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Brian De,1,* Sweet Ping Ng,1,2,* Amy Y Liu,1 Santiago Avila,1 Randa Tao,3 Emma B Holliday,1 Zachary Brownlee,4 Ahmed Kaseb,5 Sunyoung Lee,3 Kanwal Raghav,5 Jean-Nicolas Vauthey,6 Bruce D Minsky,1 Joseph M Herman,1 Prajnan Das,1 Grace L Smith,1 Cullen M Taniguchi,1 Sunil Krishnan,7 Christopher H Crane,8 Clemens Grassberger,9 Theodore S Hong,9 Steven H Lin,1 Albert C Koong,1 Radhe Mohan,1 Eugene J Koay1 1Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Department of Radiation Oncology, Austin Health, Melbourne, Victoria, Australia; 3Department of Radiation Oncology, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA; 4Department of Radiation Oncology, Tufts Medical Center, Boston, MA, USA; 5Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 6Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 7Department of Radiation Oncology, Mayo Clinic Jacksonville, Jacksonville, FL, USA; 8Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 9Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA, USA*These authors contributed equally to this workCorrespondence: Eugene J KoayMD Anderson Cancer Center, Department of Radiation Oncology, 1400 Pressler St. Unit 1422, Houston, TX, 77030, USATel + 1 713-563-2381Fax +1 713-563-2331Email EKoay@mdanderson.orgBackground: The immune system plays a crucial role in cancer surveillance. Previous studies have shown that lymphopenia associated with radiotherapy (RT) portends a poor prognosis. We sought to differentiate the effects of proton and photon RT on changes in absolute lymphocyte count (ALC) for patients with hepatocellular carcinoma (HCC).Patients and Methods: Patients with HCC treated with definitive RT from 2006 to 2016 were studied. Serial ALCs were graded according to CTCAE v4.0. Overall survival (OS), disease-free survival, and distant metastasis-free survival were analyzed using the Kaplan–Meier method. Univariableand multivariable Cox-proportional hazards analyses were used to identify predictors of OS. A cohort analysis matched for treatment volume was performed to investigate differences in ALC dynamics between photon and proton therapy.Results: Of 143 patients identified, the median age was 66 (range, 19– 90) years. The treatment modality was photon in 103 (72%) and proton in 40 (28%). Median follow-up was 17 months (95% confidence interval, 13– 25 months). The median time to ALC nadir after initiation of RT was 17 days with a median relative decrease of 67%. Those who received proton RT had a higher median ALC nadir (0.41 vs 0.32 k/μL, p=0.002) and longer median OS (33 vs 13 months, p=0.002) than those who received photon RT. Matched cohort analyses revealed a larger low-dose liver volume in the photon group, which correlated with lower ALC. On multivariable Cox analysis, Grade 3 or higher lymphopenia prior to or after RT, portal venous tumor thrombus, larger planning target volumes, Child-Pugh (CP) Class B, and increased CP score after RT were associated with a higher risk of death, whereas the use of proton therapy was associated with lower risk.Conclusion: Grade 3 or higherlymphopenia may be associated with poorer outcomes in patients receiving RT for HCC. Protons may mitigate lymphopenia compared with photons, potentially due to reduced dose exposure of sites of lymphopoiesis.Keywords: lymphocyte count, circulating lymphocytes, splenic dose, liver dose, overall survival

