Total PBMC Research Articles

Expansion, Persistence, and Characteristics of Autologous, Bhv-1100 Armored Memory-like NK Cells Infused Prior to Autologous Stem Cell Transplant in MRD+ Multiple Myeloma Patients: A First-in-Human Trial

Introduction/Background Autologous stem cell transplant (ASCT) improves MRD negativity and prolongs progression-free survival in patients with multiple myeloma (MM) in their first or second remission following induction chemotherapy. MM NK cells are dysfunctional, negatively impacting outcomes. BHV-1100, a novel Antibody Recruiting Molecule ( ARM), binds to CD38 and recruits NK cells for antibody-dependent cell cytotoxicity (ADCC) without inducing fratricide. Allogeneic, cytokine induced memory-like (CIML) NK cells effectively treat myeloid disorders, however, it is not known if autologous CIML NK cells, when coated with BHV-1100, would further improve ASCT outcomes in MM. Methods We designed a first-in-human study of autologous CIML NK cells coated ex-vivo with BHV-1100 for MRD+, MM patients undergoing ASCT following first or second remission. NK cells were isolated from non-mobilized leukapheresis on day -1 (prior to melphalan for HCT) using CD3 depletion followed by CD56 positive selection with Miltenyi's CliniMACS. The NK cells were incubated overnight (12-16 hours) with IL-12 (10ng/ml), IL-15 (100ng/ml), and IL-18 (50ng/ml) to induce CIML differentiation, washed and subsequently coated with BHV-1100 for one hour prior to infusion. The product was infused fresh on D0 after standard melphalan 200 mg/m2 myeloablative conditioning and followed by stem cell infusion. Low dose IL2 (1 mIU/m 2) was administered SQ starting on D+1, QOD for a total of 7 doses. Results This is an ongoing trial (NCT04634435) with a median follow-up of 191 days. We are herein reporting data on the in vivo expansion and functional characterization of ARMored CIML NK for the first 5 patients. CIML NK cells were manufactured with a 100% success rate and infused at a target dose of 5-10x10 6 cells/kg-body weight, 24 hours after 200 mg/m 2 melphalan administration. Patients received between 3.9-6.0x10 6/Kg stem cells. Engraftment based on recovery of neutrophil count occurred on D+12-D+14. There was a 3-fold expansion of NK cells in the peripheral blood from D+7 (from 12% to 42%) to D+28 that persisted until D+60 (25% total PBMC, Fig. 1A). Most expanded NK cells were CD56 dim, CD16 high, KIR high and CD57 high. CD57 and KIR expression increased over time from D+7 to D+60, whereas NKG2A expression decreased, indicating the expansion of mature, activated, and cytotoxic NK cells. Regulatory T cells increased by D+7 (3% vs 15% total PBMC) and returned to baseline after D+14 most likely reflecting the effect of IL-2 treatment. The functional capacity of the infused product was tested in vitro against MOLP8 MM cell line. The BHV-1100 ARMored CIML NK cells showed increased IFN (53% vs 48% at 0H and 53% vs 44% at 24H) and CD107a (Fig.1B) (26% vs 13% at 0H and 34% vs 15% at 24H) production compared to untreated CIML NK cells and the product was stable for 24 hours. Conclusion Autologous, BHV-1100 ARMored CIML NK cells have enhanced anti-MM activity as well as expand and persist in vivo peaking at D+28 after infusion. This represents an innovative approach to boost autologous cancer immunosurveillance in the context of ASCT. Aside from anticipated infusion reactions, no severe/unexpected adverse events were noted; longer follow-up is required to assess safety and efficacy.

HCMV carriage in the elderly diminishes anti-viral functionality of the adaptive immune response resulting in virus replication at peripheral sites.

Human cytomegalovirus (HCMV) infection and periodic reactivation is, generally, well controlled by adaptative immune responses in the healthy. In older people, overt HCMV disease is rarely seen despite the association of HCMV with increased risk of mortality; evidence from studies of unwell aged populations suggest that HCMV seropositivity is an important co-morbidity factor. HCMV genomes have been detected in urine from older donors, suggesting that the immune response prevents systemic disease but possibly immunomodulation due to lifelong viral carriage may alter its efficacy at peripheral tissue sites. Previously we have demonstrated that there were no age-related expansions of T cell responses to HCMV or increase in latent viral carriage with age and these T cells produced anti-viral cytokines and viremia was very rarely detected. To investigate the efficacy of anti-HCMV responses with increasing age, we used an in vitro Viral Dissemination Assay (VDA) using autologous dermal fibroblasts to determine the anti-viral effector capacity of total PBMC, as well as important subsets (T cells, NK cells). In parallel we assessed components of the humoral response (antibody neutralization) and combined this with qPCR detection of HCMV in blood, saliva and urine in a cohort of young and old donors. Consistent with previous studies, we again show HCMV specific cIL-10, IFNγ and TNFα T cell responses to peptides did not show an age-related defect. However, assessment of direct anti-viral cellular and antibody-mediated adaptive immune responses using the VDA shows that older donors are significantly less able to control viral dissemination in an in vitro assay compared to young donors. Corroborating this observation, we detected viral genomes in saliva samples only from older donors, these donors had a defect in cellular control of viral spread in our in vitro assay. Phenotyping of fibroblasts used in this study shows expression of a number of checkpoint inhibitor ligands which may contribute to the defects observed. The potential to therapeutically intervene in checkpoint inhibitor pathways to prevent HCMV reactivation in the unwell aged is an exciting avenue to explore.

