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Extracellular vesicles (EVs) are excreted from all cells in the human body and are heterogeneous lipid bilayer particles containing proteins, RNA, DNA, and other cargo. T cells are primary players of the adaptive immune system and have a significant role in anti-cancer immunotherapies. Tumor derived EVs have diverse effects on host cells including modulating the immune response. In this study, we investigate the role acute myeloid leukemia (AML) derived EVs have on modulation of gene expression levels in three different T cell populations (CD8+, CD4+, and CD4 + CD39 + T cells). AML derived EVs modulate gene expression levels in all three cell populations with transcripts associated with major immunoregulatory pathways being significantly altered. Genes whose expression were significantly upregulated or downregulated were analyzed for gene ontogeny category expression and for the impact of the changes on specific immunoregulatory pathways. CD8 + T cells were modulated to a larger extent than other T cell populations and gene expression levels associated with proliferation and differentiation were influenced by AML-derived EVs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Immunotherapeutic protocols have focused on identification of stimuli that induce effective anti-leukemic immune responses. One potent immune stimulus is the encounter with allogeneic cells. Our group previously showed that the infusion of haploidentical donor white blood cells (1-2 × 10 8 CD3+ cells/kg) into patients with refractory hematological malignancies induced responses of varying magnitude in over half of the patients. Because donor cells were eliminated within 2 weeks in these patients, it is presumed that the responses of recipient lymphocytes were critically important in achieving prolonged anti-leukemic responses., Methods: The role of patient CD3+ cells in anti-leukemic responses was examined by isolating peripheral blood mononuclear cells from newly diagnosed leukemic patients. Immunophenotyping was performed on these peripheral blood mononuclear cells. CD3+ cells were isolated from the peripheral blood mononuclear cells and tested for their ability to proliferate and lyse autologous leukemic cells when stimulated with unrelated allogeneic cells., Results: Allostimulated CD3+ cells effectively generated cytolytic responses to autologous CD3-cells in 11/21 patients. Increased numbers of CD4+ cells expressing high levels of granzyme A, B and perforin and CD8+CD39+ cells were found in nonresponsive CD3+ cells., Conclusions: These results indicate that CD3+ cells from leukemic patients are capable of generating anti-leukemic responses when stimulated with unrelated allogeneic cells. This model can be used to identify approaches using alloreactive responses by patient lymphocytes to enhance in vivo anti-leukemic responses.

H. pylori persists in the human stomach over decades and promotes several adverse clinical sequelae including gastritis, peptic ulcers and gastric cancer that are linked to the induction and subsequent evasion of chronic gastric inflammation. Emerging evidence indicates that H. pylori infection may also protect against asthma and some other immune-mediated conditions through regulatory T cell effects outside the stomach. To characterize the complexity of the CD4+ T cell response generated during H. pylori infection, computational methods were previously used to generate a panel of 90 predicted epitopes conserved among H. pylori genomes that broadly cover HLA Class II diversity for maximum population coverage. Here, these sequences were tested individually for their ability to induce in vitro responses in peripheral blood mononuclear cells by interferon-γ ELISpot assay. The average number of spot-forming cells/million PBMCs was significantly elevated in H. pylori-infected subjects over uninfected persons. Ten of the 90 peptides stimulated IFN-γ secretion in the H. pylori-infected group only, whereas two out of the 90 peptides elicited a detectable IFN-γ response in the H. pylori-uninfected subjects but no response in the H. pylori-infected group. Cytokine ELISA measurements performed using in vitro PBMC culture supernatants demonstrated significantly higher levels of TNF-α, IL-2, IL-4, IL-6, IL-10, and TGF-β1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status. Our results indicate that the human T cell responses to these 90 peptides are generally increased in actively H. pylori-infected, compared with H. pylori-naïve, subjects. This information will improve understanding of the complex immune response to H. pylori, aiding rational epitope-driven vaccine design as well as helping identify other H. pylori epitopes with potentially immunoregulatory effects.