Purpose Breast cancer tumour-infiltrating lymphocytes associate with clinico-pathological factors, including survival, although the literature includes many conflicting findings. Our aim was to assess these associations for key lymphocyte subtypes and in different tumour compartments, to determine whether these provide differential correlations and could, therefore, explain published inconsistencies. Uniquely, we also examine whether infiltrating levels merely reflect systemic lymphocyte levels or whether local factors are predominant in recruitment. Methods Immunohistochemistry was used to detect tumour-infiltrating CD20+ (B), CD4+ (helper T), CD8+ (cytotoxic T) and FoxP3+ (regulatory T) cells in breast cancers from 62 patients, with quantification in tumour stroma, tumour cell nests, and tumour margins. Levels were analysed with respect to clinico-pathological characteristics and matched circulating levels (determined by flow-cytometry). Results CD4+ lymphocytes were the most prevalent subtype in tumour stroma and at tumour edge and CD8+ lymphocytes were most prevalent in tumour nests; FoxP3+ lymphocytes were rarest in all compartments. High grade or hormone receptor negative tumours generally had significantly increased lymphocytes, especially in tumour stroma. Only intra-tumoural levels of CD8+ lymphocytes correlated significantly with matched circulating levels (p p p Conclusions Lymphocyte subtype and location define differential impacts on tumour biology, therefore, roles of tumour-infiltrating lymphocytes will only be unravelled through thorough analyses that take this into account.

Determining the magnitude of local immune response during mucosal exposure to viral pathogens is critical to understanding the mechanism of viral pathogenesis. We previously showed that vaginal inoculation of lymphocytic choriomeningitis virus (LCMV) fails to induce a robust innate immune response in the lower female reproductive tract (FRT), allowing high titer viral replication and a delay in T-cell-mediated viral control. Despite this immunological delay, LCMV replication remained confined mainly to the FRT and the draining iliac lymph node. Here, we show that rectal infection with LCMV triggers type I/III interferon responses, followed by innate immune activation and lymphocyte recruitment to the colon. In contrast to vaginal exposure, innate immunity controls LCMV replication in the colon, but virus rapidly disseminates systemically. Virus-induced inflammation promotes the recruitment of LCMV target cells to the colon followed by splenic viral dissemination by infected B cells, and to a lesser extent by CD8 T cells. These findings demonstrate major immunological differences between vaginal and rectal exposure to the same viral pathogen, highlighting unique risks associated with each of these common routes of sexual viral transmission.

Introduction Immune activation has been implicated in progression of amytrophic lateral sclerosis (ALS). Oral fingolimod reduces circulating lymphocytes. The objective of this phase IIa, randomized, controlled trial was to test the short‐term safety, tolerability, and target engagement of fingolimod in ALS. Methods Randomization was 2:1 (fingolimod:placebo). Treatment duration was 4 weeks. Primary outcomes were safety and tolerability. Secondary outcomes included circulating lymphocytes and whole‐blood gene expression. Results Thirty participants were randomized; 28 were administered a drug (fingolimod 18, placebo 10). No serious adverse events occurred. Adverse events were similar by treatment arm, as was study discontinuation (2 fingolimod vs. 0 placebo, with no statistical difference). Forced expiratory volume in 1 second (FEV1) and FEV1/slow vital capacity changes were similar in the fingolimod and placebo arms. Circulating lymphocytes decreased significantly in the fingolimod arm (P

Background: In clinical trials with immunotherapy, histological features such as tumor-infiltrating lymphocytes (TILs) are investigated as potential predictive biomarkers, with the limit of an outdated parameter for a typically dynamic element. Methods: This explorative study compared, in metastatic renal cell carcinoma (mRCC) patients, basal pathological data about TILs on diagnostic histological specimens with circulating lymphocyte subpopulations measured before and during therapy with nivolumab. Results: Of 11 mRCC patients, 5 had low presence of TILs (L-TILs), 3 moderate amount (M-TILs) and 3 high number (H-TILs). Overall, 8 patients had low intratumoral pathological CD4+/CD8+ ratio (LIPR) ≤1 and 3 cases high intratumoral pathological ratio (HIPR) ≥2. Of 8 patients with LIPR, only 2 matched with low circulating CD4+/CD8+ ratio (LCR) ≤1; 5 had high circulating ratio (HCR) ≥2. All 3 cases with HIPR (≥2) conversely had LCR (≤1). Circulating CD4+/CD8+ ratio remained unchanged during therapy (mean -0.12 in 8 weeks). The respective percentage values of CD4+ and CD8+ circulating T cells also remained stable (variation 0%); the absolute value of CD4+ was more likely to increase (mean +46.3/mm3); the level of CD8+ tended to slightly decrease (mean -6.5/mm3). No correlation of lymphocyte subpopulations with treatment outcome was found. Of note, we did not evidence correspondence between histopathological and circulating findings in terms of T-lymphocyte subpopulations, also suggesting the inconsistency of circulating data in terms of relative variations. Conclusions: Considering the likely high dynamism of TILs, rebiopsy before therapy might be proposed to assess the utility of TILs characterization for predictive purpose. (www.actabiomedica.it)

