L-leucyl-L-leucine Methyl Ester Research Articles

Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin

In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14+ monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14+ monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

Identification of Immunopotentiating Lactic Acid Bacteria that Induce Antibody Production by <i>in vitro</i> Stimulated Human Peripheral Blood Mononuclear Cells

L-leucyl-L-leucine methyl ester (LLME) is known to remove lysosome-rich cells from human peripheral blood mononuclear cells (PBMCs). To evaluate the immunopotentiating ability of lactic acid bacteria (LAB), we adopted the in vitro stimulation protocol of LLME-treated PBMCs as a model assay system and monitored the level of antibody produced by stimulated PBMCs. The results indicated that several LAB strains have immunopotentiating ability against PBMCs, as evidenced by the enhanced antibody production and increased number of antigen-specific B cells. Next, we identified T cells as the direct target cells of the immunopotentiating LAB strain L32, suggesting that L32 induced antibody production by PBMCs through T-cell activation. Finally, we tested the immunopotentiating ability of ligands for Toll-like receptor 2 (TLR2), which is known to mediate the LAB signal, and observed that both L32 and one of the TLR2 ligands, LTA-BS, induced antigen-specific antibody production by in vitro stimulated PBMC. This suggests that L32 and LTA-BS can be used as an adjuvant for stimulating immune reaction in PBMCs.

L-Leucyl-L-Leucine Methyl Ester (LLME) Treated Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for Patients With Hematological Malignancies

GVHD remains an obstacle for allogeneic HSCT. While T cell depletion of donor grafts results in much lower incidences of GVHD, rejection and poor immune recovery are often associated with this approach. To address these issues, we developed a transplant approach using LLME to test the hypothesis that the selected depletion of T cells containing cytotoxic effector granules would result in less significant GVHD while at the same time preserving GVT effects and infectious immunity. LLME preferentially kills cytotoxic effector granule bearing lymphocytes including most NK and CD8 T cells with relative sparing of CD4 T cells.

Anti-Peptide Antibody Production Elicited byin VitroImmunization of Human Peripheral Blood Mononuclear Cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-alpha as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.

Propionibacterium AcnesActs as an Adjuvant inin VitroImmunization of Human Peripheral Blood Mononuclear Cells

We have established an in vitro immunization protocol whereby human peripheral blood mononuclear cells (PBMCs) are initially treated with L-leucyl-L-leucine methyl ester (LLME) and subsequently sensitized with antigen in the presence of interleukin (IL)-2, IL-4, and adjuvant. This protocol resulted in the production of antigen-specific antibodies. PBMCs are potentiated to react with exogenous antigens upon treatment with LLME. We are using this system to investigate the immunomodulatory activity of additives. In the present study, we aimed to evaluate the immunomodulatory activity of Propionibacterium acnes (P. acnes), which is known to exhibit various immunomodulatory effects in murine models, using this in vitro immunization protocol. P. acnes was found to augment the production of antigen-specific antibodies by PBMC, possibly through increased production of inflammatory cytokines and/or increased T-B cell interaction. P. acnes hence appears to act as an adjuvant in the antibody response in in vitro immunization.

Accelerated immune recovery following LLME treated donor lymphocyte infusion

Delayed immune reconstitution is a major cause of morbidity and mortality after T cell depleted allogeneic progenitor cell transplant (PCT). To accelerate immune reconstitution without GVHD, we have administered escalating doses of L-leucyl-L-leucine methyl ester (LLME) treated lymphocytes (DLI) to 18 patients post CD34+ cell-enriched PCT in an ongoing phase I trial. LLME’s cellular toxicity occurs after polymerization by dipeptidyl peptidase, leading to selective depletion of cells with cytotoxic effector granules containing perforin.

