Circulating Lymphocyte Populations Research Articles

Pink1/Parkin deficiency alters circulating lymphocyte populations and increases platelet-T cell aggregates in rats

Parkinson’s disease (PD) is the most common progressive neurodegenerative movement disorder and results from the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Pink1 and Parkin are proteins that function together in mitochondrial quality control, and when they carry loss-of-function mutations lead to familial forms of PD. While much research has focused on central nervous system alterations in PD, peripheral contributions to PD pathogenesis are increasingly appreciated. We report Pink1/Parkin regulate glycolytic and mitochondrial oxidative metabolism in peripheral blood mononuclear cells (PBMCs) from rats. Pink1/Parkin deficiency induces changes in the circulating lymphocyte populations, namely increased CD4 + T cells and decreased CD8 + T cells and B cells. Loss of Pink1/Parkin leads to elevated platelet counts in the blood and increased platelet-T cell aggregation. Platelet-lymphocyte aggregates are associated with increased thrombosis risk suggesting targeting the Pink1/Parkin pathway in the periphery might have therapeutic potential.

Pink1/Parkin deficiency alters circulating lymphocyte populations and increases platelet-T cell aggregates in rats.

Parkinson's disease (PD) is the most common progressive neurodegenerative movement disorder and results from the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Pink1 and Parkin are proteins that function together in mitochondrial quality control, and when they carry loss-of-function mutations lead to familial forms of PD. While much research has focused on central nervous system alterations in PD, peripheral contributions to PD pathogenesis are increasingly appreciated. We report Pink1/Parkin regulate glycolytic and mitochondrial oxidative metabolism in peripheral blood mononuclear cells (PBMCs) from rats. Pink1/Parkin deficiency induces changes in the circulating lymphocyte populations, namely increased CD4 + T cells and decreased CD8 + T cells and B cells. Loss of Pink1/Parkin leads to elevated platelet counts in the blood and increased platelet-T cell aggregation. Platelet-lymphocyte aggregates are associated with increased thrombosis risk, and venous thrombosis is a cause of sudden death in PD, suggesting targeting the Pink1/Parkin pathway in the periphery has therapeutic potential.

Influence of Radiation Fractionation on Immune Repertoire Diversity in Solid Tumor Patients

Radiation (RT)-induced lymphopenia (RIL) occurs in up to 75% of patients undergoing RT and is associated with worse tumor control and survival across a spectrum of solid tumors. Patients undergoing hypofractionated RT are at lower risk of RIL compared with patients treated with more prolonged RT courses. However, it is unknown whether immune repertoire diversity is similarly affected by fractionation scheme in patients undergoing RT. This prospective study analyzed RT-induced changes in immune repertoire diversity in patients treated with conventionally (CFRT) vs hypofractionated RT (HFRT). RNA-based T and B cell receptor sequencing was performed on peripheral lymphocytes collected prospectively before RT and within 4 weeks of the last RT fraction from 23 patients (18 men, 5 women, median age 67 y) with primary solid tumors undergoing CFRT (≤3 Gy/day x ≥10 days, n = 13) or HFRT (≥5 Gy/day x ≤5 days, n = 10). Absolute lymphocyte counts (ALC; cells/μL) were obtained from clinical laboratory data. The number of unique CDR3 receptors (uCDR3) and Shannon entropy were used to monitor changes in T (TCR Vβ) and B (BCR IgH) receptor diversity. ALC decreased after RT in 90% (20/22) of patients (mean pre-RT ALC 1830 vs 1040 post-RT, p <0.001). Mean % ALC loss was greater in CFRT vs HFRT patients (44.3 vs. 35.2%). After RT, entropy in IgH and Vβ decreased in 18/23 (78%) and 17/23 (74%) patients, respectively; uCDR3 in IgH and Vβ decreased in 14/23 (61%) and 15/23 (65%). Among patients with concordant decreases in ALC and uCDR3, a moderate correlation between magnitude of ALC loss and uCDR3 levels in the T-cell receptor Vβ was observed (r = 0.64, p = 0.02). For both receptor species studied (IgH and Vβ), HFRT patients were more likely to have an increase in either entropy or uCDR3 in the face of decreased ALC (36 vs 15%, X2 p = 0.03). Furthermore, while decreases in entropy were observed among the CFRT patients for both IgH (median entropy 10.4 vs 9.4, p = 0.06) and Vβ (9.7 vs 8.1, p = 0.02), entropy did not significantly change following RT in the HFRT patients (IgH 10.6 vs 10.4, p = 0.74 and Vβ 10.9 vs 10.8, p = 0.24). RT-induced changes in immune repertoire diversity are variably reflected in the peripheral ALC. Both HFRT and CFRT depleted circulating lymphocytes, but patients undergoing HFRT were more likely to experience increases in T and B cell diversity metrics despite lymphopenia. It is therefore possible that relative sparing of repertoire diversity among patients undergoing HFRT could increase the likelihood of tumor antigen recognition by peripheral blood lymphocytes. As immune repertoire diversity is associated with the likelihood of response to immunotherapy, these findings also have implications for RT-immunotherapy combinations. Further study is required to understand the relationship between RT exposure to circulating lymphocyte populations and immune repertoire diversity.

