Late Effector Memory Research Articles

The effect of early life cytomegalovirus infection on the immune profile of children

Cytomegalovirus (CMV) infection has a life-long impact on the immune system, particularly on memory T cells. However, the effect of early life CMV infection on the phenotype and functionality of T cells in infants and especially longitudinal changes occurring during childhood have not been explored in detail.The phenotype and functionality of peripheral blood CD8+ and CD4+ T cells from children infected with CMV in early life (< 6 months of age) was analyzed using high-dimensional flow cytometry. Samples from CMV IgG-seropositive (CMV+) children were collected at 6 months and 6 years of age and compared to samples from CMV-seronegative (CMV-) children.Early life CMV infection caused multiple alterations within T cells. These include downregulation of CD28 expression and upregulation of CD57 expression within both CD27+ early and CD27- late effector memory CD8+ and CD4+ T-cells at 6 months of age. Of these changes, only alterations within the highly differentiated late effector memory compartment persisted at the age of 6 years.Early life CMV-infection has a distinct impact on developing CD8+ and CD4+ memory T cell compartments. It appears to induce both temporary as well as longer-lasting alterations, which may affect the functionality of the immune system throughout life.

A split influenza vaccine formulated with a combination adjuvant composed of alpha-d-glucan nanoparticles and a STING agonist elicits cross-protective immunity in pigs

BackgroundSwine influenza A viruses (SwIAVs) pose an economic and pandemic threat, and development of novel effective vaccines is of critical significance. We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant alone (Nano-11) [Nano11-SwIAV] or in combination with the synthetic stimulator of interferon genes (STING) agonist ADU-S100 (NanoS100-SwIAV). Specific pathogen free (SPF) pigs were vaccinated twice via intramuscular (IM) or intradermal (ID) routes and challenged with a virulent heterologous SwIAV H1N1-OH7 virus.ResultsAnimals vaccinated IM or ID with NanoS100-SwIAV had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid at day post challenge 6 (DPC6). Furthermore, NanoS100-SwIAV ID vaccinates, even at half the vaccine dose compared to their IM vaccinated counterparts, had significantly increased frequencies of CXCL10+ myeloid cells in the tracheobronchial lymph nodes (TBLN), and IFNγ+ effector memory T-helper/memory cells, IL-17A+ total T-helper/memory cells, central and effector memory T-helper/memory cells, IL-17A+ total cytotoxic T-lymphocytes (CTLs), and early effector CTLs in blood compared with the Nano11-SwIAV group demonstrating a potential dose-sparing effect and induction of a strong IL-17A+ T-helper/memory (Th17) response in the periphery. However, the frequencies of IFNγ+ late effector CTLs and effector memory T-helper/memory cells, IL-17A+ total CTLs, late effector CTLs, and CXCL10+ myeloid cells in blood, as well as lung CXCL10+ plasmacytoid dendritic cells were increased in NanoS100-SwIAV IM vaccinated pigs. Increased expression of IL-4 and IL-6 mRNA was observed in TBLN of Nano-11 based IM vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in NanoS100-SwIAV IM vaccinates by DPC6 along with reduced macroscopic lung lesions and significantly higher virus neutralization titers in lungs at DPC6. However, NanoS100-SwIAV ID vaccinates exhibited significant reduction of challenge virus titers in nasal passages and a remarkable reduction of challenge virus in lungs.ConclusionsDespite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective cell mediated immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs.Graphical

Plant derived Nano-11 particle adsorbed with stimulator of interferon genes adjuvant and split influenza virus antigens elicits the cross-protective immunity in pigs

