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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: Gamma irradiation of blood products is used to prevent transfusion-associated graft-versus-host disease by inhibiting the proliferation of lymphocytes that are implicated in the disease. Gamma irradiation also damages the red blood cells (RBCs). It is unknown whether hypoxia reduces the efficacy of gamma irradiation in inhibiting lymphocyte proliferation (LP). The objectives of the study were to investigate the effects of hypoxia on gamma irradiation-induced inhibition of LP and on the in vitro properties of RBCs., Materials and Methods: Forty-four units (300-340 ml each) of less than 8-h-old ABO-matched leukocyte reduced red cell concentrates (LR-RCC) in additive solution 3 were pooled in pairs. Peripheral blood mononuclear cells were isolated from non-leukocyte reduced RCCs and added back to the pool at a final concentration of 2 × 10 5 /ml. The pool was divided equally into a conventional storage bag A and a hypoxic processing and storage bag B. The units were gamma-irradiated at 25Gy on day 7 for the LP experiment and on either day 7 or 14 for the RBC quality experiments. LP was measured using a limiting dilution assay, and several in vitro metrics of RBCs were measured., Results: Gamma irradiation inhibited T-lymphocyte proliferation by 4.7 × 10 4 -fold reduction in both hypoxic and conventional storage. The in vitro metrics of RBC quality were better preserved in hypoxic storage., Discussion: T lymphocytes present in hypoxic RBC are equally susceptible to gamma irradiation as conventional storage. Hypoxic storage also reduces the deleterious effects of gamma irradiation on RBCs., (© 2021 AABB.)

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.

Extracellular vesicles (EV) are nano-sized membrane enclosed vehicles that are involved in cell-to-cell communication and carry cargo that is representative of the parent cell. Recent studies have highlighted the significant roles leukemia EVs play in tumor progression, and ways in which they can lead to treatment evasion, thus meriting further investigation. Leukemia EVs are involved in crosstalk between the leukemia cell and its surroundings, transforming it into a cancer favorable microenvironment. Due to the diverse biological content found in leukemia EVs, they have an assortment of effects on the cells they interact with and can be harnessed as candidates for diagnostic and therapeutic treatments. This review focuses on EVs in the context of leukemia and the means by which they modulate their microenvironment, hematopoiesis, and the immune system to facilitate malignancy. We will also address current and prospective EV-based therapeutics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although it is possible to induce remission in the majority of the patients with acute myeloid leukemia (AML), many patients still die due to disease relapse. Immunotherapy is an attractive option. It is more specific. The memory T cells induced by immunotherapy may also provide the long-term tumor immunosurveillance to prevent disease relapse. Although immunotherapy of AML started in the early 1970s, its clinical impact has been disappointing. Recent advances in tumor immunology and immunotherapeutic agents have rekindled interest. Here, we provide a review of the history of AML immunotherapy, discuss why AML is well suited for immunotherapeutic approaches and present the biological obstacles that affect the success of immunotherapy. Finally, we put forward a new paradigm of AML immunotherapy that utilizes a combination of immunotherapeutic agents sequentially to enhance the in vivo tumor immunogenicity and effective priming and propagation of tumor-specific cytotoxic T cells.

Background: Approximate 180 million people worldwide are infected with hepatitis C virus (HCV). Historically, vaccination has been the most effective strategy for controlling infections of such major health concern. Therapeutic vaccine strategies for HCV, however, have demonstrated negligible success., Aim: Demonstrate the ability of highly-conserved viral epitopes to overcome the immune dysfunction often associated with chronic HCV infections., Methods: T cells from five chronic, HCV-infected patients were immunophenotyped by flow cytometry. The ex vivo T cell responses to highly-conserved viral epitopes were assessed by ELISpot assay and cytokine bead array analysis., Results: Both HLA-DRB-1- and HLA-A2-restricted viral epitopes induced specific, T H 1-type cytokine production by T cells derived from the patients. Induction occurred despite expression of cell-surface inhibitory molecules and the presence of regulatory T cells., Conclusion: These findings support the potential ability of a broad, multi-epitope-based therapeutic vaccine to elicit virus-specific immune responses in chronic hepatitis C virus-infected patients., (Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

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.

