Long-lived Lymphocytes Research Articles

The role of circulating T cells with a tissue resident phenotype (ex-TRM) in health and disease.

Tissue-resident memory T cells (TRM) are long-lived memory lymphocytes that persist in non-lymphoid tissues and provide the first line of defence against invading pathogens. They adapt to their environment in a tissue-specific manner, exerting effective pathogen control through a diverse T cell receptor (TCR) repertoire and the expression of proinflammatory cytokines and cytolytic proteins. More recently, several studies have indicated that TRM can egress from the tissue into the blood as so-called "ex-TRM", or "circulating cells with a TRM phenotype". The numerically small ex-TRM population can re-differentiate in the circulation, giving rise to new memory and effector T cells. Following their egress, ex-TRM in the blood and secondary lymphoid organs can be identified based on their continued expression of the residency marker CD103, alongside other TRM-like features. Currently, it is unclear whether exit is a stochastic process, or is actively triggered in response to unknown factors. Also, it is not known whether a subset or all TRM are able to egress. Ex-TRM may be beneficial in health, as mobilisation of specialised TRM and their recruitment to both their site of origin as well as distant tissues results in an efficient distribution of the immune response. However, there is emerging evidence of a pathogenic role for ex-TRM, with a suggestion that they may perpetuate both local and distant tissue inflammation. Here, we review the evidence for the existence of ex-TRM and examine their potential involvement in disease pathogenesis.

JAK2V617F Impairs T Cell Differentiation in Polycythemia Vera

Introduction: Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) clonally arising from a malignant hematopoietic stem cell (HSC) bearing a driver mutation, most frequently JAK2V617F. JAK2V617F in PV has only rarely been observed in lymphocytes, which is surprising since HSCs give rise to all hematopoietic lineages. We propose two alternative hypotheses to explain this observation, either (A) lymphopoiesis in adults with PV occurs prior to the acquisition of JAK2V617F in HSCs, followed by relatively slow attrition of long-lived unmutated mature lymphocytes, or (B) JAK2V617F interferes with lymphopoiesis from HSCs. The direct and indirect effects of MPNs on lymphopoiesis is a topic of study relevant to the inflammation-mediated complications that characterize these diseases. Objective: To critically test the competing hypotheses explaining the lack of JAK2V617F entry into T lymphoid populations in a large cohort of patients with PV. Methods: Peripheral blood was collected from 50 patients with JAK2V617F PV according to IRB-approved procedures and fractionated into myeloid and lymphoid subpopulations via fluorescence-activated cell sorting (FACS). To track the malignant clone across lineages, JAK2V617F mutation allele frequency (MAF) was measured in each subpopulation by droplet digital PCR (ddPCR). Hypothesis (A) predicts that PV duration and patient age at diagnosis correlates with JAK2V617F MAF in lymphocytes, so this was tested in our cohort by linear regression. Hypothesis (B) was tested by determining the extent to which T cell differentiation is impaired in JAK2V617F mutated, primary CD34+ hematopoietic stem and progenitor cell (HSPC) samples isolated from 15 patients with PV. HSPCs were differentiated in vitro into T cell progenitors using a Notch signaling-based culture system. As a control, HSPC differentiation toward the erythroid lineage was performed in parallel for each sample. The effect of JAK2V617F on differentiation was tracked by MAF using ddPCR in progenitor subpopulations obtained by FACS along validated T cell and erythroid ontogenies (adapted from Awong et al. Blood 2009 and Fajtova et al. Leuk Lymphoma 2013, respectively). Results:JAK2V617F was readily detected in HSPCs, granulocytes (PMNs) and erythroid progenitors (EPs) across our PV cohort but was detected only at very low levels in T cells (median MAF 0.55%) and B cells (median MAF 1.3%) (Figure 1). JAK2V617F MAF in HSPCs was positively correlated with patient age (+0.77% MAF per year of age, p = 0.007) and duration of PV (+1.7% MAF per year of PV, p = <0.001) but MAF in T cells was unrelated to either (-0.003% MAF per year of age and +0.03% MAF per year of PV, p = 0.92 and 0.55, respectively). The cohort included patients 30-90 years of age with PV duration ranging from 0-30 years. Given these time frames that likely exceed the lifespans of the longest-lived lymphocytes, with recent estimates that JAK2V617F mutations are acquired in HSCs decades prior to clinical MPN diagnoses (Williams et al. Nature 2022), it is unlikely that all T lymphopoiesis occurred prior to the acquisition of JAK2V617F across our cohort. Thus, Hypothesis (A) appears implausible. We tracked JAK2V61F MAF within 3 immunophenotypically defined T cell progenitor subpopulations that developed during in vitro differentiation of PV HSPCs. All 15 PV samples could be differentiated toward T cells. However, we observed a marked decrease in JAK2V617F MAF as T cell differentiation progressed (Figure 2). In contrast, JAK2V617F MAF was maintained or increased during in vitro erythroid differentiation toward erythroblasts (EB). These results indicate that JAK2V617F exerts a selective bias against T cell commitment in PV, thereby supporting Hypothesis (B). Conclusion:JAK2V617F mutated T cells are seldom observed in patients with PV and JAK2V617F HSPCs exhibit impaired T cell differentiation in vitro. Patients with MPN appear to have deficient immune surveillance that manifests with increased risks of infection (Landtblom et al. Leukemia 2021) and second malignancies (Frederiksen et al. Blood 2011), independent of therapy. Our study suggests that impaired T cell differentiation could contribute to such risks. Further study is under way to define actionable mechanisms through which JAK2V617F interferes with T cell differentiation. Additional correlative studies will report the prognostic implications of lymphocyte JAK2V617F MAF in PV. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CLL-Derived Exosomes Turn Endothelial Cells into IL-6 Producing Cells

