Intracellular IFN-γ Production Research Articles

Enhanced Immunomodulatory Effects of Thymosin-Alpha-1 in Combination with Polyanionic Carbosilane Dendrimers against HCMV Infection.

Resistance and toxicity associated with current treatments for human cytomegalovirus (HCMV) infection highlight the need for alternatives and immunotherapy has emerged as a promising strategy. This study examined the in vitro immunological effects of co-administration of Thymosin-alpha-1 (Tα1) and polyanionic carbosilane dendrimers (PCDs) on peripheral blood mononuclear cells (PBMCs) during HCMV infection. The biocompatibility of PCDs was assessed via MTT and LDH assays. PBMCs were pre-treated with the co-administered compounds and then exposed to HCMV for 48 h. Morphological alterations in PBMCs were observed using optical microscopy and total dendritic cells (tDCs), myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs), along with CD4+/CD8+ T cells and regulatory T cells (Treg), and were characterized using multiparametric flow cytometry. The findings revealed that Tα1 + PCDs treatments increased DC activation and maturation. Furthermore, increased co-receptor expression, intracellular IFNγ production in T cells and elevated Treg functionality and reduced senescence were evident with Tα1 + G2-S24P treatment. Conversely, reduced co-receptor expression, intracellular cytokine production in T cells, lower functionality and higher senescence in Treg were observed with Tα1 + G2S16 treatment. In summary, Tα1 + PCDs treatments demonstrate synergistic effects during early HCMV infection, suggesting their use as an alternative therapeutic for preventing virus infection.

Granulocyte-Macrophage-Colony-Stimulating-Factor Combined with Prostaglandin E1 Create Dendritic Cells of Leukemic Origin from AML Patients' Whole Blood and Whole Bone Marrow That Mediate Antileukemic Processes after Mixed Lymphocyte Culture.

Although several (chemotherapeutic) protocols to treat acute myeloid leukemia (AML) are available, high rates of relapses in successfully treated patients occur. Strategies to stabilize remissions are greatly needed. The combination of the (clinically approved) immune-modulatory compounds Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) and Prostaglandine E1 (PGE-1) (Kit-M) converts myeloid blasts into dendritic cells of leukemic origin (DCleu). After stimulation with DCleu ex vivo, leukemia-specific antileukemic immune cells are activated. Therefore, Kit-M treatment may be an attractive immunotherapeutic tool to treat patients with myeloid leukemia. Kit-M-mediated antileukemic effects on whole bone marrow (WBM) were evaluated and compared to whole blood (WB) to evaluate the potential effects of Kit-M on both compartments. WB and WBM samples from 17 AML patients at first diagnosis, in persisting disease and at relapse after allogeneic stem cell transplantation (SCT) were treated in parallel with Kit-M to generate DC/DCleu. Untreated samples served as controls. After a mixed lymphocyte culture enriched with patients' T cells (MLC), the leukemia-specific antileukemic effects were assessed through the degranulation- (CD107a+ T cells), the intracellular IFNγ production- and the cytotoxicity fluorolysis assay. Quantification of cell subtypes was performed via flow cytometry. In both WB and WBM significantly higher frequencies of (mature) DCleu were generated without induction of blast proliferation in Kit-M-treated samples compared to control. After MLC with Kit-M-treated vs. not pretreated WB or WBM, frequencies of (leukemia-specific) immunoreactive cells (e.g., non-naive, effector-, memory-, CD3+β7+ T cells, NK- cells) were (significantly) increased, whereas leukemia-specific regulatory T cells (Treg, CD152+ T cells) were (significantly) decreased. The cytotoxicity fluorolysis assay showed a significantly improved blast lysis in Kit-M-treated WB and WBM compared to control. A parallel comparison of WB and WBM samples revealed no significant differences in frequencies of DCleu, (leukemia-specific) immunoreactive cells and achieved antileukemic processes. Kit-M was shown to have comparable effects on WB and WBM samples regarding the generation of DCleu and activation of (antileukemic) immune cells after MLC. This was true for samples before or after SCT. In summary, a potential Kit-M in vivo treatment could lead to antileukemic effects in WB as well as WBM in vivo and to stabilization of the disease or remission in patients before or after SCT. A clinical trial is currently being planned.

CRISPR/Cas9 Gene Editing of MIR155HG in Primary Human T Cells to Prevent Acute Graft-Versus-Host Disease

Abstract Introduction: Acute graft-versus-host disease (GVHD) mediated by donor alloreactive T cells is the leading cause of non-relapse mortality in patients post-allogeneic hematopoietic cell transplantation. Here we genetically engineered donor T cells by genomic deletion of microRNA-155 host gene (MIR155HG) to prevent GVHD. Methods: Using CRISPR/Cas9 genome editing, we targeted exon 3 (Ex3) of MIR155HG. Genomic deletion was evaluated by genomic PCR and RNA expression of mature miR-155 by qRT-PCR. In xenogeneic acute GVHD experiments, NSG mice were irradiated and transplanted with miR-155 Ex3 edited or control T cells to assess functionality in vivo. Histological analysis of target tissues (skin and liver) was performed at end of study. Weekly flow cytometric evaluation of human donor T cells in peripheral blood was performed. MOLM-13 leukemic target cells were cultured with miR-155 Ex3 edited or control CD8 +effector T cells for 72 hours and cytotoxic function was assessed by CD107a degranulation, intracellular IFN-γ production and leukemic cell death by flow cytometry. Results: Ex3 deletion was confirmed by genomic PCR resulting in downregulation of mature miR-155 expression. Recipients of Ex3 deleted human donor T cells display lower GVHD clinical scores, lower average histopathological scores and significantly improved survival compared to mice receiving control T cells. Ex3 T cells displayed potent CTL function comparable to control T cells resulting in leukemic cell death. Conclusion: CRISPR/Cas9 deletion of miR-155 in donor T cells protects against development of lethal GVHD while maintaining beneficial anti-leukemic effect. Our studies present genomic editing of donor T cells as a novel strategy to prevent GVHD. Funding for this project was provided through The Ohio State University Leukemia Research Program to PR and RG, NIH R01CA252469 to PR, and NIH T32CA090223 fellowship to KB.

