T-cell Compartment Research Articles

Identification of Melanoma-reactive CD4+ T-Cell Subsets From Human Melanoma Draining Lymph Nodes.

Our laboratory has previously demonstrated that melanoma draining lymph node (MDLN) samples from stage III patients contained both CD4 and CD8 T cells that can be readily expanded to mediate tumor cell apoptosis in vitro and improve survival in mice bearing human melanoma xenografts. In this study, we investigated whether MDLN T cells contain melanoma-reactive CD4 T-cell compartment and what they are. To test this, we performed multiparametric (11-color and 6-color) fluorescence-activated cell sorting analyses to monitor phenotypic and functional property of CD4 T cells in response to melanoma cell antigen reexposure. Our results have demonstrated that the antigen reexposure could result in a generation of CD4CCR7CD62LCD27 T-cell subsets with various effector cell-like properties. Within the CD4CCR7CD62LCD27 T-cell compartment, in response to antigen reexposure, some of the cells expressed significantly upregulated CD40L and/or CXCR5, and some of them expressed significantly upregulated interleukin-2 and/or tumor necrosis factor-α. This may suggest the existence of melanoma-reactive CD4 "effector-precursor" cells within the expanded MDLN cells and their differentiation into various effector lineages in response to antigen restimulation. Recent clinical trials have demonstrated that effective adoptive cellular immunotherapy maybe enhanced by antigen-specific CD4 T cells. Therefore, results of this study may significantly benefit innovative design of +adoptive cellular immunotherapy that can potentially mediate enhanced and durable clinical responses.

Pro- and Anti-inflammatory Cytokines in Visceral Leishmaniasis

Visceral leishmaniasis is a vector borne disease caused by Leishmania donovani complex. Pro- and anti-inflammatory cytokines play a key role in protecting from the pathogenesis of Leishmania infection, and their balance and dynamic changes may control or predict clinical outcome. Pro-inflammatory cytokines are created primarily to amplify inflammatory reactions; triggers the immune response to Leishmania infection. Even though this cytokine is necessary for a protective response, it may also cause excessive inflammation and collateral tissue damage. For this reason anti-inflammatory cytokines counteract the effects of pro-inflammatory cytokines to limit the inflammation present. However, an excessive down regulation of pro-inflammatory cytokines may favor disease progression. During VL/HIV co-infection, there is a decreased production of macrophage activating cytokines such as IFN-γ, IL-12, IL-15 and IL-18 and increased immunosuppressive cytokines like IL-4, IL-10 and TGF-β. Pro-inflammatory cytokine i.e. IL-6 and TNF-α also have been implicated in the pathogenesis of VL/HIV co-infection by inducing HIV replication. In peoples with VL and/or VL/HIV co-infection microbial translocation into the systemic circulation induces an intense pro-inflammatory response, which in turn activates lymphocyte. Thus, continuous and exaggerated activation causes exhaustion of the T-cell compartment and contributing to immunesuppression. Post kala-azar dermal leishmaniasis may also arise as a complication for VL following treatment. Its immunopathogenesis is not well understood, however, IL-10 is widely accepted as an immunosuppressive cytokine involved in the pathogenesis. Recently, IL-17 has contributed significantly to disease pathogenesis by inducing the production of TNF-α and NO.

CMV seropositivity and T-cell senescence predict increased cardiovascular mortality in octogenarians: results from the Newcastle 85+ study.

Although chronic infection with cytomegalovirus (CMV) is known to drive T lymphocytes toward a senescent phenotype, it remains controversial whether and how CMV can cause coronary heart disease (CHD). To explore whether CMV seropositivity or T-cell populations associated with immunosenescence were informative for adverse cardiovascular outcome in the very old, we prospectively analyzed peripheral blood samples from 751 octogenarians (38% males) from the Newcastle 85+ study for their power to predict survival during a 65-month follow-up (47.3% survival rate). CMV-seropositive participants showed a higher prevalence of CHD (37.7% vs. 26.7%, P = 0.030) compared to CMV-seronegative participants together with lower CD4/CD8 ratio (1.7 vs. 4.1, P < 0.0001) and higher frequencies of senescence-like CD4 memory cells (41.1% vs. 4.5%, P < 0.001) and senescence-like CD8 memory cells (TEMRA, 28.1% vs. 6.7%, P < 0.001). CMV seropositivity was also associated with increased six-year cardiovascular mortality (HR 1.75 [1.09-2.82], P = 0.021) or death from myocardial infarction and stroke (HR 1.89 [107-3.36], P = 0.029). Gender-adjusted multivariate Cox regression analysis revealed that low percentages of senescence-like CD4 T cells (HR 0.48 [0.32-0.72], P < 0.001) and near-senescent (CD27 negative) CD8 T cells (HR 0.60 [0.41-0.88], P = 0.029) reduced the risk of cardiovascular death. For senescence-like CD4, but not near-senescent CD8 T cells, these associations remained robust after additional adjustment for CMV status, comorbidities, and inflammation markers. We conclude that CMV seropositivity is linked to a higher incidence of CHD in octogenarians and that senescence in both the CD4 and CD8 T-cell compartments is a predictor of overall cardiovascular mortality as well as death from myocardial infarction and stroke.