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Comparative cellular immune responses in calves after infection with Mycobacterium avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii and M. bovis

In the present study, calves were infected with Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. avium (M. avium), Mycobacterium kansasii (M. kansasii), or Mycobacterium bovis (M. bovis) to determine differences in cellular immunity. Comparative cellular responses were assessed upon stimulation of cells with mycobacterial whole cell sonicates respective of each infection group. Antigen-specific whole blood interferon gamma (IFN-γ) responses were observed in all infection groups compared to noninfected control calves, however, responses were more robust for M. bovis calves. Upon antigen stimulation of PBMCs, secretion of IFN-γ and IL-10 was higher for M. bovis calves compared to other infection groups. In contrast, IL-12 secretion was lower for M. bovis calves compared to MAP infected calves. Within the total PBMC population, higher numbers of CD4+, CD8+, and γδ TCR + T cells were observed for MAP and M. avium calves compared to M. bovis calves. This aligned with higher expression of CD26 on these subpopulations for MAP and M. avium calves, as well. In contrast, greater expression of CD25 was observed on CD4+ and γδ TCR + T cells and natural killer cells for M. bovis calves. Overall, similarities in cellular immune responses were observed between the closely related MAP and M. avium during infection of calves. In contrast, significant differences were noted between calves infected with MAP and M. bovis. This suggests that host immune responses to different mycobacteria may impact interpretation of diagnostic tools based upon their cellular immunity.

Modified Adenovirus Prime-Protein Boost Clade C HIV Vaccine Strategy Results in Reduced Viral DNA in Blood and Tissues Following Tier 2 SHIV Challenge.

Designing immunogens and improving delivery methods eliciting protective immunity is a paramount goal of HIV vaccine development. A comparative vaccine challenge study was performed in rhesus macaques using clade C HIV Envelope (Env) and SIV Gag antigens. One group was vaccinated using co-immunization with DNA Gag and Env expression plasmids cloned from a single timepoint and trimeric Env gp140 glycoprotein from one of these clones (DNA+Protein). The other group was a prime-boost regimen composed of two replicating simian (SAd7) adenovirus-vectored vaccines expressing Gag and one Env clone from the same timepoint as the DNA+Protein group paired with the same Env gp140 trimer (SAd7+Protein). The env genes were isolated from a single pre-peak neutralization timepoint approximately 1 year post infection in CAP257, an individual with a high degree of neutralization breadth. Both DNA+Protein and SAd7+Protein vaccine strategies elicited significant Env-specific T cell responses, lesser Gag-specific responses, and moderate frequencies of Env-specific TFH cells. Both vaccine modalities readily elicited systemic and mucosal Env-specific IgG but not IgA. There was a higher frequency and magnitude of ADCC activity in the SAd7+Protein than the DNA+Protein arm. All macaques developed moderate Tier 1 heterologous neutralizing antibodies, while neutralization of Tier 1B or Tier 2 viruses was sporadic and found primarily in macaques in the SAd7+Protein group. Neither vaccine approach provided significant protection from viral acquisition against repeated titered mucosal challenges with a heterologous Tier 2 clade C SHIV. However, lymphoid and gut tissues collected at necropsy showed that animals in both vaccine groups each had significantly lower copies of viral DNA in individual tissues compared to levels in controls. In the SAd7+Protein-vaccinated macaques, total and peak PBMC viral DNA were significantly lower compared with controls. Taken together, this heterologous Tier 2 SHIV challenge study shows that combination vaccination with SAd7+Protein was superior to combination DNA+Protein in reducing viral seeding in tissues in the absence of protection from infection, thus emphasizing the priming role of replication-competent SAd7 vector. Despite the absence of correlates of protection, because antibody responses were significantly higher in this vaccine group, we hypothesize that vaccine-elicited antibodies contribute to limiting tissue viral seeding.

Abstract 1947: Development and characterization of small molecule HPK1 inhibitors

Abstract Background: Hematopoietic progenitor kinase 1 (HPK1, MAP4K1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator of T cells and dendritic cells (DC). Alteration of ERK/MAPK pathway by HPK1 in T-cells and dendritic cells is an inhibitory mechanism that negatively regulates TCR-induced IL-2 gene transcription, T cell maturation and proliferation. Inhibiting kinase activity of HPK1 results in activation of antigen presenting properties of dendritic cells and stimulates maturation and proliferation of T cells. Therefore, small molecule inhibitors of HPK1 could serve as a novel agent to transform cold, resistant tumors into sensitive hot cancers and provide additional benefit in combination with existing immunotherapies. Methods: Inhibition of HPK1 was assessed by biochemical assay with recombinant human and mouse protein. Small molecule inhibitors were tested in biochemical assay on other MAP4Ks and in addition profiled against broad kinase panel. Phosphorylation of Serine 376 and Tyrosine 128 of SLP-76 adaptor protein upon HPK1 inhibition was monitored by Western Blotting in human and murine T-cells. IL-2 release was monitored in total human PBMC, human CD3+ T cells and mouse CD3+ splenocytes. Human CD3+ T cells were isolated from PBMC, activated with plate-bound anti-CD3/anti-CD28 and exposed to compounds in the presence of PGE-2, followed by IL-2 release measurement, viability and proliferation assessment using flow cytometry. Mouse CD3+ splenocytes were isolated from Balb/c mice, activated with plate-bound anti-CD3/anti-CD28 and exposed to compounds in the presence of PGE-2, followed by IL-2 release assessment. Results: Small molecule Ryvu HPK1 inhibitors block kinase activity of recombinant mouse and human protein with nanomolar IC50 values. Ryvu compounds show broad kinome selectivity. Ryvu HPK1 inhibitors selectively engage downstream biomarkers in human and murine T cells. While inhibiting phosphorylation of Serine 376, Ryvu compounds do not affect activatory phosphorylation of Tyrosine 128 of SLP-76 in human or mouse CD3+ T cells. Ryvu HPK1 inhibitors overcome PGE-2 induced resistance following TCR activation in human PBMCs, CD3+ T-cells and mouse CD3+ T cells, inducing IL-2 release. Compounds have good druglike physicochemical properties. Conclusion: Ryvu HPK1 inhibitors promote activation of in-vitro immunostimulatory properties of both mouse and human immune cells, overcoming immunosuppression. The chemical series has the potential to show anti-tumor efficacy in syngeneic animal models as a single agent or in combination with checkpoint inhibitors. Citation Format: Stefan Chmielewski, Maciej Kujawa, Eliza Zimoląg, Paweł Guzik, Agata Dudek, Grzegorz Topolnicki, Sylwia Sudoł, Agnieszka Gibas, Marta Bugaj, Kostiantyn Krolenko, Marcin Nowogródzki, Anita Janiga, Przemysław Wyrębek, Jakub Pięta, Aleksandra Brzdonkiewicz, Grzegorz Wilkowski, Marcin Walczak, Katarzyna Maciejewska, Adam Radzimierski, Wojciech Jasnosz, Tushar Mahajan, Roberta Bartolotta, Karolina Gluza, Patryk Kret, Ewelina Rutkowska, Kinga Michalik, Katarzyna Banaszak, Adrian Podkowa, Aniela Gołas, Katarzyna Wnuk-Lipińska, Charles Fabritius, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Anna Bartosik, Monika Dobrzańska. Development and characterization of small molecule HPK1 inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1947.