Abstract 5125: Depletion of CCR8+ tumor Treg cells with SRF114 or anti-CCR8 therapy promotes robust antitumor activity and reshapes the tumor microenvironment toward a more pro-inflammatory milieu

Abstract FOXP3+ regulatory T (Treg) cells play a crucial role in orchestrating immune responses across several tissues, including the tumor microenvironment (TME). The dominant immune regulatory activity of Treg cells is evidenced, both clinically and preclinically, by the profound inflammatory dysregulation that arises in their absence. Leveraging controlled depletion of Treg cells in tumor tissue while sparing Treg cells in healthy tissue may be achieved through antibody-mediated targeting of the C-C chemokine receptor 8 (CCR8). CCR8 is a seven transmembrane G-coupled chemokine receptor protein whose expression is predominantly upregulated on Treg cells in several types of tumors compared with peripheral blood. Importantly, depletion of CCR8-expressing Treg cells in tumors provides a novel pathway to immunotherapy that is mechanistically independent of checkpoint inhibition (CPI). Using single-cell transcriptome and flow cytometry analyses, we evaluated the pharmacodynamic changes after treatment with anti-CCR8 antibodies to elucidate the effect of tumor Treg cell-targeting therapies on the downstream mechanisms of antitumor immunity within the TME. SRF114, a highly selective human anti-CCR8 afucosylated antibody, and an anti-mouse CCR8 antibody were utilized to preferentially deplete CCR8+ Treg cells in human CCR8 knock-in (hCCR8KI) or wild-type mice, respectively. Treatment with SRF114 or anti-mouse CCR8 antibody resulted in robust monotherapy activity in models susceptible to CPI. Moreover, pronounced monotherapy activity was also seen in checkpoint-resistant model(s), highlighting a mechanistically CPI-independent role for intratumoral Treg cell depletion to elicit potent antitumor activity. Treatment of tumor-bearing mice with anti-CCR8 antibodies resulted in a significant reduction of tumor-associated Treg cells, while peripheral Treg cells remained unchanged. Analysis of bulk tumor lysates showed that anti-CCR8 therapy elevated levels of pro-inflammatory cytokines and pro-angiogenic and chemoattracting factors. Evaluation of effector T cells demonstrated that anti-CCR8 therapy drives proliferation of CD8+ T cells and an increase in intracellular cytokine production of IFNγ, TNFα, and granzyme A. Additionally, the tumor myeloid compartment was strongly affected by anti-CCR8 therapy. Multiple myeloid cell subsets had increased levels of co-stimulatory molecules and expression of PD-L1. Altogether, selective depletion of intratumoral CCR8+ Treg cells results in robust antitumor activity by reshaping the TME toward a more pro-inflammatory milieu, providing an orthogonal approach to CPI for eliciting antitumor immunity and a strong rationale for evaluating SRF114 as a therapeutic agent for the treatment of cancer. SRF114 is currently in Phase 1 clinical studies. Citation Format: Marisella Panduro, Yue Ren, Ricard Masia, Yu Yang, Andrew C. Lake, Vito J. Palombella, Jonathan A. Hill, James F. Mohan. Depletion of CCR8+ tumor Treg cells with SRF114 or anti-CCR8 therapy promotes robust antitumor activity and reshapes the tumor microenvironment toward a more pro-inflammatory milieu. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5125.

Helminth species-specific effects on IFN-γ producing T cells during active and latent tuberculosis.

Interferon-γ (IFN-γ) is a key cytokine inducing protective immune responses during tuberculosis (TB) infection. Helminth-induced immune responses may affect IFN-γ production by T cells, although its connection with disease severity and immune recovery during treatment is unexplored. We investigated the species-specific effect of helminths on the IFN-γ production by T cells in relation to disease severity during active and latent TB infection (LTBI). In this study, 69 active pulmonary TB patients (PTB), 28 with LTBI and 66 healthy controls were included. Active TB was diagnosed using GenXpert MTB/RIF while QuantiFERON test (QFT) was used for the screening of healthy community controls (CCs) and for the diagnosis of LTBI. Helminth infection was identified by routine diagnosis whereas clinical disease severity was evaluated by the TB score. Intracellular IFN-γ production of T cells in stimulated peripheral blood mononuclear cells (PBMCs) was analyzed by flow cytometry using TB antigens (PPD), the polyclonal T cell activator staphylococcal enterotoxin B (SEB), or medium as unstimulated control. Helminth infected CCs and LTBI subjects showed a significant reduction of IFN-γ+ CD4+ T cells by PPD-stimulation compared to non-helminth infected control groups. The significant reduction in the frequency of IFN-γ+ T cells in both latent and active PTB patients following SEB stimulation was mostly attributed to Schistosoma mansoni infection, whereas Ascaris lumbricoides, Schistosoma mansoni, and hookworm infection contributed equally in CCs. Following anti-helminthic and anti-TB treatment for 2 months, the frequency of IFN-γ+ CD4 T cells in helminth coinfected PTB was restored to levels of helminth negative PTB before treatment. Helminth coinfected PTB patients with an intermediate and severe clinical course had reduced capacity for production of IFN-γ+ T cells compared to the corresponding non-helminth infected PTB. We found a reduction in IFN-γ producing T cells by helminth coinfection which was restored following anti-helminthic treatment. This reduction was helminth species-dependent in an exploratory sub-analysis and correlated to increased disease severity.