Abstract We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant (Nano-11) or its combination with a synthetic STING agonist ADU-S100 (NanoS100). Specific pathogen free pigs were vaccinated twice via intramuscular (IM) and intradermal (ID) routes and challenged with a virulent heterologous H1N1. Animals vaccinated IM or ID with H1N2-ADU-S100-Nano-11 had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid. There was a significant increase of IFNγ+ effector memory T-helper/memory cells and late effector cytotoxic T cells (CTLs), IL-17A+ central and effector memory T-helper/memory cells, IL-17A+ early effector CTLs, and CXCL10+ plasmacytoid dendritic cells in the PBMCs of the ID injected H1N2-ADU-S100-Nano-11 compared to H1N2-Nano-11 group. The frequency of IFNγ+ late effector CTLs and effector memory T-helper/memory cells and IL-17A+ late effectorCTLs, as well as in lungs CXCL10+ plasmacytoid dendritic cells was increased in PBMCs of IM injected H1N2-ADUS100-Nano-11 pigs. Increased expression of IL-4 and IL-6 mRNA was observed in tracheobronchial lymph nodes of IM Nano-11 based vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in IM H1N2+ADU-S100-Nano-11 vaccinates by day 6 post infection along with reduced macroscopic lung lesions. In conclusion, despite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs. Supported by USDA National Institute of Food and Agriculture, AFRI project 2019-67015-29814

Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes.

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

Clinical Evidence That Deficiency of the Methylcytosine Dioxygenase TET2 Gene Promotes the Clonal Expansion and Therapeutic Efficacy of CD19-Directed T Cells

Adoptive transfer of autologous chimeric antigen receptor (CAR) T cells that target tumor antigens can induce remissions in patients with hematologic malignancies. A major obstacle to successful clinical responses in some patient populations has been infusion with ineffective cell populations that are terminally differentiated, exhausted, poorly proliferative and short-lived. Understanding and manipulating the mechanistic pathways that sustain T cell potency will potentially unlock durable responses to tumors. Here we present an unusual case that helps clarify determinants of CAR T cell persistence. A 78-year-old individual with advanced chronic lymphocytic leukemia (CLL) was infused with CAR T cells that target CD19 (CTL019). CAR T cell expansion was delayed and not evident until 50 days following infusion and anti-tumor activity was observed in the peripheral blood, lymph nodes as well as bone marrow, accompanied by complete remission that has been sustained for over 4.5 years. Retrospective analyses revealed that at the peak of in vivo CAR T cell activity, over 94% percent of CD8+ CTL019 cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene led to monoallelic loss of TET2 . Accordingly, examination of polyadenylated TET2 RNA populations showed the appearance of new chimeric RNAs that spliced from TET2 exon 9 into the vector and terminated, truncating the encoded protein. Further analysis revealed that this patient had a germline polymorphism in the other allele of TET2 . This was a missense variant at amino acid 1879 (exon 11), converting the wildtype residue, glutamic acid, to glutamine (protein change: pE1879Q; cDNA change: c.5635G>C; allele frequency of approximately 50%). TET2 deficient CTL019 cells exhibited reduced cytosine hydroxymethylation and a chromatin accessibility profile measured by ATAC-Seq consistent with altered T cell differentiation. Furthermore, at the peak of in vivo engraftment, CAR T cells from this subject showed an early (central) memory phenotype which differs from CTL019 cells of other complete responding patients that are instead characterized by late (effector) memory differentiation at the height of their response. Experimental knock-down of TET2 in healthy donor CAR T cells altered their differentiation and augmented CTL019 effector function in a manner that was consistent with the effects of TET2 deficiency observed in this subject. Our findings suggest that the profound clonal expansion of a single TET2 deficient CAR T cell resulted in a complete and durable remission of CLL, highlighting the potential for epigenetic modulation of ineffective patient T cells to generate curative T cell products. DisclosuresFraietta:Novartis: Patents & Royalties: patents, Research Funding. Morrissette:Novartis: Consultancy. Liu:Novartis: Patents & Royalties. Young:Novartis: Research Funding. Levine:Novartis Pharmaceuticals Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding; GE Healthcare: Consultancy; Brammer Bio: Consultancy. Zhao:Novartis: Patents & Royalties. Kalos:Novartis: Research Funding. Porter:Incyte: Honoraria; Servier: Honoraria, Other: Travel reimbursement; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Immunovative Therapies: Other: Member DSMB; Novartis: Honoraria, Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding; Genentech: Honoraria. June:Tmunity Therapeutics: Equity Ownership, Research Funding; WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding.

