Balance Of Subsets Research Articles

Regulatory Effect of Iguratimod on the Balance of Th Subsets and Inhibition of Inflammatory Cytokines in Patients with Rheumatoid Arthritis.

Objective. To expand upon the role of iguratimod (T-614) in the treatment of rheumatoid arthritis (RA), we investigated whether the Th1, Th17, follicular helper T cells (Tfh), and regulatory T cells (Treg) imbalance could be reversed by iguratimod and the clinical implications of this reversal. Methods. In this trial, 74 patients were randomized into iguratimod-treated (group A) and control (broup B) group for a 24-week treatment period. In the subsequent 28 weeks, both groups were given iguratimod. Frequencies of Th1, Th17, Tfh, and Treg were quantified using flow cytometry, and serum cytokines were detected by enzyme-linked immunosorbent assay. mRNA expression of cytokines and transcriptional factor were quantified by RT-PCR. The composite Disease Activity Score, erythrocyte sedimentation rate, and C-reactive protein were assessed at each visit. Result. The clinical scores demonstrated effective suppression of disease after treatment with iguratimod. In addition, iguratimod downregulated Th1, Th17-type response and upregulated Treg. Furthermore, the levels of Th1, Th17, and Tfh associated inflammatory cytokines and transcription factors were reduced after treatment with iguratimod, while the levels of Treg associated cytokines and transcription factors were increased.

Functional and Phenotypic Plasticity of CD4(+) T Cell Subsets.

The remarkable plasticity of CD4+ T cells allows individuals to respond to environmental stimuli in a context-dependent manner. A balance of CD4+ T cell subsets is critical to mount responses against pathogen challenges to prevent inappropriate activation, to maintain tolerance, and to participate in antitumor immune responses. Specification of subsets is a process beginning in intrathymic development and continuing within the circulation. It is highly flexible to adapt to differences in nutrient availability and the tissue microenvironment. CD4+ T cell subsets have significant cross talk, with the ability to “dedifferentiate” given appropriate environmental signals. This ability is dependent on the metabolic status of the cell, with mTOR acting as the rheostat. Autoimmune and antitumor immune responses are regulated by the balance between regulatory T cells and Th17 cells. When a homeostatic balance of subsets is not maintained, immunopathology can result. CD4+ T cells carry complex roles within tumor microenvironments, with context-dependent immune responses influenced by oncogenic drivers and the presence of inflammation. Here, we examine the signals involved in CD4+ T cell specification towards each subset, interconnectedness of cytokine networks, impact of mTOR signaling, and cellular metabolism in lineage specification and provide a supplement describing techniques to study these processes.

A novel function of interferon regulatory factor-1: inhibition of Th2 cells by down-regulating the Il4 gene during Listeria infection.

Infection with certain pathogens induces a shift of the Th subset balance to a Th1 dominant state. This, in turn, results in the suppression of Th2 responses. We focused on the involvement of interferon regulatory factor-1 (IRF-1) in the suppression of Th2 cells during Listeria infection. We found that the inhibition of IL-4 production by Th2 cells is mediated by a soluble factor (LmSN) produced by Listeria-infected antigen-presenting cells. The inhibition is not observed with T cells from Irf1 gene-targeted mice. IRF-1 suppresses transcription of the Il4 gene in Th2 cells. Under the influence of the LmSN, IRF-1 binds to the 3' untranslated region (UTR) region of the Il4 gene and down-regulates Il4 gene transcription. Finally, we identified IL-1α and IL-1β as the mediator of the LmSN activity. Signaling through IL-1R induces the stabilization and/or nuclear translocation of IRF-1. We propose that IRF-1 functions to induce the T-cell subset shift via a novel mechanism. Under the influence of IL-1, IRF-1 translocates into the nucleus and acts on the 3'UTR region of the Il4 gene, thus inhibiting its transcription in Th2 cells. As a result, the immune system shifts predominantly to a Th1 response during Listeria infection, resulting in effective protection of the host.

Alterations in Peripheral Blood Follicular Helper T Cell (TFH) Subsets and TREG in Kidney Transplant Recipients With DSA.