Multiple sclerosis-disease modifying therapies affect humoral and T-cell response to mRNA COVID-19 vaccine.

The mRNA vaccines help protect from COVID-19 severity, however multiple sclerosis (MS) disease modifying therapies (DMTs) might affect the development of humoral and T-cell specific response to vaccination. The aim of the study was to evaluate humoral and specific T-cell response, as well as B-cell activation and survival factors, in people with MS (pwMS) under DMTs before (T0) and after two months (T1) from the third dose of vaccine, comparing the obtained findings to healthy donors (HD). All possible combinations of intracellular IFNγ, IL2 and TNFα T-cell production were evaluated, and T-cells were labelled "responding T-cells", those cells that produced at least one of the three cytokines of interest, and "triple positive T-cells", those cells that produced simultaneously all the three cytokines. The cross-sectional evaluation showed no significant differences in anti-S antibody titers between pwMS and HD at both time-points. In pwMS, lower percentages of responding T-cells at T0 (CD4: p=0.0165; CD8: p=0.0022) and triple positive T-cells at both time-points compared to HD were observed (at T0, CD4: p=0.0007 and CD8: p=0.0703; at T1, CD4: p=0.0422 and CD8: p=0.0535). At T0, pwMS showed higher plasma levels of APRIL, BAFF and CD40L compared to HD (p<0.0001, p<0.0001 and p<0.0001, respectively) and at T1, plasma levels of BAFF were still higher in pwMS compared to HD (p=0.0022).According to DMTs, at both T0 and T1, lower anti-S antibody titers in the depleting/sequestering-out compared to the enriching-in pwMS subgroup were found (p=0.0410 and p=0.0047, respectively) as well as lower percentages of responding CD4+ T-cells (CD4: p=0.0394 and p=0.0004, respectively). Moreover, the depleting/sequestering-out subgroup showed higher percentages of IFNγ-IL2-TNFα+ T-cells at both time-points, compared to the enriching-in subgroup in which a more heterogeneous cytokine profile was observed (at T0 CD4: p=0.0187; at T0 and T1 CD8: p =0.0007 and p =0.0077, respectively). In pwMS, humoral and T-cell response to vaccination seems to be influenced by the different DMTs. pwMS under depleting/sequestering-out treatment can mount cellular responses even in the presence of a low positive humoral response, although the cellular response seems qualitatively inferior compared to HD. An understanding of T-cell quality dynamic is needed to determine the best vaccination strategy and in general the capability of immune response in pwMS under different DMT.

Cell-associated HIV cross-presentation by pDC is potentiated by non-cognate CD8+ T cell pre-activation

Antigens (Ag) from apoptotic HIV-infected cells can be cross-presented by human plasmacytoid dendritic cells (pDC) to CD8 + T cells 1 . We studied the mechanisms leading to this cross-presentation. For this, we generated HLA-A2-restricted HIV-Gag-specific CD8 + T cell clones. HLA-A2 negative, HIV-infected or uninfected, H9 cells were used as Ag donor cells. They were irradiated by UV-C to induce apoptosis. Direct viral presentation was prevented by using saquinavir. DC populations were immunomagnetically and FACS-sorted to >95% purity, from HLA-A2+ or -donor buffy coats. CD8 + T cells IFN-γ secretion or intracellular production were assessed by ELISA or flow cytometry, respectively. Apoptotic H9-HIV cell cross-presentation by pDC led to CD8 + T cells IFN-γ secretion, which was fully MHC-class I-restricted. Surprisingly, a lower level of non-MHC restricted intracellular IFN-γ production was also induced by HLA-A2-negative pDC incubated with HIV-infected cells. Both MHC-Class I-dependent and -independent IFN-γ intracellular productions were partly dependent on TLR-7 signalization, indicating a role for stimulation of TLR7 by HIV. Indeed, H9-HIV, and not H9 cells, induced maturation and production of cytokines by pDC. Among those cytokines, IFN-α or β induced intracellular IFN-γ production when added to the CD8 + T cell clones, in the absence of pDC or H9-HIV cells. To perform cognate activation of the CD8 + T cell clones without viral stimulation, we used CD3-and CD28-bound beads: IFN-γ secretion was stimulated, and was increased by adding these cytokines. Conversely, neutralizing antibodies against these cytokines abolished the non-MHC-restricted intracellular IFN-γ production triggered by pDC incubated with by HIV-infected cells. Compared to pDC, purified cDC1 or cDC2 required additional TLR-3 or -2/4 stimulation to cross-present antigens from H9-HIV cells. Indeed, H9-HIV apoptotic cells induced maturation of pDC, but not cDC. Our work demonstrates that pDC produce type I IFNs following HIV-infected cells detection, which pre-activate CD8+ T cells by making them produce, but not release, IFN-γ. Upon additional cognate interaction, IFN-γ. release is potentiated by this previous intracellular accumulation.

SARS-CoV-2 T-Cell Responses in Allogeneic Hematopoietic Stem Cell Recipients following Two Doses of BNT162b2 mRNA Vaccine.

Background: At variance to humoral responses, cellular immunity after anti-SARS-CoV-2 vaccines has been poorly explored in recipients of allogeneic hematopoietic stem-cell transplantation (Allo-HSCT), especially within the first post-transplant years where immunosuppression is more profound and harmful. Methods: SARS-CoV-2 Spike protein-specific T-cell responses were explored after two doses of BNT162b2 mRNA vaccine in 45 Allo-HSCT recipients with a median time from transplant of less than 2 years by using INF-γ ELISPOT assay and flow-cytometry enumeration of CD4+ and CD8+ T lymphocytes with intracellular cytokine production of IFN-γ and TNF-α. Results: A strong TNF-α+ response from SARS-CoV-2-specific CD4+ T-cells was detected in a majority of humoral responders (89%) as well as in a consistent population of non-humoral responders (40%). Conclusions: T-cells are likely to participate in protection against COVID-19 viral infection, even in the absence of detectable antibody response, especially in the first years post-transplant in Allo-HSCT recipients.