Evidence of Targetable Immune Dysfunction in the Bone Marrow of Patients with Intermediate/High-Risk Myelodysplastic Syndrome Refractory to Hypomethylating Agents

Background Growing evidence indicates that a dysfunctional adaptive immune response contributes to the pathogenesis of myelodysplastic syndrome (MDS). Intermediate/high (I/H)-risk MDS is associated with immunosuppression, and median overall survival in hypomethylating agent (HMA)-refractory patients (pts) remains poor at < 6 months. Thus, new immune checkpoint-targeting therapies may be important for restoring effective anti-tumor T-cell mechanisms in these pts. Here we aim to evaluate the relationship between immunomodulatory and regulatory targets in lymphocytes and CD34+ blasts, cytogenetics and prior treatment regimens to inform future novel immunotherapy combinations.Methods Bone marrow aspirates from HMA-refractory, I/H-risk pts (n = 18) and normal donors (NDs; n = 20) were assessed by flow cytometry (FC), immunohistochemistry and RNA sequencing (RNAseq). An independent cohort of treatment-naive, I/H-risk pts (n = 9) was assessed for regulatory T-cell (Treg) counts by FC. T-cell subsets (Treg, T-naive, T-effector memory, T-central memory, T-late effector memory) were identified by FC based on established markers (CD3, CD4, CD8, CD197, CD45RO, CD127, CD25, FOXP3). Activation/proliferation status of T-cell subsets was measured by HLA-DR and Ki-67. MDS blasts were identified by CD45, CD34 and CD117. Immune checkpoint markers programmed death-1 (PD-1) and programmed death-ligands 1 and 2 (PD-L1/2) were measured on T cells and CD34+ blasts. T-cell immunoglobulin and ITIM domain (TIGIT) was also measured on CD4 and CD8 T cells. T cells (CD4, CD8) and PD-L1 were identified in decalcified bone marrow biopsies by immunohistochemistry. Whole transcriptome gene expression analysis was performed using the Illumina TruSeq RNA Access Library Prep Kit on bulk bone marrow aspirates.Results T cells were detected in bone marrow aspirates from HMA-refractory, I/H-risk pts at levels comparable to those in NDs. Treg counts (cells/µL) were increased in HMA-refractory vs treatment-naive pts (P= 0.017), with the highest Treg count associated with complex cytogenetic karyotypes (≥ 3 abnormalities). We observed increased T-cell activation and proliferation in these pts compared with NDs (MDS vs ND medians [% positive cells]; HLA-DR−/Ki-67+: 1.23 vs 0.75; HLA-DR+/Ki-67+: 1.71 vs 0.33; HLA-DR+/Ki-67−: 14.14 vs 4.80). No trends were seen in CD4-naive and memory subsets, whereas CD8-naive cells were lower and late effector memory cells were higher than in NDs (P= 0.035 each). We observed higher expression of immune checkpoint and regulatory molecules (PD-L1, PD-1, TIGIT) in MDS except for PD-L2, which was minimally detected (< 5%). Frequency (%) of PD-L1 on CD34+ blasts was higher in pts with MDS than in NDs (P= 0.02) and was associated with complex karyotype (P= 0.026) and Treg frequency (P= 0.021). PD-1+ T cells were higher in pts with MDS than in NDs (CD4, P= 0.004; CD8, P= 0.012), with a trend toward increased PD-1 expression associated with number of prior therapies. TIGIT expression on CD4 T cells correlated with PD-1 (P= 0.008) and Treg frequency (P= 0.022). Lymphocyte-activation gene 3 expression by RNAseq was also higher in MDS vs ND marrow (P < 0.001). Some signals, such as PD-L1 and Tregs, correlated by FC and RNAseq.Conclusions Bone marrow in pts with I/H-risk MDS demonstrate evidence of prolonged T-cell activation, an increase in suppressive immune cell populations (i.e., Tregs) and expression of immune checkpoint molecules (PD-L1, TIGIT) compared with normal bone marrow. Patients with complex karyotype had subtle differences, potentially relevant for identifying subsets responsive to checkpoint inhibitors. These data provide new insight into the immune landscape of HMA-refractory I/H-risk MDS. DisclosuresGreen:Genentech, Inc.: Employment. Yan:F. Hoffmann-La Roche Ltd: Employment. Nalle:Genentech, Inc.: Employment; Roche: Equity Ownership. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Zhong:F. Hoffmann-La Roche Ltd.: Employment. Krishnan:Genentech, Inc.: Employment. Phuong:Genentech, Inc.: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment, Equity Ownership. Adamkewicz:Genentech Inc.: Employment. Venstrom:Genentech, Inc.: Employment. Dail:Genentech, Inc.: Employment.

