Impact of CD8-MHC class I interaction in detection and sorting efficiencies of antigen-specific T cells using MHC class I/peptide multimers: contribution of pMHC valency.

Recombinant soluble multimeric forms of MHC class I molecules loaded with antigenic peptides (pMHC) have turned out to be particularly useful to detect and isolate specific T cells. These applications both rely on the oligomerization of pMHC monomers in order to compensate for their inherent low affinity for the TCR. In this study, we have evaluated the precise contribution of CD8-pMHC interaction on the specificity and sorting efficiency of pMHC multimers according to their degree of oligomerization.

Maximizing the retention of antigen specific lymphocyte function after cryopreservation.

The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container.

Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT.

Background: Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC), and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors.

Results: Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p View Article and Find Full Text PDF

Effect of CD8+ lymphocyte depletion on virus containment after simian immunodeficiency virus SIVmac251 challenge of live attenuated SIVmac239delta3-vaccinated rhesus macaques.

Although live attenuated vaccines can provide potent protection against simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus challenges, the specific immune responses that confer this protection have not been determined. To test whether cellular immune responses mediated by CD8+ lymphocytes contribute to this vaccine-induced protection, we depleted rhesus macaques vaccinated with the live attenuated virus SIVmac239Delta3 of CD8+ lymphocytes and then challenged them with SIVmac251 by the intravenous route. While vaccination did not prevent infection with the pathogenic challenge virus, the postchallenge levels of virus in the plasmas of vaccinated control animals were significantly lower than those for unvaccinated animals.

Low binding capacity of murine tetramers mutated at residue 227 does not preclude the ability to efficiently activate CD8+ T lymphocytes.

MHC tetramers are used to directly enumerate and visualize the antigen-specific T lymphocyte population of interest by flow cytometry, regardless of the T lymphocyte's functional capacity. Assay sensitivity can be hindered by non-specific binding activity, which is due to the inherent interactions of CD8 and MHC. Point mutations within the alpha3 loop of the HLA MHC class I heavy chain have been shown to reduce or abrogate MHC/CD8 interactions and also alleviate non-specific binding.

Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity.

In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR alpha beta pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I.

MHC class II tetramers containing influenza hemagglutinin and EBV EBNA1 epitopes detect reliably specific CD4(+) T cells in healthy volunteers.

Tracking antigen specific T cells with major histocompatibility complex (MHC) tetramers has provided us with insights into the dynamics of the adaptive immune system and holds great promise to aid in patient management and drug and vaccine development. Progress has been made primarily using MHC class I tetramers to monitor CD8(+) T cells, whereas corresponding efforts to stain CD4(+) T cells with class II tetramers have not been as successful. Two major reasons have been proposed for this lack of progress: (1).

A simian immunodeficiency virus nef peptide is a dominant cytotoxic T lymphocyte epitope in Indian-origin rhesus monkeys expressing the common MHC class I allele mamu-A*02.

The precise measurement of epitope-specific cytotoxic T lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected or vaccinated rhesus monkeys has been important in the evaluation of potential HIV vaccine strategies. This quantitation of CTL has been limited to date by the identification of only one dominant SIV/SHIV epitope in these monkeys. We have recently defined a Nef CTL epitope p199RY (YTSGPGIRY) that is recognized by CD8(+) T lymphocytes from all SIV/SHIV-infected Mamu-A*02(+) rhesus monkeys that have been evaluated.

Development of a dual monoclonal antibody immunoassay for total human kallikrein 2.

Background: Human kallikrein 2 (hK2) shares 80% sequence identity with prostate-specific antigen (PSA). Because both hK2 and hK2-alpha(1)-antichymotrypsin (hK2-ACT) complexes have been identified in patient sera, we devised an immunoassay for total hK2 [(thK2); hK2 and hK2-ACT] and evaluated it in healthy subjects and patients with prostate disease.

Methods: We developed monoclonal antibodies (mAbs) with high specificity for hK2 and hK2-ACT and minimal cross-reactivity to PSA.

BRCA1 partially reverses the transforming activity of the ras oncogene.

The BRCA1 gene is associated with hereditary breast and ovarian cancers. BRCA1 fits the model of a classic tumor suppressor gene, a hypothesis supported by recent work demonstrating that expression of BRCA1 inhibits growth of breast and ovarian cancer cell lines. The present study was designed to test the potential of BRCA1 to reverse the transforming activity of the ras oncogene.

Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay.

The prostate-specific antigen (PSA) immunoassay is an important tool for the detection and monitoring of prostate cancer. PSA exists in serum mainly as complexes with serine protease inhibitors including alpha1 antichymotrypsin (ACT) and alpha2 macroglobulin (MG). PSA-MG complex is not detected by the existing PSA immunoassays since MG (720 kDa) sequesters PSA and masks the antibody binding sites.

bcl2 and v-abl oncogenes cooperate to immortalize murine B cells that secrete antigen specific antibodies.