Ticks are notorious vectors of disease for humans, and many species of ticks transmit multiple pathogens, sometimes in the same tick bite. Accordingly, a broad-spectrum vaccine that targets vector ticks and pathogen transmission at the tick/host interface, rather than multiple vaccines against every possible tickborne pathogen, could become an important tool for resolving an emerging public health crisis. The concept for such a tick protective vaccine comes from observations of an acquired tick resistance (ATR) that can develop in non-natural hosts of ticks following sensitization to tick salivary components. Mice are commonly used as models to study immune responses to human pathogens but normal mice are natural hosts for many species of ticks and fail to develop ATR. We evaluated HLA DR3 transgenic (tg) "humanized" mice as a potential model of ATR and assessed the possibility of using this animal model for tick protective vaccine discovery studies. Serial tick infestations with pathogen-free Ixodes scapularis ticks were used to tick-bite sensitize HLA DR3 tg mice. Sensitization resulted in a cytokine skew favoring a Th2 bias as well as partial (57%) protection to infection with Lyme disease spirochetes (Borrelia burgdorferi) following infected tick challenge when compared to tick naïve counterparts. I. scapularis salivary gland homogenate (SGH) and a group of immunoinformatic-predicted T cell epitopes identified from the I. scapularis salivary transcriptome were used separately to vaccinate HLA DR3 tg mice, and these mice also were assessed for both pathogen protection and epitope recognition. Reduced pathogen transmission along with a Th2 skew resulted from SGH vaccination, while no significant protection and a possible T regulatory bias was seen in epitope-vaccinated mice. This study provides the first proof-of-concept for using HLA DR tg "humanized" mice for studying the potential tick protective effects of immunoinformatic- or otherwise-derived tick salivary components as tickborne disease vaccines.

Background: Acute myeloid leukemia (AML) and other aggressive refractory hematological malignancies unresponsive to upfront therapy remain difficult conditions to treat. Often, the focus of therapy is centered on achieving complete remission of disease in order to proceed with a consolidative stem cell transplant. At issue with this paradigm is the multitude of patients who are unable to achieve complete remission with standard chemotherapeutic options. A major benefit of transplantation is the graft versus tumor effect that follows successful engraftment. However, with this graft versus tumor effect comes the risk of graft versus host disease. Therefore, alternative treatment options that utilize immunotherapy while minimizing toxicity are warranted. Herein, we propose a novel treatment protocol in which haploidentical peripheral blood stem cells are infused into patients with refractory hematological malignancies. The end goal of cellular therapy is not engraftment but instead is the purposeful rejection of donor cells so as to elicit a potent immune reaction that appears to break host tumor tolerance., Methods/design: The trial is a FDA and institutional Rhode Island Hospital/The Miriam Hospital IRB approved Phase I/II study to determine the efficacy and safety of haploidentical peripheral blood cell infusions into patients with refractory hematological malignancies. The primary objective is the overall response rate while secondary objectives will assess the degree and duration of response as well as safety considerations. Patients with refractory acute leukemias and aggressive lymphomas over the age of 18 are eligible. Donors will be selected amongst family members. Full HLA typing of patients and donors will occur as will chimerism assessments. 1-2x108 CD3+ cells/kilogram will be infused on Day 0 without preconditioning. Patients will be monitored for their response to therapy, in particular for the development of a cytokine release syndrome (CRS) that has been previously described. Blood samples will be taken at the onset, during, and following the cessation of CRS so as to study effector cells, cytokine/chemokine release patterns, and extracellular vesicle populations. Initially, six patients will be enrolled on study to determine safety. Provided the treatment is deemed safe, a total of 25 patients will be enrolled to determine efficacy., Discussion: Cellular Immunotherapy for Refractory Hematological Malignancies provides a novel treatment for patients with relapsed/refractory acute leukemia or aggressive lymphoma. We believe this therapy offers the immunological benefit of bone marrow transplantation without the deleterious effects of myeloablative conditioning regimens and minus the risk of GVHD. Laboratory correlative studies will be performed in conjunction with the clinical trial to determine the underlying mechanism of action. This provides a true bench to bedside approach that should serve to further enrich knowledge of host tumor tolerance and mechanisms by which this may be overcome., Trial Registration: NCT01685606.

Exocytosis of granules containing the cytolytic effector (CE) molecules granzyme A (GzmA), granzyme B (GzmB), and perforin is one major pathway of lymphocyte-mediated cytotoxicity. Studies in murine models and the finding of elevated granzyme levels in the plasma of septic patients have implicated cytotoxic lymphocytes in the pathogenesis of sepsis. We sought to evaluate the role of cytotoxic cells and CE in sepsis and determine if intracellular levels of CE in cytotoxic cells correlate with disease severity. We conducted a prospective cohort study of 40 patients enrolled into one of three groups: controls (C), acutely ill nonseptic illnesses, or patients with severe sepsis (SS) (lactate, >4 mmol/L; systolic blood pressure, <90 mmHg after 2 L normal saline). Peripheral blood mononuclear cells were isolated and stained for extracellular markers for defined subpopulations and for intracellular expression of GzmA and GzmB and perforin. Levels of CE were quantified by geometric mean fluorescent intensity (GMFI) via flow cytometry. Cytotoxic T lymphocyte (CTL) expression was higher in SS (P = 0.04). The GMFI of GzmB was significantly higher in CTLs of SS patients versus acutely ill nonseptic illnesses or C. The GMFI of each GzmA and GzmB in CTLs were associated with the Acute Physiology and Chronic Health Evaluation II score (P = 0.01). A significant increase in the number of granulocytes in the peripheral blood mononuclear cells of SS patients consisted primarily of low-density neutrophils, which expressed increased levels of GzmA (P < 0.01). The results suggest that CTLs are activated in SS and express significantly higher intracellular levels of GzmB and that GzmA and B levels correlate with disease severity.