Identification of Genes Involved in the Suppression of Antibody Production from Human Peripheral Blood Lymphocytes

Pretreatment with L-leucyl-L-leucine methyl ester (LLME) is a prerequisite for peripheral blood mononuclear cells (PBMCs) to produce antigen-specific antibodies when sensitized with an antigen. Little information, however, is available regarding the mechanisms involved in LLME-induced augmentation of antibody production from PBMCs that are antigen sensitized. In the present study, we attempted to identify the genes involved in the suppression of antibody production from PBMCs that was not treated with LLME, but sensitized with an antigen. Using subtractive screening, we obtained 63 independent genes, including 17 EST genes, that are specific for LLME-nontreated PBMC. Among these genes, the expression of heavy chain ferritin (H-ferritin), CC chemokine ligand 18 (CCL18), and matrix metalloproteinase 12 (MMP12) were augmented in LLME-nontreated PBMCs, suggesting that inflammatory factors might be involved in the suppression of antibody production in LLME-nontreated PBMCs.

Complete allogeneic hematopoietic chimerism achieved by in utero hematopoietic cell transplantation and cotransplantation of LLME-treated, MHC-sensitized donor lymphocytes

Objective In utero hematopoietic cell transplantation (IUHCT) typically achieves low-level mixed hematopoietic chimerism. However, the goal of IUHCT is to achieve therapeutic levels of chimerism. We hypothesized that prenatal adoptive immunotherapy might achieve high-level donor chimerism after IUHCT. Materials and methods BALB/CE15 fetal mice were transplanted with a mixture of C57BL/6 (B6) T-cell–depleted bone marrow (TCD BM) cells and splenocytes from B6 mice presensitized to BALB/C alloantigen. The splenocytes were preincubated in L-leucyl-L-leucine methyl ester (LLME), to minimize graft vs host disease (GVHD). Recipients were followed after birth for donor cell chimerism and GVHD. Results Full donor hematopoietic chimerism following a single prenatal transplant was achieved in seven transplanted animals. Fully chimeric animals were healthy, without evidence of GVHD, and maintained their engraftment for the duration of the study (48 weeks). However, the addition of presensitized LLME-treated cells decreased survival until weaning relative to TCD BM alone, suggesting that some animals were lost to acute GVHD. Surviving chimeric animals demonstrated increased frequencies of T-regulatory cell populations in their spleen and BM, suggesting that they had successfully suppressed GVHD, allowing survival. Conclusions This study represents “proof in principle” that prenatal immunotherapeutic strategies may achieve complete hematopoietic engraftment across full MHC barriers when combined with IUHCT. However, strategies with greater hematopoietic specificity must be developed prior to consideration of clinical application.

T cell repertoire complexity is conserved after L-leucyl-L-leucine methyl ester (LLME) treatment of donor lymphocyte infusions

T cell repertoire complexity is conserved after L-leucyl-L-leucine methyl ester (LLME) treatment of donor lymphocyte infusions

IL-10 Augments Antibody Production inin VitroImmunized Lymphocytes by Inducing a Th2-Type Response and B Cell Maturation

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4(+) and CD8(+) T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4(+) and CD8(+) T cells, as well as in CD19(+) B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.

Leucyl-leucine methyl ester-treated haploidentical donor lymphocyte infusions can mediate graft-versus-leukemia activity with minimal graft-versus-host disease risk

L-leucyl-L-leucine methyl ester (LLME) prevents GVHD in several animal models by depleting dipeptidyl peptidase I (DPPI)-expressing cytotoxic cellular subsets. However, clinical application has been hampered by difficulties in stem cell engraftment following treatment of donor bone marrow inocula with LLME at the concentrations necessary to purge DPPI-expressing T-cells. Noting that T-cells can mediate graft-versus-leukemia (GVL) responses via both perforin (usually co-expressed in cytotoxic granules with DPPI) and Fas ligand pathways in a murine model, we hypothesized that LLME might be useful for treatment of delayed DLIs for potential GVL activity with a decreased risk of GVHD induction. In regard to the clinical setting, the ex vivo use of LLME for this purpose would circumvent any toxicity issues for donor stem cells, because by that time patients would have already achieved successful engraftment. For our preclinical studies, we used the haploidentical C57BL/6 (B6) (H2b) --> ((B6 x DBA/2)F1 (H(2b/d)) murine model with lethally irradiated hosts that had received transplants of T-cell-depleted bone marrow cells and were challenged with the MMD2-8 myeloid leukemia line (H2d) of DBA/2 origin. A DLI of LLME-treated donor splenocytes, from B6 mice presensitized to recipient alloantigens, was administered in varying doses 14 days post-marrow transplantation, and the potential for both GVHD and GVL activity was assessed. All mice that received any dose of LLME-treated DLI survived indefinitely, without evidence of cachexia nor B-cell hypoplasia, in contrast to the severe and lethal GVHD induced by mock-treated DLI. Histological analysis largely correlated with the symptomatic findings and revealed no GVHD-like lesions in the spleens of LLME-treated DLI recipients, although some mice displayed various degrees of hepatic mononuclear infiltration. Most notably, mice given LLME-treated DLI also experienced DLI dose-dependent increases in survival against the challenge with the MMD2-8 leukemia. LLME-treated splenocytes remained immunocompetent, as these cells could proliferate in response to mitogens and to restimulation with ovalbumin when used as a recall antigen. In conclusion, LLME-treated DLI possesses immune potential and, in particular, GVL activity without inducing clinically evident GVHD.