Lymphocyte Depletion Rate as a Biomarker of Radiation Dose to Circulating Lymphocytes During Fractionated Partial-Body Radiation Therapy

PurposeRadiation causes exponential depletion of circulating lymphocyte populations; in turn, radiation-induced lymphopenia is associated with worse survival for many solid tumors, possibly owing to attenuated antitumor immune responses. Identifying reliable and reproducible methods of calculating the radiation dose to circulating immune cells may facilitate development of techniques to reduce the risk and severity of radiation-induced toxic effects to circulating lymphocytes. Methods and MaterialsPatient-specific lymphocyte loss rates were derived from a clinical data set including 684 adult patients with solid tumors. Multivariable linear regression was used to model the relationship between the lymphocyte loss rate and physical parameters of the radiation plan that determine circulating blood dose. ResultsDuring partial-body radiation, lymphocyte loss rates are determined by physical parameters of the radiation plan that reflect radiation exposure to circulating cells, including target volume size, dose per fraction squared, and anatomic site treated. Differences in observed versus predicted lymphocyte loss rates may be partly explained by variations in concurrent chemotherapy regimens. ConclusionsWe describe a novel method of using patient-specific lymphocyte loss kinetics to approximate the effective radiation dose to circulating lymphocytes during focal fractionated photon radiation therapy. Clinical applications of these findings include the early identification of patients at particularly high risk of severe radiation-induced lymphopenia based on physical parameters of the radiation therapy plan.

MO258SAFETY, TOLERABILITY, PHARMACOKINETICS AND PHARMACODYNAMICS OF VIS649, AN APRIL-NEUTRALIZING IGG2 MONOCLONAL ANTIBODY, IN HEALTHY VOLUNTEERS: PHASE 1, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, SINGLE ASCENDING DOSE STUDY

Abstract Background and Aims Immunoglobulin A (IgA) nephropathy (IgAN) is a glomerulonephritis characterized by the presence of circulating and glomerular immune complexes containing galactose-deficient (Gd) IgA1. A proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor superfamily of ligands, is thought to play a key role in the pathogenesis of IgAN by virtue of its role in class-switching to IgA production. VIS649, a humanized immunoglobulin G (IgG2) monoclonal antibody that binds to and blocks the biological actions of APRIL, is in clinical development as a potential treatment for IgAN. The primary objective of this first-in-human study was to evaluate the safety and tolerability of VIS649 in healthy volunteers. Secondary objectives included characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) of VIS649. Method This was a Phase 1, randomized, double-blind, placebo-controlled, single ascending dose study of VIS649 in healthy adult male and female volunteers (ClinicalTrials.gov identifier: NCT03719443). The study was conducted in sequential dosing cohorts. The first four cohorts (0.5, 2.0, 6.0, and 12.0 mg/kg, respectively) each enrolled 9 participants (4 of Japanese descent and 5 of non-Japanese descent) who were randomized to VIS649 or placebo in a ratio of 7:2. In addition, a fifth cohort enrolled 15 adults randomized to receive VIS649 6.0 mg/kg or placebo (10:5), followed by tetanus/diphtheria vaccine challenge after 28 days (TENIVAC®, Sanofi Pasteur Limited; the effect of APRIL inhibition on vaccine response is described in a companion abstract). Participants received intravenous administration of study drug on Day 1, were discharged from the institution on Day 2, and were followed for 16–24 weeks on an outpatient basis. Standard safety assessments and blood sampling for PK and PD were performed at regular intervals. Results 51 participants were randomized and dosed with study drug, of whom 47 (92.2%) completed the study. VIS649 was well tolerated, with no serious adverse events (AEs) or AEs that led to study discontinuation. Most treatment-emergent AEs (TEAEs) were mild; the incidence and severity of TEAEs were not dose dependent. One participant in the 2.0 mg/kg group experienced a severe TEAE of syncope following phlebotomy that the investigator considered unlikely to be related to study drug. There was no clinically relevant effect of treatment on laboratory tests, vital signs, electrocardiogram parameters, or physical examinations. VIS649 had non-linear PK: half-life (t½) increased with dose, while drug exposure (AUC) increased in a greater than dose proportional manner. Serum IgA, Gd-IgA1, IgG, and IgM were reversibly suppressed in a dose-dependent manner following VIS649 administration. The maximum mean percentage reductions from baseline occurred at Week 12 for the 12.0 mg/kg dose: IgA, -57.2% (Figure); Gd-IgA1, -71.6% (Figure); IgG, -33.6%; and IgM, -67.2%. These reductions were reversible and showed a dose-response effect with respect to time-to-recovery. Mean free (non-VIS649 bound) serum APRIL levels decreased to the lower limit of quantification (50 pg/mL) for all VIS649 doses at Week 1, and also showed a dose-response effect with respect to time-to-recovery. No depletions in circulating lymphocyte populations were observed. There were no significant PK or PD differences between Japanese and non-Japanese participants. Conclusion A single dose of VIS649, up to 12.0 mg/kg, was safe and well tolerated in healthy adults and was able to suppress free serum APRIL to the lower level of quantification. Serum Gd-IgA1 decreased in parallel with total serum IgA and recovered in a dose-dependent manner following reappearance of free APRIL in serum. These data support the further clinical development of VIS649 as a potential treatment for IgAN.