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Comparison of Lethal and Nonlethal Mouse Models of Orientia tsutsugamushi Infection Reveals T-Cell Population-Associated Cytokine Signatures Correlated with Lethality and Protection.

The antigenic diversity of Orientia tsutsugamushi as well as the interstrain difference(s) associated with virulence in mice impose the necessity to dissect the host immune response. In this study we compared the host response in lethal and non-lethal murine models of O. tsutsugamushi infection using the two strains, Karp (New Guinea) and Woods (Australia). The models included the lethal model: Karp intraperitoneal (IP) challenge; and the nonlethal models: Karp intradermal (ID), Woods IP, and Woods ID challenges. We monitored bacterial trafficking to the liver, lung, spleen, kidney, heart, and blood, and seroconversion during the 21-day challenge. Bacterial trafficking to all organs was observed in both the lethal and nonlethal models of infection, with significant increases in average bacterial loads observed in the livers and hearts of the lethal model. Multicolor flow cytometry was utilized to analyze the CD4+ and CD8+ T cell populations and their intracellular production of the cytokines IFNγ, TNF, and IL2 (single, double, and triple combinations) associated with both the lethal and nonlethal murine models of infection. The lethal model was defined by a cytokine signature of double- (IFNγ-IL2) and triple-producing (IL2-TNF-IFNγ) CD4+ T-cell populations; no multifunctional signature was identified in the CD8+ T-cell populations associated with the lethal model. In the nonlethal model, the cytokine signature was predominated by CD4+ and CD8+ T-cell populations associated with single (IL2) and/or double (IL2-TNF) populations of producers. The cytokine signatures associated with our lethal model will become depletion targets in future experiments; those signatures associated with our nonlethal model are hypothesized to be related to the protective nature of the nonlethal challenges.

Cell-Associated HIV Cross-Presentation by Plasmacytoid Dendritic Cells Is Potentiated by Noncognate CD8+ T Cell Preactivation.

IFN-γ secretion by Ag-specific T cells is known to be tightly regulated by engagement of the TCR. Human plasmacytoid dendritic cells (pDC) can cross-present Ags from apoptotic HIV-infected cells or tumor cells to CD8+ T cells. As pDC respond to HIV virions by maturing and secreting cytokines, we hypothesized that this might affect cross-presentation from HIV-infected cells. Purified blood DC were incubated with apoptotic HIV-infected H9 cells in the presence of saquinavir, after which the activation process of HIV-specific cloned CD8+ T cells was studied. IFN-γ secretion by HIV-specific T cells was stimulated by pDC and conventional DC (cDC1) more than by cDC2 and was strictly MHC class I restricted. Surprisingly, intracellular production of IFN-γ was only partly MHC class I restricted for pDC, indicating a noncognate CD8+ T cell activation. pDC, but not cDC, matured and secreted IFN-α in the presence of apoptotic H9HIV cells. A mixture of IFN-α, IFN-β, and TNF-α induced intracellular production of IFN-γ but not granzyme B, mimicking the noncognate mechanism. Neutralization of type I IFN signaling blocked noncognate intracellular production of IFN-γ. Moreover, cognate stimulation was required to induce IFN-γ secretion in addition to the cytokine mixture. Thus, IFN-γ secretion is tightly regulated by engagement of the TCR as expected, but in the context of virus-infected cells, pDC can trigger intracellular IFN-γ accumulation in CD8+ T cells, potentializing IFN-γ secretion once CD8+ T cells make cognate interactions. These findings may help manipulate type I IFN signaling to enhance specifically Ag-specific CD8+ T cell activation against chronic infections or tumors.

Eomes+ T-betint CD8 T Cells Are Functionally Impaired and Associate with Primary Refractory Disease in Patients with Acute Myeloid Leukemia (AML)

