Multiple Lymphocyte Subsets Research Articles

Circulating Immune Landscape Profiling in Psoriasis Vulgaris and Psoriatic Arthritis by Mass Cytometry.

Psoriasis, a systemic disorder mediated by the immune system, can appear on the skin, joints, or both. Individuals with cutaneous psoriasis (PsC) have an elevated risk of developing psoriatic arthritis (PsA) during their lifetime. Despite this known association, the cellular and molecular mechanisms underlying this progression remain unclear. We performed high-dimensional, in-depth immunophenotyping of peripheral blood mononuclear cells (PBMCs) in patients with PsA and psoriasis vulgaris (PsV) by mass cytometry. Blood samples were collected before and after therapy for a longitudinal study. Then three sets of comparisons were made here: active PsA vs. active PsV, untreated PsV vs. treated PsV, and untreated PsA vs. treated PsA. Marked differences were observed in multiple lymphocyte subsets of PsA related to PsV, with expansion of CD4+ T cells, CD16- NK cells, and B cells. Notably, two critical markers, CD28 and CD127, specifically differentiated PsA from PsV. The expression levels of CD28 and CD127 on both Naïve T cells (TN) and central memory CD4+ T cells (TCM) were considerably higher in PsA than PsV. Meanwhile, after treatment, patients with PsV had higher levels of CD28hi CD127hi CD4+ TCM cells, CD28hi CD127hi CD4+ TN cells, and CD16- NK cells. In the circulation of PsA patients, the TN and CD4+ TCM are characterized with more abundant CD28 and CD127, which effectively distinguished PsA from PsV. This may indicate that individuals undergoing PsV could be stratified at high risk of developing PsA based on the circulating levels of CD28 and CD127 on specific cell subsets.

Biomarker Analysis of a Phase Ia/Ib Open-Label, Multicentre Study of Tiragolumab or Tiragolumab + Rituximab in Patients with Relapsed or Refractory (R/R) B-Cell Non-Hodgkin Lymphoma (NHL)

Background: NHL is the most common hematologic malignancy in adults, with follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) being the most common subtypes. Despite therapeutic advances, most patients will experience relapse. New treatments are therefore needed to improve the outcome of patients with R/R NHL. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a well-known immune inhibitory receptor expressed on the surface of activated T cell and natural killer (NK)-cell subsets. TIGIT is expressed at higher levels than other checkpoints in intratumoral T cells in NHL and is highly correlated with PD-1 expression and T cell infiltration. This phase Ia/Ib trial (NCT04045028) evaluated the safety and pharmacokinetics of the anti-TIGIT agent, tiragolumab, alone or in combination with rituximab.Methods: Patients were recruited with histologically confirmed B-cell NHL whose disease has relapsed or failed to respond to ≥2 prior systemic treatment regimens, had ECOG PS 0-1, adequate hematologic and end organ function, and no history of CNS lymphoma. Patients received tiragolumab 600 mg IV Q3W with or without rituximab 375 mg/m 2 IV for the initial dose and 1400 mg SC rituximab/23400 U rHuPH20 QW for 8 doses. Here, we evaluate biomarker data collected from patients with R/R NHL dosed with tiragolumab as a single agent or in combination with rituximab via flow cytometry and IHC.Results: At data cut-off (July 2021), biomarker data had been collected from 14 patients with NHL. Baseline CD8 T cell density within the tumor region evaluated via IHC for these patients was between 500-6000 per mmA 2. In the peripheral blood of the 7 patients dosed with the combination of tiragolumab and rituximab, CD8 T cell expansion observed via absolute counts by flow cytometry was seen in 2 patients. Among the 7 patients, NK/NKT CD25 expression remained unchanged and a modest increase in NK CD69 expression was sustained above baseline in 1 patient. Overall, transient NK cell activation via increased CD69 expression was observed in 2 patients, which would be expected from the addition of rituximab. Increased PD-L1 expression was observed on multiple lymphocyte subsets in 4 of 7 patients in this cohort.Of the 7 patients who received single agent tiragolumab, trends in increased CD69 expression on NKs were observed in 4 patients and NK/NKT CD25 expression in 3 patients. A modest CD8 T cell activation, via increased CD69 expression, was observed in 2 patients, though T cell counts remained unchanged. At baseline, TIGIT was abundantly expressed on peripheral blood CD8 T cells, while co-expression of exhaustion markers on CD8 T cells was less widely observed.Although one patient experienced a sustained response, no other patients achieved clinical benefit. This heavily pretreated 65-year-old female patient with FL had an objective partial response (best overall response), determined via Lugano criteria, with a response duration on single agent tiragolumab for 11 months. The patient had a two-fold upregulated CD69 expression on NKs and sixty-three-fold CD25 upregulated expression on NK/NKTs, as well as increased frequencies of PD-L1+ on immune cells over course of treatment. In this patient, relatively higher TIGIT and lower expression of exhaustion markers on CD8 T cells were observed at baseline and over treatment compared to other patients analyzed.Conclusions: In this study, tiragolumab as a single agent and in combination with rituximab was seen to result in increased PD-L1 expression on multiple lymphocyte subsets (including B cells, CD4/CD8 T cells, and NKs), which support the combination of tiragolumab with PD-L1/PD-1 inhibitors. Increases in NK/NKT CD25 expression could suggest a tiragolumab-mediated increase in proliferative potential but further investigations are needed to confirm. A patient with R/R FL in this study was observed to have the first documented objective response to single agent tiragolumab in this disease indication, suggesting biomarker-driven combination strategies may be important in this population. DisclosuresRuppert: Genentech, Inc.: Current Employment. Cuchelkar: Genentech, Inc.: Current Employment. Meng: Genentech, Inc.: Current Employment. Cho: Genentech, Inc.: Current Employment; F. Hoffmann La Roche, Ltd: Current holder of individual stocks in a privately-held company. Lear: Genentech, Inc.: Current Employment. Wong: Genentech, Inc.: Current Employment. Raval: Genentech, Inc.: Ended employment in the past 24 months; Arcus Bioscience: Current Employment, Current holder of individual stocks in a privately-held company. Nouet: F. Hoffmann La Roche, Ltd: Current Employment.