Much of the therapeutic benefit of allogeneic transplant is by a graft versus tumor effect. Further data shows that transplant engraftment is not dependant on myeloablation, instead relying on quantitative competition between donor and host cells. In the clinical setting, engraftment by competition alone is not feasible due to the need for large numbers of infused cells. Instead, low-level host irradiation has proven to be an effective engraftment strategy that is stem cell toxic but not myeloablative. The above observations served as the foundation for clinical trials utilizing allogeneic matched and haploidentical peripheral blood stem cell infusions with minimal conditioning in patients with refractory malignancies. Although engraftment was transient or not apparent, there were compelling responses in a heavily pretreated patient population that appear to result from the breaking of tumor immune tolerance by the host through the actions of IFNγ, invariant NK T cells, CD8 T cells, NK cells, or antigen presenting cells.

Granzyme K (GrK) is a member of a highly conserved group of potent serine proteases specifically found in the secretory granules of cytotoxic T lymphocytes and natural killer cells. Based on the report indicating that inter-alpha inhibitor proteins are the physiological inhibitors of GrK and on previous findings that showed a significant decrease in plasma inter-alpha inhibitor proteins in patients with sepsis, it was our aim to determine whether increased levels of uninhibited GrK would contribute to the development of sepsis. To test this hypothesis, a competitive enzyme-linked immunosorbent assay system was developed; and the levels of GrK were measured in plasma samples obtained from healthy controls and 2 sets of patients with sepsis: patients admitted to the emergency department with a putative diagnosis of sepsis and patients with severe sepsis enrolled in a clinical trial. In addition, the molecular form(s) of GrK present in these samples was analyzed by Western blot. The levels of GrK were significantly increased in emergency department patients compared with healthy controls and significantly decreased in patients with severe sepsis enrolled in a clinical trial compared with healthy controls. GrK was detected as high-molecular-weight protein complexes in healthy controls but as complexes of lower molecular weight in the septic patients. The decrease in complex size correlated with the appearance of a band at 26 kDa similar to the size of free GrK. Our results indicate that plasma levels of GrK could serve as a useful diagnostic marker to stage sepsis, permitting better classification of septic patients and enabling targeting of specific treatments, and may play a functional role in the development of sepsis.

The encounter with allogeneic major histocompatibility complex (MHC) molecules expressed on donor leukocytes during transfusion of blood products has been shown to impact the recipient's immune responses in a number of settings. To better understand the responses induced by the transfer of allogeneic cells, a murine model was used to characterize the recipient responses that control the fate of the allogeneic lymphoid cells. Recipient CD8(+) cells could rapidly eliminate a large number of donor cells within 3 days after injection. When elimination responses were studied in the absence of CD8(+) cells, it was found that alloantibody production was the secondary elimination mechanism. Optimal recipient CD8(+) and B cell responses in this model required help from CD4(+) cells that could be provided by 3 different pathways. Although recipient CD4(+) cells could provide help when activated by direct recognition of allogeneic MHC class II molecules expressed on donor cells or by indirect recognition of processed alloantigen presented on recipient antigen-presenting cells (APCs), the most rapid recipient responses were generated by help provided by donor CD4(+) cells. Purified donor CD4(+) cells were also able to induce these rapid responses, indicating that activated donor CD4(+) cells expressing allogeneic MHC molecules were able to effectively stimulate responses by both recipient CD8(+) and B cells.

