Kristine Kuus-Reichel

Effect of CD8+ Lymphocyte Depletion on Virus Containment after Simian Immunodeficiency Virus SIVmac251 Challenge of Live Attenuated SIVmac239Δ3-Vaccinated Rhesus Macaques

ABSTRACT 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 SIVmac239Δ3 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. The depletion of CD8+ lymphocytes at the time of challenge resulted in virus levels in the plasma that were intermediate between those of the vaccinated and unvaccinated controls, suggesting that CD8+ cell-mediated immune responses contributed to protection. Interestingly, at the time of challenge, animals expressing the Mamu-A*01 major histocompatibility complex class I allele showed significantly higher frequencies of SIV-specific CD8+ T-cell responses and lower neutralizing antibody titers than those in Mamu-A*01− animals. Consistent with these findings, the depletion of CD8+ lymphocytes abrogated vaccine-induced protection, as judged by the peak postchallenge viremia, to a greater extent in Mamu-A*01+ than in Mamu-A*01− animals. The partial control of postchallenge viremia after CD8+ lymphocyte depletion suggests that both humoral and cellular immune responses induced by live attenuated SIV vaccines can contribute to protection against a pathogenic challenge and that the relative contribution of each of these responses to protection may be genetically determined.

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

BackgroundCryopreservation 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.ResultsUsing CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001). The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001). Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation) and tetramer staining (p < 0.001). Roughly similar sensitivity and specificity were observed between the three assays and between shipped and cryopreserved samples for each assay.ConclusionWe conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.

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. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

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 αβ 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. Using a TCR tetramer specific for the Mamu-A*01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.

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. When inoculated i.p. into naive pristane-primed mice, these B cells generated mesenteric lymphadenopathy, intraperitoneal lymph nodules and ascites in 100% (8/8) of the mice within 36-53 days. The ascites fluid contained 69.5-122 microg/ml IgG and 2.5-13 microg/ml IgM against the immunogen. The ascites cells were passed intraperitoneally up to three times. In all passages, ascites tumors were generated, and the ascites fluid contained beta galactosidase-specific IgG and IgM, indicating that some immunoglobulin secreting B cells had been immortalized. Neither ascites nor tumors were produced when B cells infected with only one of the viruses was injected into the mice. The presence of each oncogene in ascites cells was verified by immunohistochemistry or RT-PCR. This study provides evidence for the cooperativity of an unexpected pair of oncogenes in B cell immortalization.

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). The mice were reconstituted with human PBMC on day two and immunized with 100 micrograms of tetanus toxoid (TT) on days two and fifteen. Serum hIL-6 concentrations in IL-6+ mice ranged between 2.9 and 38.1 ng/ml by days 26-33. IL-6+ mice had enlarged spleens and lymph nodes (LN). Flow cytometry and histology showed that SCIDhu PBL mouse spleen, LN and peritoneal exudate cells (PEC) contained mostly murine myeloid lineage cells. In addition, many more human B cells, T cells and IL-2R(+)-activated lymphocytes were present in spleen, LN and PEC of IL-6+ mice. Despite enhanced lymphocyte engraftment and activation, by day 14 IL-6+ mice produced up to 6-fold less TT-specific IgG relative to total IgG than either control group. TT-specific and total Ig sera concentrations were equivalent in all three groups on days 26-33. Our results suggest that sustained circulating hIL-6 enhanced human delayed-type hypersensitivity (DTH)-like inflammatory responses with consequential inhibition of TT-specific IgG production in SCIDhu PBL mice.

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 IVA039.1 can target and deliver vinca alkaloids to tissues that contain IL-2 receptor positive cells. Vinca alkaloids are lymphocytotoxic. Therapy of diabetic mice with IVA039.1 plus vincristine results in a significant decrease in the glucose levels of diabetic compared to untreated mice. The therapeutic effect of IVA039.1 plus vincristine therapy was additive but surprisingly not synergistic. The binding of IVA039.1 to vincristine has moderate affinity with a slow off rate. In vitro studies suggest that, when bound to IVA039.1, the vincristine is inactivated. We attribute the lack of an enhanced therapeutic response to bifunctional antibody therapy using IVA039.1 plus vincristine to the inaccessibility of the drug to the target cells.

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. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (pb0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 8C prior to washing, the mean viability was 69.7F12.5%, at 25 8C 92.55F3.1%, and at 37 8C 95.11F2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the noncryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum. D 2005 Elsevier B.V. All rights reserved.