Microbiome analysis combined with targeted metabolomics reveal immunological anti-tumor activity of icariside I in a melanoma mouse model

Recent studies report that the gut microbiome can enhance systemic and antitumor immunity by modulating responses to antibody immunotherapy in melanoma patients. In this study, we found that icariside I, a novel anti-cancer agent isolated from Epimedium, significantly inhibited B16F10 melanoma growth in vivo through regulation of gut microbiota and host immunity. Oral administration of icariside I improved the microbiota community structure with marked restoration of Lactobacillus spp. and Bifidobacterium spp. abundance in the cecal contents of tumor-bearing mice. We also found that icariside I improves the levels of microbiota-derived metabolites such as short-chain fatty acids (SCFAs) and indole derivatives, consequently promoting repair of the intestinal barrier and reducing systemic inflammation of tumor-bearing mice. Icariside I exhibited strong immunological anti-tumor activity, directly manifested by up-regulation of multiple lymphocyte subsets including CD4+ and CD8+ T cells or NK and NKT cells in peripheral blood of tumor-bearing mice. Collectively, these results suggest that icariside I, via its microbiome remodeling and host immune regulation properties, may be developed as an anticancer drug.

High Dose Intravenous IgG Therapy Modulates Multiple NK Cell and T Cell Functions in Patients With Immune Dysregulation.