Immunotherapy targeting T cell inhibitory mechanisms, such as the PD-1 pathway to unleash the anti-tumor immune response is a promising strategy for cancer treatment. Several studies including ours have demonstrated an involvement of PD-1 and other inhibitory T cell receptors in AML progression. Eomesodermin (Eomes) and T-bet are both T box transcription factors that are crucial in regulating T cell function. Recent studies showed that Eomes and T-bet differentially regulate PD-1 expression and T cell exhaustion in chronic viral infection. Here we examine the effect of these two transcription factors in the pathogenesis of AML using blood samples collected from a large cohort (n=59) of patients with AML. We report that Eomes+T-betintCD8 T cells are increased in AML. Importantly they are functionally impaired and associate with poor clinical outcome.We first performed flow cytometry analyses to examine the intracellular expression of Eomes and T-bet in CD8 T cells of blood samples collected from AML patients at initial diagnosis. Based on the level of Eomes vs. T-bet, three subpopulations were defined among activated CD8 T cells: Eomes- T-bethi, Eomes+ T-bethi, and Eomes+T-betint. We observed a significant increase in the percentage of Eomes+T-betint CD8 T cells from AML patients (n=59) compared to that of healthy donors (n=15) (21.97±1.85% vs. 13.47±2.92%, P=0.0367). In addition, we assessed blood samples from 11 AML patients at initial diagnosis compared to that from the same patients when they are in complete remission (CR). We found that the frequency of Eomes+T-betint CD8 T cells was significantly lower in CR. This finding suggests an association of Eomes+T-betint CD8 T cells with AML progression.We next evaluated the clinical correlation of different Eomes and T-bet expression pattern in AML. Based on the frequency of Eomes+T-betint CD8 T cells, we defined high- (Eomes+T-betint ≥21.72 %) vs. low-(Eomes+T-betint <21.72 %) subgroups in AML patients using the median value of Eomes+T-betint % as the cutoff. Among the 47 AML patients of whom the induction response was able to be evaluated, we found a significantly higher rate of primary refractory disease (failure to achieve CR) in the high-Eomes+T-betint group compared with that of the low- Eomes+T-betint group (7/24 (29.2%) vs. 1/23 (4.3%), P=0.024). Strikingly when the same analysis was applied to Eomes- T-bethi subpopulation cells, we observed an opposite trend, thus low- Eomes- T-bethi expression associates with primary refractory disease. This result indicates a reciprocal effect of Eomes and T-bet on clinical outcome of AML.We further dissected the phenotype and functional status of each T cell subpopulation. The distribution of naïve, central memory, effector memory and terminal differentiated profile was similar among Eomes+T-betint CD8 T cells compared with that of Eomes+ T-bethi and Eomes- T-bethi cells. However, intracellular production of IFN-γ upon in vitro anti-CD3/CD28 stimulation was significantly lower in Eomes+T-betint CD8 T cells compared with that in the other two subpopulations (n=47, P <0.001). In addition, Eomes+T-betint CD8 T cells displayed lower capacity for killing manifested by less expression of Perforin and GranzymeB. Thus consistent with exhaustion, Eomes+T-betint T cells are functionally deficient.To study the effect of Eomes and T-bet in leukemia antigen-specific T cells, we performed a functional assay to detect CD8 T cell response against a WT-1 (a known leukemia-associated antigen) epitope. Purified CD8 T cells derived from HLA-A*0201+ AML patients were co-cultured in vitro with T2 cells (antigen presenting cells) pulsed with HLA-A*0201-binding WT1126-134 or SV40 LT281-289 (as negative control) peptide. Intracellular production of IFN-γ was detected in 1.2% of CD8 T cells after 6 days of stimulation with WT-1 peptide, while essentially 0 CD8 cells responded to control peptide. Importantly Eomes+T-betint leukemia-specific CD8 T cells (gated on IFN-γ+) were less functional as they produced less TNF-α compared with that of the other two subpopulations.Taken together, our study demonstrates a significant association of Eomes+T-betint T cells to immune dysfunction and poor clinical outcome in AML patients. This novel finding provides a great potential to define the prognostic biomarker and therapeutic target for this devastating blood cancer. DisclosuresNo relevant conflicts of interest to declare.

Characterisation of Naive Immunological Profile of Umbilical Cord Blood: The Role of Mucosal-Associated Invariant T Cells and Functional Defects of T Cells and Natural Killer Cells

Patients undergoing allogeneic haematopoietic stem cell (HSC) transplantation are susceptible to infection and reactivation of viruses, such as cytomegalovirus, due to impairment of the T cell compartment following intensive immunosuppressive therapy. There is a growing interest in evaluating cell-mediated immunity to improve the management of these infections. Over the last two decades, umbilical cord blood (UCB) has emerged as an alternative source of cellular products for the treatment of a range of haematological disorders. UCB HSCs have a number of distinguishing characteristics which differentiate them from adult HSCs, including increased proliferation and increased in vitro colony forming capacity. The use of UCB as HSC grafts for allogeneic stem cell transplant has a number of advantages, including immediate availability and reduced risk of graft-versus-host disease despite donor-recipient HLA disparity. However, the limited number of HSCs present in a single UCB collection and incomplete immune reconstitution because of delayed HSC engraftment has restricted the use of cord blood in stem cell transplantation. In addition, UCB transplantation is associated with lack of transferred adoptive immunity, resulting in transplant related morbidity and mortality due to infectious complications. While the human neonatal immune system is considered to be immature compared with its adult counterpart, the role of the innate immune cells in the immunological naïvety is not fully understood.Here, we describe the collection and characterisation of UCB units collected from a series of Irish mothers who delivered term infants by elective caesarean at a major Irish maternity hospital. Pre analytical variables included maternal factors such as gestation at delivery, blood group, age, medical history and BMI, and neonatal factors such as birth weight and gender. Mononuclear cells (MNC) were isolated and the immunological profile was analyzed by flow cytometry for the surface markers CD3, CD4, CD5, CD8, CD19, CD34, CD45, CD56, CD161 and the Vα24Jα17, Vα7.2, Vδ1, Vδ2 and Vδ3 T cell receptors. Progenitor cell colony forming ability was assessed by CFU-GM and BFU-E assays. Stimulation of T cells, by cross-linking of CD3 and CD28 using tetrameric antibodies, and Natural Killer (NK) cells, with IL12/IL18 and target cells, was assessed by measurement of surface expression of CD107a and intracellular production of IFNγ, IL4 and IL17a.We showed no difference in the overall numbers of B cells, T cells (CD4+ and CD8+) and γδ T cells between UCB (n=23) and adult blood (n=4); however, NK cells (p<0.001) and mucosal-associated invariant T (MAIT) cells (p<0.0001) were significantly depleted in UCB. Stimulated UCB T cells and NK cells (n=17) showed significantly reduced IFNγ production compared to adult blood counterparts (n=4) (p<0.01) Following thaw of cryopreserved UCB MNC, we observed no significant change in CFU-GM, CD34+ cell concentration or immunological profile/function compared to fresh MNC (n=7).Our study suggests that reduced numbers of circulating innate immune cell populations, as well as reduced cytokine responses following stimulation, may contribute to the naïve immunological phenotype and hyporesponsiveness of UCB. Further investigation and manipulation of these innate immune cell populations could improve UCB transferred adoptive immunity potential and assist in the management of post allogeneic stem cell transplant viral infections. DisclosuresNo relevant conflicts of interest to declare.