CLL is characterized by gradual accumulation of mature appearing long-lived lymphocytes that travel in blood and reside in lymph nodes, spleen and bone marrow. In these sites, pro inflammatory humoral factors support the survival and proliferation of the neoplastic cells. Previous studies showed that levels of the proinflammatory cytokine IL-6 are at least 10 folds higher in patients with CLL compared with healthy individuals. Yet, which cells produce and secrete IL-6 and what triggers this cellular activity in CLL is unknown.Secreted by all types of cells, exosomes are nano-scaled particles that travel in blood and carry a cargo that at least partially reflects the molecular makeup of its cell of origin. Exosomes, including those originating from neoplastic cells, function as stable intercellular transport vehicles that deliver their cargo to cells that engulf them. For example, CLL-derived exosomes are taken up by mesenchymal stromal cells, transforming them to cancer associated fibroblasts.Given the appropriate stimulation, endothelial cells produce IL-6 which provides CLL cells with a survival advantage. Therefore, we hypothesized that CLL-exosomes turn endothelial cells into “IL-6-secreting cells”.To test this hypothesis, we transfected vein-derived (HUVECs) and arterial-derived (HAOEC) endothelial cells with exosomes that we isolated from the peripheral blood of 45 treatment naïve patients. We found that endothelial cells take-up CLL-exosomes in a dose- and time- dependent manner. Since CLL cells are protected from apoptosis in IL-6 rich environment, we wondered whether CLL-exosomes turn endothelial cells into IL-6-producing cells. To test this, we exposed endothelial cells to CLL-exosomes and found 50% increase in IL-6 levels, suggesting that the endothelial-exposed cells produced and secreted IL-6. Subsequently, we filtered out this growth medium and added CLL cells to this IL-6 enriched medium. After 15 minutes, STAT3 became phosphorylated and there was 40% decrease in the rate of apoptosis, indicating that IL-6 activated STAT3-dependent anti-apoptotic pathway. Phosphor-proteomics analysis of endothelial cells that were loaded with CLL-exosomes revealed 23 phosphor-proteins that were upregulated. Network analysis unraveled the central role of phosphor-b-catenin. To test whether b-catenin induces IL-6 in these cells, we transfected HUVECs with a b-catenin containing plasmid. We found by ELISA 30% increase in the levels of IL-6 in the culture medium and by chromatin immunoprecipitation assay an increased binding of 3 transcription factors (NFkB, LEF/TCF, and CEBP) to the IL-6 promoter. Taken together, we found that CLL cells communicate with endothelial cells through exosomes that they release. Once these exosomes are taken up by endothelial, they turn them into IL-6 producing cells, which in turn contributes to CLL cells' survival. DisclosuresNo relevant conflicts of interest to declare.