Intravenous immunoglobulin (IVIG) is an effective immunomodulatory treatment for immune dysregulation diseases. However, the mechanisms by which it reduces systemic inflammation are not well understood. NK cell cytotoxicity is decreased by IVIG in women with reduced fertility, but IVIG effects on NK cells in immune dysregulation are less clear. We hypothesized that IVIG modulation of lymphocyte function, especially in NK cells, is important for resolution of inflammation. Our aim was to identify IVIG-induced changes in a cohort of patients with Kawasaki disease (KD) and those that occur broadly in pediatric patients with various immune dysregulatory diseases. Peripheral blood mononuclear cells (PBMCs) of patients with KD or autoimmune/inflammatory diseases were phenotyped pre and post high dose IVIG treatment by flow cytometry. In KD patients, after IVIG infusion Treg cell frequency and the proportion of activated CD25+ immunoregulatory CD56bright NK cells was increased, and multiple lymphocyte subsets showed increased expression of the lymphoid tissue homing receptor CD62L. Importantly, IVIG treatment decreased the frequency of cells expressing the degranulation marker CD107a among cytotoxic CD56dim NK cells, which was reflected in a significant reduction in target cell killing and in decreased production of multiple pro-inflammatory mediators. Interestingly, the activating receptor CD336 was expressed on a higher proportion of CD56bright NK cells after IVIG in both KD and autoimmune/inflammatory patients while other NK receptors were increased differentially in each cohort. In autoimmune/inflammatory patients IVIG induced the proliferation marker CD71 on a higher percentage of CD56dim NK cells, and in contrast to KD patients, CD107a+ cells were increased in this subset. Furthermore, when PBMCs were stimulated ex vivo with IL-2 or Candida antigen in autologous plasma, more of the CD4+ T cells of KD patients expressed CD25 after IVIG therapy but fewer cytotoxic T cells were degranulated based on CD107a expression. In summary, IVIG treatment in patients with immune dysregulation has multiple effects, especially on NK cell subsets and CD4+ T cells, which are compatible with promoting resolution of inflammation. These novel findings provide insight into the immunomodulatory actions of IVIG in autoimmune and inflammatory conditions.

Dynamic changes to tissue-resident immunity after MHC-matched and MHC-mismatched solid organ transplantation.

The heterogeneous pool of tissue-resident lymphocytes in solid organs mediates infection responses and supports tissue integrity and repair. Their vital functions in normal physiology suggest an important role in solid organ transplantation; however, their detailed examination in this context has not been performed. Here, we report the fate of multiple lymphocyte subsets, including T, B, and innate lymphoid cells, after murine liver and heart transplantation. In major histocompatibility complex (MHC)-matched transplantation, donor lymphocytes are retained in liver grafts and peripheral lymphoid organs of heart and liver transplant recipients. In MHC-mismatched transplantation, increased infiltration of the graft by recipient cells and depletion of donor lymphocytes occur, which can be prevented by removal of recipient T and B cells. Recipient lymphocytes fail to recreate the native organs' phenotypically diverse tissue-resident lymphocyte composition, even in MHC-matched models. These post-transplant changes may leave grafts vulnerable to infection and impair long-term graft function.

LAG3: a novel immune checkpoint expressed by multiple lymphocyte subsets in diffuse large B-cell lymphoma

Blockade ofthe PD-1 axis has modest efficacy in diffuse large B-cell lymphoma (DLBCL), but data regarding LAG3 are sparse. The impact of LAG3 digital gene expression was tested in 309 patients with DLBCL treated with standard chemoimmunotherapy. Cellular distribution of LAG3 protein was determined by immunohistochemistry and flow cytometry. In tumor-infiltrating lymphocytes (TILs), LAG3 expression was highest on CD4+regulatory T cells (Tregs) and was also highly expressed on CD8+T cells compared with CD4+non-Tregs (bothP= .008). LAG3highTILs were enriched in PD-1 and TIM-3. LAG3 was also expressed on a proportion of malignant B cells, and these patients had significantly higher LAG3 messenger RNA in their biopsies (P= .03). LAG3highgene expression wasassociated with inferior survival in discovery/validation cohorts, independent of cell of origin and the international prognostic index. Patients who were PD-L1highwere fivefold more likely to be LAG3high(P< .0001). Patients who were LAG3high/PD-L1highhad an inferior progression-free survival (P= .011) and overall survival (P= .005) compared with patients who were LAG3low/PD-L1high. Digital spatial protein analysis confirms LAG3 expression on T cells and, surprisingly, tumor-associated macrophages (TAMs) at higher levels than found on CD20+B cells in the tumor microenvironment. LAG3 is frequently expressed on CD4+Tregs and CD8+TILs, typically with other immune checkpoints, and is also present in a proportion of malignant B cells in DLBCL and in areas enriched for TAMs. LAG3highexpression is associated with poor outcome independent of conventional prognosticators.