Mannose Conjugated Chitosan-based Influenza Nanovaccine Intranasal Inoculation Enhances the Cross-reactive Immunity in Maternal Antibodies Positive Pigs

Abstract Swine influenza A virus (SwIAV) causes respiratory tract infection in pigs. Parenteral administration of inactivated SwIAV vaccine in weaned piglets provides variable protection due to preexisting specific maternal derived antibodies (MDA). We developed killed SwIAV antigen (KAg) encapsulated mannose-conjugated chitosan nanoparticles (mCS NPs-KAg) vaccine which targets dendritic cells. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to control CS NPs-KAg vaccinates. In both mCS NPs-KAg and CS NPs-KAg vaccinates upon challenge with a heterologous virus observed augmented cross-reactive virus-specific sIgA antibody response in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid, and IgG antibody levels in lung lysate and BAL fluid samples. Whereas the multivalent commercial inactivated SwIAV vaccine administered intramuscular had increased only the specific serum IgG antibody response. Additionally, mCS NPs-KAg vaccine increased the specific recall lymphocyte proliferation, and the frequency of IFNγ secreting T cells, late effector and effector memory T cells, and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates. Both the mCS NPs-KAg and CS NPs-KAg vaccines efficiently cleared the challenge virus load from airways and reduced the lung lesions compared to commercial vaccine. Overall, mCS NP-KAg vaccine in MDA-positive pigs induced a robust cross-reactive immunity and offered cross-protection against influenza virus.

Vaccination and Infection of Swine With Salmonella Typhimurium Induces a Systemic and Local Multifunctional CD4+ T-Cell Response.

The gram-negative facultative intracellular bacteria Salmonella Typhimurium (STM) often leads to subclinical infections in pigs, but can also cause severe enterocolitis in this species. Due to its high zoonotic potential, the pathogen is likewise dangerous for humans. Vaccination with a live attenuated STM strain (Salmoporc) is regarded as an effective method to control STM infections in affected pig herds. However, information on the cellular immune response of swine against STM is still scarce. In this study, we investigated the T-cell immune response in pigs that were vaccinated twice with Salmoporc followed by a challenge infection with a virulent STM strain. Blood- and organ-derived lymphocytes (spleen, tonsils, jejunal and ileocolic lymph nodes, jejunum, ileum) were stimulated in vitro with heat-inactivated STM. Subsequently, CD4+ T cells present in these cell preparations were analyzed for the production of IFN-γ, TNF-α, and IL-17A by flow cytometry and Boolean gating. Highest frequencies of STM-specific cytokine-producing CD4+ T cells were found in lamina propria lymphocytes of jejunum and ileum. Significant differences of the relative abundance of cytokine-producing phenotypes between control group and vaccinated + infected animals were detected in most organs, but dominated in gut and lymph node-residing CD4+ T cells. IL-17A producing CD4+ T cells dominated in gut and gut-draining lymph nodes, whereas IFN-γ/TNF-α co-producing CD4+ T cells were present in all locations. Additionally, the majority of cytokine-producing CD4+ T cells had a CD8α+CD27- phenotype, indicative of a late effector or effector memory stage of differentiation. In summary, we show that Salmonella-specific multifunctional CD4+ T cells exist in vaccinated and infected pigs, dominate in the gut and most likely contribute to protective immunity against STM in the pig.

Direct-Acting Antiviral Treatment of HCV Infection Does Not Resolve the Dysfunction of Circulating CD8+ T-Cells in Advanced Liver Disease.