Selective modulation of trans-endothelial migration of lymphocyte subsets in multiple sclerosis patients under fingolimod treatment

Multiple sclerosis (MS) is an autoimmune disorder where auto-aggressive T cells target the central nervous system (CNS), causing demyelination. The trans-endothelial migration of leucocytes across the blood-brain barrier (BBB) is one of the earliest CNS events in MS pathogenesis. We examined the effect of the disease state and treatment with fingolimod on the transmigration of peripheral blood mononuclear cells (PBMCs) in an in vitro BBB model. Patients' leucocyte numbers, subsets and phenotypes were assessed by flow cytometry. As expected, fingolimod treatment induced a significant reduction in T cell and B cell numbers compared to untreated MS patients and healthy controls. Interestingly fingolimod led to a marked reduction of CD4+ and a significant increase in CD8+ cell numbers. In migrated cells, only CD3+ cell numbers were reduced in fingolimod-treated, compared to untreated patients; it had no effect on B cell or monocyte transmigration. T cells were then differentiated into naïve, effector and memory subsets based on their expression of CCR7. This showed that MS patients had increased numbers of effector memory CD4+ cells re-expressing CD45RA (TEMRA) and a decrease in central memory (CM) CD8+ cells. The former was corrected by fingolimod, while the latter was not. CM CD4+ and CD8+ cells migrated across BBB more efficiently in fingolimod-treated patients. We found that while fingolimod reduced the proportions of naïve CD19+ B cells, it significantly increased the proportions of these cells which migrated. When B cells were further stratified based on CD24, CD27 and CD38 expression, the only effect of fingolimod was an enhancement of CD24hiCD27+ B cell migration, compared to untreated MS patients. The migratory capacities of CD8hi Natural Killer (NK), CD8dim NK and NK-T cells were also reduced by fingolimod. While the disease-modifying effects of fingolimod are currently explained by its effect on reducing circulating auto-aggressive lymphocytes, our data suggests that fingolimod may also have a direct though differential effect on the trans-endothelial migration of circulating lymphocyte populations.

Evaluating Changes in Immune Function and Bone Microenvironment During Radium-223 Treatment of Patients with Castration-Resistant Prostate Cancer.

The effects of radium-223 on the immune system and the bone tumor microenvironment are incompletely understood. The authors describe mechanisms by which radium-223 may interact with the immune system, specifically through STAT-3 and impact on tumor and circulating lymphocyte populations. They review mechanisms through which effects of radium-223 and androgen-targeted therapy on bone microenvironment could be better elucidated. These knowledge gaps currently limit development of optimal combination therapy approaches for radium-223. Tissue based studies are currently underway in a prospective clinical trial to enhance therapeutic perspective on radium-223 treatment in the prostate cancer landscape.

Association Between Circulating Lymphocyte Populations and Outcome After Stereotactic Body Radiation Therapy in Patients With Hepatocellular Carcinoma.

Background and Objective: Radiation-induced lymphopenia has a tangible impact on overall survival (OS) in multiple solid tumors. We investigated the association between circulating lymphocyte populations (CLPs) before and after stereotactic body radiation therapy (SBRT) and OS in patients with hepatocellular carcinoma (HCC).Materials and Methods: Seventy-eight HCC patients treated with SBRT between January 2013 and June 2017 were retrospectively analyzed. Baseline and post-treatment total peripheral lymphocyte counts (TPLCs) and values of different CLPs were obtained and analyzed for clinical outcomes. Univariate and multivariate Cox regression analyses were used to explore the independent prognostic factors for patient survival.Results: The one-, two- and three-year OS rates were 94.8, 75.9, and 63.3%, respectively. The mean TPLCs before and 10 days after SBRT were 1.4 × 109/L and 0.7 × 109/L, respectively. The TPLC recovered to its baseline value 1 year after SBRT. Multivariate analysis results revealed that variables, including tumor necrosis factor-alpha (TNF-α) level <5.5 ng/mL and post-treatment TPLC <0.45 × 109/L were independent factors for inferior OS. Further analysis showed that the values of CLPs, including CD3+, CD4+, CD8+, CD19+, and CD16+56+ cells dropped profoundly 10 days after SBRT, among which CD19+ B cell count was mostly depleted and gradually recovered after 2 months. Univariate analysis showed that both baseline and post-treatment TPLC and CLP (except post-treatment B cell) counts were significantly associated with patient OS (p < 0.05 for each). Further stratified analysis performed according to OS at 2 years demonstrated that the CD16+CD56+ NK cell counts remained significantly elevated in patients with better survival (OS > 2 years) compared to those in short-term survivors at 10 days, 1 month, and 2 months after SBRT (p < 0.05 for each). In addition, there were significant differences in TPLC and CD8+ T cell counts in patients with long-term and short-term OS at 2 months after SBRT (p < 0.05).Conclusions: Peripheral lymphopenia after SBRT might be an independent prognostic factor for poorer outcome in HCC patients. Post-treatment lymphocyte subsets, including CD8+ T cell and NK cell counts were also associated with 2-year OS rates.

