34+ cells have been collected. Therefore, it is of interest to know both the CD3+ and the CD34+ cell count of allogeneic PBSC. This is the first study comparing the performance of a modified ISHAGE protocol allowing additional quantification of CD3+ cells on two different flow cytometers, the FACSCalibur and the FACSVerse, respectively. METHODS: CD45+ and CD34+ cell concentrations were measured using a standard and a modified ISHAGE protocol including CD3+ cell quantification on both machines. All cell concentrations were measured using a Trucount bead based stem cell enumeration kit. The FACSVerse machine can additionally be equipped with a sample volume sensor allowing cell quantification without using beads. The samples analysed were taken from granulocyte-colony-stimulating factor mobilized peripheral blood stem cell apheresis procedures (pre- and post-apheresis, and apheresis concentrate). RESULTS: There were no significant differences in cell concentrations measured by the standard and modified ISHAGE protocol, regardless of which machine had been used when using bead quantification. No significant differences between the results of the two flow cytometers using the modified ISHAGE protocol were observed. Pearson´s correlation was always > 0.96, and regression coefficients were higher than 0.93. The only significant differences were observed between bead quantification and volume sensor quantification on the FACSVerse machine. CONCLUSIONS: The modified ISHAGE protocol can effectively be used on both flow cytometers tested, especially if bead quantification is used.