Recipient Hematopoietic, but Not Non-Hematopoietic, Antigen-Presenting Cells Are Indispensable for CD4+ T-Cell-Mediated Xenogeneic Graft-Versus-Host Disease

BackgroundAs a major drawback of conventional GVHD models using inbred mouse strains, they are not suitable for assessing the safety and effectiveness of current human-specific therapies. To overcome this limitation, xenogeneic GVHD models using highly immunodeficient mice are currently being developed and are widely used to study human diseases. However, the underlying mechanisms of human immune responses against host are not fully understood. We have previously reported that human CD4+ T-cells and, to a lesser extent, CD8+ T-cells play a critical role in the induction and development of xenogeneic GVHD. (Kawasaki et al. Blood Abstract 2016 #701). Donor antigen-presenting cells (APCs) were not necessarily required for T-cell activation. In this context, the present study focused on the immunobiology of CD4+ T-cell-mediated GVHD, and defined the individual role of host hematopoietic, and non-hematopoietic, APCs in inducing human immune responses in NOD/Shi-scid-IL2rg null (NOG) mice.MethodsA xenogeneic GVHD model was established by intravenously injection of human CD4+ T-cells into NOG mice. Bone marrow (BM) chimeras were generated as summarized in Figure 1A. Briefly, BM cells from wild-type (MHC+/+) or MHC−/− NOG mice, respectively, were transplanted into other mice, in which only non-hematopoietic cells including target organs, or only hematopoietic cells including APCs, respectively, were deficient in MHC molecules. After human CD4+ T-cells were injected into these BM chimeras, survival and clinical GVHD scores were monitored.ResultsControl mice (MHC+/+ → MHC+/+) uniformly developed progressive GVHD, and they all died within 12 weeks following the injection of human CD4+ T-cells. The former chimeras (MHC+/+ → MHC−/−) showed similar GVHD severity and mortality, whereas the latter chimeras (MHC−/− → MHC+/+) showed significantly less body weight loss and lower clinical GVHD scores than the control (Figure 1B, C), which resulted in longer survival (Figure 1D). These results indicate that host hematopoietic, but not non-hematopoietic, APCs play a critical role in the development of xenogeneic GVHD.To verify these results, we then analyzed T-cell subsets in the lungs and spleen of NOG mice one week after the injection of purified human naïve (CD45RA+CCR7+) CD4+ T-cells. Flow cytometric analysis showed a markedly increased percentage of central memory (CD45RA−CCR7+) and effector memory (CD45RA−CCR7−) CD4+ T-cells in the lungs and spleen of MHC+/+ mice. Notably, the memory differentiation was also observed in MHC−/− mice. However, the proportion of terminally differentiated effector memory (CD45RA+CCR7−) CD4+ T-cells was significantly increased in MHC−/− mice, suggesting that a certain number of effector T-cells failed to develop a memory phenotype due to a lack of antigen-specific stimulation. Splenectomized NOG mice also showed rapid memory differentiation of CD4+ T-cells in the lungs during early GVHD. These findings suggest that neither MHC molecule expression on host tissues nor host hematopoietic APCs are required for the memory differentiation of CD4+ T-cells in lungs during early GVHD in xenogeneic chimeras.Focusing on memory T-cells during early xenogeneic GVHD, we revealed that most memory CD4+ T-cells in lungs were less activated and showed a slowly proliferative phenotype (CD69low and CFSElow) compared to those in spleen. Intracellular IFN-γ production was also decreased in these cells. These findings suggest that most memory T-cells in lungs have less potential to cause sustained inflammation, and are therefore less associated with the pathogenesis of GVHD. Increased susceptibility of T-cells to peripheral apoptosis appears to be one of the characteristic properties of spontaneous proliferation. Consistent with this, we revealed that the memory CD4+ T-cells in lungs showed a significantly decreased expression of Bcl-2 compared to those in the spleen, indicating that these cells spontaneously acquire the memory phenotype, but not in response to non-hematopoietic APCs.ConclusionsOur data clearly indicate that the immunobiology of xenogeneic GVHD is highly dependent on human CD4+ T-cells and mouse hematopoietic, but not non-hematopoietic, APCs. We believe that our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow CD8 T Cells Express High Frequency of PD-1 and Exhibit Reduced Anti-Leukemia Response in AML Patients