Could Lymphocyte Profiling be Useful to Diagnose Systemic Autoimmune Diseases?

Considering the implications of B, T, and natural killer (NK) cells in the pathophysiology of systemic autoimmune diseases, the assessment of their distribution in the blood could be helpful for physicians in the complex process of determining a precise diagnosis. In primary Sjögren's syndrome, transitional and active naive B cells are increased and memory B cells are decreased compared to healthy controls and other systemic diseases. However, their utility to improve the accuracy of classification criteria has not been proven. In early untreated rheumatoid arthritis, proportions of regulatory T cells are constantly reduced, but other patterns are difficult to determine given the heterogeneity of published studies. In systemic lupus erythematosus, the lack of studies using large cohorts of patients and the diversity of the possible pathological mechanisms involved are also important impediments. Nevertheless, transitional B cell and plasma cell proportions are increased in most of the studies, the CD4/CD8 ratio is decreased, and the number of NK cells is reduced. Despite the low number of studies, anomalies of lymphocyte subset distribution was also described in ANCA-associated vasculitis, systemic scleroderma, and myositis. For now, flow cytometric analysis of lymphocyte subsets has focused mainly on specific subpopulations and is more useful for basic and translational research than for diagnostics in clinical practice. However, new modern methods such as mass cytometry and bioinformatics analyses may offer the possibility to simultaneously account for the relative proportions of multiple lymphocyte subsets and define a global profile in homogeneous groups of patients. The years to come will certainly incorporate such global lymphocyte profiling in reclassification of systemic autoimmune diseases.

Inosine Acedoben Dimepranol promotes an early and sustained increase in the natural killer cell component of circulating lymphocytes: A clinical trial supporting anti-viral indications

Inosine Acedoben Dimepranol (IAD), licensed for the treatment of cell-mediated immune deficiencies associated with viral infections, has been reported to impact a variety of immune parameters both in vitro and in vivo. Here we report the results from a clinical trial where multiple lymphocyte subsets – CD19+ B cells, CD3+ T cells, CD4+ T-helper cells, FoxP3hi/CD25hi/CD127lo regulatory T cells (Tregs), CD3−/CD56+ NK cells, and CD3+/CD56+ NKT cells – were, together with serum immunoglobulins and IgG subclasses, followed during 14days of IAD administration to ten healthy volunteers; these selected from 27 individuals pre-screened in vitro for their capacity to respond to IAD as gauged by increases in the percentage of Treg and/or NKT cells arising in PHA-stimulated cultures. While a transient spike and dip in Treg and T-helper fractions, respectively, was noted, the outstanding consequence of IAD administration (1g po, qds) was an early and durable rise in NK cells. For half the cohort, NK cells increased as a percentage of total peripheral blood lymphocytes within 1.5h of receiving drug. By Day 5, all but one of the volunteers displayed higher NK cell percentages, such elevation – effectively a doubling or greater – being maintained at termination of study. The IAD-induced populations were as replete in Granzyme A and Perforin as basal NK cells. The novel finding of IAD boosting phenotypically competent NK numbers in healthy individuals supports the drug's indicated benefit in conditions associated with viral infection and reinforces the potential for uplift where immune performance may be compromised.

Changes in peripheral blood lymphocyte subsets during arthritis development in arthralgia patients