Adoptive immunotherapy in the form of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a treatment modality for acute and chronic leukemias that has been in practice for several decades. Drawbacks to transplantation include toxicity from chemotherapy/radiation conditioning regimens, additional toxicity from graft versus host disease, and reliance on appropriate human leukocyte antigen matched donors. Newer modalities with increased specificity of donor cells to tumor cells in addition to therapies that do not require engraftment for anti-tumor effect reduce the risk of graft versus host disease and may create a more robust graft versus leukemia response. Without the need for engraftment, or at the very least in the absence of a 100% engraftment requirement, conditioning regimens may be minimized. Three methods of adoptive immunotherapy that may offer some of these advantages over traditional transplantation are donor lymphocyte infusions (DLI), chimeric antigen receptor modified T cells (CAR T cells), and cellular immunotherapy. DLIs and cellular therapy consist of transfusing T lymphocytes from the donor to recipient in an unmanipulated form. Alternatively, donor T lymphocytes can be modified with addition of chimeric antigen receptors for specific antigen directed killing of tumor cells. Significant responses and survival benefit have been reported with these modalities. Herein, we review the mechanisms for these newer adoptive immune therapies, clinical indications for their use, and potential future directions.

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.

Background: Exposure of blood products to gamma irradiation is currently the standard of care in the prevention of transfusion-associated graft-versus-host disease (TA-GVHD). Regulatory, technical, and clinical challenges associated with the use of gamma irradiators are driving efforts to develop alternatives. Pathogen reduction methods were initially developed to reduce the risk of microbial transmission by blood components. Through modifications of nucleic acids, these technologies interfere with the replication of both pathogens and white blood cells (WBCs). To date, systems for pathogen and WBC inactivation of products containing red blood cells are less well established than those for platelets and plasma., Study Design and Methods: In this study, the in vitro and in vivo function of WBCs present in whole blood after exposure to riboflavin plus ultraviolet light (Rb-UV) was examined and compared to responses of WBCs obtained from untreated or gamma-irradiated blood by measuring proliferation, cytokine production, activation, and antigen presentation and xenogeneic (X-)GVHD responses in an in vivo mouse model., Results: In vitro studies demonstrated that treatment of whole blood with Rb-UV was as effective as gamma irradiation in preventing WBC proliferation, but was more effective in preventing antigen presentation, cytokine production, and T-cell activation. Consistent with in vitro findings, treatment with Rb-UV was as effective as gamma irradiation in preventing X-GVHD, a mouse model for TA-GVHD., Conclusion: The ability to effectively inactivate WBCs in fresh whole blood using Rb-UV, prior to separation into components, provides the transfusion medicine community with a potential alternative to gamma irradiation., (© 2012 American Association of Blood Banks.)

The transfusion of blood products can result in a variety of consequences, including transfer of pathogens and the induction of immune responses, such as the almost invariably fatal transfusion-associated graft-versus-host disease (TA-GVHD). Exposure of blood products to γ-irradiation is currently the standard of care for the prevention of TA-GVHD. Regulatory, technical and clinical challenges associated with the use of γ-irradiators are driving efforts to develop alternatives. Initially, pathogen reduction methods were developed to reduce the risk of microbial transmission by blood components. Through modifications of nucleic acids, these technologies interfere with the replication of both pathogens and leucocytes. These methods have been found to be as effective as γ-irradiation in preventing T lymphocyte proliferation and TA-GVHD responses. Additional benefits of pathogen reduction protocols potentially include inhibition of antigen presentation, cytokine production and activation of lymphocytes., (© 2012 Blackwell Publishing Ltd.)

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.

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1-TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase β plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.