CLL-Derived Exosomes Educate Endothelial Cells to Become CLL-Supportive Cells

Background: CLL is characterized by a gradual accumulation of mature appearing long-lived lymphocytes. Several pro-survival pathways that protect CLL cells from apoptosis are activated in these cells. For example, IL-6 dependent phosphorylation of the signal transducer and activator of transcription 3 (STAT3) provides CLL cells with a survival and a proliferative advantages. What is the source of IL-6 is currently unknown. Within lymphoid organs CLL cells engage in complex molecular interactions. In these sites, CLL cells are not simply the seeds that grow on the supportive soil of the microenvironment but play an active role in shaping its surrounding. Secreted by all types of cells, exosomes are nano-scaled particles that travel in blood and function as stable intercellular transport vehicles that deliver their cargo to cells that engulf them. For example, CLL-derived exosomes (herein CLL-exosomes) are taken up by mesenchymal stromal cells, transforming them to cancer associated fibroblasts. Given the appropriate stimulation, endothelial cells produce IL-6. Therefore, we hypothesized that CLL-exosomes actively recruit endothelial cells to become IL-6-secreting cells.Methods: CLL cells from 37 patients were included in this study. CLL-exosomes were isolated by ultracentrifugation. Electron microscopy, NanoSight tracking analysis, flow cytometry and Western immunoblotting were used to characterize CLL exosomes. Exosomal uptake by HUVECs was assessed by flow cytometry and fluorescent microscopy. The phosphor-protein profiling of exposed HUVECs was analyzed by mass spectrometry. RT-PCR and Western immunoblotting were used to determine the expression profile of HUVEC-exposed cells. HUVECs were transfected with β-catenin containing plasmid using DNA transfection reagent. Cytokine levels were determined by ELISA and CHIP assay was used to identify activated transcription factors .Results: First, we isolated CLL-exosomes from 37 treatment naïve patients. For that purpose we grew CLL cells and collected the secreted exosomes after 72 hours by ultracentrifugation. By NanoSight tracking system we identified large amount of exosomes and verified the presence of the typical cap shaped vesicles by electron microscopy. Western immunoblotting confirmed the presence of CD63 and CD81 exosomal markers and by flow we detected CD19/CD5 on these particles. Next, we exposed HUVECs to an increasing amount of CLL-exosomes and verified by two distinct methods that these particles are up taken by HUVECs in a dose- and time- dependent manner. By mass spectrometry we found 53 phosphorproteins that were at least 2 folds upregulated and none that were downregulated in HUVEC-exposed cells. Pathway analysis unraveled the central position of β-catenin. Immunoprecipitation studies verified that levels of phosphor-β-catenin are upregulated while levels of total β-catenin remained unchanged, suggesting that CLL-exosomes induced phosphorylation rather than the generation of newly formed β-catenin and leaving us wondering whether upregulation of phosphor β-catenin is somehow beneficial to their parental neoplastic cells. Because the IL-6 promoter binds 3 transcription factors that are activated by β-catenin we assumed that by activating the β-catenin pathway, endothelial cells become "micro factories" for the production of humoral IL-6. To verify the role of β-catenin in promoting production of IL-6 molecules in endothelial cells we transfected HUVECs with β-catenin containing plasmid. By Western immunoblotting we verified that the protein levels of β-catenin were upregulated. Then, by ChIP assay we found that 3 different transcription factors, namely LEF/TCF, CEBP and NFkB increased their binding to the IL-6 promoter region by 1.5 to 12 folds in HUVECs that were transfected with β-catenin ORF. Finally we show that intracellular STAT3 of CLL cells that were grown on this IL-6-rich medium are phosphorylated on tyrosine residues and that the rate of CLL cells in active apoptosis was markedly decreased. Conclusions: CLL cells shape their own fate. They do so by sending exosomes that activate the β-catenin axes. In this way CLL cells reprogram endothelial cells to become IL-6 producing cells and IL-6 contributes to CLL cells' survival. Disclosures No relevant conflicts of interest to declare.

γδ T cells: A disappearing act with a big reveal.

In this issue of JEM, Sandrock et al. (https://doi.org/10.1084/jem.20181439) compare the origin of IL-17-producing γδ T cells (Tγδ17) with other γδ T cell populations and demonstrate the role Tγδ17 cells play in skin pathology. Using two genetically modified mouse models, one with inducible γδ T cell-specific labeling and the other with conditional γδ T cell depletion, the authors find that Tγδ17 are mostly long-lived lymphocytes and that depleting γδ T cells protects mice from psoriasis.

Genetic models reveal origin, persistence and non-redundant functions of IL-17-producing γδ T cells.

γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell-deficient Tcrd -/- mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive Tcrd -/- mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin-mediated conditional γδ T cell depletion. In contrast to IFN-γ-producing γδ T cells, IL-17-producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17-driven skin pathology.

Abstract CT130: Clinical trial of P-BCMA-101 T stem cell memory (Tscm) CAR-T cells in relapsed/refractory (r/r) multiple myeloma (MM)

Abstract Background: P-BCMA-101 is a novel chimeric antigen receptor (CAR)-T cell therapy designed to increase efficacy while minimizing toxicity through reduced immunogenicity, lack of tonic signaling, a safety switch, high purity (&amp;gt95% CAR+) and a Tscm phenotype (Tscm are long-lived lymphocyte stem cells that appear to gradually differentiate into effectors, improving efficacy, durability and safety of a CAR-T therapeutic). The P-BCMA-101 CAR utilizes an anti-BCMA (B-Cell Maturation Antigen) Centyrin™ fused to a second-generation CAR scaffold (a CARTyrin) rather than antibody fragments. Centyrins are fully human and have high binding affinities, but are smaller, more stable and potentially less immunogenic. This product is made using the piggyBac™ (PB) transposon system, requiring only mRNA and plasmid DNA. PB eliminates the need for virus, preferentially producing the desired Tscm phenotype and reducing manufacturing costs. The higher cargo capacity of PB also permits the incorporation of other genes, such as a safety switch and a selection gene in P-BCMA-101. The former allows for in vivo depletion of P-BCMA-101 if severe adverse events were to occur and the latter allows enrichment of CAR+ cells leading to greater consistency and purity to produce a better therapeutic index. Efficacy of P-BCMA-101 in NSG mice bearing aggressive human MM.1S and p53 -/- MM.1S MM was reported (Hermanson, AACR 2016). Whereas control animals died early, tumor burden was reduced to the limit of detection after P-BCMA-101 treatment, and recurrences were spontaneously re-controlled. Objectives &amp; Methodology: Thus, a 3+3 dose escalation Phase 1 clinical trial was initiated in patients with r/r MM to assess the safety and efficacy of P-BCMA-101 (NCT03288493). Patients are apheresed to harvest T cells, then P-BCMA-101 CARTyrin T cells are manufactured and administered to patients as a single dose after a standard cyclophosphamide/fludarabine conditioning regimen. Preliminary Results: Two heavily pretreated patients are thus far evaluable at a dose of 0.75 x 106 CARTyrin+ cells / kg. P-BCMA-101 manufactured for patients were primarily Tscm, and demonstrated robust and specific in vitro killing and cytokine production against BCMA+ tumor cells. Both patients' MM was BCMA+. Patient 1, a 54yo female with λ light chain myeloma had rapid progression in spite of chemotherapy, with free light chains (FLC) spiking to 3290 mg/L and renal failure prior to P-BCMA-101 treatement. After P-BCMA-101 administration a partial response was reported, with FLC rapidly decreasing from 1308 mg/L at baseline to 305 mg/L. No cytokine release syndrome (CRS) was seen, though there were mild elevations in CRS markers. Patient 2 is a 50yo female with oligosecretory κ light chain myeloma with plasmacytomas. Likewise, no CRS was reported. PET/CT is pending. P-BCMA-101 CARTyrin T cells markedly expanded and persisted in both patients, comprising 14-40% of circulating T cells within 3 weeks of infusion. Both patients continue in good condition, and additional patients have been enrolled. Clinical trial data are early, but appear consistent with the preclinical findings for P-BCMA-101. Funding for this study was provided by Poseida Therapeutics and CIRM Citation Format: Tara K. Gregory, Jesus G. Berdeja, Krina K. Patel, Syed Abbas Ali, Adam D. Cohen, Caitlin Costello, Eric M. Ostertag, Nishan de Silva, Devon J. Shedlock, Michelle Resler, Matthew A. Spear, Robert Z. Orlowski. Clinical trial of P-BCMA-101 T stem cell memory (Tscm) CAR-T cells in relapsed/refractory (r/r) multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT130.

Mass Cytometric Analysis of HIV Entry, Replication, and Remodeling in Tissue CD4+ T Cells

To characterize susceptibility to HIV infection, we phenotyped infected tonsillar Tcells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ Tcells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ Tcells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool.