Changes of Circulating Lymphocyte Populations and Its Association with Outcome after Hypofractionated Radiation Therapy in Patients with Non-small-cell Lung Cancer

Hypofractionated radiation therapy may have significant immunomodulatory effects and could enhance anti-tumor immune effect. The purposes of this study were to investigate changes of peripheral blood lymphocytes at different time points during and after radiotherapy and their association with survival. We collected serial blood samples before treatment and about day 14 of radiotherapy, at the end of radiotherapy, 1 month and 3 months after radiotherapy from 42 patients with non-small-cell lung cancer from November 2016 to October 2018 . 40.5% (17/42) of the patients received 60-84Gy in 10-12 fractions of Stereotactic ablation radiotherapy. Other patients (59.5%, 25/42) were treated with Partial Stereotactic Ablative Boost Radiotherapy，the GTVb（GTV-boost）received 18-32Gy in 3-4 fractions, then the PTV was treated with conventional fractionated radiotherapy to a median dose of 55.44 Gy（48-60Gy）. We used flow cytometry to measure the changes of total CD3+, CD4+, CD8+ T cells, CD3+HLADR+ T cells, CD19+ B lymphocytes and CD16+CD56+ natural killer cells. The median follow up time was 10.6 months(range, 3.3 to 31.3). The one-year and two-year PFS was 73.0% and 58.0%, the one-year and two-year OS was 80.0% and 68.5%, and the one-year and two-year locoregional failure-free survival rate was 87.7% and 71.4%, respectively. The number of all the lymphocytes decreased gradually at the beginning of radiotherapy, reached the minimum at the end of radiotherapy, and gradually returned to the baseline level after radiotherapy. However, the fractions of CD3+HLADR+ T cells（activated T lymphocytes） decreased slightly at the beginning of radiotherapy, then gradually increased during radiotherapy until 3 months after radiotherapy. Moreover, 1 month after radiotherapy, the proportion of CD4+T cells was lower than the baseline, and the proportion of CD8+T cells was higher than the baseline, and the ratio of the two was lower than the baseline. Longer OS was significantly correlated with higher CD4+/CD8+Tcell ratio after radiotherapy (p＜0.05). And we found hypofractionated radiation therapy of patients with large tumor size produced more significant immune effects, while large tumor size may lead to worse OS, which still needs further exploration. The proportion of CD8+T cells and CD3+HLADR+ T cells（activated T lymphocytes）increased one month after the hypofractionated radiation therapy. We found longer OS was significantly correlated with higher CD4+/CD8+Tcell ratio after radiotherapy (p＜0.05), and this may be because the larger irradiation volume related to significant immunological effects of the tumor. These results may provide reference for future studies to determine the optimal treatment schedules when combining irradiation with specific immunotherapy approaches.

Differential Association Between Circulating Lymphocyte Populations With Outcome After Radiation Therapy in Subtypes of Liver Cancer

Irradiation may have significant immunomodulatory effects that impact tumor response and could potentiate immunotherapeutic approaches. The purposes of this study were to prospectively investigate circulating lymphoid cell population fractions during hypofractionated proton therapy (HPT) in blood samples of liver cancer patients and to explore their association with survival. We collected serial blood samples before treatment and at days 8 and 15 of HPT from 43 patients with liver cancer-22 with hepatocellular carcinoma (HCC) and 21 with intrahepatic cholangiocarcinoma (ICC)-enrolled in a phase 2 clinical trial. All patients received 15 fractions of proton therapy to a median dose of 58Gy (relative biological effectiveness).We used flow cytometry to measure the changes in the fractions of total CD3+, CD4+, and CD8+ T cells; CD4+ CD25+ Tcells; CD4+ CD127+ T cells; CD3+ CD8+ CD25+ activated cytotoxic T lymphocytes (CTLs); and CD3- CD56+ natural killer cells. With a median follow-up period of 42months, median overall survival (OS) in the study cohort was 30.6months for HCC and 14.5months for ICC patients. Longer OS was significantly correlated with greater CD4+ CD25+ T-cell (P=.003) and CD4+ CD127+ T-cell (P=.01) fractions at baseline only in ICC patients. In HCC patients, the fraction of activated CTLs mid treatment (at day 8) was significantly associated with OS (P=.007). These findings suggest a differential relevance of immunomodulation by HPT in these liver cancers. Antitumor immunity may depend on maintenance of a sufficiently high number of activated CTLs during HPT in HCC patients and CD4+ CD25+ T cells and CD4+ CD127+ T cells prior to treatment in ICC patients. These results could guide the design of future studies to determine the optimal treatment schedules when combining irradiation with specific immunotherapy approaches.

Impact of gefitinib in early stage treatment on circulating cytokines and lymphocytes for patients with advanced non-small cell lung cancer.