BackgroundMultiple lymphocyte subsets like T and B cells have been connected to joint infiltration and inflammation in rheumatoid arthritis (RA). Identification of leucocyte subsets that are dysregulated in arthritis development could provide insight into the aetiology of RA. This study aimed to investigate the composition of the peripheral blood components, i.e. CD14+ monocytes, CD4+ and CD8+ T lymphocytes (CD3+), CD80+, C-X-C chemokine receptor 3 (CXCR3)+ and CD27+ B lymphocytes (CD19+), CD16+CD56+CD3− natural killer (NK) cells and activated CD56+CD3+ T cells, for association with arthritis development in patients with arthralgia.MethodsPeripheral blood was collected from 89 patients with early RA (disease duration <6 months), 37 healthy controls (HC) and 113 patients with arthralgia (22 developed arthritis within ≤1 year, 18 developed arthritis after >1 year and 73 did not develop arthritis). Absolute numbers of monocytes and lymphocyte subsets in whole heparinized blood were determined with flow cytometry using quantification beads in combination with fluorescent labelled antibodies for T cells, B cells, monocytes, NK cells and activated T cells.ResultsIn patients with early RA, significant decreases in numbers of (activated) T cells, CD80+ and memory B cells and a trend towards smaller numbers of CD8+ T cells was observed compared to HC. Similar differences were seen in patients with arthralgia who developed or did not develop arthritis (non-converters), with significantly decreased CD8+ T cells and memory B cells. Patients with arthralgia who developed arthritis were split into groups that developed arthritis within 1 year (early converters) or after 1 year (late converters). Late converters had a significantly decreased number of CD8+ T cells compared to non-converters; early converters had a decreased number of memory B cells. Longitudinal analysis of converters showed a significant relative increase in CD80+ B cells towards the conversion time point compared to 24 months prior to conversion.ConclusionsThis study revealed that patients with arthralgia who develop arthritis demonstrate a change in cellular immune parameters apparent in the periphery, starting with a decrease in cytotoxic T cells 24 months prior to arthritis development, followed by a decrease in the number of memory B cells 12 months prior to disease onset.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1102-2) contains supplementary material, which is available to authorized users.

Apoptotic cell responses in the splenic marginal zone: a paradigm for immunologic reactions to apoptotic antigens with implications for autoimmunity.

Apoptotic cells drive innate regulatory responses that result in tolerogenic immunity. This is a critical aspect of cell physiology as apoptotic cells expose potentially dangerous nuclear antigens on the surface in apoptotic blebs, and failure in their recognition, phagocytosis, or destruction can cause dramatic autoimmunity in experimental models and is linked to development and progression of systemic pathology in human. The marginal zone is a specialized splenic environment that serves as a transitional site from circulation to peripheral lymphoid structures. The marginal zone serves a key role in trapping of particulates and initiation of innate responses against systemic microbial pathogens. However in recent years, it has become clear the marginal zone is also important for initiation of immune tolerance to apoptotic cells, driving a coordinated response involving multiple phagocyte and lymphocyte subsets. Recent reports linking defects in splenic macrophage function to systemic lupus erythematosus in a manner analogous to marginal zone macrophages in lupus-prone mice provide an impetus to better understand the mechanistic basis of the apoptotic cell response in the marginal zone and its general applicability to apoptotic cell-driven tolerance at other tissue sites. In this review, we discuss immune responses to apoptotic cells in the spleen in general and the marginal zone in particular, the relationship of these responses to autoimmune disease, and comparisons to apoptotic cell immunity in humans.

Why the immune system takes its chances with randomness

In their recent Opinion article (Lymphocyte fate specification as a deterministic but highly plastic process. Nature Rev. Immunol. http://dx.doi.org/10.1038/nri3734 (2014))1, Reiner and Adams presented a fascinating deterministic interpretation of how lymphocytes acquire different fates. They propose that the generation of multiple lymphocyte subsets from each precursor occurs via an inevitable developmental pathway. This deduction is based on the premise that the system is too important to be left to stochastic processes. To account for recent evidence to the contrary, stochastic processes are suggested to only appear under conditions in which artificially large numbers of responding precursors might relax the deterministic programme (as used in Refs 2,3) or under in vitro conditions in which the usual three-dimensional (3D) arrangement of externally delivered signals that channel fates is removed (as used in Ref. 4). In other words, stochastic mechanisms only occur when experimental conditions happen to support the role of randomness.

Abstract A88: Identification of factors modulating sensitivity to T cell infiltration in the breast cancer setting via next-generation sequencing.