Donor-specific anti-HLA antibodies (DSA) represent a key risk factor for acute and chronic rejection, and are a major cause of long-term graft failure after kidney transplantation (KTx). The immunological mechanisms that lead to the development of DSA are not entirely understood. We postulated that an imbalance between TFH (CD4+CD45RO+CXCR5+), which provide key help for antibody production by B cells, and TREG (CD4+CD25highFoxP3+CD127-) correlates with the development of DSA after Tx. Twenty-six thymoglobulin-induced KTx patients participating in an ongoing prospective study were monitored for DSA occurrence in the first year after Tx. During this period, 7 patients were identified as DSA positive by Luminex Single Antigen Bead Assay (cut-off 1000 MFI). Three patients developed DSA by day 30 post-Tx, while 3 presented DSA after day 180. Moreover, one patient had DSA pre-Tx that showed a 4 fold increase by day 30 post-Tx. In addition, 6 healthy control (HC) subjects and 5 DSA negative KTx recipients were included as controls. Whole blood was obtained at regular intervals before and after Tx, and analyzed by flow cytometry.We observed that the overall frequency of circulating TFH cells was not significantly different among the three groups: KTx recipients with DSA, KTx recipients with no DSA, and HC. However, the % of circulating Th1-TFH (CD4+CD45RO+CXCR3+CXCR5+) cells was significantly increased and the % of circulating TREG was decreased in KTx patients with DSA, as compared to their own pre-Tx values and to HC, resulting in a significantly increased Th1-TFH:TREG ratio. In addition, KTx patients with no DSA displayed comparable levels of Th1-TFH and TREG to those of HC, but significantly lower % of Th1-TFH compared to DSA positive patients.In conclusion, patients who develop DSA display a significant skewing of TFH cell subsets toward a Th1-TFH phenotype, accompanied by a concomitant decrease in TREG that results in elevated Th1-TFH:TREG ratio. This finding suggests that an altered balance of TFH cell subsets and TREG is associated with DSA generation after KTx.

ETO family protein Mtg16 regulates the balance of dendritic cell subsets by repressing Id2.

Dendritic cells (DCs) comprise two major subsets, the interferon (IFN)-producing plasmacytoid DCs (pDCs) and antigen-presenting classical DCs (cDCs). The development of pDCs is promoted by E protein transcription factor E2-2, whereas E protein antagonist Id2 is specifically absent from pDCs. Conversely, Id2 is prominently expressed in cDCs and promotes CD8(+) cDC development. The mechanisms that control the balance between E and Id proteins during DC subset specification remain unknown. We found that the loss of Mtg16, a transcriptional cofactor of the ETO protein family, profoundly impaired pDC development and pDC-dependent IFN response. The residual Mtg16-deficient pDCs showed aberrant phenotype, including the expression of myeloid marker CD11b. Conversely, the development of cDC progenitors (pre-DCs) and of CD8(+) cDCs was enhanced. Genome-wide expression and DNA-binding analysis identified Id2 as a direct target of Mtg16. Mtg16-deficient cDC progenitors and pDCs showed aberrant induction of Id2, and the deletion of Id2 facilitated the impaired development of Mtg16-deficient pDCs. Thus, Mtg16 promotes pDC differentiation and restricts cDC development in part by repressing Id2, revealing a cell-intrinsic mechanism that controls subset balance during DC development.

Cancer-associated Autoantibodies as Biomarkers for Early Detection and Prognosis is Cancer: An Update

Spontaneous autoantibody responses against tumor-associated antigens have been detected in all types of cancer analyzed so far. Recent studies have shown that cancer-associated autoantibodies can be found in the serum of patients even several years before the clinical diagnosis. Furthermore, they may at least partially reflect the antigenic profile of cancer and the balance of immune cell subsets in the tumor microenvironment. Therefore, cancer associatedautoantibodies represent attractive biomarkers for the development of non-invasive serological tests for the diagnosis or early detection of cancer, prognosis of survival and prediction of the clinical benefit of immunotherapy. Still, the frequency of responses against each particular antigen is generally low and the autoantibody repertoire is highly heterogeneous and overlapping with that elicited by viral infections and autoimmune disorders, hence precluding the clinical use of single autoantibody biomarkers. This can, however, be overcome by analyzing the autoantibody responses to multiple antigens simultaneously. In this review, we discuss the diagnostic relevance of the recently identified cancer-associated autoantibody signatures and the challenges in the development of clinically applicable biomarker assays. In addition, the putative functional role and the significance of IgG subclasses in the anti-tumor immune response and their prognostic value are discussed. Keywords: Autoantibodies, B cells, biomarkers, early detection of cancer, proteomics, tumor antigens.