Chronic hepatitis C virus (HCV) infection disrupts immune functions, including that of cytotoxic CD8+ T-cells which are important mediators of immune response. While HCV cure aims to eliminate long term sequelae of infection, whether direct-acting antiviral (DAA) treatment results in immune reconstitution remains unclear. We and others have reported generalized CD8+ T-cell dysfunction in chronic HCV infection and our research suggests that the degree of liver damage is a factor in this process. Our recent research indicates that liver fibrosis is not readily reversed after DAA-mediated clearance of chronic HCV infection. We therefore examined the function of circulating CD8+ T-cell subsets in chronic HCV infection in the context of liver fibrosis severity, determined by ultrasound elastography and Metavir F-score system. We observed progressive shifts in CD8+ T-cell subset distribution in HCV-infected individuals with advanced liver fibrosis (F4) compared to minimal fibrosis (F0-1) or uninfected controls, and this remained unchanged after viral cure. Impaired CD8+ T-cell function was observed as a reduced proportion of CD107+ and perforin+ late effector memory cells in HCV+(F4) and HCV+(F0-1) individuals, respectively. In HCV+(F4) individuals, nearly all CD8+ T-cell subsets had an elevated proportion of perforin+ cells while naïve cells had increased proportions of IFN-γ+ and CD107+ cells. These exaggerated CD8+ T-cell activities were not resolved when evaluated 24 weeks after completion of DAA therapy and HCV clearance. This was further supported by sustained, high levels of cell proliferation and cytolytic activity. Furthermore, DAA therapy had no effect on elevated concentrations of systemic inflammatory cytokines and decreased levels of inhibitory TGF-β in the plasma of HCV+(F4) individuals, suggesting HCV infection and advanced liver disease result in a long-lasting immune activating microenvironment. These data demonstrate that in chronic HCV infection, liver fibrosis severity is associated with generalized hyperfunctional CD8+ T-cells, particularly with perforin production and cytotoxicity, and this persists after viral clearance. Whether DAA therapy will eliminate other related long-term sequelae in HCV+(F4) individuals remains an important research question.

A novel simplified method of generating cytomegalovirus-specific cytokine-induced killer cells of high specificity and superior potency with GMP compliance

We describe a method of rendering polyclonal cytokine-induced killer cells (CIK) specific against cytomegalovirus (CMV), focusing on GMP compliance. Peripheral blood mononuclear cells (PBMNC) are stimulated with pooled CMV peptides pp65 and IE-1 for 16–24 h and the reactive T cell subset which up-regulate CD137 is further co-stimulated with anti-CD137, followed by expansion in G-Rex flasks under standard CIK culture condition. This method generates a large number CMV-specific CIK with superior potency compared to published method currently in clinical trials. The cytotoxicity as measured by chromium release assay correlates with the upregulation of CD107a upon peptide re-challenge as measured by flow cytometry. CMV-CIK at maturity consist of mainly late effector memory CD8 T cells and have a skewed TCR repertoire with preferential expansion of a few families. Such CMV-CIK retain their function after freezing and thawing. CMV-CIK thus generated is ready for clinical trial against drug-resistant CMV disease.

New Insights into Lymphocyte Differentiation and Aging from Telomere Length and Telomerase Activity Measurements.

αβ CD8+, γδ, and NK lymphocytes are fundamental effector cells against viruses and tumors. These cells can be divided into multiple subsets according to their phenotype. Based on progressive telomere attrition from naive to late effector memory cells, human CD8+ T cell subsets have been positioned along a pathway of differentiation, which is also considered as a process of lymphocyte aging or senescence. A similar categorization has not been clearly established for γδ and NK cell populations. Moreover, the distinction between the aging of these populations due to cellular differentiation or due to the chronological age of the donor has not been formally considered. In this study, we performed systematic measurements of telomere length and telomerase activity in human αβ CD8+, γδ, and NK lymphocytes based on subset division and across age to address these points and better understand the dichotomy between differentiation and temporal aging. This approach enables us to position phenotypically distinct γδ or NK subsets along a putative pathway of differentiation, such as for CD8+ T cells. Moreover, our data show that both cellular differentiation and donor aging have profound but independent effects on telomere length and telomerase activity of lymphocyte subpopulations, implying distinct mechanisms and consequences on the immune system.