Background: During the transfusion of blood products, the transfer of allogeneic donor white blood cells (WBCs) can mediate adverse reactions in recipients. To minimize reactions, blood components are leukoreduced and/or exposed to gamma irradiation. Nucleic acid-targeted pathogen reduction technologies (PRTs) processes are well suited for WBC inactivation. The Mirasol PRT system (CaridianBCT Biotechnologies) uses riboflavin and ultraviolet light to reduce the active pathogen load and inactivate residual WBCs in blood products used for transfusion. The aim of this study was to compare the effect of PRT treatment to gamma irradiation., Materials and Methods: WBCs were resuspended in 100% plasma or in plasma containing 65% platelet additive solution (SSP+). A single product was split into three aliquots: control, PRT treatment, or gamma irradiation. After treatment, peripheral blood mononuclear cells were isolated from products, and cell viability and functionality were assessed in limiting dilution assays (LDAs), by CD69 expression, by proliferation, and by cytokine accumulation after lipopolysaccharide addition., Results: PRT treatment and gamma irradiation reduced the ability of the T cells to proliferate by similar degrees as evidenced by a 6 log or greater reduction in the LDA and a lack of response to several stimuli. However, gamma-irradiated T cells were still capable of up regulating surface CD69, inducing a proliferative response in allogeneic responder cells, and producing levels of proinflammatory cytokines similar to those of untreated control cells, responses that PRT-treated T cells were incapable of mediating., Conclusions: These in vitro results demonstrate that PRT treatment is more effective than gamma irradiation at abrogating selected WBC immune functions., (© 2010 American Association of Blood Banks.)

Functional white blood cells (WBCs) in blood components may be responsible for a number of adverse transfusion effects, including transfusion-associated graft-versus-host disease (TA-GVHD), alloimmunization, and alloimmune platelet (PLT) refractoriness. TA-GVHD occurs when functional lymphocytes are transfused into a patient who is unable to mount an immune response to the human leukocyte antigen (HLA) due to HLA compatibility or immunosuppression. Alloantibodies against HLA antigens on donor WBCs and PLTs are the major cause of refractoriness to PLT transfusions in patients receiving repeated blood transfusions. Attempts to reduce these undesirable effects have included leukoreduction filters and gamma irradiation. Studies have shown that exposure of PLT concentrates to riboflavin and light (Mirasol pathogen reduction technology [PRT], CaridianBCT Biotechnologies) causes irreparable modifications of nucleic acids that result in inactivation of a wide range of pathogens as well as inhibition of the immunologic responses mediated by WBCs present in PLT concentrates. This article summarizes these studies and also reports on additional findings from the Trial to Reduce Alloimmunization to Platelets (TRAP) and Mirasol Clinical Evaluation (MIRACLE) trials. Data from in vitro studies and this clinical trial suggest that PRT treatment may be as effective as gamma irradiation in preventing TA-GVHD and more effective than leukoreduction in preventing alloimmunization., (© 2010 American Association of Blood Banks.)

Objective: The purpose of this study was to determine in a mouse model whether uterine natural killer (uNK) cell cytotoxic activation induces infection/inflammation-associated preterm labor and delivery., Study Design: Wild type or interleukin (IL)-10(-/-) mice were injected intraperitoneally with lipopolysaccharide on gestational day 14. Mice were either killed for collection of uteroplacental tissue, spleen, and serum or allowed to deliver. Uteroplacental tissue was used for histology and characterization of uNK cells., Results: Low-dose lipopolysaccharide treatment triggered preterm labor and delivery in IL-10(-/-), but not wild type mice, in a manner independent of progesterone levels. Preterm labor and delivery in IL-10(-/-) mice was associated with an increased number and placental infiltration of cytotoxic uNK cells and placental cell death. Depletion of NK cells or tumor necrosis factor (TNF)-alpha neutralization in these mice restored term delivery. Furthermore, TNF-alpha neutralization prevented uNK cell infiltration and placental cell apoptosis., Conclusion: The uNK cell-TNF-alpha-IL-10 axis plays an important role in the genesis of infection/inflammation-induced preterm labor/delivery.

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.