T Cells from Chronic Lymphocytic Leukemia (CLL) Patients Display Dysregulated Expression of Immune Checkpoint Molecules and Activation Markers Mainly Restricted to CD4+ Cells and Correlated with Disease Activity

Abstract Background CLL patients (pts) have impaired humoral and cellular immune functions, which is largely due to profound defects of T-cells. Regulation and activation of T lymphocytes depend not only on T cell receptor signaling but also on co-signaling receptors delivering either inhibitory or stimulatory signals, known as immune checkpoints. CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) is transiently expressed on activated T cells, binding the same ligands as CD28, inhibiting T-cell activation. Similarly, programmed cell death protein 1 (PD-1) is expressed on activated CD4+ and CD8+ T cells inhibiting T-cell functions upon binding to the ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). CD137 is an inducible costimulatory receptor expressed by activated T cells. Dysregulated expression of immune checkpoint receptors on T cells of CLL pts may have an impact on T-cell responsiveness and might be a mechanism for the immune deficiency in the disease. Aim To evaluate the expression of the immune checkpoint molecules CTLA-4, PD-1 and CD137 as well as of the cell proliferation marker Ki67, the activation marker CD69 and of CD103, a marker expressed on regulatory T cells, in T cells from CLL pts in different disease phases. Methods Peripheral blood samples were obtained from 69 CLL pts and 13 healthy control donors (HD). Pts were sub-grouped according to disease phase: indolent vs progressive (i.e. fulfilling criteria for active disease). The expression of CTLA-4, PD-1, PD-L1, CD69, CD103, CD137 and Ki-67 was assessed by flow-cytometry on CD4+ and CD8+ T cells. We also analysed the change in expression of these markers on T cells after 72 hours of PHA stimulation. Results CLL pts (n=17) had a significanty higher percentage of proliferating (Ki67+) CD3+ cells compared to HD (n=7) (median 3.7% in progressive vs 1.7% in indolent CLL vs 0.9% in HD, p=0.004 and p=0.04, respectively) (Fig.1). Progressive CLL pts had a significantly higher percentage Ki67+ CD4+ compared to indolent pts as well as HD (p=0.007 and p=0.001, respectively). Both indolent and progressive pts had higher percentage of Ki67+ CD8+ T cells compared to HD (p=0.01 and p=0.03, respectively). The percentage of CTLA-4+ CD4+ and CTLA-4+ CD8+ cells was low in CLL pts as well as in HD. However, the percentage of PD-1+ CD4+ T cells was significantly higher in progressive (n=32) as compared to indolent (n=35) CLL pts (median 40.3% vs 23.3%, p&lt;0.0001) and HD (n=13) (median 21.5%, p&lt;0.0001) (Fig.2) and correlated positively to the white blood cell counts (WBC) at the time of testing (r=0.29, p=0.03), while no difference was found with regard to the percentage of PD-1+ CD8+ T cells. No difference was observed between CLL pts and HD regarding the expression of PD-L1 on T cells. Both the percentage of CD69+ CD4+ and CD137+ CD4+ T cells were significantly higher in progressive as compared to indolent disease and correlated positively to WBC while no difference was found seen in CD8+ T cells. The percentage of CD103+ T cells was significantly lower in progressive compared to and HD within both the CD4+ (p=0.02) and the CD8+ subpopulations (p=0.02). After 72-hrs of PHA stimulation, PD-1 and CTLA-4 expression increased in CD4+ and CD8+ cells to a similar extent in CLL pts and HD, while PD-L1 increased in HD but not in progressive CLL pts (p=0.03 and p=0.007 for CD4+ and CD8+ cells, respectively). CD69 expression increased to a similar extent in CLL pts and HD, while CD137 expression increased more in T cells from progressive pts compared to HD (p=0.03 and 0.01 for the CD4+ and CD8+ cells, respectively). No increase in CD103 on CD8+ T-cells was observed in CLL pts compared to HD (p=0.04 and p=0.01 for the indolent and progressive pts, respectively). Conclusions Progressive CLL pts have more proliferating (Ki67+) T cells in both the CD4+ and CD8+ compartments compared to HD. CD4+ T-cells in progressive CLL pts display an activated phenotype (CD69+) and express the immune co-stimulatory molecule CD137 at a significantly higher level compared to indolent pts and HD. Nevertheless, the expression of the inhibitory immune checkpoint molecule PD-1 is so high that it is reasonable to assume that these cells are heavily impaired in their immune functions. The differences observed in the expression of immune checkpoints and activation markers between CLL pts in different phases of the disease suggest that major changes occur in the CD4+ T-cell compartment during disease progression. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Hansson: Jansse Cilag: Research Funding. Österborg:Janssen, Pharmacyclics, Gilead: Consultancy, Research Funding; Novartis: Research Funding.