Binding of programmed death-1 (PD-1) with its ligands (PD-L1/2) transmits a co-inhibitory signal in activated T-cells that promotes T-cell exhaustion, leading to tumor immune evasion. The efficacy of antibodies targeting PD-1 and PD-L1 has led to a paradigm shift in lung cancer treatment but the prognostic and predictive value of tumor PD-L1 expression remains controversial. Evaluating PD-1, PD-L1/2 expression in peripheral blood cells may serve as a potential biomarker for prognosis and response to therapy. In this prospective observational study, plasma cytokine levels and PD-1, PD-L1 and PD-L2 expression was evaluated in circulating CD3+, CD3+CD4+ and CD3+CD8+ cells from 70 treatment-naive patients with advanced NSCLC (Stage IIIB and IV) and from 10 healthy donors. The primary objective was to assess OS according to PD-1, PD-L1, PD-L2 expression status on PBMCs and lymphocyte subsets. Our results indicate that the percentage of PD-L1+CD3+, PD-L1+CD3+CD8+ PD-L2+PBMCs, PD-L2+CD3+, PD-L2+CD3+CD4+ cells was higher in patients than in healthy donors. Survival was decreased among patients with a high percentage of either PD-1+PBMCs, PD-1+CD3+, PD-L1+CD3+, PD-L1+CD3+CD8+, PD-L2+CD3+, PD-L2+CD3+CD4+, or PD-L2+CD3+CD8+ cells. IL-2 and TNF-α showed the strongest association with PD-L1 and PD-L2 expression on specific subsets of T-lymphocytes. Our findings suggest that increased PD-1/PD-L1/PDL-2 expression in PBMCs, particularly in T-cells, may be an additional mechanism leading to tumor escape from immune control. This study is registered with ClinicalTrials.gov, number NCT02758314.

In this retrospective case-control study, we compared the number of circulating lymphocyte subsets among 31 healthy controls, 18 naive and 40 antiretroviral-treated HIV patients, 28 untreated and 18 treated relapsing-remitting multiple sclerosis (MS) patients. Lymphocyte subsets were no different between untreated MS patients and controls. Untreated and treated MS had a lower CD8+ count and a higher CD4+ number compared to untreated and treated HIV patients except similar CD4+ number between treated MS and HIV patients. CD4/CD8 ratio was lower in female HIV non-responders and in relapsing MS women compared, respectively, to female HIV responders and remitting MS women, and there were no differences in men.

Animal productions (i.e. fish, eggs, milk and dairy products) represent the major source of exposure to dioxins, furans, and dioxin-like (DL) polychlorobiphenyls for humans. The negative effects of these highly toxic and persistent pollutants are mediated by the activation of the aryl hydrocarbon receptor (AHR) that elicits the transcriptional induction of several genes, including those involved in xenobiotic metabolism. Previously we demonstrated the presence and functioning of the AHR signaling pathway in primary cultures of bovine blood lymphocytes. The aim of the present study was to investigate by real time PCR the expression and the inducibility of selected target genes (i.e. AHR, AHR nuclear translocator (ARNT), AHR repressor, CYP1A1 and CYP1B1) in uncultured cells from dairy cows naturally exposed to DL-compounds. The study was carried out on two groups of animals bred in a highly polluted area and characterized by a different degree of contamination, as assessed by bulk milk TEQ values, and a control group reared in an industry free area. Bovine lymphocytes expressed only AHR, ARNT and CYP1B1 genes to a detectable level; moreover, only CYP1B1 expression appeared to be correlated to TEQ values, being higher in the most contaminated group, and decreasing along with animal decontamination. Finally, lymphocytes from exposed cows displayed a lower inducibility of both CYP1A1 and CYP1B1 after the in vitro treatment with a specific AHR ligand. In conclusion, our results indicate that DL-compound contaminated cows may display significant changes in AHR-target gene expression of circulating lymphocytes.