In this manuscript, a general strategy was designed and used to rapidly test whether any combination(s) of p53, v-abl, bcl2 and ras oncogenes could act cooperatively to immortalize B cells. Here we report that only the combination of v-abl and bcl2 was successful. Splenic B cells from beta galactosidase-immunized mice were stimulated in vitro with lipopolysaccharide and dextran sulphate for 48 h and co-infected with ecotropic A-MuLV (v-abl) and amphotropic pZip-bcl2 (human bcl2) viruses.

Large-scale propagation of recombinant adherent cells that secrete a stable form of human glandular kallikrein, hK2.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease that is expressed predominantly in the prostate epithelium and has 78% aa identity with prostate-specific antigen (PSA). hK2 has been recognized as a potential prostate cancer marker and has been demonstrated to be highly expressed in prostate cancer compared to benign prostatic tissue. Purification and characterization of hK2 have been impeded due to its lower expression in bodily fluids and tissues compared to PSA and its ability to autodegrade.

The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia.

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera.

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

Objectives: Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility.

Methods: We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA.

Human IL-6 enhances human lymphocyte engraftment and activation but not human antibody production in SCIDhu PBL mice.

The SCIDhu PBL model of human Ig production was modified by using human interleukin-6 (hIL-6) secreting tumors for continuous hIL-6 production, in vivo. On day one, SCID mice were injected subcutaneously with 200 microliters PBS (control mice), 10(4) SP2/0-Ag14 cells (IL-6+ mice) or 10(4) hIL-6 secreting SP2/0-hIL6.17 cells (IL-6- mice).

Generation of murine monoclonal antibodies in serum-free medium.

Traditional hybridoma fusion technology requires complete medium with serum supplements to support the growth of hybridoma cells. Serum is also required for subcloning of hybridoma cells to support low density cell growth. IL-6 has been shown to enhance the growth of hybridomas and stimulate antibody production by B cells.

Expression of human glandular kallikrein, hK2, in mammalian cells.

The human kallikrein family consists of three members, hK1, hK2, and hK3 [prostate-specific antigen (PSA)]. PSA is a widely accepted marker for prostate cancer. The mRNAs for both hK2 and PSA are localized predominantly to prostate epithelium and are regulated by androgens.

Evaluation of Bcl-2/B cell transgenic mice (B6) for hybridoma production.

Bcl-2 is an oncogene associated with prevention of apoptosis in a variety of cell types. Bcl-2 expression in B lymphoid cells prolongs antibody production, in vitro and in vivo. A line of transgenic mice (B6) has been developed that expresses human Bcl-2 in the B cells of SWR/SJL mice.

Identification of human glandular kallikrein hK2 from LNCaP cells.

Based on studies indicating that human glandular kallikrein (hK2) mRNA is present in the prostate, we prepared a monoclonal antibody to a synthetic peptide corresponding to the 41-56 region of hK2 to try to identify the hK2 protein. Although prostate-specific antigen (PSA) and hK2 share 80% homology, the 41-56 amino acid sequence of hK2 is only 50% homologous with PSA. A monoclonal antibody, HK1A523, was identified that demonstrates high specificity for hK2.

Therapy of streptozotocin induced diabetes with a bifunctional antibody that delivers vinca alkaloids to IL-2 receptor positive cells.

IVA039.1 is a bifunctional antibody with specificity for the murine IL-2 receptor and vinca alkaloids. Biodistribution studies show that IVA039.

Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies?

While monoclonal antibodies show promise for use in the treatment of a variety of disease states, including cancer, autoimmune disease, and allograft rejection, generation of anti-antibody responses still remains a problem. For example, 50% of the patients who receive OKT3 produce blocking antibodies that interfere with its binding to T cells, thus decreasing the therapeutic effect (51). HAMA responses have also interfered with tumor imaging (39,40) and radioimmunotherapy (56).

Production and in vivo characterization of a bifunctional antibody (IVA039.1) with specificity for the mouse interleukin-2 receptor and vinca alkaloids.

The autoreactive T cell plays a pivotal role in the pathogenesis of type I diabetes in humans and in rodent animal models. Elimination or attenuation of these cells may provide a means to treat the disease. The use of antibodies directed to T cells has shown varying degrees of effectiveness in the treatment of autoimmune disease.

Production of IgG monoclonal antibodies to the tumor-associated antigen, CA-195.

The isotype of a monoclonal antibody is closely associated with its biologic activity. Certain immunoglobulin subclasses are more effective than others regarding their ability to execute complement-mediated lysis of cells, antibody-dependent cell-mediated cytotoxicity, and tumor localization. Many potential targets for cancer immunotherapy are tumor-associated antigens with high percentages of carbohydrate.