Immunological Derangement in Follicular Lymphoma Patients Developing Severe Side Effects during Treatment with PI3K Inhibitors

IntroductionPhosphoinositide-3-kinases (PI3Ks) are pivotal in various cellular functions including proliferation and survival, cell differentiation, intracellular trafficking and immunity. The PI3Kδ isoform is highly expressed in cells of hematopoietic origin, and is often dysregulated in various hematologic malignancies.New drugs targeting PI3Ks have emerged as promising treatment options for patients (pts) with CLL and indolent NHL, but a clear understanding of their effects on pts' immunity is lacking. Herein we performed detailed immunological analysis in two follicular lymphoma (FL) pts treated with PI3K inhibitors and developing severe immune-mediated side effects.Patients and MethodsTwo of 9 pts with relapsed FL treated with PI3Kδ (pt1) and PI3Kα/δ (pt2) inhibitors developed severe adverse events (SAE). Both pts were heavily pretreated including autologous transplantation and discontinued therapy due to SAE. Pt1 showed grade 3 transaminase elevation, esophagitis and diarrhea requiring hospitalization after 3 cycles of therapy. Diarrhea worsened with fever and weight loss, in spite of transient discontinuation and dose reduction of PI3k inhibitor. Colonoscopic biopsy showed GVHD-like colitis. He died 3 months later. Pt2 has developed, after the first cycle and over a period of one year, febrile neutropenia, erythroderma, ulcerative keratitis requiring corneal transplant, pure red cell aplasia, endocrine abnormalities, grade 3 diarrhea with weight loss, and inflammatory bowel disease-like colitis.Detailed T-cell subpopulations immunophenotyping and T-cell receptor (TCR) spectratyping were performed in samples obtained at 6 months and 1 year after the start of treatment.ResultsCompared to healthy controls (HC) both pts showed marked abnormalities in T-cell subsets including a very low percentage of CD4+CD45RA+CCR7+CD31+ recent T-emigrants (RTE) and of CD4+CD45RA+CCR7+ naïve T cells in both CD4+ and CD8+ populations; CD4+CD45RA-CCR7+ central memory were significantly decreased, while CD4+CD45RA-CCR7- effector memory T cells were increased. Natural CD4+CD25+CD127low/- regulatory T cell (Treg) were low in both pts and the defect equally involved naïve, central and effector memory Treg. On the contrary, inducible Treg (Tr1) did not differ between FL pts and HC.The predominance of effector T cells strongly biased the TCR repertoire. Cells expressing all TCR beta variable (BV) chains were clonally or oligoclonally expanded at the spectratyping analysis because their profiles deviated from the Gaussian patterns found in polyclonal repertoires of HC.DiscussionThe clinical picture of the two FL pts was reminiscent of congenital immunodeficiencies with defects of Treg activity (e.g. IPEX caused by FoxP3 gene mutation). Similar symptoms and T-cell abnormalities are also present in pts with inadequate thymic function and immunologic reconstitution due to severe chronic GVHD. The associated T-cell immunological derangement was characterized by: 1) a marked reduction of the thymic output; 2) a clear defect in natural Treg; 3) an increase of peripheral oligoclonal effector memory T cells. Preclinical data on the role of PI3kδ support its possible pathogenetic involvement: PI3Kδ is known to play critical roles during thymopoiesis and its deletion/inactivation results in a block at the CD4 CD8 double negative stage of T-cell development. In mice with a mutation of PI3Kδ subunit, Treg were increased in the thymus but decreased in all peripheral immune organs, suggesting an impaired emigration of these cells from the thymus. Moreover, mice expressing an inactive form of PI3Kδ develop inflammatory bowel disease.These preliminary data suggest that small molecule-mediated inhibition of PI3Kδ may cause severe alterations within the T-cell compartment in selected pts, which may derive from a defect in the thymic output, including that of Treg. The generation of mature, oligoclonal T cells can be either the result of an homeostatic T-cell proliferation or of a strong (auto)antigenic stimulation. To better elucidate such mechanisms, further studies are in progress on a larger number of pts showing different levels of PI3k inhibitor toxicity. Nevertheless, we suggest that a close follow-up of lymphocyte subpopulation changes should be worthwhile since it may anticipate the onset of these severe clinical manifestations. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impaired Response to Influenza Vaccination in AML Patients Post-Chemotherapy Associated with a Highly Atypical B-Cell Profile