Background Chronic graft-versus-host disease (cGVHD) is a major factor of morbidity and mortality for allogeneic stem cell transplantation (aSCT). The skin and internal organ involvement is the most common systemic complication of cGVHD and closely resembles systemic sclerosis (SSc). Circulating lymphocytes characterize the autoimmune nature of both conditions. Therefore we hypothesized that the common clinical manifestation (systemic organ and skin injury) and the common underlying players (lymphocytes) justify the combined meta-analysis of these diseases. Results The aSCT and SSc datasets were uploaded from Gene Expression Omnibus (GEO), a public functional genomics data repository. The available microarray studies of peripheral blood mononuclear cells (PBMCs) and isolated lymphocytes were limited to well established microarray platforms (Affymetrix, Agilent, Canvac, and Illumina) and experimental settings with ≥10 patients per group. The resulting pools of data were merged by unique gene identifier and analyzed by the expression genome-wide association studies (eGWAS) coupled with the subtraction of the cGVHD+ and cGVHD− molecular signatures. The eGWAS was applied to 47 and 50 lymphocyte profiles from aSCT and SSc patients, respectively. The identified 35 candidates were represented by 8 known cGVHD genes (including CXCR4, LTBR and PML) and 28 new candidate genes (including SEPX1 and DNJGB1). The further mutual subtraction of cGVHD+ and cGVHD− candidates and pathway analysis identified a list of 25 genes. Seven of these genes belong to the fibroblast development and function pathway, consisting of the well known cGVHD genes CCND1, JUN, and FOS, and the new molecular targets MMP2, FOSB, TNFAIP8, and DUSP1. These genes become primary candidates for a potential link of systemic effects of cGVHD and SSc. Conclusions We designed a new approach for meta-analysis by combining data from different diseases using common clinical manifestation as a linker. This allowed us to power up the insufficient standalone meta-analysis of aSCT microarray studies, by adding SSc samples to the data pool. This new method has successfully identified novel molecular targets for systemic effects of both aSCT and SSc. We believe that this approach is generalizable and can be applied to an array of diseases with common clinical manifestations.

Circulating lymphocytes of obese individuals with and without type 2 diabetes have derangements of pyruvate dehydrogenase (PDH) that are described as reflecting a disorder underlying systemic insulin resistance, namely basal activity below normal and, in vitro, unresponsiveness to insulin at 33 pmol/l and activation at 330 pmol/l instead of activation and inhibition as in controls.To explore whether the above enzyme derangements are overcome in obese individuals on dexfenfluramine treatment, known to improve poor peripheral insulin sensitivity.Fifteen obese diabetic patients and 15 age-matched euglycaemic obese subjects with normal glucose tolerance were enrolled for a trial composed of two 21-day periods; in the first (D-21-D0), participants received a placebo, and in the second (D0-D21), dexfenfluramine (30 mg/day). At D-21, D0 and D21 participants were evaluated for weight, BMI, fasting glycaemia (FG), fasting insulinaemia (FI), fasting insulin resistance index (FIRI), area under the glycaemic (G-AUC) and insulinaemic (I-AUC) curves from an OGT test, and for PDH activity assayed in their circulating lymphocytes before (basal activity) and after incubation with 33 or 330 pmol/l insulin. At D2, basal PDH activity and clinical parameters were assayed.In both groups of participants at D0 all parameters tested were constant with respect to D-21; at D2, only basal PDH activity rose significantly; at D21, basal and insulin stimulated PDH activities were normalized and weight decreased significantly, as did FG, FI, FIRI and G-AUC in the diabetic, and FI, FIRI, G-AUC and I-AUC in the non-diabetic participants.In obese, non-diabetic and diabetic individuals on dexfenfluramine treatment, amelioration of clinical parameters and indexes of poor insulin sensitivity of blood glucose homeostasis are preceded by correction, in their circulating lymphocytes, of PDH derangements described as reflecting a disorder underlying insulin resistance.