Background: Mirasol PRT (Navigant Biotechnologies) treatment utilizes exposure to light in the presence of riboflavin to introduce irreparable lesions to nucleic acids thereby inhibiting pathogen and WBC replication. The ability of Mirasol PRT-treated mononuclear cells (MNCs) to generate xenogeneic graft-versus-host disease (GVHD) responses was used to model transfusion-associated GVHD (TAGVHD)., Study Design and Methods: Pairs of human MNCs from five different individual donors that had or had not received Mirasol PRT treatment and contained 30 x 10(6) CD3+ cells were injected intraperitoneally into sublethally irradiated (350 cGy) Rag2(-/-)gamma c(-/-) double-knockout mice. Recipient mice were weighed and observed regularly and euthanized when they exhibited symptoms of GVHD or at termination of the experiment. Recipient lymphoid compartments were collected and phenotyped for the presence of human lymphoid cells. The presence of human cytokines and/or immunoglobulins in the recipient plasma was also used to detect the presence of human cells., Results: Twelve of 14 mice injected with untreated cells developed xenogeneic GVHD, whereas 0 of 14 mice injected with Mirasol PRT-treated cells developed xenogeneic GVHD. End-stage xenogeneic GVHD in the recipients of untreated cells was characterized by the presence of splenic human cytolytic CD4+ and CD8+ cells, with high levels of interferon-gamma, interleukin-10, and xenoreactive antibodies in the plasma., Conclusion: Mirasol PRT treatment of the donor MNCs abolished xenogeneic GVHD responses, indicating that the use of Mirasol PRT treatment of blood products should prevent the development of TAGVHD.

Background: The presence of white blood cells (WBCs) in blood products has been shown to contribute to the development of a variety of immune responses including both donor antirecipient and recipient antidonor responses. Mirasol PRT treatment is a pathogen reduction protocol that is based on the inactivation of nucleic acid replication after exposure to riboflavin and light and was tested for its ability to inactivate nucleated WBC function., Study Design and Methods: Sets of paired WBCs from different donors who either had received Mirasol treatment or were untreated were tested for their ability to be activated in response to phorbol myristic acetate (PMA); to proliferate in response to stimuli including phytohemagglutinin PHA, anti-CD3+ anti-CD28, or allogeneic stimulator cells; to stimulate proliferation by allogeneic responder cells; and to produce cytokines in response to lipopolysaccharide (LPS) and anti-CD3+ anti-CD28., Results: Although Mirasol PRT treatment did not significantly change the distribution of human lymphocyte subpopulations, there were significant functional changes. Mirasol PRT treatment inhibited activation in response to PMA and completely inhibited proliferation in response to PHA, anti-CD3+ anti-CD28, or allogeneic stimulator cells. Mirasol PRT treatment also prevented the cells' ability to act as antigen-presenting cells and the ability to produce cytokines in response to stimuli such as LPS or anti-CD3+ anti-CD28., Conclusions: Mirasol PRT treatment is able to functionally inactivate WBCs in blood products along with inactivating pathogens and should prevent immunological consequences resulting from the presence of WBCs in blood products.

Specialized NK cells are recruited in high numbers to the mammalian embryo implantation sites, yet remain pregnancy compatible. It is not well understood whether uterine NK (uNK) cells become adversely activated and mediate fetal demise, a common complication of early pregnancy. In this study we show that mating of IL-10(-/-) mice resulted in fetal resorption or intrauterine growth restriction in response to very low doses of LPS. Pregnancy in congenic wild-type mice was normal even at 10-fold higher LPS doses. Fetal resorption in IL-10(-/-) mice was associated with a significant increase in uNK cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells, TNF-alpha neutralization, or IL-10 administration rescued pregnancy in LPS-treated IL-10(-/-) animals. Our results identify an immune mechanism of fetal demise involving IL-10 deficiency, NK cells, and inflammation. These results may provide insight into adverse pregnancy outcomes in humans.