Splenic microenvironment is important in the survival and growth of Chronic Lymphocytic Leukemia in mice

Abstract Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the western world, is characterized by accumulation of clonally expanded CD5+CD19+ B lymphocytes in the peripheral blood and secondary lymphoid organs. CLL cell survival is dependent on B cell receptor signaling and cross-talk within the leukemia microenvironment. CLL presents with enlargement of the spleen, and lymph nodes due to accumulation of long-lived lymphocytes with impaired apoptotic mechanisms. The Eμ-Tcl1 mice, with a B cell specific overexpression of the oncogene, T cell Leukemia 1 (Tcl1), serve as an excellent model to study CLL as they develop a CLL like disease by 9–13 months of age. They exhibit splenomegaly, enlarged lymph nodes and a high peripheral blood white count. Adoptive transfer of primary CD5+CD19+ CLL cells from the Eμ-Tcl1 mice into syngeneic recipients leads to rapid disease development. Ultrasonography of recipient mice showed a dramatic enlargement of the spleen, which preceded detection of CLL cells in the blood. Unlike various genetic modifications, which can only delay the onset of CLL, splenectomy in these models, cures or significantly delays disease development from 30–35 days to 250–270 days. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for prostate apoptotic response-4 (Par-4) compared to C57BL/6 wild type mice. Par-4 is a known tumor suppressor protein that can be secreted and induce apoptosis selectively in cancer cells but not in normal cells. Par-4 appears to regulate a specific microenvironment required for CLL growth. Presently we are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche.

The microtubule-associated protein Lis1 regulates T lymphocyte homeostasis and differentiation

Abstract The microtubule-associated protein Lissencephaly 1 (Lis1) is a key regulator of cell division during stem cell renewal and differentiation. We examined the role of Lis1 in T lymphocyte homeostasis and fate diversification in response to microbial infection. T cell-specific deletion of Lis1 resulted in depletion of the peripheral CD4+ and CD8+ T lymphocyte pool, owing to a selective loss of homeostatic, cytokine-induced proliferation. By contrast, cognate antigen-triggered proliferation was relatively unaffected in CD8+ T cells, enabling Lis1-deficient T cells to differentiate into terminal effector cells in response to microbial infection. Strikingly, however, Lis1-deficient CD8+ T cells failed to develop into long-lived memory lymphocytes. Taken together, these findings suggest that Lis1 plays an important role in T cell homeostasis and the generation of memory T lymphocytes.

Role of Prostate Apoptosis Response-4 Tumor Suppressor in the Survival and Growth of Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of clonally expanded population of CD5+ CD19+ B lymphocytes in peripheral blood and secondary lymphoid organs, with a majority of circulating cells in a non-dividing resting stage. However CLL is no longer considered a static disease that results from simple accumulation of long-lived lymphocytes, but rather, is a dynamic process with a birth rate of about 0.1-2% of the entire CLL clone per day. The Eµ-Tcl1 mouse serves as an excellent model for the development of CLL as they develop a CLL like disease by 9-13 months of age, due to overexpression of the oncogene, T cell Leukemia 1 (Tcl1), in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5+ CD19+ CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to development of a CLL like disease within 3-5 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, ultrasonography of spleen and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers. Par-4 has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. Although we find that Eµ-Tcl1 CLL cells constitutively secrete Par-4, they are resistant to Par-4 mediated apoptosis. We show that CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling with FDA approved drugs (i.e. Dasatinib, Ibrutinib, and Fostamatinib) causes a decrease in Par-4 protein and mRNA levels and increases apoptosis. Interestingly, systemic Par-4 appears to inhibit CLL growth in vivo, since adoptively transferred Eµ-Tcl1 CLL cells grew better in Par-4 null mice, despite excellent Par-4 expression and secretion by the transferred cells. Thus there were three times more CLL cells in the bone marrow and twice as many in the spleens of Par-4 null mice compared to their wild type counterparts. We conclude that even though Par-4 is pro-apoptotic in the CLL microenvironment, intracellular Par-4 is either rendered inactive or is potentially pro-survival in Eµ-Tcl1 CLL cells. (Supported in part by NIH grants) DisclosuresNo relevant conflicts of interest to declare.