Aims In circulating lymphocytes of NIDDM patients pyruvate dehydrogenase (PDH), the major determinant in glucose consumption through oxidative pathways, is poorly active. The aim of this study is to examine whether sulphonylurea drug treatment revives PDH activity in circulating lymphocytes from NIDDM patients. Methods Twenty normal-weight individuals with NIDDM were enrolled in this study. They had maintained their glycaemic levels close to normal by means of a restricted diet that had no longer been successful in the proceeding 2 months. The treatment protocol consisted in 160 mg gliclazide daily for 5 weeks. Twenty healthy subjects, matched for age, body mass index and gender, were enrolled as a control group. Patients, before and after treatment, as well as controls were tested for PDH activity in their circulating lymphocytes. Nine other untreated patients and nine healthy subjects, with the above mentioned characteristics, were recruited for the assay of PDH activity in their circulating lymphocytes before and after exposure, in vitro, to gliclazide, to insulin, and to gliclazide and insulin in combination. Results In gliclazide-treated NIDDM patients, PDH activity in circulating lymphocytes recovered. In vitro, in circulating lymphocytes of untreated patients and controls insulin at 5 μU ml−1 was ineffective and highly effective, respectively, in raising enzyme activity; gliclazide at 10 ng ml−1 was ineffective on PDH in both groups, but in combination with insulin at 5 μU ml−1 in both groups PDH was as active as in cells of controls exposed to insulin only. In cells of controls, gliclazide alone at 25–50 ng ml−1 caused enzyme activation, whereas above 50 ng ml−1 it caused inhibition; in cells of patients below 50 ng ml−1 it had no effects, but at 50 ng ml−1 and above raised enzyme activity to the basal level of controls. Conclusions This study suggests that free gliclazide concentrations determine recovery of PDH activity in circulating lymphocytes of treated patients through drug-mediated enhanced insulin control over PDH or through the drug alone.

Acute exercise sessions produce an immune system response, possibly leading to cessation, loss of continuity or performance decrement during the training period, caused by the increased upper respiratory infections (IVASs) susceptibility. Considering that lymphocyte cells have to fight infections and that sedentary subjects in poor physical condition should enroll physical programs of low intensity and volume, the aim of this study was to evaluate the amount of circulating leukocytes and lymphocytes as well as the mesenteric lymphocytes tissues, and the proliferative capacity of tissues lymphocytes in male Wistar rats submitted to acute sets of 5 and 15 minutes of low and moderate intensities. Results show leukocytosis and lymphocytosis in both groups 5 and 15 minutes of low and moderate intensities, increased tissue lymphocytes counting in the 5 minutes group of low intensity and in the 5 and 15 minutes groups of moderate intensity, in general, no alteration in the T cellular proliferative capacity, however with reduction in B lymphocytes in the 5 and 15 minutes groups of low intensity and in the 5 minutes of moderate intensity. The relevance and the clinical meaning of these alterations need to be well clarified for a better understanding of the complex triad: physical exercise, immunological responses and infections susceptibility after submaximal sets of short term. Sessões agudas promovem respostas no sistema imunológico, podendo acarretar desistência, não continuidade ou queda no desempenho nos programas de exercícios, pela aumentada susceptibilidade às infecções das vias aéreas superiores (IVASs). Considerando que os linfócitos desempenham funções no combate às infecções e que indivíduos sedentários com baixo nível de aptidão física, devam iniciar programas de treinamento com baixo volume e intensidade, o estudo teve como objetivo avaliar a contagem de leucócitos e linfócitos circulantes e dos linfócitos teciduais mesentéricos, bem como a capacidade proliferativa dos linfócitos teciduais em ratos machos "Wistar" submetidos a sessões agudas de cinco e 15 minutos nas intensidades leve e moderada. Os resultados mostram leucocitose e linfocitose nos grupos cinco e 15 minutos em ambas as intensidades, aumentada contagem de linfócitos teciduais nos grupos cinco minutos leve bem como cinco e 15 minutos moderado, de forma geral, nenhuma alteração na resposta proliferativa celular T, porém com diminuição na resposta celular B nos grupos cinco e 15 minutos leve e cinco minutos moderado. A relevância e o significado clínico dessas alterações precisam ser mais bem esclarecidos para o melhor entendimento da complexa tríade: exercício físico, respostas imunológicas e susceptibilidade a infecções, após sessões submáximas de curta duração.