Abstract 5641: CD28 expression on multiple myeloma cells enhances the cytotoxic activity of CD38/CD28xCD3 trispecific T cell engager

Abstract SAR442257 (CD38/CD28xCD3) is a trispecific T cell engager designed for the treatment of multiple myeloma (MM). Its cytotoxic activity and anti-tumor efficacy is dependent on the expression of CD38 by malignant plasma cells and the recruitment and activation of T cells via CD3 and CD28. Here we show that MM cells also express CD28 on their membrane and investigate how this expression impacts the cytotoxic activity of the trispecific antibody. Using CRISPR, CD28 knock-out (KO) models were generated from two MM cell lines that have different receptor densities of CD38 and CD28: KMS-11 (CD38 ~3 000/cell; CD28 ~35 000/cell) and RPMI8226 (CD38 ~129 000/cell; CD28 ~84 000/cell). T-cell-mediated cytotoxicity was evaluated in vitro using either effector CD8+ T cells or total PBMC isolated from healthy donors. Binding of SAR442257 to tumor cells in the presence of saturating concentrations of anti-CD38 antibodies was evaluated by flow cytometry using APC-labeled SAR442257 and FITC-labeled anti-CD38 antibodies. In vitro killing assays showed that SAR442257 is active on both CD38 high and CD38 low MM cell lines (EC50 in the pM range for WT cell lines; Table 1). Following CD28 KO, SAR442257 remained active but displayed reduced cytotoxic activity as compared to WT CD38+ CD28+ cells. The reduced cytotoxic activity was noticeable in both CD38 low and CD38 high MM models suggesting that the expression of CD28 by MM cells contributes to the cytotoxic activity of the trispecific antibody. We further show that when other anti-CD38 antibodies are bound to CD38, binding of SAR442257 to tumor cells and T-cell mediated cytotoxicity are rescued by CD28 as evidenced by loss of binding and cytotoxic activity in CD28KO cells. Overall, these data show that SAR442257 is active on both CD38 high and low MM models and that CD28 expressed on MM tumor cells can be directly targeted by SAR442257, enhancing its cytotoxicity and allowing it to bind MM cells when CD38 is occupied by other anti-CD38 antibodies Table 1.T cell mediated killing assays. EC50rel, Relative EC50; CI, Confidence interval.EC50rel (pM) [±CI95%]KMS-11RPMI8226WTCD28KOWTCD28KOEffector CD8+ T cells4.1 [2.9 ; 5.9]30.2 [16.9 ; 54.0]0.2 [0.1 ; 0.3]0.5 [0.3 ; 0.8]Total PBMC26.0 [7.8 ; 87]>250032.0 [14.8 ; 69.2]304.0 [118.0 ; 787.0] Citation Format: Nizar El-Murr, Christophe Henry, Elisa Francesconi, Florence Attenot, Angela Virone-Oddos, Laurent Vidard, Lan Wu, Zhi-Yong Yang, Marielle Chiron. CD28 expression on multiple myeloma cells enhances the cytotoxic activity of CD38/CD28xCD3 trispecific T cell engager [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5641.

Rapid protocol for in vitro induction of cytokine associated with Th2 and Th17 cells from PBMC

Abstract T helper cell is a key component of the immune system. They mediate humoral and cellular immune responses against pathogens and other diseases like cancer by inducing and activating effector cells such as B cells, cytotoxic T cells and innate cells. They are also involved in the regulation of immune responses by controlling the persistence and magnitude of the response. Activation of naïve T cell is initiated by interaction with antigen presenting cells. The downstream signal transduction and the cytokine milieu can lead to their proliferation and differentiation into different T cell subtypes. To facilitate the study of Th2 and Th17 cells, we present a rapid protocol to efficiently induce cytokine associated with Th2 and Th17 cells from total PBMC. Canonical anti-CD3 and anti-CD28 clones are used to activate the purified cells. Different sets of bioassay-validated cytokines, including IL-12, IL-4, IL-6, IL-1β, IL-2, and TGF-β1 are used to induce cytokine associated with Th2 and Th17 cells. Hallmark cytokine and transcription factor expression profile of different lineages are evaluated by Flow Cytometry. In summary, we have successfully developed a rapid protocols to induce cytokine expression in vitro associated with Th2 and Th17 using high quality reagents developed in BioLegend.

Correlation of immune-related adverse events with peripheral baseline immune markers and overall survival in patients with metastatic melanoma on ipilimumab and chemotherapy.

