Eugene Storozynsky

In Vivo Behavior of Peptide-Specific T Cells During Mucosal Tolerance Induction: Antigen Introduced Through the Mucosa of the Conjunctiva Elicits Prolonged Antigen-Specific T Cell Priming Followed by Anergy

The mucosa of the conjunctiva is an important site of entry for environmental Ags as well as Ags emanating from the eye itself. However, very little is known about T cell recognition of Ag introduced through this important mucosal site. We have characterized the in vivo process of CD4 T cell recognition of Ag delivered via the conjunctival mucosa. Application of soluble OVA to the conjunctiva of BALB/c mice induced potent T cell tolerance. APC-presenting OVA peptide in vivo was only found in the submandibular lymph node and not in other lymph nodes, spleen, or nasal-associated lymphoid tissue. Similarly, in TCR transgenic DO11.10 adoptive transfer mice, OVA-specific CD4+ T cell clonal expansion was only observed in the submandibular lymph node following conjunctival application of peptide. These experiments thus define a highly specific lymphatic drainage pathway from the conjunctiva. OVA-specific T cell clonal expansion peaked at day 3 following initiation of daily OVA administration and gradually declined during the 10-day treatment period, but remained elevated compared with nontreated adoptive transfer mice. During this period, the T cells expressed activation markers, and proliferated and secreted IL-2 in vitro in response to OVA stimulation. In contrast, these cells were unable to clonally expand in vivo, or proliferate in vitro following a subsequent OVA/CFA immunization. These results suggest that Ag applied to a mucosal site can be efficiently presented in a local draining lymph node, resulting in initial T cell priming and clonal expansion, followed by T cell anergy.

Interleukin‐3 and granulocyte–macrophage colony‐stimulating factor enhance the generation and function of dendritic cells

Dendritic cells, well‐known for their potent antigen‐presenting activity, are generally present at very low frequency in the spleens of naive mice. We examined the ability of mice to generate functional dendritic cells (DC) following exposure to the cytokines interleukin‐3 (IL‐3) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Tumours secreting these cytokines provided a continuous stimulus resulting in a greatly increased number and frequency of DC in the spleen. These cells were purified by conventional DC isolation techniques and were found to exhibit many of the characteristics of DC from unmanipulated mice, including high allo‐stimulatory activity in mixed lymphocyte reactions and expression of many similar cell surface markers. Using ovalbumin‐peptide specific class I‐ and class II‐restricted hybridomas containing the lacZ reporter gene, we found that these cytokine‐generated DC had a greatly increased efficacy in the uptake and processing of particulate antigen. These cells appear to have retained the ability to ingest antigen that is generally associated with immature DC, but also exhibit the peptide/major histocompatibility complex (MHC)‐presenting capabilities of mature DC. Development of an assay to measure the activity of a single DC revealed that these dual activities were the properties of the majority of the cytokine‐generated DC. These findings indicate that exposure in vivo to the cytokines IL‐3 and GM‐CSF can result in the generation of large numbers of DC with increased capability of stimulating T cells. Thus, these cells may be important in vivo in the process of cross‐priming and the subsequent generation of tumour‐reactive cytotoxic T lymphocytes (CTL).

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes

Abstract Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans.

IL-3-mediated enhancement of particulate antigen presentation by macrophages.

Mice were exposed to interleukin- (IL-) 3 in vivo by injection of tumor cells transfected with the IL-3 gene. At 10 days post tumor injection, bone marrow cells were recovered, pulsed with particulate antigen in the form of ovalbumin (Ova)-coated magnetic beads, and tested for their ability to present antigen via class I to an Ova/class I-restricted T cell hybridoma. Cells from IL-3-stimulated mice exhibited a marked increase in antigen presentation compared with cells from mice injected with control non-cytokine-secreting tumor cells. These cells were markedly more efficient at presenting particulate Ova antigen than in presenting soluble Ova. Based on adherence, radiation resistance, and surface markers, the cells presenting antigen appear to be in the macrophage cell lineage. These cells are susceptible to lysis by antigen-specific cytotoxic T lymphocytes, which may contribute to limiting the effectiveness of antitumor responses.

Multimodality assessment and treatment of chemotherapy-induced cardiotoxicity

*University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA; eugene_storozynsky@urmc.rochester.edu

Review of Ambulatory Pulmonary Artery Catheterization in the Management of Advanced Heart Failure

The use of pulmonary artery catheterization (PAC) has declined secondary to associated complications and lack of demonstrable efficacy in the inpatient setting. Few studies have been published on the use of PAC in nonacute heart failure (HF) patients. The purpose of this study was to review the use of PAC in guiding advanced therapy in nonacute ambulatory HF patients. A retrospective observational study assessing our groups practice pattern with regard to the use of PAC in 515 ambulatory HF patients, outcomes, and adverse events that resulted from its use was performed. A total of 159 ambulatory HF patients were referred for PAC; 7% underwent heart transplant, 6% had ventricular assist device (VAD) placement, 18% underwent inotropic therapy, and 48% had addition of therapy while 14% had subtraction of therapy. Adverse events occurred in 4% of ambulatory PAC. Patients who underwent heart transplant, VAD, or inotropic therapy had significantly elevated pulmonary capillary wedge pressures, mean pulmonary artery pressures, and depressed cardiac index. Patients selected for inotropic therapy also had significantly elevated right atrial pressures and depressed ejection fractions. PAC use safely guided medical therapy in more than half of the nonacute ambulatory patients.