Background: Development and characterization of methods for preventing transfusion-associated GVHD have utilized in vitro studies with human WBCs and in vivo studies in animal models. The limitation of these assays is that the in vivo GVHD response of treated human WBCs has not been tested directly., Study Design and Methods: PBMNCs isolated from nonleukoreduced RBC units exposed to gamma irradiation, treated with PEN110 or PBS, were tested for their ability to induce xenogeneic GVHD when injected into severe combined immunodeficient (SCID) mice., Results: These studies showed that the SCID mice injected with PBS-treated PBMNCs developed serum levels of human immunoglobulin that were followed by weight loss and display of ruffled fur characteristic of xenogeneic GVHD in these mice. In contrast, SCID mice injected with PEN110-treated or gamma-irradiated PBMNCs did not exhibit any of these responses., Conclusions: In these studies PEN110 treatment and gamma irradiation were equally effective at preventing in vivo GVHD responses when the treated cells were injected into SCID recipients. These results are consistent with previous results obtained when these two treatment methods were compared with in vitro studies with PBMNCs and in vivo studies in mouse models.

Background: WBCs in blood components are a major factor contributing to immune responses such as GVHD and alloimmunization that follow transfusion. Irradiation of blood components has been used to regulate the contribution of donor WBCs to these immune responses. A systematic comparison of how the exposure of lymphoid cells to gamma-irradiation, UVB light, or psoralen + UVA light (PUVA) effects immune response was conducted to better define the best type of irradiation to be used in different clinical settings., Study Design and Methods: Murine spleen cells were irradiated with varying doses and tested for their in vitro ability to be activated, to proliferate in response to mitogen or allogeneic stimulator cells, or to serve as stimulator cells. Irradiated donor cells were also tested for in vivo generation of GVHD, induction of alloantibody production, induction of restricted cytolytic T lymphocytes, and persistence of irradiated cells., Results: In general, increasing amounts of irradiation resulted in increased inhibition of in vitro and in vivo responses, although the doses required for inhibition differed from assay to assay. In contrast, irradiation of donor cells did not always result in inhibition of recipient alloantibody responses but was dependent on the donor-recipient combination that was studied., Conclusion: Control of the in vivo outcomes of transfusing allogeneic cells is regulated by the type and dose of irradiation used and by the genotype of the donor and/or recipient.

Background: The presence of WBCs in blood components is the primary factor influencing the immunologic consequences of transfusion, such as GVHD and alloimmunization. Depletion or inactivation of WBCs can reduce the deleterious responses. Because treatment with PEN110 (Inactine, V. I. Technologies), an ethyleneimine derivative that disrupts nucleic acid replication, was shown to inactivate in vitro human PBMNC function, the ability of PEN110-treated cells to trigger GVHD or alloantibodies was studied with in vivo murine models., Study Design and Methods: In vitro assays were employed to confirm that PEN110 treatment inactivated murine splenocyte function as effectively as for human PBMNCs. In vivo experiments in mice examined the ability of PEN110-treated cells to induce GVHD responses in a parent into F1 hybrid GVHD model, to induce alloantibodies, to stimulate MHC-restricted cytolytic T lymphocyte responses, and to persist after injection., Results: PEN110-treated murine splenocytes did not respond or induce responses in any in vitro or in vivo assay. The PEN110-treated cells were eliminated from blood and secondary lymphoid organs much more rapidly than were untreated cells., Conclusion: PEN110 treatment prevents the development of GVHD and alloantibody production following WBC transfusion in a murine model system, supporting the continued development of PEN110 treatment of cellular blood components as an alternative to gamma irradiation for the prevention of GVHD.