9561 Background: Very few predictive markers have been validated for immune-related adverse events (irAE), which are the number one reason for cessation of treatment and treatment-related deaths on checkpoint inhibitors. In a phase II trial of combined ipilimumab (ipi) with carboplatin/paclitaxel (C/P) (NCT01676649), we sought to determine baseline peripheral blood markers predictive of irAEs and to correlate these adverse with subsequent survival. Methods: Thirty patients with untreated unresectable/metastatic melanoma were treated with C (AUC = 6) and P (175mg/m2) every 3 weeks x 5 and Ipi (3mg/kg) every 3 weeks x 4. irAEs were graded according to the CTCAE v5.0. Median follow-up was 60 months. Baseline blood markers was assessed by a 37 multiplex cytokine-chemokine platform (Meso Scale Discovery) and multiparameter flow cytometry. Results: Grade 2 or higher irAE occurred in 11/30 of patients (4 colitis, 2 hepatitis, 2 hypophysitis, 3 rash). Median survival was 43.8 months in patients with irAE and 14.5 months for those without irAE (p = 0.001). Pts with irAE had lower baseline circulating levels of IL-6 (3.6 vs. 19.8 pg/mL, p = 0.049), TNF alpha (7.6 vs. 11.6 pg/mL, p = 0.003), CXCL9 (83.4 vs. 116.8 pg/mL, p = 0.042) and CCL3 (52.3 vs. 76.4 pg/mL, p = 0.012), but higher baseline circulating dendritic cells (DC) (4.9 vs. 2.9 % of total PBMC, p = 0.002) and a lower level of myeloid-derived suppressor cells (MDSC) (0.8 vs. 1.7 %, p = 0.049). There was no correlation between baseline B cell circulating numbers or differentiation and irAEs. Conclusions: Although results from previous studies of ipi monotherapy vary, our small study shows a highly significant correlation between irAEs and improved long term outcome for patients treated with ipi/C/P. Our exploratory analysis also identifies putative predictive immune and cytokine-chemokine markers of irAEs, which are associated with: 1) Antigen presentation dysregulation (high DC cells) and lack of adequate auto-immune suppression (low MDSC), as also seen in classic autoimmune diseases; and 2) A low inflammatory baseline signature (low TNF, IL-6, CCL3, CXCL9) consistent with possible immune exhaustion and reduced lymphocytic homeostatic fitness. Our combined markers depict a dysregulated immune landscape favorable to the expansion of self-reactive immune cells under CTLA-4 blockade. Our results are hypothesis generating and require further evaluation in a larger group of patients, including PD-1 treated cohorts. Clinical trial information: NCT01676649.

Identification of T-cell subsets associated with muscle strength in people with cancer: A pilot study

Abstract Background Immune deficiency and muscle wasting are both associated with poor prognosis in people with cancer. Survival and quality of life can be improved by attenuating either the immune response or muscle mass, suggesting a relationship between them. Previous data show that mice injected with precursor CD4 T cells maintain muscle mass by restoring immune homeostasis. These data are consistent with the hypothesis that robust anti-cancer T cell immunity leads to improved muscle health. As a first step, this study aims to determine if circulating T cell subsets correlate with muscle strength in people with cancer. Methods The frequency(freq) of T cell subsets was quantified in 11 men with gastrointestinal cancer, and 9 men without cancer, using flow cytometry. Total PBMC protein and gene expression was analyzed using qPCR and Western blots. Clinical measures were assessed using: DEXA scan (body composition); Hand-grip (HGS) test and Knee extension test (Muscle Strength); Stair Climb test (Performance) and Karnofsky and ECOG scales (functional impairment). Data was analyzed using Spearman’s correlation and ANOVA. Results Our data show significant correlations between i) higher freq of CD8 naïve and EM T cells, and lower freq of CD8 CM T cells with stronger HGS, ii) lower freq of Tregs with greater lean mass index, iii) lower freq of CD8 cells expressing CD95 with greater Stair Climb Power, iv) higher freq of CD197+CD45RA+ T cells and greater knee extension strength, and iv) higher expression of CD4 in whole blood with greater functional impairment in people with cancer. Conclusions We have identified significant correlations between levels of T cell populations and muscle strength, performance, and body composition in people with cancer.

A-104 Tracing the intracellular journey of HTLV-1 HBZ during infection: From asymptomatic carriers to HAM/TSP ending to ATL: A one-way ticket?

