Biomedical subjects

Albert D Donnenberg

Publications and source records attributed to Albert D Donnenberg.

9 recordsLinked to original sources

PubMed2005

p53 as an immunotherapeutic target in head and neck cancer.

Squamous cell carcinomas of the head and neck (HNSCCs) are characterized by a high frequency of mutations in the p53 gene often leading to p53 protein accumulation. Since accumulation of p53 is associated with enhanced presentation of wild-type sequence (wt) p53 peptides to immune cells, the development of ' pan' vaccines against HNSCC has focused on wt p53 epitopes. We used the HLA-A2.1-restricted wt p53 264-272 epitope pulsed on autologous dendritic cells to generate cytotoxic T lymphocytes (CTLs) ex vivo from circulating precursor T cells of HLA-A2.1+ patients with HNSCC. CTLs specific for the wt p53 264-272 peptide were generated from leukocytes obtained from a cohort of patients with HNSCC (group A). Paradoxically, none of those patients had tumors which adequately presented the epitope, i.e. accumulated p53. In contrast, patients who did not generate CTLs (group B) had tumors which accumulated altered p53 and potentially could present the p53 264-272 epitope. When p53 264-272-specific T cells were directly enumerated in the peripheral circulation of patients with HNSCC using tetrameric p53 264-272/HLA-A2.1 complexes by multicolor flow cytometry, group A had high and group B low percentages of tetramer+ CD3+ CD8+ T cells. These findings suggested that in vivo p53-specific CTLs in group A might play a role in the elimination of tumor cells expressing the p53 264-272 epitope ('immunoselection'), leading to the outgrowth of 'epitope loss' tumor cells. On the other hand, precursor CTLs specific for the wt p53 264-272 peptide in group B are unresponsive to the p53 antigen. Unresponsiveness of CTLs specific for the wt p53264-272 peptide detected in group B could be reversed by using more immunogenic variant peptides of the p53 264-272 epitope. In vivo, immunoselection of tumors which become resistant to anti-p53 immune responses has important implications for future p53-based vaccination strategies. It calls for modified approaches, in which altered peptide variants of the wt sequence p53 264-272 epitope are used in a vaccine in order to overcome unresponsiveness of T lymphocytes to the native epitope.

Cancer Vaccines↗

PubMed2005

Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis.

The failure to eradicate cancer may be as fundamental as a misidentification of the target. Current therapies succeed at eliminating bulky disease but often miss a tumor reservoir that is the source of disease recurrence and metastasis. Recent advances in the understanding of tissue development and repair cause us to revisit the process of drug resistance as it applies to oncogenesis and tumor heterogeneity. The cancer stem cell hypothesis states that the cancer-initiating cell is a transformed tissue stem cell, which retains the essential property of self-protection through the activity of multiple drug resistance (MDR) transporters. This resting constitutively drug-resistant cell remains at low frequency among a heterogeneous tumor mass. In the context of this hypothesis, the authors review the discovery of MDR transporters in cancer and normal stem cells and the failure of MDR reversal agents to increase the therapeutic index of substrate antineoplastic agents.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2004

P-glycoprotein activity is decreased in CD4+ but not CD8+ lung allograft-infiltrating T cells during acute cellular rejection.