Altered CD4+ T cell subset balance in lymphoid organs of mouse model of colorectal cancer (397.2)

The adenomatous polyposis coli (APC) gene is a known tumor suppressor gene, and mutations resulting in APC inactivation leads to the formation of multiple intestinal neoplasms (Min) within mutant mice (APCMin/+). Thus, the APCMin/+ mouse is used as a model of human colorectal cancer (CRC). The intestinal compartment is especially susceptible to tumor formation due the high turnover rate of epithelial cells. Also, the presence of the intestinal microbiota makes the intestines a very immunologically active site. Reports show that tumor associated CD4+ T cell derived IL‐17 promotes intestinal tumor development in APCMin/+ mice. To further investigate the role of the APCMin/+ mutation on CD4+ T cell function, we performed flow cytometric, histochemical, and immunofluorescent studies on both wild type (APC+/+) and APCMin/+ mice. We identified increased levels of IFN‐+ CD4+ and IL‐17A+ CD4+ cells, and decreased levels of IFN‐+IL‐17+ double positive CD4+ cells in the mesenteric lymph nodes and Peyer’s patches of APCMin/+mice. Culture of naïve CD4+ T cells from the spleens of APCMin/+mice lead to increased regulatory T cell development and lower IL‐17A expression. These results suggest an altered T cell balance in APCMin/+ mice. This intrinsic imbalance in CD4+ T cell subsets may disrupt the intestinal immune environment, inhibiting intestinal tumor destruction in the APCMin/+mouse.Grant Funding Source: UAB Comprehensive Cancer Center

Deciphering the Stromal and Hematopoietic Cell Network of the Adventitia from Non-Aneurysmal and Aneurysmal Human Aorta

Aneurysm is associated to a complex remodeling of arteries that affects all their layers. Although events taking place in the intima and the media have received a particular attention, molecular and cellular events taking place in the adventitia have started to be deciphered only recently. In this study, we have precisely described the composition and distribution of stromal and hematopoietic cells in human arterial adventitia, both at steady state and in the setting of aortic aneurysm. Using polychromatic immunofluorescent and flow cytometry analyses, we observed that unlike the medial layer (which comprises mostly macrophages and T cells among leukocytes), the adventitia comprises a much greater variety of leukocytes. We observed an altered balance in macrophages subsets in favor of M2-like macrophages, an increased proliferation of macrophages, a greater number of all stromal cells in aneurysmal aortas. We also confirmed that in this pathological setting, adventitia comprised blood vessels and arterial tertiary lymphoid organs (ATLOs), which contained also M-DC8+ dendritic cells (slanDCs) that could participate in the induction of T-cell responses. Finally, we showed that lymphatic vessels can be detected in aneurysmal adventitia, the functionality of which will have to be evaluated in future studies. All together, these observations provide an integrative outlook of the stromal and hematopoietic cell network of the human adventitia both at steady state and in the context of aneurysm.

Targeting Innate Receptors with MIS416 Reshapes Th Responses and Suppresses CNS Disease in a Mouse Model of Multiple Sclerosis

Modification of the innate immune cell environment has recently been recognized as a viable treatment strategy for reducing autoimmune disease pathology. MIS416 is a microparticulate immune response modifier that targets myeloid cells, activating cytosolic receptors NOD2 and TLR9, and has completed a phase 1b/2a trial for the treatment of secondary progressive multiple sclerosis. Using a mouse model of multiple sclerosis, we are investigating the pathways by which activation of TLR9 and NOD2 may modify the innate immune environment and the subsequent T cell-mediated autoimmune responses. We have found that MIS416 has profound effects on the Th subset balance by depressing antigen-specific Th1, Th17, and Th2 development. These effects coincided with an expansion of specific myeloid subpopulations and increased levels of MIS416-stimulated IFN-γ by splenocytes. Additionally, systemic IFN-γ serum levels were enhanced and correlated strongly with disease reduction, and the protective effect of MIS416 was abrogated in IFN-γ-deficient animals. Finally, treatment of secondary progressive MS patients with MIS416 similarly elevated the levels of IFN-γ and IFN-γ–associated proteins in the serum. Together, these studies demonstrate that administration of MIS416, which targets innate cells, reshapes autoimmune T cell responses and leads to a significant reduction in CNS inflammation and disease.