HTLV-1 is the etiological agent of Adult T cell Leukemia (ATL) and of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) neurologic disorder. Although substantial progress has been made in understanding some cellular and molecular mechanisms underlying the pathology of the neoplastic and neurological diseases, still the intimate reason why some HTLV-1 infected patients develop HAM/TSP and others progress toward the neoplastic state are largely elusive. Two viral proteins, Tax-1 and HBZ, play an important role in both diseases. For example, Tax-1, the crucial transactivator needed for viral replication, has been implicated in the initiation but not in the maintenance of neoplastic process since leukemic cells may not express it. On the other hand, HBZ seems to be expressed throughout the infection and, importantly, is always expressed in leukemic cells. Nevertheless, the precise mechanism through which HBZ participates to initiation and/or maintenance of leukemic state is still unclear, as it is unclear the association of HBZ expression with initiation and/or maintenance of the inflammatory syndrome leading to HAM/TSP. Studies of this aspect have been partially prevented by the lack of suitable reagents to detect endogenous HBZ protein during the various phases of HTLV-1 infection and disease development. The recent isolation of a specific monoclonal antibody against HBZ, the 4D4-F3 mAb, by our group has allowed to precisely quantify and determine the subcellular distribution of the viral protein. We have demonstrated that HBZ, previously considered an exclusive nuclear protein, is mainly localized in the cytoplasm of PBMCs from HAM/TSP patients. Here, the analysis of a larger panel of patients has added new relevant informations on this point. PBMC from 13 HTLV-1 infected asymptomatic carriers (AC), 14 HAM/TSP and 4 ATL patients were analysed. Expression and subcellular distribution of endogenous HBZ and Tax-1 proteins were assessed by confocal microscopy with the 4D4-F3 and A51-2 mAbs, respectively. HBZ expression was confined to the cytoplasm of PBMC of HAM/TSP patients, appearing as discrete dots. The percentage of HBZ-positive cells ranged between 1% and 10% of total PBMC with the exception of one negative patient. Tax-1 was expressed in all HAM/TSP cases analyzed, ranging from 2% to 15% of total PBMC. Tax-1 was preferentially localized in the cytoplasm with fewer speckle-like dots in the nucleus. Interestingly, only in 1 case co-expression of HBZ and Tax-1 was observed in the same cells. In this unique case, however, although 8% and 15% of the cells were positive for HBZ and Tax-1, respectively, only 1% of cells co-expressed the 2 viral proteins. Of particular interest was the finding that PBMC of 6 out of 13 AC expressed HBZ (1%–4% of the cells) and again with an exclusive cytoplasmic localization. Tax-1 was expressed in 10 out of 13 cases (1%–11% of the cells). Only in 2 AC, HBZ and Tax-1 were found to be co-expressed in a very limited proportion of cells. The study of 4 distinct cases of ATL demonstrated, instead, that HBZ protein was expressed in the vast majority of the leukemic cells and importantly with an exclusive nuclear localization. Interestingly, none of these ATL cases were found to express Tax-1 protein. These results extend our previous observation on the dichotomy of HBZ localization between HAM/TSP and ATL, pointing to the exclusive either cytoplasmic or nuclear localization in the 2 diseased states, respectively. Moreover, they show a rather selective expression in distinct cells of either HBZ or Tax-1. The unprecedented observation that in AC, HBZ is expressed only in the cytoplasm strongly suggests a progressive modification of HBZ localization during the disease states associated to HTLV-1infection. Future studies will clarify whether the distinct HBZ intracellular localization is simply a marker or a causative event of disease evolution.

The In Vivo infected Clones Have Mixed Immunophenotype and Malignant Transformation Is Characterized By Dominant Growth of CD4+CCR4+CD26-CD7- Cells in Human T Lymphotropic Virus Type 1 Infection

Aggressive Adult T cell leukemia/lymphoma (ATL), a human T lymphotropic virus type 1 (HTLV-1) -associated disease, has a poor prognosis. There is an urgent need for effective prevention and treatment. Aggressive ATL develops in patients with non-malignant HTLV-1 (asymptomatic carriers (AC) and patients with HTLV-1 associated myelopathy (HAM)) over decades with evolution from high proviral load (PVL) and non-dominant clonal growth through emergence of dominant clones and indolent to aggressive ATL. A single dominant clone is a major contributor to HTLV-1 infection burden in ATL despite a polyclonal background whilst hundreds of small clones contribute relatively equally to infection burden in non-malignant HTLV-1 infection(1). CD4+CCR4+CD26-CD7- is the dominant immunophenotype of lymphocytes in patients with ATL (ATL cells) but infected cells with similar immunophenotype (‘ATL-like’ cells) are also present in patients with non-malignant HTLV-1 infection(2). We hypothesize that ‘ATL-like’ infected cells harbor: larger HTLV-1 infected clones detected in total PBMC and are more oligo-clonal compared to total PBMC in patients with high PVL non-malignant HTLV-1 infection and; only the dominant clone detected in total PBMC in patients with ATL. The aim is to study the relationship between clonality and immunophenotype in patients with high PVL non-malignant HTLV-1 infection and ATL.Clonality analysis was performed on total PBMC by ligation mediated PCR followed by high throughput sequencing (LMPCR-HTS) and on sorted CD4+CCR4+CD26-CD7- cells by LMPCR-HTS and T-cell receptor (TCR) sequencing in ACs (four and nine respectively), patients with HAM (four and nine respectively) and patients with ATL (four and nine respectively). Clonality analysis within CD4+CCR4+CD26-CD7- cells, similar to PBMC, showed hundreds of non-dominant clones (relative abundance of largest clone ≤ 25%) in 83% of patients with high PVL non-malignant HTLV-1 infection and a dominant clone (relative abundance of largest clone ≥ 25%) over a background of non-dominant clones in 17% and 100% of patients with non-malignant HTLV-1 infection and ATL respectively. CD4+CCR4+CD26-CD7- cells contributed a median of 95% of the largest PBMC clone in patients with ATL and a median of 23% of the largest PBMC clone in 63% of patients with non-malignant HTLV-1 infection. Cells which do not have ‘ATL-like’ immunophenotype (CD4+CCR4- and CD4+CCR4+CD26-CD7+) were also detected alongside CD4+CCR4+CD26-CD7- cells within individual clones in patients with non-malignant HTLV-1 infection as shown in figure 1. Three out of eighteen patients with high PVL non-malignant HTLV-1 infection had two or more dominant clones, one of which showed signs of overt ATL 12 months after the sample data. Clonality analysis in an patient with incident aggressive ATL showed presence of two dominant clones with two integrated provirus each 3 months prior to diagnosis and single dominant clone at presentation, remission and relapse as shown in figure 2. There was no significant difference in distribution of viral integration site by LMPCR-HTS or TCR repertoire in patients with non-malignant HTLV-1 infection and ATL.In summary, ‘ATL-like’ cells are derived from relatively small non-dominant clones in the majority of patient with high PVL non-malignant HTLV-1 infection and from a dominant clone over a non-dominant background in patients with ATL. The dominant clone in patients with ATL was comprised almost universally of ‘ATL-like’ cells whilst the large PBMC clones in patients with non-malignant HTLV-1 infection had cells with a mixed immunophenotype including ‘ATL-like’ and non- ‘ATL-like’ cells. Oligoclonal proliferation of ‘ATL-like’ cells precedes monoclonal proliferation at clinical presentation of ATL. Clonality analysis in patient with high PVL non-malignant HTLV-1 infection might help in further prognostication in malignant risk.Cook LB, Melamed A, Niederer H, Valganon M, Laydon D, Foroni L, et al. The role of HTLV-1 clonality, proviral structure, and genomic integration site in adult T-cell leukemia/lymphoma. Blood. 2014;123(25):3925-31.Kagdi H, Demontis MA, Ramos JC, Taylor GP. Switching and loss of cellular cytokine producing capacity characterize in vivo viral infection and malignant transformation in human T- lymphotropic virus type 1 infection. PLoS pathogens. 2018;14(2):e1006861. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immortalization of splenic and peripheral blood macrophages using a multi-cistronic v-Raf/v-Myc lentivirus.