The immunotherapy of cancer is arguably the most promising therapy under development, and vaccination against cancer antigens is a promising strategy to stimulate adaptive immune responses against malignant clones. However, the ability of patients treated with chemotherapy to respond appropriately to immune challenge may be suboptimal. This study was designed to determine the functional capacity of the immune system in adult acute myeloid leukemia (AML) patients who have completed chemotherapy and are potential candidates for immunotherapy. We used the response to influenza vaccination as a surrogate for the health of the immune system in AML patients in a complete remission (CR) post-chemotherapy.Ten adult AML patients in CR after completion of intensive chemotherapy were recruited to the clinical protocol J1293. They were on average 37 weeks post-treatment (range 4-148) when receiving the 2012-2013 inactivated seasonal influenza vaccine. Peripheral blood samples were collected at baseline and 30 days post-vaccination. Ten age and sex matched healthy donors (HD) served as baseline controls. Serological response to vaccination was assessed via microneutralization assays; multi-parameter flow cytometry was used to characterize lymphocyte subsets. ELISPOT assays were used to evaluate lymphocyte function, microarrays were used to assess gene expression, and deep sequencing of the B-cell receptor heavy chain (IGH) was performed to determine expansion and clonality of B-cells. Wilcoxon rank-sum tests were used to assess statistical significance.Only 2/10 (20%) patients seroconverted (AML responders, or AML-R) with a four-fold increase or greater in influenza-specific antibody. One responder was 148 weeks post-treatment; the other had acute promyelocytic leukemia (APL) and was 4 weeks post-treatment. Deep immunophenotyping revealed no striking differences in T-cell compartments between AML at baseline and HD, indicating rapid T-cell recovery after chemotherapy. In contrast, we observed a highly atypical B-cell profile. AML non-responders (AML-NR) at day 0 versus HD had significantly reduced frequencies of mature IgA+ (4.5% vs. 11%) and IgG+ (2.4% vs. 5.9%) B-cells (as a percentage of CD19+CD20+). Further dissection exposed markedly higher frequencies of CD10+CD27- transitional B-cells (36% vs. 16%, p<0.005) and dramatically fewer memory (resting, tissue-like, activated) B-cells (5% vs. 25%, p<0.005) (as a percentage of CD19+CD20+) in AML-NR at day 0 versus HD. There were no significant changes in any B-cell population at day 30 over baseline in any patient. Examining frequencies of transitional, naive, and memory B-cells in AML patients at day 0 when ranked by time since treatment showed a decrease in transitional B-cells with a corresponding increase in naive B-cells over time but no concurrent increase in memory B-cell frequencies (Fig 1). These data suggest B-cell deficiencies of several types: loss of the memory B-cell compartment due to chemotherapy, a subsequent excess of transitional B-cells, and a lack of naive B-cell development into specific, class-switched effectors of the antibody response, which together likely lead to humoral immune incompetence.Interestingly, functional T-cell assays revealed that of 5 evaluable patients, including the 2 AML-R and 3 AML-NR, 5/5 (100%) patients had an increase in influenza-specific cytokine production (1.24 - 4.40x higher on day 30 over baseline), suggesting a functional T-cell response even with deficient influenza-specific antibody production. Supervised clustering of microarray data identified many upregulated genes in AML-NR related to apoptosis, BCR, IL2, IL-4, IL-8, and IL-12 signaling pathways, indicative of developing B-cells. IGH sequencing demonstrated AML-NR had greater variability in CDR3 length than seen in HD, consistent with an antigen inexperienced B-cell repertoire.These data suggest that while some aspects of cellular immunity recover comparatively quickly, the humoral immune system is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. Abnormal frequencies of transitional and memory B-cells may explain the poor response to vaccination often seen in patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immune capacity observed here might have implications for the success of immunotherapies based on vaccination. [Display omitted] DisclosuresNoonan:Celgene: Speakers Bureau. Borrello:Celgene: Research Funding.

Ex Vivo Modulation of Donor Cells Results in Enhanced Survival and Reduced Gvhd Mortality