Abstract A123: Skewed CD4 and CD8 T-cell differentiation in pancreatic cancer patients

Abstract To understand potential immune system alterations in newly diagnosed, untreated, pancreatic cancer patients and provide a foundation for immunotherapy, we profiled PBMC from pancreatic ductal adenocarcinoma (PDA) patients and age-matched healthy controls using high-dimensional CyTOF analysis. We developed two immune profiling panels: a broad profiling panel that includes 45 phenotypic markers that together permit the identification and enumeration of the main innate and adaptive immune cell subsets in humans, and a deep profiling panel that includes 45 features focusing on T-cell phenotype and biology. We report a 2-fold increase in monocytes, more regulatory T-cells, and more plasmocytes in circulation in pancreatic cancer patients as compared to age-matched controls, as well as a bias towards cytokine-producing NK cells. Using high-dimensional approaches, we observed skewed T-cell differentiation in pancreatic cancer patients, with peripheral blood CD8 T-cells being biased towards more CD45RA-positive CD27-positive CCR7-positive CD95-positive CD49d-positive stem cell memory cells (P=7x10-5), more CD45RA-negative CD27-positive CCR7-negative effector memory cells (P=0.002) and less CD45RA-positive CD27-negative CCR7-negative late effector memory cells (P=0.003) as compared to age-matched controls. Strikingly, when examinating T-cell differentiation in human spleens, we found increased proportions of late effector memory T-cells in the splenic CD8 T-cell compartment of pancreatic cancer patients as compared to age-matched controls. These results suggest an impaired T-cell trafficking in pancreatic cancer patients, with late memory T-cells being retained in the spleen. We are now investigating the mechanisms underlying these observations, as well as their impact on T-cell immunity of the cancer patients. Our goal is to understand the nature of the skewing and how any changes in baseline immune health of the T-cell compartment relate to disease progression and/or response to therapy. These studies should provide a foundation for improving therapy in pancreatic cancer patients. Citation Format: Cecile Alanio, Bertram Bengsch, Josephine R. Gies, Sarah Henrickson, Nan Ping Weng, Janáe A Ritz-Romeo, Mark O'Hara, Joseph J Melenhorst, Simon Lacey, Regina M Young, Carl H June, E. John Wherry. Skewed CD4 and CD8 T-cell differentiation in pancreatic cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A123.

Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56hi versus CD56dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.

Peripheral CD8 effector-memory type 1 T-cells correlate with outcome in ipilimumab-treated stage IV melanoma patients.

The role of the assessment of peripheral T-cell phenotypes in predicting overall survival (OS) after ipilimumab treatment is unclear. Here, we analysed mononuclear cells in the blood before and at different time points during treatment with ipilimumab in 137 late-stage melanoma patients. The proportions of baseline naïve and memory T-cells were measured by flow cytometry and correlated with OS, with an emphasis on PD-1 expression. High frequencies (>13%) of CD8 effector-memory type 1 (EM1) T-cells at baseline correlated with longer OS (p=0.029) and higher clinical response rates (p=0.01). The frequency of these EM1 cells and the M category had independent impacts on OS (hazard ratio=1.5, p=0.033; and hazard ratio=1.9, p=0.007). In contrast, high baseline frequencies of late stage-differentiated effector memory CD8 cells (>23.8%) were negatively associated with OS (p=0.034) but did not correlate with clinical response. Following treatment, a decrease of CD8 cells from baseline to the time of the second drug dose and at later time points was strongly and consistently correlated with a high clinical response rate. Our observations thus suggest an important predictive role of baseline CD8 EM1 cells and changes in CD8 cells for clinical response of ipilimumab. Further validation of these biomarker candidates is warranted.

Baseline Differences in Minor Lymphocyte Subpopulations may Predict Response to Fingolimod in Relapsing-Remitting Multiple Sclerosis Patients.

Fingolimod, oral treatment for relapsing-remitting multiple sclerosis (RRMS), is an agonist of sphingosine and its metabolite S1P that binds their receptors, blocking the egress of lymphocytes from lymph nodes. The aim of this study was immunomonitoring of minor peripheral lymphocyte subpopulations in RRMS patients under treatment with fingolimod and correlation with treatment response. Prospective study. T- and B-cell subpopulations were analyzed using multiparametric flow cytometry in peripheral blood from 14 RRMS patients under treatment with fingolimod at baseline, +1, +3, +6, +9, and +12 months of follow-up. Response to therapy was assessed at month +12. Most changes in minor lymphocyte subpopulations occurred in the first month of treatment and were maintained until the end of follow-up. The basal percentages of recent thymic emigrants (RTEs) and transitional B cells were lower in responder patients than in nonresponders. After 1 month of follow-up, the percentages of late effector memory CD4(+) T cells in peripheral blood were higher in responder patients. If confirmed in a bigger cohort of patients, analysis of percentages of minor lymphocyte subpopulations in peripheral blood of patients with RRMS prior and after +1 month of treatment might predict clinical response to fingolimod.