Background: The presence of WBCs in blood components is the major factor influencing the immunologic consequences of transfusion. Attempts to ameliorate these responses have used WBC reduction or inactivation by ionizing radiation. PEN110 (Inactine, V. I. Technologies) is a chemical that inhibits the replication of infectious pathogens by modifying their nucleic acids. These experiments compared effects of PEN110 treatment or gamma irradiation on WBC function., Study Design and Methods: Aliquots of non-WBC-reduced RBC units were treated with PEN110 or gamma irradiation with appropriate controls, and PBMNCs from these units were tested with in vitro assays. The assays included immunophenotyping, activating T cells with phorbol ester, proliferation of cells in response to mitogens or allogeneic cells, and determining the ability of cells to stimulate proliferative responses and to produce IL-8. DNA fragmentation following PEN110 treatment was examined by PCR and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assays., Results: Treatment of non-WBC-reduced RBC units with PEN110 functionally inactivated WBC in all in vitro assays used. In contrast, while gamma irradiation inhibited proliferation of the WBCs, it did not or only partially inhibited the ability of WBC to function in the other assays. PEN110, but not gamma irradiation, rapidly induced fragmentation of cellular DNA., Conclusion: Because PEN110 was as effective as gamma irradiation at inhibiting WBC proliferation, PEN110 treatment could potentially be used to prevent the development of GVHD following transfusion.

Recent studies have defined several phenotypic and molecular changes associated with the maturation of naive human B cells within the milieu of germinal centers. Although naive B cells serve as natural precursors to germinal center (GC)/memory (M) subpopulations, little is known about the physiological requirements for the survival of the naive B cell pool in the absence of cell-cell contact or Ag-mediated activation. Because IL-4 induces expression of several membrane receptors such as CD23 which are uniquely present on resting human naive B lymphocytes, we hypothesized that these cells might be intrinsically programmed to respond to IL-4 in the absence of cell division. Using buoyant density-dependent isolation and further enrichment by negative/positive selection of human naive and GC/M subpopulations, we characterized cytokine receptor moieties on these cells and analyzed their survival and growth in the presence of IL-4 or IL-10. Resting naive B cells expressed significantly higher IL-4 receptor alpha-chain on their cell surface than the combined GC/M subpopulation. The IL-10 receptor and the IL-2 receptor gammac chain were almost equally expressed on both subpopulations. When cultured in vitro, the addition of IL-4, but not IL-10, protected naive B cells from apoptosis in the absence of activation and growth. However, IL-4 exerted no such effect on resting GC/M B cells. These data support the hypothesis that IL-4 plays a pivotal role in the survival and maintenance of resting human naive B cells.

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.

Cytokine responses to lymphocytic choriomeningitis virus infections were evaluated, and CD8+ T cell, CD4+ T cell, and IL-2 contributions delineated. In immunocompetent mice, lymphocytic choriomeningitis virus induced both IFN-gamma and IL-4 as well as IL-2. Experiments in mice either beta2-microglobulin-deficient, lacking MHC class I molecules and CD8+ T cells, or A beta(b)-deficient, lacking MHC class II molecules and CD4+ T cells, demonstrated that mixtures of T cell responses were required for optimal ex vivo cytokine productions. Intracellular cytokine expression analyses of cells from immunocompetent and immunodeficient mice showed that CD8+ T cells were predominant IFN-gamma producers, and that expansion of CD8+ T cells primed to make IFN-gamma was independent of CD4+ T cells in vivo. Studies in IL-2-deficient mice demonstrated that this cytokine promoted IFN-gamma and IL-4 responses, and ex vivo experiments showed that exogenous IL-2 was required to maintain high-level IFN-gamma production by in vivo-primed CD8+ T cells. Conditions associated with cytokine decreases were accompanied by reduced detectable plasma Ab responses. The results indicate that, although IL-2-dependent CD8+ T cell proliferation does not require endogenous CD4+ T cells, IL-2 production by the CD4+ T cells may promote continued cytokine release from activated CD8+ T cells. By defining these critical steps in cellular and cytokine interactions for shaping endogenous immune responses, the studies advance understanding of the unique conditions regulating CD8+ T cell responses to viral challenges.