Different individual immune responses elicited by in vitro immunization.

We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo.

Cell-Type Specificity of l-Leucyl l-Leucine Methyl Ester

l-Leucyl l-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent which is converted to a membranolytic compound by dipeptidyl peptidase I and kills human leukocytes such as CD8+ T cells and monocytes but not B cells. The reagent has also been used in mice on the assumption that the cell-type specificity to murine leukocytes is the same as that to human leukocytes. During study on the effect of LeuLeuOMe on antigen-driven IL-2 production using murine splenocytes as antigen-presenting cells, however, we noticed that murine B cells were sensitive to LeuLeuOMe. We therefore examined the cell-type specificity using murine splenocytes and peritoneal macrophages. Flow cytometric analysis revealed that the most sensitive cells to LeuLeuOMe were CD8+ cells and that CD19+ cells (B cells) were as sensitive as CD3+ cells (T cells). Murine splenic B cells, which were either positively or negatively sorted with a cell sorter, were also sensitive to LeuLeuOMe, whereas human peripheral blood B cells, which were positively sorted, were not. Peritoneal macrophages were the most insensitive to LeuLeuOMe. Thus, this study demonstrated that the cell-type specificity to murine leukocytes is different from that to human leukocytes.

Stimulation of human B cells specific for Candida albicans for monoclonal antibody production

The stimulation and immortalisation of human peripheral blood B lymphocytes specific for Candida albicans antigen were investigated. An in vitro immunisation system was employed which involved pretreatment of mononuclear cells with L-leucyl L-leucine methyl ester which removes the suppressive effects of CD8 + T cells, NK cells and monocytes. The remaining cells, CD4 + T cells, B cells and dendritic cells, were cultured with antigen and a mixture of cytokines. A mixture of IL-2, −4 and −6 was found to be optimal for antibody production as determined by an Elispot assay. Transformation of the activated B cells by Epstein Barr virus was found to be optimal after 2 days and lines secreting anti- Candida antibodies were established. These lines could form the basis for specific monoclonal antibody production by generating hybridomas, or by a newly described technique whereby cDNA encoding antibody Fab regions is transferred into phage display libraries. The overall strategy might be generally applicable for the generation of human monoclonal antibodies to infectious agents.

Stimulation of human B cells specific for Candida albicans for monoclonal antibody production.

The stimulation and immortalisation of human peripheral blood B lymphocytes specific for Candida albicans antigen were investigated. An in vitro immunisation system was employed which involved pretreatment of mononuclear cells with L-leucyl L-leucine methyl ester which removes the suppressive effects of CD8+ T cells, NK cells and monocytes. The remaining cells, CD4+ T cells, B cells and dendritic cells, were cultured with antigen and a mixture of cytokines. A mixture of IL-2, -4 and -6 was found to be optimal for antibody production as determined by an Elispot assay. Transformation of the activated B cells by Epstein Barr virus was found to be optimal after 2 days and lines secreting anti-Candida antibodies were established. These lines could form the basis for specific monoclonal antibody production by generating hybridomas, or by a newly described technique whereby cDNA encoding antibody Fab regions is transferred into phage display libraries. The overall strategy might be generally applicable for the generation of human monoclonal antibodies to infectious agents.