Children diagnosed with both Type 1 diabetes and Celiac disease - An Immunological challenge

Type 1 diabetes (T1D) and celiac disease are both characterized by an autoimmune feature. As T1D and celiac disease share several common risk factors such as environment, genetics and immune dysregulation, patients have risk of developing the other disease subsequently. Patients with manifest T1D may have had a latent celiac disease, which is activated parallel to the anti-islet immune reactivity during the development of T1D. Contrary, a low prevalence of β-cell autoimmunity is found in young patients with celiac disease. The role of antigen-specific T cells and their relation to cytokines and chemokines is not well characterized in children with combination of T1D and celiac disease. Defective regulation and an impaired ability of responder T cells to be suppressed are suggested to contribute. We have previously shown that children suffering from these two immunological diseases in combination have a suppressed immune response to several antigens for example food antigens like gluten. Low percentages of both early and late effector memory CD8 + cells together with observations of immune aberrancies seen in the gut, in children who are prone to T1D, may suggests poor development of oral tolerance that may predispose for development of celiac disease. This review highlights the immunological complexity in these two common pediatric immunological disorders that indicates that the combination of type 1 diabetes and celiac disease is an immunological challenge. It is obvious that we are far from understanding the immunological impact of these two autoimmune diseases in combination. This immunological challenge therefore needs to be elucidated to be able to predict and prevent these autoimmune diseases.

Protective effector memory CD4+ T cells are generated from an early effector memory precursor (MPF6P.739)

Abstract Effector memory CD4+ T cells (Tem) are the predominant memory subset in chronic infections such as malaria and tuberculosis. Tem respond quickly by making effector cytokines. Therefore, understanding their mechanism of generation and the role of persistent antigen is essential for vaccination strategies. The current paradigm of Tem generation is that they develop progressively from mature effector T cells in the contraction phase, however this remains to be established. In order to identify a precursor of Tem we identified three effector subsets with an Early CD127-CD62Lhi CD4+ memory precursor effector T cell (MPEC) population, and short-lived CD62Llo Teff (SLEC). In adoptive transfer experiments, we show the potential of Early Teff to diverge into either terminal Teff or memory cells depending on the environment, while mature Teff lose their memory potential, in contrast to the progressive differentiation model. Furthermore, we demonstrate that resting central memory T cells can become Tem in the absence of further stimulation. All three effector subsets protect immunocompromised animals, however, only Late Effector memory (TemL) improves both infection and pathology. Based on our data we propose an early decision point for generation of protective effector memory T cells, suggesting that memory development occurs in the early phase of activation. This work also suggests that TemL represent a correlate of immunity in chronic infection and can improve vaccine design studies.

Dasatinib promotes Th1-type responses in granzyme B expressing T-cells

Tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of chronic myeloid leukemia (CML). Besides inhibiting target kinases in leukemic cells, 2nd generation TKI dasatinib also inhibits off-targets in immune effector cells resulting in atypical immune responses in some patients. Dasatinib has been described to increase the proportion of late effector memory T-cells, however, to date no follow-up studies have been performed in first-line patients. In this study, we explored the functional properties of T-cells using primary samples from CML patients (n = 28) on TKI therapy. Granzyme B (GrB) was used as a marker for late phase antigen experienced CD4+ and CD8+ T-cells. Dasatinib treatment increased the numbers of both GrB expressing memory CD4+ and CD8+ T-cells when compared with healthy controls. Functionally, the GrB+CD4+ T-cells were highly active and differentiated into Th1-type T-cells capable of producing IFN-γ, which is important for tumor control. Similar kind of increase was not observed during imatinib or nilotinib therapy. These data support the dual mode of action of dasatinib: potent BCR-ABL1 inhibition in leukemic cells is accompanied by the enhancement of cellular immunity, which may have implications in the long-term control of leukemia.