Chronic and acute graft-versus-host disease (cGVHD and aGVHD) result from donor cells responding to host disparate MHC alleles. In cGVHD (H-2d-->H-2bd), heightened polyclonal immunoglobulin production is due to the interaction of donor allospecific helper T cells (Th) and the host B cells. In vivo administration of antibody to the ligand for CD40, gp39, blocked cGVHD-induced serum anti-DNA autoantibodies, IgE production, spontaneous immunoglobulin production in vitro, and associated splenomegaly. Antibody production remained inhibited for extended periods of time after termination of anti-gp39 administration. Antiallogeneic CTL responses induced in a GVHD were also prevented by the in vivo administration of anti-gp39 as was the associated splenomegaly. These data suggest that CD40-gp39 interactions are critical in GVHD and that CD40-gp39 may be a valuable ligand-receptor pair for targeting immunotherapeutic agents to control GVHD.

Pharmacologic differentiation of the promyelocytic leukemia HL60 is associated with an increase in cellular tyrosine phosphatase activity. We asked (a) if this increase might, at least in part, be due to changes in a transmembranous protein-tyrosine phosphatase, CD45; and (b) if CD45 changes similarly in other differentiating leukemias. Differentiation of HL60, several chronic myelogenous leukemias, a monocytic leukemia (THP-1), and a monoblastoid leukemia (U-937) could be induced by phorbol ester, 1,25-dihydroxy vitamin D3, dimethyl sulfoxide, or cyclic AMP analogues. This differentiation was associated with a marked increase in (a) total cellular tyrosine phosphatase activity (2-4-fold as measured by the ability to dephosphorylate a tyrosine-phosphorylated peptide); (b) CD45-specific tyrosine phosphatase activity (2-4-fold); (c) CD45 cell surface expression by flow cytometry (2-5-fold); (d) synthesis of both exon B-dependent M(r) 205,000 and exon ABC- M(r) 185,000 CD45 proteins, as revealed by immunoprecipitation with antisera specific for CD45 isoforms. Both isoforms have enhanced electrophoretic mobility when isolated from the differentiated cells. This enhanced mobility did not appear to be due to decreased stoichiometry of CD45 phosphorylation on serine/threonine residues. Interestingly, 12-O-tetradecanoylphorbol-13-acetate transiently reduced CD45 protein-tyrosine phosphatase activity in the chronic myelogenous leukemia cell RWLeu4 without altering the CD45 amount (as measured by cell surface immunofluorescence). Modulation of CD45 tyrosine phosphatase activity (and protein levels) may play a role in differentiation or in maintaining cells in a nonproliferative state or may represent a phenotypic marker of differentiation.

For the first time, a screening procedure for antitumor monoclonal antibodies (MOABs) has been developed in which the ability of MOABs to mediate the indirect action of another immunotoxin is the primary criterion for selection of hybridomas for expansion and cloning. Use of the indirect immunotoxin makes it possible to screen MOABs for cytotoxic potential without the necessity of covalently coupling toxin to each individual MOAB. Hybridomas derived from spleens of immunized mice were screened for the synthesis of monoclonal antibodies able to participate in the delivery of a pharmacologically active, indirect immunotoxin conjugate to H69 lung cancer cells. The indirect immunotoxin conjugate was goat anti-mouse immunoglobulin disulfide linked to pokeweed antiviral protein. There was no correlation between the results of the screening with the indirect immunotoxin and an enzyme-linked immunosorbent assay screen for binding of MOAB to tumor cell membranes. An indirect immunotoxin prepared from Fab' fragments of goat anti-mouse immunoglobulin was also effective as an indirect immunotoxin conjugate. Each screening of more than 300 hybridomas was performed in 3 days. The indirect immunotoxin screen detected antitumor MOABs which were not detected by the conventional enzyme-linked immunosorbent assay method. A MOAB selected by the indirect immunotoxin screen was conjugated to pokeweed antiviral protein; the conjugate mediated immunocytotoxicity in a standard direct toxicity assay.

Nine patients with either chronic progressive or relapsing/remitting multiple sclerosis were treated with several courses of lymphopheresis. The lymphocytes obtained by this procedure showed a significantly lower percentage of OKT3+ cells than normal controls at the beginning of therapy. This percentage increased to normal levels after five lymphopheresis treatments and then decreased during the last stages of treatment. Patients had normal levels of OKT4+ and OKT8+ cells compared to controls and these percentages showed no statistically significant changes during the course of treatment. The proliferative response of patient's lymphocytes after stimulation with concanavalin A, PHA and alloantigen was not significantly different from normal controls and these responses were unchanged during the lymphopheresis treatment. Natural killer cell activity was also normal in our patients. The results reported in this study do not suggest a basic T cell abnormality in our patients with multiple sclerosis. Furthermore, the lymphopheresis treatments did not induce any significant change in T cell numbers or functional activity in vitro.