Acute myeloid leukemia (AML) is a devastating blood cancer with 5- year survival of only 25%. Optimizing anti-leukemia immune response to improve clinical outcome is a promising strategy for novel effective leukemia therapeutics. However due to limited accessibility, the majority of studies have been restricted to evaluate samples from peripheral blood. The immune response in bone marrow of AML is poorly understood. In this study, we aim to investigate the T cell immune response in bone marrow samples derived from AML patients.Samples collected from a cohort of patients with newly diagnosed AML (n=19) were used in this study. Mononuclear cells of bone marrow and peripheral blood were isolated from clinical samples before multi-channel flow cytometry analysis. We observed a comparable frequency of CD3+ T cells among lymphocytes in bone marrow and peripheral blood. Similarly there is no difference of CD4, CD8 T cell frequency, or CD4/CD8 ratio. However, when T cell subsets for each differentiation stage were analyzed based on the expression of CD45RA and CCR7, we found significantly increased effector memory T cells (CD45RA-CCR7-) among CD8 T cells in bone marrow compared with that in peripheral blood (43.88±5.012% vs. 35.15±5.104%, P =0.0088). Whereas the percentage of terminally differentiated effector cells (CD45RA+CCR7-) was significantly lower in bone marrow (20.36±5.025% vs. 28±5.947%, P =0.0481). No difference was found in the frequency of naive (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) subsets.Several studies including ours have demonstrated an involvement of the inhibitory receptors PD-1, TIM-3 and TIGIT in AML progression. Most of the studies were performed using clinical samples from peripheral blood. To evaluate whether the effect of inhibitory pathways is different between bone marrow and peripheral blood, we assessed the expression of these inhibitory receptors on T cells derived from both sites. We observed comparable expression of TIGIT and TIM-3 in bone marrow and peripheral blood. The frequency of PD-1 expression on CD8 T cells was however significantly higher in bone marrow (39.55±3.809% vs. 32.8±3.512%, P =0.0058). We further dissected the intracellular expression of Eomesodermin (Eomes) and T-bet, the two T box transcription factors that are crucial in regulating T cell function. We found that the frequency of Eomes+T-betint CD8 T cells was significantly higher in bone marrow than in peripheral blood (17.01±1.843% vs. 14.95±1.682%, P =0.0405), indicating a predominance for late stage of T cell exhaustion status in bone marrow. This result suggests a relatively more suppressive environment in bone marrow compared with that of peripheral blood.We next examined the functional status of CD8 T cells. Intracellular IFN-γ and TNF-α production was assessed upon in vitro stimulation with anti-CD3/CD28. Intriguingly no significant difference between bone marrow and peripheral blood was detected. Consistently we observed no difference between the two sites in term of the expression of Ki67, Granzyme B, and Perforin, suggesting a comparable proliferation and killing capacity of CD8 T cells between bone marrow and peripheral blood. We further investigated the function of leukemia-reactive CD8 T cells by testing T cell response against a WT-1 peptide. WT-1 is a well known tumor-associated antigen, in which HLA-A*0201 restricted WT-1126-134 is one of the mostly studied epitope in AML. Purified CD8 T cells derived from bone marrow or blood of newly diagnosed HLA-A*0201 AML patients were co-cultured with T2 cells (used as antigen presenting cells) pulsed with WT-1126-134. We found a significantly lower production of IFN- γ by CD8 T cells from bone marrow compared with that from peripheral blood (0.4325±0.1612% vs, 0.7425±0.2809%, P =0.0472). These data demonstrate that although the total CD8 T cells in bone marrow remain functional, the leukemia-reactive CD8 T cells are less functional in bone marrow compared with that in peripheral blood.Collectively, our data demonstrate that bone marrow CD8T cells from AML patients express higher frequency of PD-1 and exhibit reduced anti-leukemia response. Our study suggests that bone marrow is a unique anatomical location fostering negative immune response in AML, thus targeting the negative regulatory pathways in bone marrow is of great potential to develop novel effective leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.

Polymorphisms of Monocyte-Activating Cytokine/Chemokine Genes Affect the Susceptibility to, and Severity of Chronic ITP

Introduction: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder characterized by the production of platelet antibodies, resulting in the destruction of platelets and inhibition of their production. A shift toward Th1 and Th17 cells together with impaired Tregs has been reported using quantitative RT-PCR and flow cytometry method. Further, recent reports have shown that CD16 (+) monocytes in ITP patients are polarized to secrete the Th1 cytokine IL-12, promoting Th1 responses. Enhanced phagocytosis of splenic macrophages was also observed in ITP patients. Thus, monocytes/macrophages are also involved in the pathogenesis of ITP. However, it remains unclear whether genetic factors of cytokines/chemokines activating monocytes increase the risk of chronic ITP. We investigated the impact of monocyte-related gene polymorphisms: TNF-α -857C/T, IFN-γ +874T/A, and MCP1+2518G/A on the susceptibility and clinical feature of chronic ITP (cITP) and Th1/Th2 ratio in peripheral blood of cITP patients,Patients and methods: In this study, 126 Japanese cITP patients (92 females and 34 males with a median age of 47.7 [range: 2.4-82.3] years), as well as 202 race-matched healthy subjects were examined. The diagnosis of cITP was defined according to International Working Group criteria. Genotyping of the polymorphisms was determined by PCR based technique and direct sequencing. We evaluated Th1/Th2 ratio in peripheral blood of 15 normal donors and 25 ITP patients. Intracellular IL-4 (Th2 cytokine) and IFN-γ (Th1 cytokine) production was assessed in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin by flow cytometry.This study was approved by the Institutional Review Board of Gunma University Hospital.Results: The platelet count ranged from 1×109/L to 98×109/L with a mean count of 32×109/L at the initial diagnosis. Steroid treatment was given to 86 patients (58.1%), and splenectomy was performed in only 18 patients (12.2%). The genotype distributions of these three polymorphisms in healthy subjects were similar to those observed in previous studies of Japanese populations. Patients with cITP had a significantly higher frequency of the TNF-α -857 TT genotype (high producer type) and MCP1+2518 G allele (high-producer type) compared to healthy controls (14.0% vs 5.4%, p < 0.01; 67.6% vs. 60.6%, P < 0.01). However, no significant difference in the genotype frequencies of IFN-γ was observed between cITP patients and healthy controls. Thus, it was suggested that the TNF-α and MCP-1 polymorphisms shifted toward monocyte activation in cITP patients. We examined the association between these polymorphisms and the clinical characteristics of cITP patients. cITP patients with IFN-γ +874 non-AA genotype (high expression type) showed a lower minimum platelet count than those with an AA genotype (12.9 × 109/L ± 10.7 ×109/L vs 19.4×109/L ± 18.9 ×109/L, P < 0.05). We also examined the association between these polymorphisms and Th1/Th2 ratio in both normal donors and cITP patients. The median Th1/Th2 ratio in patients with cITP was significantly higher than that of healthy controls (31.4, range 0.6-98.8 vs. 17.8, range, 2.2-52.5, P < 0.001). Patients with the IFN-γ +874 non-AA genotype (high expression type) had a significantly higher Th1/Th2 ratio compared to those with the IFN-γ +874 AA genotype (low expression type) (71.5, range 29.4-92.8 vs. 27.5, range 0.6-98.8, P < 0.05). However, there was no significant association between the Th1/Th2 ratio and other polymorphism in cITP patients.Conclusion: These findings suggested that the high expression type of monocyte-activating cytokine/chemokine genes increased the susceptibility to, and severity of chronic ITP DisclosuresMurakami:Ono: Honoraria; BMS: Honoraria; Fujimoto: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Celgene: Honoraria.

