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

Alloantigen Bound to Agarose Beads and Syngeneic Carrier Cells Are Capable of Stimulating Mouse Cytolytic T Lymphocytes <i>in Vitro</i>

Abstract Bead-bound antigen was prepared by coupling alloantigen covalently to agarose beads. Alloantigen-bearing syngeneic carrier cells were prepared by dilution of detergent solubilized alloantigen in the presence of syngeneic spleen cells. Both types of antigen were compared to spleen cells and reconstituted membrane fragments for the ability to stimulate cytolytic thymus-dependent lymphocytes in vitro. All these types of antigen could stimulate immune but not nonimmune spleen cells to form cytolytic T lymphocytes. The amount of lytic activity obtained with the bead-bound antigen was found to be only dependent upon the amount of H-2 antigen present in the culture and independent of the number of beads.</jats:p

Dissociated and Reconstituted Subcellular Alloantigen Capable of Stimulating Mouse Cytotoxic T Lymphocytes <i>in Vitro</i>

Abstract Reconstituted subcellular membrane fragments were prepared from sonicated murine lymphoid tissue membrane fragments by detergent solubilization and dialysis. The reconstituted subcellular antigen was compared with sonicated cell membranes and x-irradiated cells for the ability to stimulate the formation of allogeneic cytotoxic thymus-dependent lymphocytes in vitro. The results showed that the reconstituted subcellular antigen had properties similar to those reported for the sonicated antigen. Both types of antigen could stimulate immune spleen cells but not normal spleen cells. Furthermore, soluble H-2 antigens, even of membrane origin, were incapable of the stimulation unless reconstituted into membrane fragments large enough to be centrifuged at 48,000 × G for 30 min.</jats:p

Dissecting the use of recipient alloreactive responses to achieve anti-leukemic effect

Abstract Harnessing alloreactive responses to incur anti-leukemic effects has focused on the donor lymphocyte response directed toward recipient antigens. Limited studies have examined the anti-leukemic potential of recipient alloreactive responses to donor cell antigens. An example is the infusion of large numbers of haploidentical, G-CSF mobilized, donor T cells (1 – 2 × 108 CD3+ cells/kg) into patients with refractory hematological malignancies who had received100 cGy total body irradiation prior to infusion. Strong recipient immune responses were generated capable of eliminating the donor cells within two weeks (thereby preventing GVHD) with side effects of high fever (104°F), malaise, rash, and diarrhea. More than half of these patients also generated responses to the hematological malignancies(14/26, 9 major), including some complete remissions. The protocol was modified in the reopened clinical trial by elimination of both the G-CSF mobilization of donor cells and the recipient 100 cGy total body irradiation. These patients exhibited milder, self-resolving fevers and limited anti-leukemic responses (1/5 with transient response). Analysis of blood samples obtained at several different timepoints after infusion revealed diminished IL-6 levels. Increased levels of plasma IL-2 and granzyme B and increased expression of granzyme B and PD-1 by activated recipient T cells correlated with elimination of donor cells. Increased expression of PD-1 ligands on leukemic cells provides one explanation for the lack of anti-leukemic response. These results indicate that appropriate manipulation of both donor cells and patients in this protocol should improve the anti-leukemic responses of recipient lymphocytes.</jats:p