Role of Prostate apoptosis response-4 tumor suppressor in the survival and growth of Chronic Lymphocytic Leukemia

Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of clonally expanded population of CD5 + CD19 + B lymphocytes in peripheral blood and secondary lymphoid organs, with a majority of circulating cells in a non-dividing resting stage. However CLL is no longer considered a static disease that results from simple accumulation of long-lived lymphocytes, but rather, is a dynamic process with a birth rate of about 0.1-2% of the entire CLL clone per day. The Eµ-Tcl1 mouse serves as an excellent model for the development of CLL as they develop a CLL like disease by 9-13 months of age, due to overexpression of the oncogene, T cell Leukemia 1 (Tcl1), in B cells through the Ig VH promoter and Eµ enhancer (Bichi et al. PNAS. 2002). In an adoptive transfer model, intravenous or intraperitoneal injection of primary CD5 + CD19 + CLL cells from the Eµ-Tcl1 CLL mouse into recipient syngeneic mice leads to development of a CLL like disease within 3-5 weeks of transfer. We have characterized the growth of CLL cells in these mice by periodic submandibular bleeding, ultrasonography of spleen and flow cytometry. We find that Eµ-Tcl1 CLL cells express more Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, than normal B-1 or B-2 cells in mice. Par-4 is silenced by promoter methylation in more than 30% of all cancers. Par-4 has been shown to be secreted and to induce apoptosis selectively in various types of cancer cells but not in normal cells. Although we find that Eµ-Tcl1 CLL cells constitutively secrete Par-4, they are resistant to Par-4 mediated apoptosis. We show that CLL cells have constitutively active B-cell receptor signaling and that inhibition of BCR signaling with FDA approved drugs (i.e. Dasatinib, Ibrutinib, and Fostamatinib) causes a decrease in Par-4 protein and mRNA levels and increases apoptosis. Interestingly, systemic Par-4 appears to inhibit CLL growth in vivo, since adoptively transferred Eµ-Tcl1 CLL cells grew better in Par-4 null mice, despite excellent Par-4 expression and secretion by the transferred cells. Thus there were three times more CLL cells in the bone marrow and twice as many in the spleens of Par-4 null mice compared to their wild type counterparts. We conclude that even though Par-4 is pro-apoptotic in the CLL microenvironment, intracellular Par-4 is either rendered inactive or is potentially pro-survival in Eµ-Tcl1 CLL cells. (Supported in part by NIH grants) Disclosures No relevant conflicts of interest to declare.

Peripheral Prepositioning and Local CXCL9 Chemokine-Mediated Guidance Orchestrate Rapid Memory CD8+ T Cell Responses in the Lymph Node

After an infection, the immune system generates long-lived memory lymphocytes whose increased frequency and altered state of differentiation enhance host defense against reinfection. Recently, the spatial distribution of memory cells was found to contribute to their protective function. Effector memory CD8+ T cells reside in peripheral tissue sites of initial pathogen encounter, in apparent anticipation of reinfection. Here we show that within lymph nodes (LNs), memory CD8+ T cells were concentrated near peripheral entry portals of lymph-borne pathogens, promoting rapid engagement of infected sentinel macrophages. A feed-forward CXCL9-dependent circuit provided additional chemotactic cues that further increase local memory cell density. Memory CD8+ T cells also produced effector responses to local cytokine triggers, but their dynamic behavior differed from that seen after antigen recognition. These data reveal the distinct localization and dynamic behavior of naive versus memory T cells within LNs and how these differences contribute to host defense.

Research brief

Long-lived memory lymphocytes generate short-lived terminal progeny and also replenish the long-lived memory pool upon reinfection. Chronic viral infections strain the renewal capacity of antiviral T-cell populations, however, which can lead to failure of long-term immunity. Researchers have now identified two distinct subsets of antiviral CD8 T cells in mice that are characterised by differential expression of the T-bet and Eomesodermin transcription factors and that cooperate to contain chronic viral infections.

Lupus nephritis: lessons from murine models

Lupus nephritis is a challenging clinical condition for which current therapies are unsatisfactory with respect to both remission induction and unwanted toxic effects. Despite intervention, the rates of end-stage renal disease seem to be increasing in the USA. Discoveries over the past decade have greatly improved our understanding of immune activation and effector inflammatory pathways in lupus nephritis; however, this increased understanding has not yet translated into the approval of an effective new therapeutic agent. An analysis of the mechanisms of action of novel immunomodulatory drugs in multiple models of murine lupus clearly shows that interacting networks of immune and effector pathways are recruited as the disease progresses. Reversing established disease by targeting a single cell population or inflammatory pathway is, therefore, difficult once long-lived autoreactive lymphocyte populations are present and peripheral organs are inflamed. Data from murine models of lupus suggest that we need to consider new paradigms for the management of systemic lupus erythematosus that include earlier immune intervention, long-term maintenance therapies and protection of target organs.

