Background, biology and significance of human granzymes

The human granzymes (Grz) are a highly conserved group of potent peptidases that are found, together with a pore forming protein-perforin in specialized granules of cytotoxic cells such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Granule exocytosis (perforin/Grz) pathway is used by these cells to defend organism against virus-infected and tumor cells by inducing them to undergo apoptosis. While the pro-apoptotic functions of Grz have been well established, it has recently become apparent that Grz also possess important extracelular activities which are now being extensively investigated. Soluble Grz are found extracellularly in normal plasma suggesting their constitutive secretion in healthy individuals via a granule independent biosynthetic pathway. The potent activities of extracellular Grz appear to be controlled by highly abundant plasma derived serine peptidase inhibitors. However, unregulated activities of proteolytic Grz have been shown to result in disease pathology especially, in the absence of their corresponding inhibitors. To date, most of the studies have concentrated on the structure and function of granzyme A (GrA) and GrB while very little work has been done on the remaining Grz which include GrM, GrH and GrK in humans. In this report, we discuss the current knowledge of Grz biochemistry, biology, functions, activity regulation and their role in human pathology with special emphasis on the significance of human GrK in this field

Nonengraftment Haploidentical Cellular Therapy for Hematologic Malignancies

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

Human Immune Responses to \u3cem\u3eH. pylori\u3c/em\u3e HLA Class II Epitopes Identified by Immunoinformatic Methods

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

HCV Epitope, Homologous to Multiple Human Protein Sequences, Induces a Regulatory T Cell Response in Infected Patients

Background & Aims: Spontaneous resolution of hepatitis C virus (HCV) infections depends upon a broad T cell response to multiple viral epitopes. Most patients fail to clear infections spontaneously, however, and develop chronic disease. The elevated number and function of CD3+CD4+CD25+FoxP3+ regulatory T(reg) cells in HCV-infected patients suggest the role of Treg cells in impaired viral clearance. The factors contributing to increased Treg cell activity in chronic hepatitis C cases remain to be delineated. Methods: Immunoinformatics tools were used to predict promiscuous, highly-conserved HLA-DRB1- restricted immunogenic consensus sequences (ICS), each composed of multiple T cell epitopes. These sequences were synthesized and added to cultures of peripheral blood mononuclear cells (PBMCs) derived from patients who resolved HCV infection spontaneously, patients with persistent infection, and non-infected individuals. The cells were collected following 5 days incubation, quantified and characterized by flow cytometry. Results: One ICS, HCV_G1_p7_794, induced a marked increase in Treg cells in PBMC cultures derived from infected patients, but not patients who spontaneously cleared HCV or non-infected individuals. An analogous human peptide (p7_794), on the other hand, induced a significant increase in Treg cells among PBMCs derived from both HCV infected and non-infected individuals. JanusMatrix analyses determined that HCV_G1_p7_794 is comprised of Treg cell epitopes that exhibit extensive cross-reactivity with the human proteome. Conclusion: A virus-encoded peptide (HCV_G1_p7_794) with extensive human homology activates cross-reactive CD3+CD4+CD25+FoxP3+ nTreg cells, which potentially contribute to immunosuppression and chronic hepatitis C

Cellular Immunotherapy: Using Alloreactivity to Induce Anti-Leukemic Responses without Prolonged Persistence of Donor Cells

A goal of cancer immunologists is to harness cellular immune responses to achieve anti-cancer responses. One of the strongest activating stimuli for the immune system is the encounter with cells expressing allogeneic HLA molecules. While alloreactive responses can negatively impact of the outcome of hematopoietic stem cell transplant because of graft-versus-host disease (GVHD), these same responses can have anti-leukemic effects. Donor lymphocyte infusions have been used in an attempt to harness alloreactive responses to achieve anti-leukemic responses. Because this protocol is usually carried out in the absence of recipient anti-donor responses, this protocol often induces GVHD as well as anti-leukemic responses. A recent study indicated the infusion of large number of haploidentical donor cells (1–2 × 108 CD3+ cells/kg) into patients with refractory hematological malignancies (100 cGy total body irradiation) resulted in 14 (7 major) responses/26 patients. A rapidly developing cytokine storm was observed, while no persisting donor cells could be detected at two weeks after infusion eliminating the possibility of GVHD. Characterization of the effector mechanisms responsible for the anti-leukemic responses in this protocol, should guide new approaches for achieving enhanced anti-leukemic responses using this protocol

Induction of anti-leukemic responses by stimulation of leukemic CD3+ cells with allogeneic stimulator cells

Abstract 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 × 108 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