The Th17/Treg balance is disturbed during aging

Aging is associated with multiple changes in the proliferative and functional abilities of the immune system which are not related to any pathology but consequences in immunosenescence and inflammaging.T helper (TH) 17 cells have been implicated in the development of autoimmune and chronic inflammatory diseases in humans. Additionally, a reciprocal relationship between these pro-inflammatory TH17 and the anti-inflammatory regulatory T cells (Tregs) has been described. Recent studies reported an increase of TH17 cells in aged humans and aged mice, but the role of TH17 cells and their relation to Tregs is poorly understood in human aging. This study investigated the proportion of TH17 (CD4+ IL23 receptor(R)+) cells and Tregs (CD4+ Foxp3+) as well as Interleukin (IL)-17 and IL-10 production in four different age groups from human healthy donors. The data revealed a continual increase of basal CD4+ IL23R+ cell amounts in the different age groups. By analyzing the balance of both T-cell subsets it was observed that, on a basal resting level, TH17 cells were significantly increased in older individuals whereas Tregs were reduced. However, the TH17/Treg ratio decreased age-dependently after stimulation and was accompanied by elevated Foxp3 mRNA and IL-10 protein expressions.In conclusion, changes of the TH17/Treg ratios in combination with altered cytokine expression during aging may contribute to an imbalance between the pro-inflammatory and the anti-inflammatory immune response. This indicates a higher susceptibility to develop inflammatory diseases with increasing age.

Differential expression of the proteome of myeloid dendritic cells in severe aplastic anemia

Severe aplastic anemia (SAA) is a syndrome of severe bone marrow failure with high mortality. Our previous studies have demonstrated that both immature and activated DC1 increased in the bone marrow of SAA patients, and the balance of DC1 subsets shifted the stable form to active one, which might promote Th0 cells to polarize to Th1 cells and cause the over-function of T lymphocytes and hematopoiesis failure in SAA. So we assumed myeloid dendritic cells (mDCs) may be the key immune cells that cause destruction of hematopoietic cells in SAA, but the mechanism of activation of mDCs is unclear. Here, we investigated the proteome of mDCs in SAA patients to further explore the pathogenesis of SAA and the possible antigen that leads to immune activation in SAA. mDCs from 12 SAA patients, 12 remission patients and 12 controls were sorted by flow cytometry and examined by two-dimensional gel electrophoresis and mass spectrometry. Intensity changes of 41 spots were detected with statistical significance. Nine of the 41 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels were found in the SAA group. These changes reveal that abnormal expression of cofilin, glucose-6-phosphate dehydrogenase and pyruvate kinase enzyme M2 in mDCs from SAA patients may be the reason for mDC hyperfunction.

Reduced numbers of regulatory B cells are negatively correlated with disease activity in patients with new-onset rheumatoid arthritis

This study is aimed at determining the numbers of circulating Treg and Breg cells in patients with new-onset rheumatoid arthritis and during subsequent drug therapies. Patients were treated orally with 10 mg methotrexate weekly, and 20 mg leflunomide and 60 mg common threewingnut root daily (Lei Gong Teng) for 12 weeks, but received no steroid therapy. Basal measurements were performed of serum C-reactive protein, anticyclic citrullinated peptide antibody, and erythrocyte sedimentation rate, and the numbers of cluster of differentiation CD4(+)CD25(+)Foxp3(+) T cells, interleukin 10 (IL10)-expressing on CD5(+)CD1d(+) and TIM1(+) B cells. Compared with the healthy controls, patients exhibited significantly less numbers of circulating CD19(+)TIM1(+)IL10(+), CD19(+)CD5(+)CD1d(+)IL10(+) B cells and CD4(+)CD25(+)Foxp3(+) T cells (P < 0.001, all). Drug therapy modulated the balance of different subsets of Breg and Treg cells. The numbers of CD19(+)TIM1(+)IL10(+) and CD19(+)CD5(+)CD1d(+)IL10(+) B cells correlated positively with the numbers of CD4(+)CD25(+)Foxp3(+) T cells in these patients (r = 0.707, P = 0.001; r = 0.481, P = 0.007, respectively). The values of DAS28 were negatively correlated with the numbers of CD19(+)TIM1(+)IL10(+) and CD19(+)CD5(+)CD1d(+)IL10(+) B cells, and CD4(+)CD25(+)Foxp3(+) T cells (r = -0.533, P = 0.023; r = -0.442, P = 0.016; and r = -0.444, P = 0.014, respectively). Of note, TIM1(+) B cells identified more circulating IL10(+) B cells than CD5(+)CD1d(+) B cells. Our data indicate that Breg and Treg cells have a potentially crucial role in controlling disease activity in rheumatoid arthritis patients, and TIM1(+) Breg cells may be a viable therapeutic target for these patients.