Allogeneic hematopoietic stem cell transplant (HSCT) represents a potential curative treatment for a number of life-threatening blood malignancies. The utility of this treatment regimen, however, is limited by a number of serious complications including graft versus host disease, which occurs in approximately half of all transplant patients. Standard-of-care for treating acute GvHD has remained unchanged for several decades and consists of high doses of steroids, which are only effective in approximately 35 percent of the cases. Therefore, the reduction of GvHD represents a large unmet medical need, and new approaches are needed to effectively attenuate GvHD. Here we present a fundamentally novel strategy for potentially reducing GVHD - by modulating donor mobilized peripheral blood cells with small molecules prior to HSCT, a programmed mobilized peripheral blood (mPB) allogeneic graft, with reduced T-cell alloreactivity, can be administered as the hematopoietic cell source for HSCT.To this end, we applied our screening platform to identify a combination of small molecule modulators (FT1050, FT4145) that promote the activation of genes implicated in cell cycle, immune tolerance and anti-viral properties of T cells, as well as in the survival, proliferation and engraftment potential of CD34+ cells. Genome-wide expression analysis of the T-cell compartment of mobilized peripheral blood following treatment with FT1050+FT4145 revealed the induction of genes involved in cell cycle (e.g., CCND1, CCNE1), immune tolerance (e.g., ALDH, AREG) and anti-viral properties (e.g., EFNB2). To further assess the therapeutic impact of ex vivo programming with FT1050 and FT4145, a number of T cell assays to assess T cell phenotype and function were conducted on mPB. Overall, ex vivo programming of mPB resulted in reduced allogeneic T cell responses and was accompanied by reduced capacity of modulated T cells to produce Interferon Gamma (IFN-ɣ). Concomitantly, the ability of the modulated T cells to make Interleukin 4 (IL-4) and 10 (IL-10) was enhanced, suggesting a polarization of these cells towards a less inflammatory functional state. This was further evidenced by increased surface expression of an immune-inhibitory molecule, PD1, and reduced expression of the activation markers 41BB and ICOS.We next examined the potential beneficial role of ex vivo programming with FT1050+FT4145 in a major histocompatibility complex (MHC) mis-matched HSCT mouse model. Briefly, lethally irradiated BALB/c mice received bone marrow and splenocytes from C57BL/6 donor mice pulse treated with vehicle or FT1050+FT4145. Significantly less GvHD, as determined by survival, weight loss, GVHD score (diarrhea, inactivity, hunched posture, ruffled fur, eye lesion, snout swelling/skin integrity), cytokine production and histopathology of GvHD target organs was observed in recipients receiving FT1050+FT4145 treated cells as compared to those receiving vehicle treated cells. In addition, we observed increased levels of donor T regulatory cells (Tregs) in secondary lymphoid organs concomitant with decreased levels of circulating IFN-ɣ in recipients receiving FT1050+FT4145 treated cells. Based on the attenuation of alloreactive T-cell responses in these preclinical studies, we believe our findings provide a compelling scientific basis to support the clinical evaluation of ex vivo programmed mobilized peripheral blood in patients undergoing HSCT for the treatment of hematologic malignancies. DisclosuresLevin:Fate Therapeutics, Inc: Employment, Equity Ownership. Shoemaker:Fate Therapeutics Inc: Employment.

Treatment of Post- HSCT Immunodeficiency By Infusion of Ex Vivo- Generated T Cell Precursors from Adult and Cord Blood Hematopoietic Stem and Progenitor Cells

Non-HLA identical hematopoietic stem cell transplantation (HSCT) provides a corrective therapy for most life-threatening primary immunodeficiencies (PID) and some malignant hemopathies. Despite advances made, severe complications following the treatment such as the prolonged persistence of T cell immunodeficiency still limit the use of this partially incompatible HSCT. After HSCT, the reconstitution of a functional T cell compartment relies on the availability of T cell precursors to rapidly seed the thymus and differentiate into mature T cells.We have previously demonstrated that an in vitro culture system based on the use of a modified Delta-like-4 (DLL4) Notch ligand and T cell cytokines allows for the effective generation of human T cell precursors from cord blood within 7 days. Moreover, once injected into NOD/SCID/gcko mice, T cell precursors generated in this system were able to colonize the thymus and generate a diversified and functional T-cell compartment. Here, we aimed at testing the capacity of adult HSPCs in this reconstitution system. We found that, like their CB- derived counterparts, T cell precursors generated from adult HPSCs phenotypically resembled thymic CD34+CD7+ cells with high in vitro T-cell differentiation potential. Interestingly, the peak of T cell progenitors for adult HSPCs occurred around day 3, compared to day 7 in CB. At this timepoint, T cell precursors derived from adult HSPC already expressed all critical genes for T cell lineage development, as well as the major chemokine receptors implicated in thymus homing. The introduction of retronectin further improved differentiation and proliferation of T cell progenitors from both HPSC sources in our in vitro system. Comparative molecular analysis of adult- and CB- derived progenitors suggested, that differential requirements for Notch receptor/ligand interactions may explain the differences in kinetics observed during the culture of the two types of HSPC. It remains to be further evaluated, whether targeted modifications of the Notch signaling pathway can improve the outcome of this in vitro T cell differentiation system for adult HPSCs.Overall our results suggest that adult HSPCs, like their CB- derived counterparts, provide an effective source of in vitro cultured T cell progenitors harboring all the necessary requirements for the in vivo -reconstitution of a functional T cell compartment. This is particularly important in the context of future clinical applications in HSCT where adult HSPCs are more available and more frequently used than CB HSPCs. Based on our results, we propose that upon injection into a patient, DLL4- cultured T cell precursors from both HSPC sources could significantly accelerate the reconstitution of the adaptive immune system after a partially HLA-incompatible HSCT. Currently, we are translating these results into a phase I clinical trial including adult and pediatric patients transplanted for malignant hemopathies or PIDs requiring an allogeneic HSCT from a HLA-partially mismatched donors. DisclosuresNo relevant conflicts of interest to declare.

B Cell Follicle Sanctuary Permits Persistent Productive Simian Immunodeficiency Virus Infection in Elite Controllers

Y Fukazawa, R Lum, AA Okoye. Nat Med. 2015;21:132–140 A small percentage of HIV-infected patients (elite controllers, EC) appear to be capable of controlling HIV replication, and this is most likely mediated by CD8+ cytotoxic T cells. T follicular helper cells (TFH) are antigen-specific CD3+ CD4+ “helper” T cells that are localized to B-cell follicles of peripheral lymphoid tissue, such as lymph nodes. They are characterized by specific surface antigen expression (PD-1, CXCR5, CD200, ICOS), a specific transcription factor (Bcl6), and specific functions (generation and maturation of antibody responses). In 2012, Perreau et al reported that TFH cells serve as the major CD4+ T-cell compartment for …

Characterization of γδ T-cells via flow cytometry.