Abstract Clinical and pre-clinical trials have established that killer (CD8+) T cells can recognize and destroy tumors. The challenge of our time is to address barriers to T cell infiltration into solid tumors and thereby make cancer immunotherapy a powerful, broadly applicable treatment modality. Bilateral breast cancer represents a unique opportunity to identify those proteins that modulate immune cell entry into solid tumors. Bilateral synchronous breast cancer is when tumors arise independently in separate breasts of the same patient at the same time. Similar to the powerful use of “twin studies” in epidemiology, bilateral tumors provide an opportunity to explore two independent tumorigenic events against a common genetic (and immunological) background. We hypothesise that the sensitivity of breast cancers to T cell infiltration can be predicted on the basis of their molecular profiles. To assess this question, a cohort of bilateral and unilateral breast cancer tissues has been assembled in collaboration with CTRNet-affiliated biobanks. Our objective was to identify 10 tumor pairs with differential T cell infiltration. Here we present the novel protocol for multi-colour brightfield immunohistochemistry (IHC) which was used to quantitate multiple lymphocyte subsets in these precious specimens at the same time. A workflow for automated image collection is presented, which is geared to the analysis of whole-tumour sections. Our results demonstrate that bilateral breast tumors often show differential lymphocyte infiltration despite being exposed to the same level of systemic immunity. Paired tumors will be subjected to whole transcriptome sequencing and lymphocyte receptor spectratyping to identify proteins that distinguish infiltrated from non-infiltrated tumors. Thus, patients with bilateral breast cancer have provided a powerful, translationally-relevant opportunity to study local barriers to T cell infiltration in solid tumors. Citation Format: Sally Amos, Ron de Leeuw, Nikita Kuklev, Juzer Kakal, Sindy Babinsky, Katy Milne, Sara Kost, Doug Freeman, Rob Holt, Peter Watson, Brad Nelson. Identification of factors modulating sensitivity to T cell infiltration in the breast cancer setting via next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A88.

Protein Kinase C-θ Clustering at Immunological Synapses Amplifies Effector Responses in NK Cells

In lymphocytes, stimulation of cell surface activating receptors induces the formation of protein microclusters at the plasma membrane that contain the receptor itself, along with other signaling molecules. Although these microclusters are generally thought to be crucial for promoting downstream cellular responses, evidence that specifically links clustering potential to signaling output is lacking. We found that protein kinase C-θ (PKCθ), a key signaling molecule in multiple lymphocyte subsets, formed microclusters in activated NK cells. These microclusters coalesced within the immunological synapse between the NK cell and its target cell. Clustering was mediated by the regulatory region of PKCθ and specifically required a putative phosphotyrosine-binding site within its N-terminal C2 domain. Whereas expression of wild-type PKCθ rescued the cytokine production defect displayed by PKCθ-deficient NK cells, expression of a PKCθ point-mutant incapable of forming microclusters had little to no effect. Hence, PKCθ clustering was necessary for optimal effector function. Notably, only receptors containing ITAMs induced PKCθ microclusters on their own, explaining previous observations that ITAM-coupled receptors promote stronger activating signals and effector responses than do receptors lacking these motifs. Taken together, our results provide a cell biological basis for the role of PKCθ clustering during NK cell activation, and highlight the importance of subcellular compartmentalization for lymphocyte signal transduction.

Low Blood Counts of Memory/Effector CD8 T Cells, Gamma/Delta T Cells, Memory B Cells and Plasmacytoid Dendritic Cells and High Counts of Th2 Cells in Systemic Sclerosis