Abstract We have created an improved system for immortalizing mature macrophages by modifying the original v-raf/v-myc (J2) virus. Previously, these J2 viruses required a producer co-culture condition and maintained replicating virus. This provided more complexity, reagents, and biosafety risks. Our improved virus is replication deficient. Its lentiviral genome contains the v-raf/v-myc oncogenes and a thy1.1 surface marker, each of which is separated by a self-cleaving peptide. This virus, therefore, eliminates the issues described above. We transduced our modified virus into total murine PBMC isolated from murine peripheral blood or spleen. We found the transduction and successful cell line production can be done with as few as 1×105 PBMC. The resulting cytokine independent macrophage cell line from both spleen and blood expressed mature macrophage markers (F4-80+CD11b+) but lacked early myeloid progenitor markers (Sca1−c-kit−). In addition, we found these macrophages were functional as demonstrated by their ability to undergo phagocytosis and ability to upregulate MHCII in response to IFNγ. These findings demonstrate that the v-raf and v-myc oncogenes in our new system can immortalize macrophages from spleen but uniquely, and perhaps more importantly, can immortalize macrophages from blood. This advance provides a non-invasive method to obtain the starting tissue needed to make these macrophage cell lines. Having the v-raf/v-myc oncogenes in a lentivirus means it is likely this method will work on blood from humans and other species. Additionally, the cytokine independence associated with cell lines made with our virus make it ideal for creating macrophage cell lines from mammals without commercially available growth factors.

IL-28B Genetic Variants Determine the Extent of Monocyte-Induced Activation of NK Cells in Hepatitis C.

BackgroundImmuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B genotype and NK cell activity.MethodsA total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used.ResultsFollowing stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals.ConclusionsOur data indicate that monocytes from carriers of an IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions.

An optimized IFN-γ ELISpot assay using T-activated ® proteins for determination of CMV-specific cell-mediated immunity

Abstract In healthy individuals, Cytomegalovirus (CMV) infections are controlled by CMV-specific cell-mediated immunity (CMI). However, functional impairment of the CMI in immunocompromized individuals can lead to uncontrolled CMV-replication. Thus, monitoring CMV-specific CMI is highly relevant for the prognosis of CMV-associated diseases. Here we show the optimization and technical validation of a monitoring tool for the determination of CMV-specific CMI. Phenotype, quantity and cytokine expression profile of T cells from 19 CMV-seropositive healthy donors reactivated by CMV (IE-1, pp65) -specific pools of overlapping 15mer peptides and the corresponding T-activated proteins® were compared using a 10-parameter flow cytometry-based intracellular cytokine staining assay. Subsequently, an IFN-g ELISpot protocol using T-activated proteins® was established and validated. Pp65 peptide pool stimulation revealed significant higher CD8+ T-cell responses as the corresponding T-activated proteins® (p=0.006). CD4+ T-cell responses however were comparable. Testing of T-activated proteins® in seronegative donors revealed a specificity of 100%. Use of T-activated proteins® with the optimized IFN-g ELISpot resulted in coefficients of variation for inter-assay, inter-operator and inter-site variations of less than 22 %. A linear correlation between the amount of CMV protein-reactive cells and total PBMC counts was observed. Given the natural antigen processing of T-activated proteins® along the cross-presentation pathway, reflecting the in vivo situation, these proteins in combination with the optimized IFN-g ELISpot serve as a valuable tool to monitor the CMV-specific CMI.

CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel.

BackgroundChronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection.MethodologyCD4+CD25hiFOXP3+ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE.Principal Findings S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only.Conclusions Schistosoma-associated CD4+CD25hiFOXP3+Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.

Ibrutinib Treatment of Relapsed CLL Following Allogeneic Transplantation: Sustained Disease Response and Promising Donor Immune Modulation