ObjectivesThe impact of epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKIs) on the human immune system remains undefined. This study illustrates the immunomodulatory effect of gefitinib in patients with advanced non-small cell lung cancer (NSCLC) and its relevant prognostic significance.Patients and methodsPeripheral blood samples were collected from 54 patients at baseline and after 4 weeks of gefitinib treatment. Circulating lymphocyte populations and cytokine levels were measured. Pilot investigation of the impact of gefitinib on programmed cell death ligand-1 (PD-L1) expression was conducted by immunohistochemistry (IHC).Results and conclusionA significant increase of peripheral natural killer cells and interferon-gamma (INF-γ) after 4 weeks of gefitinib treatment (P=0.005 and 0.02, respectively). In addition, circulating interleukin (IL)-6 was significantly decreased, especially in patients sensitive to gefitinib (P<0.001). Higher levels of IL-6 at baseline independently correlated with poorer progression-free survival. Experiments with NSCLC specimens illustrated that PD-L1 expression were downregulated after 4 weeks of gefitinib treatment. In summary, it was found that gefitinib treatment can alter circulating cytokines and lymphocytes. Dynamic changes of circulating lymphocytes, cytokines, and even PD-L1 IHC expression around gefitinib treatment support the specific immunomodulatory effect of this agent for advanced NSCLC.

Treatment-Induced Changes in Circulating Lymphocyte Populations Associate With Survival of Patients With Hepatocellular Carcinoma (HCC) and Intrahepatic Cholangiocarcinoma (ICC) Treated With Hypofractionated Proton Therapy (HPT)

Treatment-Induced Changes in Circulating Lymphocyte Populations Associate With Survival of Patients With Hepatocellular Carcinoma (HCC) and Intrahepatic Cholangiocarcinoma (ICC) Treated With Hypofractionated Proton Therapy (HPT)

Abstract P156: Fiber-rich Diet Suppresses Lactobacillus And Increases Blood Pressure In The Shr Independently Of T-lymphocyte Immune Responses

Introduction: Gut dysbiosis has been linked to hypertension in both rodents and humans. Microbial metabolites such as propionate have been shown to regulate blood pressure (BP), while butyrate, one of the major fermented end-products of fiber, reportedly produces beneficial anti-inflammatory effects in multiple dysbiosis-related diseases. Therefore, we tested the impact of a fiber-rich, butyrolytic diet on BP regulation and immune responses in the spontaneously hypertensive rats (SHR). Methods: SHR (5 wo) were placed on either the fructooligosaccharides/inulin-rich diet (Fiber, N=6), or its calorie-matched control diet (Control, N=6) (Research Diets, Inc.) for 10 weeks. Baseline BP was measured by tail cuff every week for the duration of the study. Fecal samples were collected for HPLC analysis of butyrate, and Lactobacillus population by QPCR. Manganese-enhanced magnetic resonance imaging was used to monitor neural activity in cardioregulatory brain regions. Blood was analyzed for circulating lymphocyte populations previously implicated in BP control in the SHR (CD3 + CD45 + , CD4 + CD25 + , CD8 + ). Results: Fiber-rich diet produced an increase in fecal butyrate levels as early as five weeks (Control vs. Fiber, 4.9umol/g vs. 9.7umol/g, p=0.068, N=6). This was associated with contraction of fecal Lactobacillus (47.5% vs. 5.9%, p=0.0008, N=6). However, we observed significantly higher systolic BP (181.4mmHg vs. 201.7 mmHg, p=0.0088, N=6) in the fiber group compared with control, beginning with week 9 post-diet switch. Changes in neural activation were observed in the paraventricular nucleus of hypothalamus (PVN) (3.3 voxel vs. 7.3 voxel, p=0.26) and amygdala (93.3 voxel vs. 31 voxel, p=0.0059, N=3). No changes in circulating T-lymphocytes were observed between the two groups: CD3 + CD45 + (32.4% vs. 32.1% lymphocytes); CD4 + CD25 + (1.05% vs. 0.78% lymphocytes); CD8 + (18.7% vs. 16.2% lymphocytes, N=6) at week 10 post-diet switch. Conclusion: Fiber-rich diet suppression of Gut Lactobacillus is associated with increase in BP in the SHR, independently of T-lymphocyte responses. The observed higher neural activity in PVN and lower in amygdala in the fiber group suggest direct effects of gut bacterial metabolites on brain cardioregulatory regions.

