An enhanced and scalable process for the purification of SIV Gag-specific MHC tetramer.

A recently developed method for the identification and quantitation of antigen-specific T lymphocytes involves the use of complexes of biotinylated major histocompatibility complex (MHC) and avidin conjugated to a fluorescent reporter group. This complex, dubbed the "tetramer," binds to antigen-specific T lymphocytes in vitro, which can then be sorted and counted by fluorescence-activated flow cytometry to measure immune response. Our research has focused on developing the purification process for preparing tetramer reagent. Our goal was to reengineer a published lab-scale purification process to reduce the number of processing steps and to make the process scalable. In our reengineered process, recombinant MHC alpha chain is isolated from Escherichia coli as inclusion bodies by tangential flow filtration. The purified MHC alpha chain is refolded with beta-2-microglobulin and the target peptide antigen to form the class I MHC. The resulting MHC is purified by hydrophobic interaction chromatography (HIC) and biotinylated enzymatically, and the biotinylated MHC is purified by a second HIC step. The tetramer is prepared by mixing biotinylated MHC with an avidin-fluorophore conjugate. The tetramer is further purified to remove any excess MHC or avidin components. Analysis by flow cytometry confirmed that the tetramers generated by this new process gave bright staining and specific binding to CD3+/CD8+ cells of vaccinated monkeys and led to results that were equivalent to those generated with tetramer produced by the original process.

Evaluation of cytotoxic T-lymphocyte responses in human and nonhuman primate subjects infected with human immunodeficiency virus type 1 or simian/human immunodeficiency virus.

Cytotoxic T-lymphocyte (CTL) responses have been implicated as playing an important role in control of human immunodeficiency virus (HIV) infection. However, it is technically difficult to demonstrate CTL responses consistently in nonhuman primate and human subjects using traditional cytotoxicity assay methods. In this study, we systematically evaluated culture conditions that may affect the proliferation and expansion of CTL effector cells and presented a sensitive method for detection of cytotoxicity responses with bulk CTL cultures. We confirmed the sensitivity and specificity of this method by demonstration of vigorous CTL responses in a simian-HIV (SHIV)-infected rhesus macaque. The expansion of epitope-specific CTL effector cells was also measured quantitatively by CTL epitope-major histocompatibility complex tetramer complex staining. In addition, two new T-cell determinants in the SIV gag region are identified. Last, we showed the utility of this method for studying CTL responses in chimpanzee and human subjects.

Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination.

With accumulating evidence indicating the importance of cytotoxic T lymphocytes (CTLs) in containing human immunodeficiency virus-1 (HIV-1) replication in infected individuals, strategies are being pursued to elicit virus-specific CTLs with prototype HIV-1 vaccines. Here, we report the protective efficacy of vaccine-elicited immune responses against a pathogenic SHIV-89.6P challenge in rhesus monkeys. Immune responses were elicited by DNA vaccines expressing SIVmac239 Gag and HIV-1 89.6P Env, augmented by the administration of the purified fusion protein IL-2/Ig, consisting of interleukin-2 (IL-2) and the Fc portion of immunoglobulin G (IgG), or a plasmid encoding IL-2/Ig. After SHIV-89.6P infection, sham-vaccinated monkeys developed weak CTL responses, rapid loss of CD4+ T cells, no virus-specific CD4+ T cell responses, high setpoint viral loads, significant clinical disease progression, and death in half of the animals by day 140 after challenge. In contrast, all monkeys that received the DNA vaccines augmented with IL-2/Ig were infected, but demonstrated potent secondary CTL responses, stable CD4+ T cell counts, preserved virus-specific CD4+ T cell responses, low to undetectable setpoint viral loads, and no evidence of clinical disease or mortality by day 140 after challenge.

Functional implications for signaling via the IL4R/IL13R complex on bovine cells.

IL-4 and IL-13 share a wide range of activities on monocytes, epithelial cells and B cells and thus play an important role in host defense. Many of these activities are not conserved among species as human, but not murine, B cells are thought to be responsive to IL-13. We previously demonstrated that human IL-13 is highly conserved at the nucleic acid level with a candidate bovine IL-13 cDNA homologue. Moreover, recombinant human IL-13 stimulates Ig secretion by appropriately activated bovine B cells. These studies have been extended to examining Ig class switching at both the protein and mRNA levels in addition to examining other markers of cellular activation. Our results suggest that IL-13 influences B cell differentiation by enhancing IgM, IgG1, and IgE production. IL-13 stimulation alone increases MHC class II expression and progression through cell cycle, although at lower levels in comparison to rboIL-4. The biology of the receptors for IL-4 and IL-13 is complex and raises several key questions with regard to IL-4-dependent and -independent mechanisms of host immunomodulation. Recent studies suggest that at least four chains are involved. These include the p140 IL-4 binding chain (IL-4Ralpha), the common gamma chain (gammac chain), IL-13 receptor alpha- chain (IL-13Ralpha-1) and the IL-13 receptor alpha-2 chain (IL-13Ralpha-2). We have recently cloned cDNAs for the bovine homologues of the IL-13Ralpha-1 and IL-4Ralpha chains and evaluated mRNA expression for a variety of cell types following stimulation. The expression patterns and their implications for receptor chain utilization in signaling via these key TH2 signature cytokines will be discussed.

Immunoregulatory roles of interleukin-13 in cattle.

Interleukin-13 (IL-13) is produced predominantly by helper T lymphocytes of the Th2 phenotype and mediates its effects on several immune cells, including B lymphocytes and macrophages, stimulating their proliferation, differentiation, and effector functions. IL-13 activates human B cells but has no detectable activity on murine B lymphocytes, suggesting that the activity of IL-13 varies among species. Our studies show that IL-13 enhances proliferation and differentiation of bovine B cells and upregulates cell surface major histocompatibility complex (MHC) class II expression. We examined mRNA expression of the putative signaling component of the bovine IL-13Ralpha1 homolog in several peripheral blood populations. After stimulation with calcium ionophore and phorbol ester, IL-13Ralpha1 mRNA levels appeared to be downmodulated in T cells, upregulated in macrophages and B cells, and unchanged in neutrophils. Together, these studies begin to provide insight into the relative importance of IL-13 in immunoregulation in cattle.