Abstract 4917 Introduction:Systemic sclerosis (SSc) is presumed to result from aberrant activation of autoreactive T cells. However, the exact pathogenesis of SSc is not known. Patients and Methods:To contribute to the understanding of the immunopathology of systemic sclerosis (SSc), we compared blood counts of multiple lymphocyte subsets between 20 adult SSc patients not treated with immunomodulatory drugs and healthy controls. The patients had to fit entry criteria for SCOT trial (Scleroderma – Cyclophosphamide or Transplantation?, www.sclerodermatrial.org), i.e, 1. symptoms for no longer than 5 years (except for Raynaud’s phenomenon), 2. diffuse scleroderma, and 3. either moderate lung involvement (forced vital capacity (FVC) or diffusion of carbon monoxide (DLCO) between 45 and 70% predicted) or moderate kidney involvement (history of hypertensive renal crisis, but normal renal function at study entry). Multiparameter flow cytometry was used for the determination of the lymphocyte subset counts. Results:Counts of the following subsets were significantly lower in the patients compared to the controls: total T cells (median 1316 vs 2088/ul, p=0.015), total CD8 T cells (273 vs 580/ul, p<0.001), central memory CD8 T cells (23 vs 87/ul, p<0.001), effector memory CD8 T cells (17 vs 39/ul, p=0.015), effector CD8 T cells (28 vs 68/ul, p=0.001), gamma/delta T cells (31 vs 77/ul, p<0.001), switched (IgM/DàIgG/A isotype switched) memory B cells (6 vs 26/ul, p<0.001), non-switched memory B cells (7 vs 17/ul, p=0.004), and plasmacytoid dendritic cells (2 vs 6/ul, p=0.002). Counts of Th2-biased (producing interleukin-4 upon polyclonal stimulation) CD4 as well as CD8 T cells were significantly higher in the patients compared to the controls (248 vs 139/ul for CD4, p=0.002, and 259 vs 164/ul for CD8, p<0.001). Conclusion:Immunopathology of SSc is complex. Low blood counts of memory/effector CD8 T cells, gamma/delta T cells, memory B cells and plasmacytoid dendritic cells and Th2-biased T cells may play a role in the pathogenesis of SSc. However, cause and effect relations need to be established. Given previous reports of increased numbers of CD8 and gamma/delta T cells in the affected tissues of patients with systemic sclerosis and increased numbers of plasmacytoid dendritic cells in the affected tissues of patients with autoimmune diseases (compared to healthy individuals) (Prescott RJ et al: J Pathol 166 (1992) 255–63, Atamas SP et al: Arthritis Rheum 42 (1999) 1168–78, Giacomelli R et al: Arthritis Rheum 41 (1998) 327–34, Yurovski VV et al: J Immunol 153 (1994) 881–91, Nestle FO et al: J Exp Med 202 (2005) 35–43, Farkas L et al: Am J Pathol 159 (2001) 237–43), it is possible that the low blood counts of CD8 T cells, gamma/delta T cells and plasmacytoid dendritic cells result from redistribution of these cells from blood to affected tissues. Disclosures:No relevant conflicts of interest to declare.

Immune Mediators of Rotavirus Antigenemia Clearance in Mice

The immunological mediators that clear rotavirus antigenemia or viremia remain undefined. Immunodeficient mice and antibody transfer were used to test whether lymphocytes or rotavirus-specific serum antibodies are essential for resolving antigenemia. Clearance of antigenemia required lymphocytes, but neither T nor B lymphocytes were absolutely required. Transfer of convalescent-phase or nonneutralizing rotavirus-specific serum antibodies to the systemic compartment of severe-combined-immunodeficient (SCID) mice temporarily suppressed the onset or level of chronic rotavirus antigenemia. Our findings provide the first report demonstrating that clearance of rotavirus antigenemia and possibly viremia are mediated by multiple effector lymphocyte subsets and serum antibodies.

Kinetics of T lymphocyte apoptosis and the cellular immune response in SIVmac239‐infected rhesus macaques

Although increased apoptosis is a central feature of AIDS, little is known about its kinetics or relationship to the early host response in acute HIV/SIV infection. Ex vivo apoptosis in freshly isolated peripheral blood and lymph node lymphocytes was monitored longitudinally in SIVmac239-infected rhesus macaques by flow-cytometric detection of active caspase-3, cleaved poly (ADP-ribose) polymerase, and fragmented DNA. Increased apoptosis of multiple lymphocyte subsets was observed in the first 2 weeks following SIV infection. Apoptosis of CD4+ T lymphocytes was of low magnitude but peaked earlier than other T lymphocyte subsets. A 10- to 36-fold increase in CD8+ T lymphocyte apoptosis coincided temporally with onset of the SIV-specific cellular immune response and enrichment of caspase-3-positive cells within recently proliferating, activated CD8+ T lymphocytes. The virus-specific T lymphocyte response to primary infection and generalized non-specific immune activation contribute to the pathogenesis of apoptosis in acute SIV infection.