Roscovitine Triggers Apoptosis in B‐Cell Chronic Lymphocytic Leukemia Cells with Similar Efficiency as Combinations of Conventional Purine Analogs with Cyclophosphamide

B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation in peripheral blood of many long-lived lymphocytes that do not die because of the deregulation of apoptosis. Most CLL cells are quiescent, and therefore the leukemic lymphocytes are resistant to conventional chemotherapy. The aim of this study was to evaluate in vitro the chemosensitivity of CLL cells to cladribine or fludarabine used alone or in combinations with mafosfamide (Mf; the active form of cyclophosphamide) as well as to roscovitine, a potent inhibitor of cyclin-dependent kinases with proapoptotic potential. The results of flow cytometry revealed that tested agents differentially reduced the viability of leukemic cells. Interestingly, roscovitine exerts a similar cytotoxic effect as the combinations of the used purine analogs with Mf, but with other kinetics. Roscovitine kills leukemic cells after a much shorter exposure time. Immunoblotting analysis showed that the reduction of the number of living cells coincides with marked changes of the balance between pro- and antiapoptotic factors. The latter were markedly reduced. The activation of proapoptotic proteins became evident especially after exposure of cells to roscovitine alone or to combinations of purine analogs and Mf. Furthermore, exposure of CLL cells to tested drugs degraded p27(KIP1) protein. Our findings demonstrate that roscovitine alone significantly reduces the number of viable CLL cells by inducing them to undergo apoptosis, and it acts earlier than clinically applied combinations of purine analogs with Mf/cyclophosphamide. These results confirm the high efficacy of roscovitine against CLL cells.

The effects of sildenafil citrate on malignant B-cells in patients with chronic lymphocytic leukemia

7076 Background: Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western Hemisphere, with over 10,000 cases diagnosed annually in the United States. It is characterized by progressive accumulation of functionally incompetent long-lived lymphocytes, shown to be secondary to a defect in programmed cell death or apoptosis. The phosphodiesterase inhibitor sildenafil induces capsase dependent apoptosis of malignant B lymphocytes in vitro. This study will test the hypothesis that sildenafil reduces the expression of BCL-2 and increases the spontaneous apoptosis rate of malignant B-cells in patients with CLL. Methods: Thirteen patients with Rai Stage 0 CLL were enrolled. Nine of the patients were aged sixty-five years or older and received sildenafil 25 mg weekly; four patients were under the age of sixty-five and received sildenafil 25 mg weekly. All patients took the medication for a total of three months. Lymphocyte counts, BCL-2 expression, caspase 3 activity, and apoptosis rate were monitored on enrollment and monthly for duration of the study. Results: The median age of patients enrolled in the study was 74 with a median white blood cell count of 18 x103/mL. Twelve of the 13 patients completed three months of therapy. While one patient withdrew due to blehparitis, not felt to be a side effect of sildenafil, all other patients tolerated the medication well without any adverse effects. There was no significant decrease in white blood cell count or Bcl-2 expression; capsase 3 activity and apoptosis rates remained undetectable on presentation and throughout treatment. Conclusions: At a dose of 25 to 50 mg weekly, sildenafil does not appear to have any effects on the malignant B cells in CLL. While this dose may not produce a measurable clinical or cellular response, higher doses may still have an effect on the malignant B cells of CLL. The dose of sildenafil was based on a case series of patients with Viagra who had decreases in IgM levels while taking sildenafil 25–50 mg weekly. Subsequent studies in have shown a greater reduction in IgM in patients taking sildenafil 100 mg daily and patients did not report any significant side effects. A follow-up study using sildenafil 100 mg daily is warranted. No significant financial relationships to disclose.