Phenotypic and Functional Characteristics of NK Cells Associated with Cytomegalovirus (CMV) Reactivation after Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

s / Biol Blood Marrow Transplant 20 (2014) S128eS150 S146 virus (EBV) is a gammaherpes virus that is reactivated from latent to lytic cycle and can cause aggressive lymphomas known as post transplant lymphoproliferative disorder (PTLD) in immunocompromised hosts. We attempted to distinguish immune reconstitution profiles in double UCBT recipients who developed EBV viremia from those who did not. Thirty-one patients with hematologic malignancies received dUCBT with melphalan, fludarabine, ATG conditioning and tacrolimus plus sirolimus for GvHD prophylaxis. During the first 12 months after dUCBT, 14 of 31 (45%) patients developed EBV viremia and four (13%) developed PTLD. At one month after dUCBT, patients with EBV viremia displayed higher numbers of CD19+ B cells (p1⁄40.04) and CD4+CD25+ Treg cells (p1⁄40.03) compared with patients who never became viremic. Surprisingly, EBV viremia correlated with increased numbers of CD3+ (p1⁄40.04), CD4+ (p1⁄40.015) and CD8+ (p1⁄40.021) T cells, but this response was ineffective at controlling the virus. One explanation for this unexpected result might be skewing towards a late effector memory T cell phenotype, a stage in which T cells are incapable of mounting protective immune responses. We examined naive vs. memory T cell distribution and determined that patients who developed EBV reactivation had higher numbers of memory cell subsets (CD4+CD45RO+, p1⁄40.023; CD8+CD45RO+, p1⁄40.019) at two months after dUCBT. Analysis of repertoire diversity by deep-sequencing on PCR-amplified CDR3 regions of the TCRb gene using the ImmunoSEQ assay showed a more diverse TCR repertoire, as determined by higher entropy (p1⁄40.03) and lower clonality (p1⁄40.03), among patients who did not develop EBV reactivation. Assessment of SCF and IL-7, critical regulators of hematopoietic stem cell and naive T cell homeostasis, respectively, showed that patients without EBV viremia had higher levels of SCF (p1⁄40.0016) and IL-7 (p1⁄40.046) compared with patients who developed viremia and PTLD. Our findings suggest that control of EBV reactivation after dUCBT might be linked to the support of naive T cell homeostasis, which enables maintenance of a diverse TCR repertoire.

Long-Term Immune Reconstitution and Infection Burden after Mismatched Hematopoietic Stem Cell Transplantation

Mismatched unrelated donor (MMUD) or umbilical cord blood (UCB) can be chosen as alternative donors for allogeneic stem cell transplantation but might be associated with long-lasting immune deficiency. Sixty-six patients who underwent a first transplantation from either UCB (n = 30) or 9/10 MMUD (n = 36) and who survived beyond 3 months were evaluated. Immune reconstitution was prospectively assessed at sequential time points after transplantation. NK, B, CD4+, and CD8+ T cells and their naïve and memory subsets, as well as regulatory T cells (Treg), were studied. Detailed analyses on infections occurring after 3 months were also assessed. The 18-month cumulative incidences of infection-related death were 8% and 3%, and of infections were 72% and 57% after MMUD and UCB transplantation, respectively. Rates of infection per 12 patient-month were roughly 2 overall (1 for bacterial, .9 for viral, and .3 for fungal infections). Memory, naïve CD4+ and CD8+T cells, naïve B cells, and Treg cells reconstitution between the 2 sources were roughly similar. Absolute CD4+T cells hardly reached 500 per μL by 1 year after transplantation and most B cells were of naïve phenotype. Correlations between immune reconstitution and infection were then performed by multivariate analyses. Low CD4+ and high CD8+T cells absolute counts at 3 months were linked to increased risks of overall and viral (but not bacterial) infections. When assessing for the naïve/memory phenotypes at 3 months among the CD4+ T cell compartment, higher percentages of memory subsets were protective against late infections. Central memory CD4+T cells protected against overall and bacterial infections; late effector memory CD4+T cells protected against overall, bacterial, and viral infections. To the contrary, high percentage of effector- and late effector-memory subsets at 3 months among the CD8+ T cell compartment predicted higher risks for viral infections. Patients who underwent transplantation from alternative donors represent a population with very high risk of infection. Detailed phenotypic analysis of immune reconstitution may help to evaluate infection risk and to adjust infection prophylaxis.