A Multicenter Phase I/Ib Study of Ipilimumab for Relapsed Hematologic Malignancies after Allogeneic Hematopoietic Stem Cell Transplantation

▪ BACKGROUNDThe prognosis for patients (pts) with hematologic malignancies (HM) who relapse after alloHSCT is dismal, and novel treatment options are urgently needed. Checkpoint blockade has the potential to augment the graft-vs-tumor (GVT) effect pharmacologically, but may increase the risk of graft-vs-host disease (GVHD). We conducted a multicenter, open-label phase I/Ib study of the CTLA4 blocking antibody ipilimumab in pts with relapsed HM after alloHSCT. METHODSThe primary objectives were to determine the MTD and evaluate safety. Secondary objectives were to assess efficacy and changes in circulating lymphocyte populations. Ipilimumab induction was given IV every 3 weeks for 4 cycles, followed by maintenance dosing every 12 weeks up to 1 year. Disease-specific response evaluations were performed at the mid-point (7 weeks), end of induction (13 weeks), and throughout maintenance. Immunophenotyping was assessed by 8-color flow cytometry. Two dose levels (3 mg/kg and 10 mg/kg) were studied in phase I. The 10 mg/kg dose was chosen for expansion on the basis of safety and efficacy results. RESULTSA total of 28 patients were enrolled. In phase I, 6 pts were treated at 3 mg/kg and 7 pts were treated at 10 mg/kg. In the phase Ib expansion cohort, 15 additional pts were treated at 10 mg/kg. Histologies included AML (n=12, including 3 pts with leukemia cutis), HL (n=7), NHL (n=4), and MDS (n=2), and 1 pt each had MM, MPN, and ALL. The median age at enrollment was 58 yrs. (range 22-75). The median number of prior therapies excluding transplant was 3 (range 2-11), and 19/27 (70%) of pts had received prior therapy for post transplant relapse. In the toxicity analysis, immune-related adverse events (irAEs) typical of ipilimumab were observed in 4 pts, including pneumonitis (n=1 gr2, n=2 gr4), diarrhea (n=2 gr 1), colitis (n=1 gr 3), and ITP (n=1 gr2). These irAEs were generally reversible with steroids, and 3 pts were able to resume ipilimumab. Five DLTs leading to discontinuation occured, including cGVHD (n=3, all liver, gr 3), aGVHD (n=1, gut, gr 2), and TRM (n=1) due to presumed sepsis in the context of gr4 pneumonitis and gr3 colitis. Seventeen pts discontinued due to progressive disease, and 6 pts remain on active treatment. In the efficacy analysis, none of the 5 evaluable pts treated at 3 mg/kg achieved a formal response by disease specific criteria. In the 21 evaluable patients treated at 10 mg/kg, 10 patients (48%) had disease reduction. The overall response rate (ORR) by formal disease specific criteria was 33%. Responders included a HL pt with a PR with dramatic reduction in widespread bony disease and a marrow CR by 7 weeks, and a MM pt with a highly refractory lung plasmacytoma who achieved a PR. Notably, 5/12 (42%) pts with AML achieved CR, including all 3 pts with leukemia cutis (see example in Fig. 1) and one pt with myeloid sarcoma. The responses in two of the three leukemia cutis pts were durable, with both pts still in CR at 6 and 8 months on study. With a median follow-up time among survivors of 8.5 mo., the 6 mo. OS is 67%. Immunophenotyping studies revealed that the absolute numbers of T cells (including CD3+, CD4+, CD8+, Tcon, and Treg) peaked at 4 weeks after initial dosing and trended back down by 8 weeks. The ratio of Treg/Tcon decreased between 24 to 41% in the 15 pts with samples available at each time point. In the 5 pts who achieved CR, the Treg/Tcon ratio was similar to the rest of the cohort at baseline (0.07 vs. 0.08, p=0.57), but by 8 weeks there was a trend toward a decreased ratio in CR pts (0.05 vs. 0.15, p=0.14). The absolute number of NK cells was lower in responders than in non-responders by the 8 week time point (p = 0.04). CONCLUSIONRepeated dosing of ipilimumab is feasible in pts with relapsed HM after alloHSCT, although at 10 mg/kg both irAEs and GVHD were observed on treatment. Ipilimumab in this setting can provide substantial anti-tumor activity in pts with both lymphoid and myeloid malignanices. Interestingly, chemorefractory extramedullary myeloid diseases such as leukemia cutis and myeloid sarcoma appeared to respond particularly well. The Treg/Tconv cell ratio decreased with treatment, consistent with enhancement of GVT. Checkpoint blockade is a promising new therapeutic approach for post alloHSCT relapse, and is worthy of exploration in future studies. [Display omitted] DisclosuresDavids:Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Other: ad board. Chen:Bayer: Consultancy, Research Funding. Armand:Sequenta, Inc.: Research Funding; Merck: Consultancy, Research Funding; BMS: Research Funding; Infinity: Consultancy, Research Funding. Soiffer:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

The Immunology of a Healing Response in Cutaneous Leishmaniasis Treated with Localized Heat or Systemic Antimonial Therapy