Semaphorin 6D regulates the late phase of CD4 + T cell primary immune responses

The semaphorin and plexin family of ligand and receptor proteins provides important axon guidance cues required for development. Recent studies have expanded the role of semaphorins and plexins in the regulation of cardiac, circulatory and immune system function. Within the immune system, semaphorins and plexins regulate cell-cell interactions through a complex network of receptor and ligand pairs. Immune cells at different stages of development often express multiple semaphorins and plexins, leading to multivariate interactions, involving more than one ligand and receptor within each functional group. Because of this complexity, the significance of semaphorin and plexin regulation on individual immune cell types has yet to be fully appreciated. In this work, we examined the regulation of T cells by semaphorin 6D. Both in vitro and in vivo T cell stimulation enhanced semaphorin 6D expression. However, semaphorin 6D was only expressed by a majority of T cells during the late phases of activation. Consequently, the targeted disruption of semaphorin 6D receptor-ligand interactions inhibited T cell proliferation at late but not early phases of activation. This proliferation defect was associated with reduced linker of activated T cells protein phosphorylation, which may reflect semaphorin 6D regulation of c-Abl kinase activity. Semaphorin 6D disruption also inhibited expression of CD127, which is required during the multiphase antigen-presenting cell and T cell interactions leading to selection of long-lived lymphocytes. This work reveals a role for semaphorin 6D as a regulator of the late phase of primary immune responses.

Human Cytomegalovirus–Specific Memory CD8+and CD4+T Cell Differentiation after Primary Infection

The development of human cytomegalovirus (HCMV)-specific T cell immunity after primary infection and its correlation with virus transmission to the fetus were investigated. The membrane phenotype (CCR7 and CD45RA expression) of and intracellular cytokine (interferon [IFN]-gamma and interleukin-2) production by HCMV-specific T cells (stimulated with HCMV-infected dendritic cells) were investigated in 21 immunocompetent pregnant women (12 transmitters and 9 nontransmitters) and in 5 nonpregnant subjects during the first year after infection. IFN-gamma-producing CD4+ and CD8+ T cells were readily detected during the first month, and their levels did not significantly change with time. CCR7 expression was negligible during both the early and the late stage of infection. Among CCR7- cells, those reexpressing CD45RA progressively increased until they reached median levels of 33% (range, 7%-51%) and 51% (range, 22%-76%) for HCMV-specific CD4+ and CD8+ T cells, respectively, similar to those observed in subjects with remote infection. CD45RA reexpression correlated with HCMV disappearance from blood. The level of HCMV-specific CD45RA+ T cells during the first months after infection was significantly lower in mothers who were transmitters than in those who were nontransmitters. After primary infection, circulating HCMV-specific effector T cells revert to the CD45RA+ phenotype, which appears to be associated with control of viremia and vertical transmission. Thus, these cells may represent long-lived true memory lymphocytes in the HCMV-specific pool.

Current key developments

Inflammatory bowel disease (IBD) is associated with extraintestinal manifestations which occur either at the same time as bowel inflammation (joint, skin and eye) or run an independent course (autoimmune hepatitis and primary sclerosing cholangitis (PSC)). It has been suggested that eye, skin and joint manifestations are driven by the trapping of gut-derived effector cells in capillaries in these sites; however, this cannot explain the liver diseases that develop when bowel inflammation is quiescent or even after colectomy.1,2 This led us to propose that long-lived memory lymphocytes that arise as a consequence of bowel inflammation express homing receptors that direct their subsequent migration not only to the gut but also the liver.3 Such cells could recirculate between the liver and gut without causing damage for many years but if they subsequently encounter an antigen in the liver this could result in their activation and the promotion of tissue damage and disease. This could explain how a patient can develop liver disease many years after their IBD has become quiescent. In order to prove the hypothesis we needed to: • demonstrate that lymphocytes in the liver of patients with PSC were originally activated in the gut • provide a mechanism to explain how these cells are recruited to the liver • show that they are critical for disease pathogenesis. Over the last nine years we have answered the first two questions and thus the hypothesis is still valid.4 When lymphocytes are activated by dendritic cells (DC) in gut-associated lymphoid tissues (GALT) they are not only programmed to respond to antigen but are also imprinted with a homing phenotype which directs their subsequent trafficking back to the gut.5 After antigen has been cleared, a population of long-lived memory cells remain that retain gut tropism and thereby provide immune surveillance against the same pathogen entering the gut in the future. The molecular basis of this tissuespecific homing has recently been elucidated. Lymphocytes are recruited into tissues from the blood by sequential interactions with adhesion molecules and chemotactic cytokines called chemokines presented on the endothelium lining the vessels in the target tissue. Adhesion molecules allow lymphocytes with an appropriate receptor to recognise and bind the endothelium and chemokines can then direct migration through the endothelium CURRENT KEY DEVELOPMENTS Roger Williams