Stervbo et al. report on the influences of ageing on a variety of peripheral innate and adaptive immune populations in two publications in the October issue of this journal (Stervbo et al. 2015a; Stervbo et al. 2015b). These articles surveyed peripheral blood mononuclear cell phenotypes from donors recruited in the cross-sectional Protective Immunity in Ageing (PRIMAGE) study. The focus of that study was the investigation of fundamental immunologic questions of the natural ageing process using seasonal influenza vaccination as a model (PRIMAGE 2015). Both papers also nicely combined phenotypic and functional assessment of age-related differences in major compartments of the cellular immune system in peripheral blood. Unlike many earlier studies, Stervbo et al. also focused on the underinvestigated γδ T cell compartment (Stervbo et al. 2015a). This is valuable because there are very few data on the impact of age on this conserved minor T cell subset. It is therefore especially important to be certain of the exactitude of the published results. However, we would like to sound a note of caution in this regard due to the use of the pan-γδ antibody clone B1, which may not be able to identify every γδ T cell when used in complex antibody panels applied in polychromatic flow cytometry. These issues were addressed by us in a carefully reviewed and validated antibody panel recently published in the journal Cytometry Part A’s “OMIP” series of standardized antibody panels (Wistuba-Hamprecht et al. 2014). A possible explanation could be steric hindrance effects because the epitopes recognized by anti-CD3 antibodies and the B1-clone might be in close proximity, which is not the case for the 11F2-clone that for example reliably detects the γδ T-cell compartment. In parallel to publication of these findings, we informed the manufacturers and one of the largest companies immediately revised their technical data sheet in accordance with these results (Becton Dickinson 2015). It might therefore be problematic to perform subset analysis on the basis of a B1-positive cell population. We felt that readers of Stervbo et al. 2015a, 2015b should be aware of these potential limitations.

Peripheral blood T-cell signatures from high-resolution immune phenotyping of γδ and αβ T-cells in younger and older subjects in the Berlin Aging Study II.

BackgroundAging and latent infection with Cytomegalovirus (CMV) are thought to be major factors driving the immune system towards immunosenescence, primarily characterized by reduced amounts of naïve T-cells and increased memory T-cells, potentially associated with higher morbidity and mortality. The composition of both major compartments, γδ as well as αβ T-cells, is altered by age and CMV, but detailed knowledge of changes to the γδ subset is currently limited.ResultsHere, we have surveyed a population of 73 younger (23–35 years) and 144 older (62–85 years) individuals drawn from the Berlin Aging Study II, investigating the distribution of detailed differentiation phenotypes of both γδ and αβ T-cells. Correlation of frequencies and absolute counts of the identified phenotypes with age and the presence of CMV revealed a lower abundance of Vδ2-positive and a higher amount of Vδ1-positive cells. We found higher frequencies of late-differentiated and lower frequencies of early-differentiated cells in the Vδ1+ and Vδ1-Vδ2-, but not in the Vδ2+ populations in elderly CMV-seropositive individuals confirming the association of these Vδ2-negative cells with CMV-immunosurveillance. We identified the highest Vδ1:Vδ2 ratios in the CMV-seropositive elderly. The observed increased CD4:CD8 ratios in the elderly were significantly lower in CMV-seropositive individuals, who also possessed a lower naïve and a larger late-differentiated compartment of CD8+ αβ T-cells, reflecting the consensus in the literature.ConclusionsOur findings illustrate in detail the strong influence of CMV on the abundance and differentiation pattern of γδ T-cells as well as αβ T-cells in older and younger people. Mechanisms responsible for the phenotypic alterations in the γδ T-cell compartment, associated both with the presence of CMV and with age require further clarification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0052-x) contains supplementary material, which is available to authorized users.

Temporal fate mapping reveals age-linked heterogeneity in naive T lymphocytes in mice

Understanding how our T-cell compartments are maintained requires knowledge of their population dynamics, which are typically quantified over days to weeks using the administration of labels incorporated into the DNA of dividing cells. These studies present snapshots of homeostatic dynamics and have suggested that lymphocyte populations are heterogeneous with respect to rates of division and/or death, although resolving the details of such heterogeneity is problematic. Here we present a method of studying the population dynamics of T cells in mice over timescales of months to years that reveals heterogeneity in rates of division and death with respect to the age of the host at the time of thymic export. We use the transplant conditioning drug busulfan to ablate hematopoetic stem cells in young mice but leave the peripheral lymphocyte compartments intact. Following their reconstitution with congenically labeled (donor) bone marrow, we followed the dilution of peripheral host T cells by donor-derived lymphocytes for a year after treatment. Describing these kinetics with mathematical models, we estimate rates of thymic production, division and death of naive CD4 and CD8 T cells. Population-averaged estimates of mean lifetimes are consistent with earlier studies, but we find the strongest support for a model in which both naive T-cell pools contain kinetically distinct subpopulations of older host-derived cells with self-renewing capacity that are resistant to displacement by naive donor lymphocytes. We speculate that these incumbent cells are conditioned or selected for increased fitness through homeostatic expansion into the lymphopenic neonatal environment.