Abstract Background: Treatment of relapsed chronic lymphocytic leukemia (CLL) with ibrutinib results in high rates of progression-free and overall survival. Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase (BTK), is FDA-approved for use in relapsed CLL. Ibrutinib may also modulate donor T cell alloimmunity via inhibition of interleukin-2-inducible kinase (ITK). Here we report the effects of ibrutinib salvage therapy in 5 CLL patients who relapsed following allogeneic hematopoietic cell transplantation (allo-HCT). In addition to minimal residual disease (MRD) response measurements, we also assessed donor T cell chimerism and donor B cell immune reconstitution following ibrutinib therapy. Methods: Five patients with high-risk CLL relapsed 1-8.5 yrs following allo-HCT. Four patients had never achieved donor CD3 T cell chimerism >95% following reduced-intensity transplant. Ibrutinib 420mg daily was started 1 mo-2 yrs after clinical relapse. Four of the 5 patients remain on ibrutinib with treatment courses ranging from 3-17 mos. CLL MRD was measured by IgH high-throughput sequencing (HTS) using the ClonoSIGHTTM test (Sequenta, Inc.), which has the sensitivity to detect 1 CLL clone per million leukocytes. Lymph node (LN) size was assessed by CT scan and reported as the sum of the products of the LN diameters (SPD). Donor B cell reconstitution was determined by IgH HTS quantification of total IgH molecules and unique IgH clonotypes. Results: Lymphocytosis was observed in all 5 patients following initiation of ibrutinib treatment, consistent with previous reports. In 2 patients who received >1 yr of ibrutinib treatment, lymphocytosis peaked at 3 wks and 8 wks after initiation of treatment and slowly declined thereafter, fully resolving within 1 yr (Fig 1A). All 4 patients with pathologic lymphadenopathy prior to treatment experienced dramatic LN reduction (Fig 1B; 68% average reduction in LN size after 3 mos on ibrutinib). The longest duration of follow-up is reported for patient SPN 3975, who had a 17p deletion and received ibrutinib for 39 mos. Treatment was discontinued after CLL MRD became undetectable by ClonoSIGHT (Fig 1C). Evidence of donor T cell immune modulation included achievement of full donor CD3 chimerism after 1 yr and resolution of oral and skin chronic graft-versus-host disease (GVHD) after 6 mos. Although this patient has been off ibrutinib for >8 mos, full donor chimerism persists and CLL MRD remains undetectable (Fig 1C). Before ibrutinib treatment, donor B cells (excluding the patient’s CLL clone) accounted for 1% of PBMC (Fig 1D). Furthermore, recovering B cells have diverse, low frequency IgH clonotypes (Fig 1E). Together, our findings show rapid, sustained, and diverse immune reconstitution without CLL recurrence following ibrutinib discontinuation. Conclusions: Ibrutinib provides effective salvage therapy for CLL relapse following allo-HCT. Post-transplant CLL relapse is often extra-nodal and our experience shows ibrutinib is effective in clearing both nodal and extra-nodal disease. Here we present one patient who stopped therapy after achieving MRD negativity and maintains undetectable disease 8 mos following ibrutinib discontinuation. Ibrutinib treatment demonstrates promising donor immune modulation by promoting full donor chimerism and resolution of chronic GVHD. Our five patient experience supports using ibrutinib in relapsed CLL patients following allo-HCT. Clinical trials are needed to determine the best duration of ibrutinib therapy following post-allo-HCT relapse, chronic GVHD treatment efficacy, and the role of ibrutinib maintenance following allo-HCT. Download : Download high-res image (120KB) Download : Download full-size image Figure 1 . (A) Percent change in absolute lymphocytic count (ALC) for 2 patients who received ibrutinib treatment for >1 yr. SPN = Stanford Patient Number. (B) Percent reduction in LN size, as reported by the sum of the product of LN diameters (SPD) for 4 patients following initiation of ibrutinib. (C) CLL MRD (reported as a percentage of WBCs) and blood donor CD3 T cell levels shown for patient SPN 3975. (D) B cells (excluding the CLL clone) as percent of total PBMC for patient SPN 3975 as measured by IgH HTS. (E) Total IgH molecules and unique IgH clone counts for patient SPN 3975 at different time points (D=day) post allo-HCT. Disclosures Kong: Sequenta, Inc.: Employment, Equity Ownership. Klinger: Sequenta, Inc.: Employment, Equity Ownership. Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Coutre: Pharmacyclics, Inc.: Consultancy, Research Funding. Miklos: Sequenta, Inc.: Research Funding.

Ibrutinib Treatment of Relapsed CLL Following Allogeneic Transplantation: Sustained Disease Response and Promising Donor Immune Modulation

Background: Treatment of relapsed chronic lymphocytic leukemia (CLL) with ibrutinib results in high rates of progression-free and overall survival. Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase (BTK), is FDA-approved for use in relapsed CLL. Ibrutinib may also modulate donor T cell alloimmunity via inhibition of interleukin-2-inducible kinase (ITK). Here we report the effects of ibrutinib salvage therapy in 5 CLL patients who relapsed following allogeneic hematopoietic cell transplantation (allo-HCT). In addition to minimal residual disease (MRD) response measurements, we also assessed donor T cell chimerism and donor B cell immune reconstitution following ibrutinib therapy.Methods: Five patients with high-risk CLL relapsed 1-8.5 yrs following allo-HCT. Four patients had never achieved donor CD3 T cell chimerism >95% following reduced-intensity transplant. Ibrutinib 420mg daily was started 1 mo-2 yrs after clinical relapse. Four of the 5 patients remain on ibrutinib with treatment courses ranging from 3-17 mos. CLL MRD was measured by IgH high-throughput sequencing (HTS) using the ClonoSIGHTTM test (Sequenta, Inc.), which has the sensitivity to detect 1 CLL clone per million leukocytes. Lymph node (LN) size was assessed by CT scan and reported as the sum of the products of the LN diameters (SPD). Donor B cell reconstitution was determined by IgH HTS quantification of total IgH molecules and unique IgH clonotypes.Results: Lymphocytosis was observed in all 5 patients following initiation of ibrutinib treatment, consistent with previous reports. In 2 patients who received >1 yr of ibrutinib treatment, lymphocytosis peaked at 3 wks and 8 wks after initiation of treatment and slowly declined thereafter, fully resolving within 1 yr (Fig 1A). All 4 patients with pathologic lymphadenopathy prior to treatment experienced dramatic LN reduction (Fig 1B; 68% average reduction in LN size after 3 mos on ibrutinib). The longest duration of follow-up is reported for patient SPN 3975, who had a 17p deletion and received ibrutinib for 39 mos. Treatment was discontinued after CLL MRD became undetectable by ClonoSIGHT (Fig 1C). Evidence of donor T cell immune modulation included achievement of full donor CD3 chimerism after 1 yr and resolution of oral and skin chronic graft-versus-host disease (GVHD) after 6 mos. Although this patient has been off ibrutinib for >8 mos, full donor chimerism persists and CLL MRD remains undetectable (Fig 1C). Before ibrutinib treatment, donor B cells (excluding the patient’s CLL clone) accounted for <0.2% of total PBMC as determined by IgH HTS. Following discontinuation of ibrutinib, the percentage of donor B cells increased within 6 months, and now comprises >1% of PBMC (Fig 1D). Furthermore, recovering B cells have diverse, low frequency IgH clonotypes (Fig 1E). Together, our findings show rapid, sustained, and diverse immune reconstitution without CLL recurrence following ibrutinib discontinuation.Conclusions: Ibrutinib provides effective salvage therapy for CLL relapse following allo-HCT. Post-transplant CLL relapse is often extra-nodal and our experience shows ibrutinib is effective in clearing both nodal and extra-nodal disease. Here we present one patient who stopped therapy after achieving MRD negativity and maintains undetectable disease 8 mos following ibrutinib discontinuation. Ibrutinib treatment demonstrates promising donor immune modulation by promoting full donor chimerism and resolution of chronic GVHD. Our five patient experience supports using ibrutinib in relapsed CLL patients following allo-HCT. Clinical trials are needed to determine the best duration of ibrutinib therapy following post-allo-HCT relapse, chronic GVHD treatment efficacy, and the role of ibrutinib maintenance following allo-HCT. [Display omitted] DisclosuresKong:Sequenta, Inc.: Employment, Equity Ownership. Klinger:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Coutre:Pharmacyclics, Inc.: Consultancy, Research Funding. Miklos:Sequenta, Inc.: Research Funding.