Soluble Interleukin (IL)-15Rα Is Generated by Alternative Splicing or Proteolytic Cleavage and Forms Functional Complexes with IL-15

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor alpha (IL-15Ralpha) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15Ralpha (sIL-15Ralpha) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15Ralpha gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon gamma production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15Ralpha ectodomain released by tumor necrosis factor-alpha-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15Ralpha generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

Soluble Interleukin-15 Receptor α (IL-15Rα)-sushi as a Selective and Potent Agonist of IL-15 Action through IL-15Rβ/γ: HYPERAGONIST IL-15·IL-15Rα FUSION PROTEINS

Interleukin-15 (IL-15) is crucial for the generation of multiple lymphocyte subsets (natural killer (NK), NK-T cells, and memory CD8 T cells), and transpresentation of IL-15 by monocytes and dendritic cells has been suggested to be the dominant activating process of these lymphocytes. We have previously shown that a natural soluble form of IL-15R alpha chain corresponding to the entire extracellular domain of IL-15R alpha behaves as a high affinity IL-15 antagonist. In sharp contrast with this finding, we demonstrate in this report that a recombinant, soluble sushi domain of IL-15R alpha, which bears most of the binding affinity for IL-15, behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R beta/gamma heterodimer, whereas it does not affect IL-15 binding and function of the tripartite IL-15R alpha/beta/gamma membrane receptor. Our results suggest that, if naturally produced, such soluble sushi domains might be involved in the IL-15 transpresentation mechanism. Fusion proteins (RLI and ILR), in which IL-15 and IL-15R alpha-sushi are attached by a flexible linker, are even more potent than the combination of IL-15 plus sIL-15R alpha-sushi. After binding to IL-15R beta/gamma, RLI is internalized and induces a biological response very similar to the IL-15 high affinity response. Such hyper-IL-15 fusion proteins appear to constitute potent adjuvants for the expansion of lymphocyte subsets.

Expression of chemokine receptors on intrahepatic and peripheral lymphocytes in chronic hepatitis C infection: its relationship to liver inflammation.

Intrahepatic lymphocytes are believed to be directly involved in the immunopathogenesis of chronic liver diseases. Little is known about the trafficking of lymphocytes into the liver and their role in chronic hepatitis C infection. The expression of 4 chemokine receptors and an activation marker on multiple lymphocyte subsets in paired liver biopsy and peripheral blood specimens from 23 patients with chronic hepatitis C infection were analyzed by a 6-color flow-cytometric assay. CCR5, CXCR3, and CXCR6 were expressed on intrahepatic CD4+ and CD8+ T cells, natural killer (NK) T cells, NK cells, and B cells at significantly higher frequencies than on peripheral lymphocyte subsets. The expression of these receptors and the activation marker CD38 tended to increase with the severity of liver inflammation. This increase was significant for several intrahepatic lymphocytes subsets. Correlations in expression differed among pairs of these extralymphoid homing receptors on the intrahepatic T cells. The homing program for intrahepatic lymphocytes involves multiple extralymphoid chemokine receptors that are regulated by >1 pathway. The expression of homing receptors on intrahepatic lymphocytes is associated with the immunopathogenesis of chronic hepatitis C disease. These preliminary results indicate that confirmational studies with larger sample sizes are warranted.

Different Immunological Requirements for Protection Against Acute versus Persistent Friend Retrovirus Infections

The propensity of retroviruses to rapidly establish persistent infections poses a formidable problem in vaccination strategies. In the current study, we use a live attenuated vaccine to study protection against acute and persistent Friend virus infections in mice. Adoptive transfers of immune CD8+ T cells combined with passive immunizations with virus-neutralizing antibodies increased protection against acute disease compared with either treatment alone, but there was no protection against the establishment of persistent infection. In addition, the protection against acute disease elicited by the combination treatment was dependent on endogenous CD4+ T cells as no protection was achieved in CD4+ T-cell-depleted mice. Quantitative studies showed that doubling the numbers of immune lymphocytes used in adoptive transfer experiments increased protection against acute disease depending on the type of lymphocyte subset used in the transfer. CD8+ T cells were the most potent subset for the transfer of such protection. However, even high numbers of immune CD8+ T cells gave no protection against the establishment of persistent infections. The data indicate that strengthening the numbers of specific immune cell subsets may have a beneficial effect on protection against acute disease, but protection from establishment of persistence requires complex immune responses involving multiple lymphocyte subsets.