Long noncoding RNAs in T lymphocytes.

Long noncoding RNAs are recently discovered regulatory RNA molecules that do not code for proteins but influence a vast array of biologic processes. In vertebrates, the number of long noncoding RNA genes is thought to greatly exceed the number of protein-coding genes. It is also thought that long noncoding RNAs drive the biologic complexity observed in vertebrates compared with that in invertebrates. Evidence of this complexity has been found in the T-lymphocyte compartment of the adaptive immune system. In the present review, we describe our current level of understanding of the expression of specific long or large intergenic or intervening long noncoding RNAs during T-lymphocyte development in the thymus and differentiation in the periphery and highlight the mechanisms of action that specific long noncoding RNAs employ to regulate T-lymphocyte function, both in vitro and in vivo.

Reduced naïve CD8(+) T-cell priming efficacy in elderly adults.

SummaryAging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8+ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8+ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8+ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8+ T‐cell responses specific for a model antigen. Reduced CD8+ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8+ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.

Memory re-differentiation and reduced lymphocyte activation in chronic HCV-infected patients receiving direct-acting antivirals.

Recently, the treatment of HCV has advanced significantly due to the introduction of direct-acting antivirals (DAAs). Studies using interferon (IFN)-containing regimens failed to consistently show restoration of immunologic responses. Therefore, IFN-free DAA formulations provide a unique opportunity to dissect the immunologic effect of HCV cure. This study investigates the restoration of the immune compartment as a consequence of rapid viral clearance in patients successfully treated with DAAs and in the absence of IFN and ribavirin. Here, we evaluate the immunologic changes that occurred following DAA-mediated HCV cure. Peripheral blood from nineteen previously treatment-naïve patients with chronic HCV genotype 1a/1b who received an IFN and ribavirin-free regimen of daclatasvir, asunaprevir and BMS-791325 was evaluated. Immune reconstitution occurs in patients in whom HCV was successfully eradicated via DAA therapy. Restoration of the CD4(+) T-cell compartment in the peripheral blood and a re-differentiation of the T lymphocyte memory compartment resulted in a more effector memory cell population and a reduction in expression in the co-inhibitory molecule TIGIT in bulk T lymphocytes. Furthermore, we observed a partial reversal of the exhausted phenotype in HCV-specific CD8(+) T cells and a dampening of the activation state in peripheral NK cells. Collectively, our data provide the groundwork for dissecting the effect of DAA therapy on the immune system and identifying novel mechanisms by which chronic HCV infection exerts immunosuppressive effects on T cells through the recently described co-inhibitory molecule TIGIT.

Abstract A86: Harnessing T-cell homeostasis to diagnose and treat solid and liquid tumors

Abstract T-cell populations are subject to homeostatic control from cytokines and microenvironmental signaling. Disruption of homeostasis can cause changes to the dynamics of the system that have implications for the progression of cancer. Here we present two mathematical models that examine the progression of tumors in the context of T-cell homeostasis and provide therapeutic decision support in the clinic. Model 1: During a chronic disease such as cancer, T cells often become tolerant to the antigens presented by the disease. This tolerant state effectively limits the response of the immune system to the tumor. Experimental evidence has shown that depletion of T-cells can lead to a loss of T-cell tolerance. During the homeostatic phase of T-cell compartment repopulation, there is a temporary window of opportunity during which T cells lose their tolerant state, allowing them to respond to tumor antigens. In addition, clonal expansion of the tumor-specific T-cell clone may be enhanced during the regrowth phase due to increased stimulation. We use an ordinary differential equation (ODE) model to explore the effect of T-cell depletion and homeostatic repopulation on the loss of tolerance in the T-cell compartment and subsequent effectiveness of immune-mediated tumor cytotoxicity. The model predicts different outcomes for the tumor and T-cell compartment, dependent on the strength and schedule of the depletion therapy. The optimal regimen can lead to tumor control in some cases, but T-cell exhaustion is also common dynamic predicted by the model. By understanding the effects of T-cell depletion, immune depleting therapies can be optimized to enhance immune potential. Model 2: Large Granular Lymphocytic Leukemia (LGLL) is a T-cell lymphoproliferative disorder that exhibits clonal expansion of a subset of T cells. Since there are no clinical biomarkers to predict the aggressiveness of the disease, treatment decisions are often made on a watch and wait approach. Using a set of ODEs, we develop a model of LGLL that uses clinical patient data from diagnosis to predict the timeframe for progression of the disease. Our experimental results have suggested that the disease is caused by a change in sensitivity to both positive and negative regulators of T-cell homeostasis. The model incorporates these cell-specific mechanisms to investigate their effect when placed in a homeostatic setting. The level of dysregulation as measured from patient-specific data determines the rate of outgrowth of the diseased T-cell clone, and therefore serve as a useful predictive tool for managing treatment decisions in the clinic. Citation Format: Mark Robertson-Tessi, Derek Park, Kimberly Luddy, Adam Mailloux, Pearlie K. Burnette, Alexander Anderson. Harnessing T-cell homeostasis to diagnose and treat solid and liquid tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A86.