Switch to raltegravir‐based regimens and HIV DNA decrease in patients with suppressed HIV RNA

IntroductionRaltegravir intensification is associated with an increase in 2-LTR episomal HIV DNA= circles, indicating a persistent low-level replication, in some individuals in ART with suppressed HIV RNA. We aimed at monitoring residual plasma HIV RNA and cellular HIV DNA in virologically suppressed patients switching to a raltegravir-based regimen.Materials and MethodsForty-six HIV-infected subjects on PI or NNRTI based-regimens, with plasma HIV RNA level <40 copies/mL for ≥6 months and CD4 >200 cells/µL for ≥12 months were enrolled. Thirty-four patients switched to raltegravir-based regimen (RASTA study group) and 12 continued a PI or NNRTI based-regimen (control group). Ultrasensitive HIV residual viremia and total PBMC HIV DNA were assessed at baseline (W0), 24 (W24) and 48 (W48) weeks. HIV RNA levels were determined by an ultrasensitive test derived from a commercial real time PCR (limit of detection 5 copies/ml). A real time PCR was used to quantify HIV DNA copy numbers in PBMCs.ResultsAt W0, HIV DNA was detected in all patients while at W48 it was detectable in 82.3% of RASTA group vs 100% of controls (p=0.01). The difference between the average values of HIV DNA log10 copies/10°6 CD4 at W0 (median 3.11, IQR 2.70–3.45) and W48 (median 2.87, IQR 2.24–3.38) was statistically significant for RASTA group (p=0.035). Male gender (mean difference −0.37 log10 copies/10°6 PBMC, p=0.023) and previous PI based-ART (mean difference +0.39 log10 copies/10°6 PBMC, p=0.036) were predictive of HIV DNA level at W0. After adjusting for previous PI based-ART, male gender was the only variable independently associated with HIV DNA size at W0 (mean difference −0.326 log10 copies/10°6 PBMC, 95% CI −0.641, −0.011 p=0.043). Ultrasensitive HIV-1 RNA was detectable at W0 in 50% of RASTA group versus 66.7% of controls and at W48 in 32.4% versus 45.5%, respectively. No differences were found between HIV RNA levels at W0 and W48 within and between the two groups.ConclusionsSwitching to raltegravir-based regimens may be associated with a decrease of HIV reservoir, as measured by total PBMC HIV DNA. A larger sample size is required to confirm this finding.

Clonality of HTLV-1 differs between infected CD8+ T cells and infected CD4+ T cells in vivo

HTLV-1 selectively infects CD4+ T cells in vivo, with a minor population carried in CD8+ T cells. We previously established a method for the analysis of the clonality (clone frequency distribution) of infected cells by mapping and quantifying HTLV-1 proviral integration sites using high-throughput sequencing (Gillet et al, Blood, 2011; Cook et al, Blood, 2012; Melamed et al, PLoS Pathogens, 2013). To test the hypothesis that the clonality of HTLV-1 differs between infected CD8+ T cells and infected CD4+ T cells in natural infection, we magnetically sorted CD4+ and CD8+ T cells from PBMCs of 12 HTLV-1-infected individuals: 6 patients with HAM/TSP and 6 asymptomatic HTLV-1 carriers. We then used our high-throughput sequencing technique to quantify HTLV-1 clonality in each cell population. The median proviral load in CD4+ T cells and CD8+ T cells was 12.3 copies (range 6.0-30.2) and 2.0 (1.1-6.2) copies per 100 cells, respectively. The median proportion of the load carried by the infected CD8+ cells was 5.0% (2.3%-35.3%). Proviral load in CD8+ cells and in CD4+ cells significantly correlated with proviral load in total PBMC (p<10^-6 and p<10^-3, respectively). The clone frequency distribution was significantly more oligoclonal in CD8+ cells than in CD4+ cells: infected CD8+ clones were significantly over-represented among the most abundant clones in the blood. We conclude that HTLV-1-infected CD8+ T cells have a clonal distribution distinct from infected CD4+ cells. These results show that infected CD8+ T cells contribute disproportionately to the high PVL seen in HTLV-1 infection in vivo.