BackgroundThe effectiveness of systemic antimonial (sodium stibogluconate, Pentostam, SSG) treatment versus local heat therapy (Thermomed) for cutaneous leishmaniasis was studied previously and showed similar healing rates. We hypothesized that different curative immune responses might develop with systemic and local treatment modalities.MethodsWe studied the peripheral blood immune cells in a cohort of 54 cutaneous Leishmania major subjects treated with SSG or TM. Multiparameter flow cytometry, lymphoproliferative assays and cytokine production were analyzed in order to investigate the differences in the immune responses of subjects before, on and after treatment.ResultsHealing cutaneous leishmaniasis lead to a significant decline in circulating T cells and NKT-like cells, accompanied by an expansion in NK cells, regardless of treatment modality. Functional changes involved decreased antigen specific CD4+ T cell proliferation (hyporesponsiveness) seen with CD8+ T cell depletion. Moreover, the healing (or healed) state was characterized by fewer circulating regulatory T cells, reduced IFN-γ production and an overall contraction in polyfunctional CD4+ T cells.ConclusionHealing from cutaneous Leishmaniasis is a dynamic process that alters circulating lymphocyte populations and subsets of T, NK and NKT-like cells. Immunology of healing, through local or systemic treatments, culminated in similar changes in frequency, quality, and antigen specific responsiveness with immunomodulation possibly via a CD8+ T cell dependent mechanism. Understanding the evolving immunologic changes during healing of human leishmaniasis informs protective immune mechanisms.

Review: Lymphocytes, cytokines, chemokines and the T-helper1-T-helper2 balance in canine atopic dermatitis.

The development of atopic dermatitis (AD) and other cutaneous hypersensitivities involves the activation and differentiation of allergen-specific lymphocytes. Although hypersensitivity is often considered to be a 'T-helper2-polarized' lymphocyte response, recent evidence suggests that clinical disease is associated with the development of multiple lymphocyte phenotypes. The purpose of this paper is to review recent advances in the understanding of the roles of lymphocytes, cytokines and noncytokine factors in the pathogenesis of canine AD. Citation databases, abstracts and proceedings from international meetings published between 2001 and 2013 were reviewed in this update. Where necessary, older articles were included for background information. The development of canine AD is associated with changes in both cutaneous and circulating lymphocyte populations. These lymphocyte responses are characterized by the production of a complex variety of cytokines, including not only T-helper2 but also T-helper1, T-helper17 and regulatory T-cell responses. In addition, microarray gene expression analysis has enabled the identification of a number of noncytokine factors that appear to be associated with atopic inflammation. These include the calcium-binding protein S100A8, serum amyloidA and a number of protease inhibitors, as well as genes involved in epidermal barrier formation, innate immunity receptors, cell cycle proteins and apoptosis. The development of AD in dogs is characterized by the development of a delicate balance between a variety of T-cell phenotypes and inflammatory mediators, including cytokines, chemokines and noncytokine factors.

Reduction of Specific Circulating Lymphocyte Populations with Metabolic Risk Factors in Patients at Risk to Develop Type 2 Diabetes

Low-grade inflammation, characterized by increased pro-inflammatory cytokine levels, is present in patients with obesity-linked insulin resistance, hyperglycemia and hyperlipidemia and considered to play a leading role to progression into type 2 diabetes (T2D). In adipose tissue in obese patients and in pancreatic islets in T2D patients cellular inflammation is present. However, the systemic leukocyte compartment and the circulating endothelial/precursor compartment in patients at risk to develop T2D has so far not been analyzed in detail. To address this, peripheral blood cells from a cohort of 20 subjects at risk to develop diabetes with normal to impaired glucose tolerance were analyzed by flow cytometry using a wide range of cellular markers and correlated to known metabolic risk factors for T2D i.e. fasting plasma glucose (FPG), 2 h plasma glucose (2 h PG), HbA1c, body mass index (BMI), homeostasis model assessment of β-cell function (HOMA-B), homeostasis model assessment of insulin sensitivity (HOMA-IS) and fasting insulin (FI). The four highest ranked cell markers for each risk factor were identified by random forest analysis. In the cohort, a significant negative correlation between the number of TLR4+ CD4 T cells and increased FPG was demonstrated. Similarly, with increased BMI the frequency of TLR4+ B cells was significantly decreased, as was the frequency of IL-21R+ CD4 T cells. Unlinked to metabolic risk factors, the frequency of regulatory T cells was reduced and TLR4+ CD4 T cells were increased with age. Taken together, in this small cohort of subjects at risk to develop T2D, a modulation of the circulating immune cell pool was demonstrated to correlate with risk factors like FPG and BMI. This may provide novel insights into the inflammatory mechanisms involved in the progression to diabetes in subjects at risk.

Oral vitamin D increases the frequencies of CD38+ human B cells and ameliorates IL‐17‐producing T cells

Vitamin D deficiency (serum 25-hydroxyvitamin D < 50 nm) has been associated with the onset of immunological diseases including atopic dermatitis (AD), cutaneous or systemic lupus erythematosus and allergic asthma. In this study, we assessed whether oral vitamin D (cholecalciferol) supplementation leads to a systemic modulation of the phenotype of circulating lymphocyte populations and whether a defined serum 25-hydroxyvitamin D (25(OH)D) concentration can be related to the effects on lymphocytes. Cholecalciferol was administered in a dose-escalation setting to vitamin D-deficient individuals from 2000 up to 8000 IU daily for 12 weeks. Individuals without cholecalciferol intake served as controls. Peripheral B cells and T cells were examined by multicolour flow cytometric analysis. The mean serum 25(OH)D concentrations increased upon cholecalciferol intake up to 159 ± 28.7 nm, and remained low in the control group 30.0 ± 12.5 nm. Following cholecalciferol intake, the frequencies of circulating CD38 expressing B cells were significantly increased and IFN-γ+ , and/or IL-17+ CD4+ T helper cells were decreased. These data were identified to correlate with the serum 25(OH)D levels by applying two different analysis approaches (ROC and a nonlinear regression analysis). Our data indicate that increasing 25(OH)D serum concentrations are associated with an increased expression of CD38 on B cells and a decreased T-cell-dependent proinflammatory cytokine production. The therapeutical role of our findings in systemic immunological diseases should be explored in the future by further controlled clinical studies.