Poor functional immune recovery in aged HIV-1-infected patients following successfully treatment with antiretroviral therapy

Aging is now a well-recognized characteristic of the HIV-infected population and both AIDS and aging are characterized by a deficiency of the T-cell compartment. The objective of the present study was to evaluate the impact of antiretroviral (ARV) therapy in recovering functional response of T cells to both HIV-1-specific ENV peptides (ENV) and tetanus toxoid (TT), in young and aged AIDS patients who responded to ARV therapy by controlling virus replication and elevating CD4+ T cell counts. Here, we observed that proliferative response of T-cells to either HIV-1-specific Env peptides or tetanus toxoid (TT) was significantly lower in older antiretroviral (ARV)-treated patients. With regard to cytokine profile, lower levels of IFN-γ, IL-17 and IL-21, associated with elevated IL-10 release, were produced by Env- or TT-stimulated T-cells from older patients. The IL-10 neutralization by anti-IL-10 mAb did not elevate IFN-γ and IL-21 release in older patients. Finally, even after a booster dose of TT, reduced anti-TT IgG titers were quantified in older AIDS patients and it was related to both lower IL-21 and IFN-γ production and reduced frequency of central memory T-cells. Our results reveal that ARV therapy, despite the adequate recovery of CD4+ T cell counts and suppression of viremia, was less efficient in recovering adequate immune response in older AIDS patients.

Age-Associated Differences in MiRNA Signatures Are Restricted to CD45RO Negative T Cells and Are Associated with Changes in the Cellular Composition, Activation and Cellular Ageing.

MicroRNAs (miRNAs) have emerged as important players in the regulation of T-cell functionality. However, comprehensive insight into the extent of age-related miRNA changes in T cells is lacking. We established miRNA expression patterns of CD45RO- naïve and CD45RO+ memory T-cell subsets isolated from peripheral blood cells from young and elderly individuals. Unsupervised clustering of the miRNA expression data revealed an age-related clustering in the CD45RO- T cells, while CD45RO+ T cells clustered based on expression of CD4 and CD8. Seventeen miRNAs showed an at least 2-fold up- or downregulation in CD45RO- T cells obtained from young as compared to old donors. Validation on the same and independent samples revealed a statistically significant age-related upregulation of miR-21, miR-223 and miR-15a. In a T-cell subset analysis focusing on known age-related phenotypic changes, we showed significantly higher miR-21 and miR-223 levels in CD8+CD45RO-CCR7- TEMRA compared to CD45RO-CCR7+ TNAIVE-cells. Moreover, miR-21 but not miR-223 levels were significantly increased in CD45RO-CD31- post-thymic TNAIVE cells as compared to thymic CD45RO-CD31+ TNAIVE cells. Upon activation of CD45RO- TNAIVE cells we observed a significant induction of miR-21 especially in CD4+ T cells, while miR-223 levels significantly decreased only in CD4+ T cells. Besides composition and activation-induced changes, we showed a borderline significant increase in miR-21 levels upon an increasing number of population doublings in CD4+ T-cell clones. Together, our results show that ageing related changes in miRNA expression are dominant in the CD45RO- T-cell compartment. The differential expression patterns can be explained by age related changes in T-cell composition, i.e. accumulation of CD8+ TEMRA and CD4+ post-thymic expanded CD31- T cells and by cellular ageing, as demonstrated in a longitudinal clonal culture model.

Induction of Regulatory T Cells by Intravenous Immunoglobulin: A Bridge between Adaptive and Innate Immunity.

Intravenous immunoglobulin (IVIg) is a polyclonal immunoglobulin G preparation with potent immunomodulatory properties. The mode of action of IVIg has been investigated in multiple disease states, with various mechanisms described to account for its benefits. Recent data indicate that IVIg increases both the number and the suppressive capacity of regulatory T cells, a subpopulation of T cells that are essential for immune homeostasis. IVIg alters dendritic cell function, cytokine and chemokine networks, and T lymphocytes, leading to development of regulatory T cells. The ability of IVIg to influence Treg induction has been shown both in animal models and in human diseases. In this review, we discuss data on the potential mechanisms contributing to the interaction between IVIg and the regulatory T-cell compartment.

Transgenic mice overexpressing arginase 1 in monocytic cell lineage are affected by lympho-myeloproliferative disorders and disseminated intravascular coagulation.

Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.