9 p.-6 fig.-1 tab., The integrin alpha(4)beta(7) is the cell adhesion receptor for the mucosal vascular addressin MAdCAM-1, and this interaction is dominant in lymphocyte homing to Peyer's patch high endothelial venules, and plays key roles in lymphocyte recruitment at sites of inflammation. To identify alpha(4) subunit amino acids important for alpha(4)beta(7)/MAd-CAM-1 interaction, we expressed mutant alpha(4) and wild type beta(7) chains in K562 cells and analyzed the effect of the mutations on cell adhesion to a soluble MAdCAM-1 (sMAdCAM-1-Ig), Transfectants expressing mutated alpha(4) at Tyr(187) displayed a substantial decrease in adhesion to this ligand, which was associated with a reduced alpha(4)beta(7)/sMAdCAM-1-Ig interaction, as determined by soluble binding assays. Addition of Mn2+ to the adhesion assays did not restore the impaired adhesion. Mutations at alpha(4) Gln(152)Asp(153) also affected transfectant adhesion to sMAdCAM-1-Ig, but did not involve an alteration of alpha(4)beta(7)/MAdCAM-1 binding, and adhesion was restored by Mn2+. Instead, mutations at alpha(4) Asn(123)Glu(124) did not affect this adhesion. Mutation of alpha(4) Tyr(187) abolished alpha(4)beta(7)-mediated cell adhesion to CS-1/fibronectin, an additional ligand for alpha(4)beta(7), while alpha(4) Gln(152)Asp(153) transfectant mutants showed a reduced adhesion, These results identify alpha(4), Tyr(187) as a key residue during receptor alpha(4)beta(7)/ligand interactions, indicating that it plays important roles in alpha(4)beta(7)-mediated leukocyte adhesion, and provide a potential target for therapeutic intervention in several inflammatory pathologies., This work was supported by Grants PM95-0017 and SAF99-0057 from the plan General del Conocimiento-Ministerio de Educaciòn y Ciencia (Spain).

The synthetic pentapeptide (Arg-Lys-Asp-Val-Tyr; TP-5) corresponding to the active site of the hormone thymopoietin, was given at the dose of 300 mg/m2/day (1 day), higher than the usually administered, to a group of 27 immunodepressed patients in order to determine the tolerability and the immunomodulatory activity. The examination of a series of hematological parameters including counts of differential clustering of lymphocytes by cytofluorimetric analysis was performed 24 hr and 48 hr after treatment, and repeated at different intervals up to 14 days after treatment. TP-5 caused a significant increase of circulating lymphocytes and particularly of CD3+CD4+ and CD3+CD8+ subtypes, peaking at 48 hr and maintaining the increased values up to the last examination on day 14 from treatment. A faster increase (zenith at 24 hr) was observed for CD4+ cells, in comparison with CD8+ cells (zenith at 48 hr). The number of patients that increased total lymphocytes or lymphocyte subset after treatment ranged between 52.6 (CD4+ cells) and 69.2% (NK cells), whereas about 7.7% (NK cells) to 36.9% (CD4+ cells) remained unchanged and a smaller amount of 10.5% (CD4+ and CD8+ cells) or 23.1% (NK cells) showed a decrease greater than 10% of their respective basal value. No significant relationship between responders and non-responders can be found on the basis of previous treatments, cancer type, sex or age.