EV PD-L1 Contributes to Immunosuppressive CD8+ T Cells in Peripheral Blood of Pediatric Wilms Tumor.

Wilms tumor (WT) is the most common renal cancer and the most prevalent abdominal cancer in children. Children with recurrent or progressive forms of WT could benefit from novel immune-targeted approaches. While the immune status of these patients, especially the immunosuppression of peripheral T cells, was rarely reported. The present study enrolled a consecutive series of 14 Chinese WT children and 14 age- and gender-matched healthy controls. We demonstrated that plasma extracellular vesicular (EV) PD-L1 levels significantly increased in WT patients than in healthy controls. EV PD-L1 significantly inhibited the activation of human CD8+ T cells by down-regulating the cell surface CD69 expression and the intracellular IFNγ and TNFα production in vitro. In peripheral CD8+ T cells of WT patients, the intracellular IFNγ and TNFα production significantly decreased than healthy controls. The level of plasma EV PD-L1 significantly correlated with the intracellular TNFα production in peripheral CD8+ T cells of WT patients. In conclusion, the significantly increased plasma EV PD-L1 in WT patients contributed to the immunosuppression of peripheral CD8+ T cells. Monitoring the level of plasma EV PD-L1 will be helpful for the selection of immune-targeted therapies for WT patients.

Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children.

Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children limits the size of the virus reservoir, but whether the time of treatment initiation (TI) can durably impact host immune responses associated with HIV infection is still unknown. This study was conducted in PBMC of 20 HIV-infected virally suppressed children on ART (mean age 9.4 y), classified as early treated (ET; age at ART initiation ≤0.5 y, n = 14) or late treated (LT; age at ART initiation 1-10 y, n = 6). Frequencies and functions of Ag-specific CD4 (CD40L+) and CD8 (CD69+) T cells were evaluated by intracellular IL-2, IFN-γ, and TNF-α production with IL-21 in CD4 or CD107a, granzyme B and perforin in CD8 T cells following stimulation with HIV gp140 protein (ENV) or GAG peptides by multiparameter flow cytometry. ET showed a higher proportion of cytokine-producing ENV- and GAG-specific CD4 and CD8 T cells compared with LT. In particular, ET were enriched in polyfunctional T cells. RNA sequencing analysis showed upregulation of immune activation pathways in LT compared with ET. Our results suggest that timing of TI in HIV-infected children has a long-term and measurable impact on the quality of the HIV-specific T cell immune responses and transcriptional profiles of PBMC, reinforcing the importance of early TI.

The expression of PD-1 alone by T cells is associated with cell activation while the co-expression of TIM-3 indicates exhausted subsets both in peripheral blood and bone marrow of multiple myeloma patients

Abstract Background Multiple myeloma (MM) is a lymphoid neoplasm characterized by the accumulation of malignant clones of plasma cells, usually within the bone marrow (BM). Despite the increase of T cells expressing inhibitory signal molecules (ISMs) — PD-1, TIM-3 etc. — having been described in MM, the first results of anti-PD-1 therapy for MM remain modest. ISMs are expressed on T cells and modulate the functional activity. A stable expression of ISMs on T cells is associated with T cell exhaustion, the condition of severe decrease of both cytotoxicity and cytokine secretion. At the same time, ISMs are also expressed by activated T cells. We studied the expression of PD-1 and TIM-3 by circulating and bone marrow (BM) T cells as a manifestation of T cell exhaustion treated MM patients. Methods Peripheral blood (PB) and BM samples of 45 MM patients were obtained during routine diagnostic procedures. The expression of PD-1 and TIM-3 by CD4+ and CD8+ T cells, intracellular production of IFNγ and intracellular expression of Ki-67 by T cells and Granzyme B production by CD8+ T cells were assessed using flow cytometry. Results Relative counts of PD-1+ subset of CD8+ T cells and both PD-1+ and TIM-3+ subsets of CD4+ T cells were higher in BM compared with PB. BM samples also contained higher amounts of double-positive PD-1+TIM-3+ subsets of CD8+ and CD4+ T cells compared with PB. Percentage of PB CD8+PD-1+, CD4+PD-1+, CD4+TIM-3+, CD8+PD-1+TIM-3+ and CD4+PD-1+TIM-3+ T cells correlated with the content of respective subsets in BM. Both PB and BM CD8+PD-1+ and CD4+PD-1+ T cells of MM patients retain a cytotoxic, proliferative and cytokine-producing potential appropriate to PD-1-negative subsets. On the contrary, the functional activity of CD8+PD-1+TIM-3+ and CD4+PD-1+TIM-3+ T cells was significantly reduced compared with PD-1- and TIM-3-negative subsets. Conclusion PD-1+ T cells in MM patients are related to activated rather than exhausted populations. T cell exhaustion is associated with cells co-expressing PD-1 and TIM-3. It is recommended to evaluate T cell subsets co-expressing PD-1, TIM-3 and other ISMs, and to study their functional properties. Legal entity responsible for the study The authors. Funding The Russian Science Foundation (grant no. 18-75-00050). Disclosure All authors have declared no conflicts of interest.

Alteration of the mare's immune system by the synthetic progestin, altrenogest.

Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines. In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1β in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1β expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares. P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.