Accelerating ordered subsets image reconstruction for X-ray CT using spatially nonuniform optimization transfer.

Statistical image reconstruction algorithms in X-ray computed tomography (CT) provide improved image quality for reduced dose levels but require substantial computation time. Iterative algorithms that converge in few iterations and that are amenable to massive parallelization are favorable in multiprocessor implementations. The separable quadratic surrogate (SQS) algorithm is desirable as it is simple and updates all voxels simultaneously. However, the standard SQS algorithm requires many iterations to converge. This paper proposes an extension of the SQS algorithm that leads to spatially nonuniform updates. The nonuniform (NU) SQS encourages larger step sizes for the voxels that are expected to change more between the current and the final image, accelerating convergence, while the derivation of NU-SQS guarantees monotonic descent. Ordered subsets (OS) algorithms can also accelerate SQS, provided suitable "subset balance" conditions hold. These conditions can fail in 3-D helical cone-beam CT due to incomplete sampling outside the axial region-of-interest (ROI). This paper proposes a modified OS algorithm that is more stable outside the ROI in helical CT. We use CT scans to demonstrate that the proposed NU-OS-SQS algorithm handles the helical geometry better than the conventional OS methods and "converges" in less than half the time of ordinary OS-SQS.

Overexpression of the Mesenchymal Stem Cell Cxcr4 Gene in Irradiated Mice Increases the Homing Capacity of These Cells

The efficiency of the intravascular delivery of mesenchymal stem cells (MSCs) homing to bone marrow has been largely limited. This study aimed to evaluate the homing efficacy in irradiated mice of MSCs that have been engineered to overexpress the murine Cxcr4 gene. Mouse MSCs were infected by a lentivirus vector carrying Cxcr4. MSC migration was detected by an in vitro transwell migration assay. EGFP-positive MSCs were systemically injected into BALB/c mice and detected in bone marrow samples by flow cytometry. The concentration of mouse stromal-derived factor 1 was detected by ELISA. The plasma concentration of the inflammatory cytokines, interleukin (IL)-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70, were determined by cytometric bead array. MSCs that overexpressed Cxcr4 displayed enhanced migration toward a stromal-derived factor 1 gradient. The transplantation of Cxcr4-overexpressing MSCs into irradiated mice leads to increased homing to the bone marrow. Moreover, the frequency of the EGFP-positive cells in a bone marrow infusion 24 h after total body irradiation was 2.2-fold more than at 4 h after irradiation. The concentration of both plasma and bone marrow stromal-derived factor 1 increased after irradiation, and this was positively correlated with the number of Cxcr4-overexpressing MSCs homing to the bone marrow. Moreover, compared with the control groups, the plasma levels of IL-6, IFN-γ, TNF-α, and MCP-1 and IL-12p70 in recipients infused with Cxcr4-overexpressing MSCs was significantly decreased. The level of IL-10 was increased, which correlated with changes in the Th1 and Th2 subset balance. MSCs that overexpressed Cxcr4 and were injected into irradiated mice had an enhanced homing capacity which was related to the bone marrow level of stromal-derived factor 1.