Effects of L‐leucyl‐L‐leucine methyl ester on human marrow and protection of progenitor cells by IL‐1

In murine models, L-leucyl-L-leucine methyl ester (LLME) is effective as an ex vivo purging agent for the prevention of graft-versus-host disease. However, reduction of progenitor cells by LLME raises concerns about the engraftment potential of LLME-treated marrow. Therefore, the effects of LLME on human marrow were investigated. Concentrations of LLME from 0.005 mM to 5 mM were used to examine the effects of LLME on marrow cells. Concentrations of LLME less than or equal to 0.05 mM had no effect on recovery of nucleated marrow cells, mononuclear cells (MNC), myeloid cells or monocytes. At 0.5 mM, nucleated marrow cell recovery was 25%, and MNC recovery was 93%. As determined by cytochemical stains, 0.5 mM LLME eliminated myeloid cells and monocytes. At 0.005 mM, LLME had little effect on marrow progenitor cells; progenitor cell recovery with 0.05 mM LLME was 35% for granulocyte-macrophage colony forming units (CFU-GM) and 23% for erythroid burst forming units (BFU-E) (p less than 0.05 compared to 0.005 mM). At higher LLME concentrations, recoveries of both CFU-GM and BFU-E were less than 1%. To determine if earlier hematopoietic cells remained after treatment with LLME, progenitor cell experiments were repeated with 0.5 mM LLME using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) or recombinant human interleukin 3 (rhIL-3) plus or minus recombinant human mast cell growth factor (rhMGF) as sources of colony-stimulating activity. In this system, both IL-3 and GM-CSF induced rare progenitor cells (less than 6/5 x 10(4) cells plated).(ABSTRACT TRUNCATED AT 250 WORDS)

CHARACTERIZATION OF THE IN VITRO SENSITIVITY OF HUMAN LYMPHOID AND HEMATOPOIETIC PROGENITORS TO L-LEUCYL-L-LEUCINE METHYL ESTER

L-leucyl-L-leucine methyl ester (LLME) is a lysosomotropic agent that in microMolar concentrations has been found to be selectively toxic to human and murine precursor and effector cytotoxic cells, irrespective of their surface membrane phenotype. We describe a new method of synthesis of LLME and evaluate the effects of this preparation on human lymphoid and hematopoietic progenitor cells. The new method of synthesis did not change the previously characterized activities of LLME. Consistent with previous observations, NK effectors, LAK precursors and effectors, and allospecific CTL (aCTL) effectors were completely ablated by treatment with 50-250 microM LLME, while the activities of helper T cells and B cells were preserved after treatments of up to 1000 microM LLME. The effects of LLME treatment on human marrow-derived erythroid, myeloid, and monocyte progenitors have not been previously described. We found that the growth of each of these committed precursors was reduced or eliminated following treatment with 100-250 microM LLME. Admixture of LLME-treated marrow with marrow depleted of T cells and other mature cellular elements resulted in increased growth of myeloid and erythroid colonies suggesting that cells that could provide colony-enhancing activities were preserved. In contrast to previous studies in humans, we found a minority of individuals to have aCTL precursors that were partially resistant to LLME. PBL from 10 of 15 individuals tested showed nearly complete ablation of aCTL precursors following treatment with 375 microM LLME. The remaining 5 individuals demonstrated significant aCTL precursor activity after identical treatment. The resistance to LLME was restricted to aCTL precursors, and neither increasing the dose of LLME nor prolonging the time of treatment completely overcame the resistance. The pattern of susceptibility (sensitive versus resistant) was found to be independent of the degree or type of HLA disparity between responder and stimulator. LLME-treated cultures with and without CTL activity contained a predominance of CD4+ T cells. However, in the subjects tested LLME-resistant aCTL was shown to be CD8+. In vitro priming of aCTL precursors from sensitive individuals did not consistently result in the development of resistance to LLME. These data indicate that further studies are needed to evaluate the effects of LLME on human stem cells and to determine the potential role of resistant aCTL precursors in GvHD prior to application of this technique as a form of selective T cell depletion in humans.