Controlled Epstein-Barr Virus Reactivation After Allogenic Transplantation Improves Survival and Is Associated To An Expansion Of Natural Killer Cells (NK)

Background B-lymphocytes infected by the Epstein-Barr virus (EBV) are strongly controlled by the immune system, with the persistence of low counts of EBV-infected B lymphocytes in 98% of healthy individuals. Both T- and Natural Killer (NK)-lymphocytes play a main role in immune surveillance of EBV infection. EBV-reactivation (R) frequently occurs in patients having allogeneic transplantation (AT). Aims We evaluated the impact of controlled EBV-R on survival of allografted adult patients with haematological malignancies and analyzed the different circulating lymphocyte populations. Methods 190 consecutive adult patients were included in this study. Patients received fludarabine, busulfan, ATG for reduced intensity conditioning regimen or cyclophosphamide plus TBI for myeloablative conditioning regimen. Prophylaxis of acute GVHD was conventional. EBV viral load (EBV-VL) was monitored in PB, for all the patients after AT, weekly for the first 6 months, then monthly using standard PCR technique. EBV-R was defined by an EBV-VL ³500 copies/ml, increasing 1 week later. This cut-off level of EBV-VL was used to decide therapy, first reducing immunosuppression followed by rituximab if no EBV-VL decrease (375mg/m2, 4 injections weekly). Lymphocyte immunophenotyping (CD3,4,8,3+56+,3-56+,19,25,DR) was performed weekly for 3 months, then monthly using standard technique. In order to show interdependency between the parameters, a multivariate analysis was performed for OS and PFS including parameters like EBV-R, ATG usage. All analysis was performed with SAS 9.3. Results There were 105 acute leukaemias (26 ALL, 71 AML) and myelodysplastic syndromes (8), 71 lymphoproliferative disorders (17 lymphomas, 8 CLL and 46 MM), 5 aplastic anaemia and 9 CML. There were 76 females and 114 males (median age: 51y, range 18-69). 86 (45%) patients presented EBV-R, with 74 patients having EBV-VL&gt;1000 copies/mL. There was no statistical difference between the 2 groups of patients (EBV-R and no EBV-R), for age, sex, cancer type, conditioning regimen, particularly fludarabine, type of graft, and number of infused CD34+ hematopoietic progenitors. Patients with EBV-R received more frequently ATG (P =0.004). The median time of EBV-R detection was 73 days (IQR: 40-210) after transplantation. In case of EBV-R, the dose of immunosuppressive drugs was reduced before stopping. 69/86 patients received rituximab. Patients with no EBV-R did not receive rituximab. Within the 3 months after rituximab, GVHD intensity was decreased, particularly for acute GVHD, which was less frequent. The median follow up was 36.6 months (95%IC 31.5-45.7). Patients with EBV-R had a longer PFS than those with no EBV-R (at 2 years 51% vs. 69%, at 5 years 47% vs 38%) (P&lt;.04). Similarly, OS was longer in patients with EBV-R than that observed for patients with no EBV-R (at 2 years 76% vs. 64% and at 5 years 63 % vs. 47%, P&lt;.001). OS and PFS were not different between patients according to the type of hematological malignancy (respectively, P=.46 and P=.49). The use of ATG was not a significant factor on OS (univariate (p=0.93) and multivariate analysis (p=0.81)). Similarly, it was not a significant factor on PFS (univariate (p=0.47) or multivariate analysis (p=0.68)). Among the patients with EBV-R, OS and PFS were not significantly different for patients receiving or not rituximab (respectively p=0.67 and p=0.88). The median duration of lymphocyte measurement was 94 days (IQR 59-248) after AT. B-lymphocytes and particularly T-lymphocytes, including CD4+ and CD8+ lymphocytes were not significantly different between the 2 groups, respectively P=.29 and P=.48. Only circulating NK cells were significantly different, with a median increased by 22% (P=.03) in patients with EBV-R. CMV co-infection did not impact NK cell counts (P=.058). NK cell count was not different according the use of rituximab (P=.22). The use of rituximab did not modify NK cell counts at 3 and 6 months after stopping treatment (both P=.99). Conclusions Controlled EBV-R in allografted cancer patients is associated with better OS and PFS, in association with a significant increase in circulating NK cells. A prospective study is ongoing for analyzing different subpopulations of NK cells. Disclosures: No relevant conflicts of interest to declare.

Sparing Circulating Lymphocyte Populations With Endorectal Brachytherapy

Sparing Circulating Lymphocyte Populations With Endorectal Brachytherapy