ZBTB7B (Th-POK) Regulates the Development of IL-17–Producing CD1d-Restricted Mouse NKT Cells

CD1d-dependent NKT cells represent a heterogeneous family of effector T cells including CD4(+)CD8(-) and CD4(-)CD8(-) subsets that respond to glycolipid Ags with rapid and potent cytokine production. NKT cell development is regulated by a unique combination of factors, however very little is known about factors that control the development of NKT subsets. In this study, we analyze a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of ZBTB7B and demonstrate that this mutation has dramatic, intrinsic effects on development of NKT cell subsets. Although NKT cell numbers are similar in Zbtb7b mutant mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of ZBTB7B mutant NKT cells in the thymus are retinoic acid-related orphan receptor γt positive, and a high frequency produce IL-17 while very few produce IFN-γ or other cytokines, sharply contrasting the profile of normal NKT cells. Mice heterozygous for the helpless mutation also have reduced numbers of CD4(+) NKT cells and increased production of IL-17 without an increase in CD8(+) cells, suggesting that ZBTB7B acts at multiple stages of NKT cell development. These results reveal ZBTB7B as a critical factor genetically predetermining the balance of effector subsets within the NKT cell population.

Kinetics and Characteristics of Monocyte Subsets in Kidney Transplant Recipients

Although a major contribution of monocyte-macrophage cell lineage to kidney graft rejection and injury has been documented, it is unclear how the monocyte-macrophage related responses develop after kidney transplantation. Macrophages are present in large numbers in acutely rejecting grafts. Monocyte accumulation in peritubular capillaries is one of the main features of acute humoral rejection. The correlates of innate immunity to graft rejection and survival remain to be elucidated. Here, we analyzed the kinetics and characteristics of peripheral blood monocyte subsets after kidney transplantation. By flow cytometry the phenotypic characteristics of classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocytes were measured. To study the activation status, cell surface expression of co-stimulatory molecules CD40 and CD80 and MHC class II molecule HLA-DR was determined. Whole blood analysis was performed in a cohort of 30 healthy individuals, 30 renal transplant recipients at the time of transplantation, 17 recipients at 3 months, and 16 recipients at 6 months after transplantation in a cross-sectional approach. Our data reveal that, as a sign of triggered innate immunity, the percentage of CD14/16+ intermediate-non classical proinflammatory monocytes was significantly increased at the time of transplantation compared to healthy individuals (22.3% ± 2.3 and 15.1% ± 0.9 respectively; p≤0.001). Remarkably, despite potent immunosuppressive drugs, this increase in proinflammatory monocytes, and the concomitant decrease in the classical subset (69.3% ± 3.4 and 74.9% ± 1.5 respectively; p≤0.001) was retained during the post transplant course when the kidney function was recovered. Although the percentage of CD40 and CD80 expressing monocytes and the level of cell surface expression of CD40, CD80 and HLA-DR within the different monocyte subsets did not differ between kidney transplant recipients and healthy individuals, the percentage of HLA-DR expressing monocytes was significantly increased at the time of transplantation. This reached a minimum at 3 months and was followed by a recovery towards normal range after 6 months. In conclusion, in kidney transplant recipients the balance in monocyte subsets is skewed towards intermediate and non-classical subsets. This shift could be one of major cellular drivers of early post-transplant immunity that may determine the outcome of kidney transplant.

Peripheral CD8(+) T cell proliferation is prognostic for patients with advanced thoracic malignancies.

There is a complex interplay between the immune system and a developing tumor that is manifest in the way that the balance of Tcell subsets in the local tumor environment reflects clinical outcome. Tumor infiltration by CD8(+) Tcells and regulatory Tcells (Treg) is associated with improved and reduced survival, respectively, in many cancer types. However, little is known of the prognostic value of immunological parameters measured in peripheral blood. In this study, peripheral CD8(+) T cells and Treg from 43 patients with malignant mesothelioma or advanced non-small-cell lung cancer scheduled to commence palliative chemotherapy were assessed by flow cytometry and evaluated for association with patient survival. Patients had a higher proportion of peripheral Treg, proliferating CD8(+) Tcells and CD8(+) Tcells with an activated effector phenotype compared with age-matched healthy controls. Higher proportions of Treg and proliferating CD8(+) Tcells were both associated with poor survival in univariate analyses (hazard ratio [HR] 3.81, 95% CI 1.69-8.57; p<0.01 and HR 2.86, 95% CI 1.26-6.50; p<0.05, respectively). CD8(+) Tcell proliferation was independently predictive of reduced survival in multivariate analysis (HR 2.58, 95% CI 1.01-6.61; p<0.05). These findings suggest that peripheral CD8(+) T cell proliferation can be a useful prognostic marker in patients with thoracic malignancies planned for palliative chemotherapy.

