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.

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry.

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

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.

Putative immunodominant human immunodeficiency virus-specific CD8(+) T-cell responses cannot be predicted by major histocompatibility complex class I haplotype.

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.

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.

Kinetics of expression and subset distribution of the TNF superfamily members CD40 ligand and Fas ligand on T lymphocytes in cattle.

CD40 and Fas are members of the tumor necrosis factor receptor (TNFR) superfamily. CD40 and Fas play key roles in T cell-B cell interactions. Cross linkage of these molecules induces cell activation and cell death, respectively. The interaction of CD40 with its ligand (CD40L), which is expressed on activated T cells, plays a pivotal role in the generation of the T-dependent (TD) immune response, and FasL-bearing T cells, which have been shown to be predominantly of either the TH0 or TH1 type, have the potential to induce the apoptotic death of Fas expressing B cells. We investigated bovine CD40L mRNA expression in established T cell clones by RT-PCR and Southern blotting. T cells analyzed included CD4+ TH0 and TH1 cell subpopulations, CD8+, and gamma/delta T cells stimulated with either specific antigen or Con A. All CD4+ clones but not all CD8+ or gamma/delta T cell receptor (TCR)-bearing clones expressed mRNA for CD40L. To determine the activation requirements for CD40L expression in cattle, we examined the kinetics and induction requirements for CD40L transcription in peripheral blood T cells using a phorbol ester and/or ionomycin, immobilized mouse anti-bovine CD3, or Con A. Our results demonstrate that CD40L mRNA appears relatively early after activation (1 h) and peaks at 2-4 h poststimulation. A rise in intracellular calcium concentration mediated by ionomycin treatment alone was sufficient to induce CD40L mRNA expression at relatively high levels. Ionomycin treatment in combination with other agonists (anti-CD3, PMA) did not enhance CD40L mRNA expression above levels obtained with ionomycin alone. The bovine Fas ligand gene was partially cloned and mRNA expression determined by RT-PCR in a panel of T cell clones. Our results demonstrate that TH0 and TH1 bovine T cell clones expressed Fas ligand transcripts although only one gamma/delta T cell clone did. This expression was upregulated within 3 h after mitogen stimulation and reduced by 24 h.