Blood transfusion can result in a variety of immunologic responses, including alloimmunization, transfusion-associated graft-versus-host disease, and immunosuppression that results in increased postoperative infection rate, and can also result in increased survival of allografts. One of the many factors influencing the resulting immunologic responses from a blood transfusion is the persistence of the donor leukocytes. A recent study has shown that almost all (99.9%) allogeneic leukocytes are removed within 2 days following transfusion but did not characterize the mechanism responsible for the rapid removal of allogeneic donor cells. Using a murine model these studies show that it is recipient CD8+ T cells that are responsible for the rapid elimination of allogeneic lymphocytes in naive recipients. Effective elimination was dependent on the donor/recipient combination and required that the donor cells express at least one MHC class I disparity to be recognized by the recipient CD8+ cells and that the donor cells also be able to induce additional responses that were needed by the CD8+ cells to manifest full activity. The perforin pathway was the predominant pathway used by the recipient CD8+ cells to mediate this elimination.

In vivo NK cell responses to lymphocytic choriomeningitis virus were studied in CD8+ T cell-deficient mice. On day 7 after infection, dramatically elevated splenic NK cell activities were observed in both beta 2-microglobulin-negative (beta 2-m-/-) mice deficient in CD8+ T cells and anti-CD8-treated C57BL/6 animals. The enhanced responses could be attributed to increased numbers of activated NK1.1+CD3- cells. The day 7 NK cell responses in beta 2-m-/- mice, but not in normal C57BL/6 animals, were cyclosporin A sensitive and coincided with IL-2 production and high affinity IL-2R expression on NK cells. Proof that IL-2 played an essential role in day 7 responses was provided by the observation that IL-2-/- x beta 2-m-/- mice lacked the late NK cell activation. Taken together, these results showed that NK cells can be activated and expanded by an IL-2-dependent pathway. Because these responses can only be measured in the absence of CD8+ T lymphocytes, an exciting model of networking between T and NK cells in response to viruses is postulated.

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.

The regulation of in vivo cytolytic response is important in a model of murine graft-vs-host disease induced by the injection of parental splenocytes into unirradiated B6D2F1 recipients. Injection of C57BL/6J spleen cells into B6D2F1 recipients results in an acute form of graft-vs-host disease that is characterized by the presence of CTL and suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells into B6D2F1 recipients results in a chronic form of graft-vs-host disease that is characterized by the lack of in vivo CTL and hyperproduction of Ig and autoantibodies that results in an SLE-like syndrome. One reason for the lack of donor antirecipient CTL after injection of DBA/2J donor cells is that B6D2F1 recipient cells functionally inactivate the donor DBA/2J CTL precursor cells by expressing veto activity. These B6D2F1 veto cells are radiosensitive, inhibited by anti-CD8 antibodies, found primarily in lymph nodes, and were further characterized by testing the response of these inhibitory cells to lymphokines. These studies indicate that IL-2 can potentiate the activity of the veto cells induced in vivo and veto cells with a similar phenotype can be generated by in vitro incubation of naive lymph node cells with IL-2. These cells have been designated as IL-2-activated veto cells or LAV cells. IL-2 did not increase inhibitory activity by increasing the number of CD8+ cells or the number of CD8 molecules on the LAV cell surface but by altering the activation state of the LAV cell. The inhibitory capabilities of antibodies binding various cell surface molecules indicated that CD2 and intercellular adhesion molecule-1 molecules in addition to CD8 molecules played a role in the function of LAV cells.

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.

Injection of parental spleen cells into unirradiated F1 hybrid recipients is frequently used for the induction of murine graft-vs-host disease (GVHD). Injection of C57BL/6 spleen cells into B6D2F1 recipients results in the acute form of GVHD whereas injection of DBA/2J cells into the same recipients results in a chronic form of GVHD. In vivo studies have suggested that injection of DBA/2J cells into B6D2F1 recipients results in the chronic form of GVHD instead of acute GVHD because of recipient B6D2F1 effector cells that inhibit DBA/2J CD8+ T cell function. The present report describes the development of an in vitro assay that measures the activity of these B6D2F1 effector cells. The results obtained using this assay indicate that these B6D2F1 effector cells are found primarily in the LN, need to be activated in order to function, are inhibited by anti-CD8 antibodies, and mediate their function as a result of DBA/2J cells binding to Ag expressed on their cell surface.