The use of sequential staining for detection of heterogeneous intracellular response of individual Jurkat cells to lysophosphatidylcholine

Living cells are known to exhibit great morphological, functional, spatial and temporal heterogeneity. Hence, the study of cells in a bulk, whether this bulk is homogenous or heterogeneous, does not provide sufficiently detailed or interpretable results. An advantageous approach would rather be a comprehensive study of cell biological activity in single isolated living cells. In this study, we present an imaging approach for studying pre-apoptotic and very early apoptotic events, during cell death induced by lysophosphatidylcholine (LPC) at the single cell level. The aim of this study is to investigate intracellular events, such as the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) formation, before and immediately after LPC introduction to the lymphocytes at the level of individual cells. A new protocol of sequential staining was developed to study the relation between early apoptosis signs (PS externalization), MMP changes and intracellular ROS production rates at an individual Jurkat cell resolution. Simultaneous kinetic assessments of MMP, intracellular ROS levels and phosphatidylserine (PS) externalization were performed at a single cell resolution, using Optical LiveCell™ Array technology and image analysis. The parameters were measured and analyzed both before and during exposure to inducers in a Jurkat cell population, including three groups of single cells: spontaneous apoptotic cells, induced apoptotic cells and fully functional living cells. Exogenous LPC caused a heterogeneous intracellular response among Jurkat cells immediately after its introduction. Subgroups of cells with opposite changes of MMP and different kinetics of ROS increase, were revealed within the whole cell population. The subset of apoptosis-induced Jurkat cells, which became apoptotic within 3h after the LPC introduction, exhibited higher initial MMP compared to fully functional or spontaneous apoptotic cells. LPC-induced apoptosis was accompanied by a concomitant increase in intracellular ROS levels.In the present study, a method is described to assess the intracellular events in cells which were initially different in their physiological status. The individual T lymphocytes (Jurkat cells) in vitro have various susceptibilities to LPC effects at the very early stage of contact with the inducer. The apoptotic effect of LPC in individual Jurkat cells is associated with a relatively higher initial MMP before the introduction of the inducer and with a faster ROS formation within the affected cells. Such divergence may be significant in regulating the balance of lymphocyte subsets in pathological sites, either maintaining or preventing the inflammation components of atherosclerosis.We conclude that the presented approach provides the researcher, not only with the cell retaining methodology, but with opportunities to observe and find the distinctive cell subsets within the whole cell population as well, thus helping to define more exactly the role and importance of such sub-populations in physiological or pathological conditions.

Immunogenicity and Safety of Seasonal Influenza Vaccination in Patients with Classic Kaposi's Sarcoma

Classic Kaposi's sarcoma (cKS) is a human herpesvirus-8 (HHV-8)-associated lympho-angioproliferative tumor typically occurring in the elderly. It is associated with HHV-8-driven perturbed balance of peripheral B-cell subsets, which may have an impact on immune responses to antigenic stimulation. We took advantage of the common practice of cKS patients to undergo seasonal influenza vaccination because of advanced age and analyzed the immunogenicity and safety of licensed trivalent influenza vaccine in 46 cKS patients and 44 matched controls. Licensure criteria for immunogenicity were fulfilled in both groups. Four weeks after vaccination, hemagglutination-inhibition antibody titers against each viral strain contained in the vaccine increased in patients and controls (all P<0.001). Protection against at least one strain was achieved by 96% of cKS and 91% of control subjects. Protection against all strains persisted after 12 weeks, demonstrating a long-lasting response to vaccination. The vaccine was equally well tolerated by patients and controls, as assessed by evaluating solicited local and systemic reactions to the vaccine, and appearance or increase of antinuclear autoantibodies. HHV-8 virological rebound was observed in four cKS patients, but was not accompanied by progression of KS lesions. We conclude that seasonal influenza vaccine given to cKS patients is immunogenic and safe.

Tolerance and Withdrawal of Immunosuppressive Drugs in Patients Given Kidney and Hematopoietic Cell Transplants

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.