Aging induces a number of changes in the immune system, including the involution of the thymus which results in the loss of thymic hormone production and alteration in T cell function. One age-dependent change in immune response is the increasing risk of developing acute or chronic form of graft-versus-host disease (GVHD) following bone marrow transplantation as the age of the recipient increases. A murine model of GVHD that has been extensively studied is one in which injection of C57BL/6 spleen cells into unirradiated B6D2F1 mice results in an acute form of GVHD characterized by cytolytic T lymphocytes (CTL), suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells results in a chronic form of GVHD characterized by a lack of CTL and hyperproduction of immunoglobulin and autoantibodies. This study shows that the GVHD response of DBA/2J spleen cells is dependent on the age of the donor DBA/2J mice. If spleen cells from DBA/2J mice older than 3 months are injected into B6D2F1 recipients, CTL and lack of immunoglobulin production indicative of acute GVHD result. Administration of thymosin fraction 5, a collection of thymic hormones, to DBA/2J mice older than 3 months caused spleen cells from these treated mice to give a GVHD response characteristic of the chronic form of GVHD in B6D2F1 recipients. Thus, thymic hormones were able to modulate the changes in GVHD responses of DBA/2 lymphocytes that occur as the mice age. Preliminary fractionation of TF5 has indicated that there are at least two active thymic peptides present in TF5.

Non-MHC loci have been shown to play an important role in the development and regulation of graft-vs-host disease (GVHD). In the murine model of GVHD under study, injection of C57BL/6 spleen cells into unirradiated (C57BL/6 x DBA/2)F1 hybrid recipient mice results in an acute form of GVHD characterized by CTL, suppressor cells, and runting. In contrast, injection of DBA/2 spleen cells into the same recipients results in a chronic form of GVHD that is characterized by a lack of CTL and hyperproduction of Ig and autoantibodies. After preliminary studies with the use of congenic mice showed that non-MHC loci were controlling GVHD responses in this model, genetic analysis of GVHD response of BXD recombinant inbred strains and (B10.D2 x DBA/2) X DBA/2 BC mice identified a single locus, Gvh, on chromosome 7 that controls whether acute or chronic GVHD results from injection of parental lymphocytes into unirradiated (C57BL/6 x DBA/2)F1 recipient mice.

A cell type in addition to T lymphocytes and macrophages was found to be necessary for the in vitro stimulation of mouse anti-Sendai viral T lymphocytes in response to subviral antigens. This cell type was found to be glass and nylon wool adherent, to bear no surface immunoglobulin or Thy 1.2 antigen, and to be radiosensitive. This type of cell was not needed when the stimulatory antigen was virus-infected cells.

The previously described NK inhibitory monoclonal antibody 13.1 is shown to immunoprecipitate a series of high m.w. glycoproteins homologous with the murine T-200/Ly-5 molecules. Not all antibodies to the human T-200 molecule, however, have an inhibitory effect on NK cell function. A comparison is made between two noninhibitory anti-T-200 antibodies, 13.5 and 13.6, and two inhibitory anti-T-200 antibodies, 13.1 and 13.3. All antibodies are of the IgG1 subclass. Sequential immunoprecipitation experiments show that these antibodies react with the same set of molecules. The differences in NK-blocking activity could not be explained by the amount of antibody bound per cell in NK-enriched populations, nor by the avidity with which they bound. It is shown by competitive radiobinding assays that the 13.1 and 13.3 antibodies define a region, termed region A, distinct from that defined by the nonblocking antibodies 13.5 and 13.6, termed region B. Region B is shown to reside between the membrane and region A. These findings show that the inhibition of NK lysis by anti-T-200 antibodies is a function of the site on that molecule to which these antibodies bind. This may also explain the ability of antibodies to the A region of T-200 to block selectively the lysis of myeloid and erythroid tumor targets, with no effect on the lysis of T lymphoma targets.

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.

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.