BACKGROUND: Modulation of P-glycoprotein (P-gp) activity in graft-infiltrating T cells may alter their susceptibility to immunosuppression. METHODS: P-gp activity was measured by rhodamine efflux in T-cell subsets from bronchoalveolar lavage (BAL) of five healthy volunteers and 27 lung allograft recipients. The effect of T-cell activation on P-gp activity was modeled by stimulation of peripheral blood mononuclear cells with staphylococcal enterotoxin B. RESULTS: Most BAL T cells expressed memory-effector markers. Patients had a lower proportion of CD4 T cells (P = 0.005), whereas control subjects had CD4-to-CD8 ratios similar to peripheral blood. In controls, basal P-gp activity was greatly increased in both CD4 (35% P-gp active) and CD8 (63%) lung T cells compared with peripheral T cells. Basal P-gp activity was elevated in patient BAL T cells but was lower than control BAL activity (CD4, P = 0.07; CD8, P = 0.03). Lung T cells from transplant patients had modest (CD4) or marked (CD8) increases in substrate-induced P-gp activity compared with normal lung, indicating that P-gp was not irreversibly inhibited. Patients with acute cellular rejection (ACR) had reduced P-gp activity in CD4, but not CD8, BAL T cells compared with patients without ACR (P = 0.004). To determine the relationship between T-cell activation on P-gp modulation, P-gp activity was measured in staphylococcal enterotoxin B-stimulated peripheral blood mononuclear cells. P-gp activity was abrogated in CD71 cycling cells but remained high in a persistent but minor population of resting naive T cells. CONCLUSIONS: Lung T cells have increased in vivo P-gp activity and therefore may eliminate substrate drugs, resulting in local resistance to immunosuppressive therapy. However, P-gp function is reduced during T-cell activation, providing a window of susceptibility to treatment during ACR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Identification, rare-event detection and analysis of dendritic cell subsets in broncho-alveolar lavage fluid and peripheral blood by flow cytometry.

Dendritic cells (DCs) and their precursors play important roles, not only as antigen presenting cells, but components of the immunoregulatory network. Depending on their lineage, activation and differentiation state, DCs can promote a strong T-cell response or a state of anergy, and can polarize the T helper response in the direction of TH1 or TH2. The lung comprises one of the major interfaces between the host and the environment and regularly comes into contact with antigens, allergens and pathogens. Inflammation in response to these stimuli must be very carefully regulated in the lung since perturbations, especially of a chronic nature, can result in immunopathology that interferes with the lungs' critical function of gas exchange. Especially in disease states such as chronic obstructive pulmonary disease, which has been associated with a chronic TH1 response, and asthma, which is TH2-driven, the ability to access and characterize DCs and their precursors is critical to the understanding of immune modulation in these processes. In this report we will demonstrate that mature DCs and their monocytoid and plasmacytoid precursors can be sampled in the lung by the minimal invasive procedure of broncho-alveolar lavage (BAL) despite their relative scarcity and can be detected by rare event multiparameter flow cytometry.

Antigens, CD↗

PubMed2003

Dendritic cell subsets in blood and lymphoid tissue of rhesus monkeys and their mobilization with Flt3 ligand.

We provide phenotypic and functional evidence of premonocytoid dendritic cells (DCs) and preplasmacytoid DCs in blood and of corresponding DC subsets in secondary lymphoid tissue of rhesus monkeys. Subsets were identified and sorted by 4-color flow cytometry using antihuman monoclonal antibodies cross-reactive with rhesus monkey. To mobilize pre-DC subsets, fms-like tyrosine 3 kinase ligand (Flt3L; 100 microg/kg subcutaneously) was administered for 10 days. Presumptive pre-DC subsets were identified within the lineage- (Lin-) major histocompatibility complex (MHC) class II+ fraction of blood mononuclear cells. Premonocytoid DCs were CD11c+CD123- (interleukin-3Ralpha- [IL-3Ralpha-]). Preplasmacytoid DCs were characterized as CD11c-CD123++ Flt3L increased the CD11c+ pre-DC (7-fold) and CD123++ pre-DC subsets (3-fold) in blood. The freshly isolated CD11c+ pre-DC subset induced modest proliferation of naive allogeneic T cells. After overnight culture with granulocyte macro-phage-colony-stimulating factor (GMCSF) and CD40L, both subsets up-regulated surface costimulatory molecules, and CD11c+ pre-DCs became potent allostimulators. Freshly isolated CD123++ pre-DCs showed typical plasmacytoid morphology and, when cultured with IL-3 and CD40L for 72 hours, developed mature DC morphology. Following stimulation with CD40L, CD11c+ pre-DCs secreted increased levels of IL-12p40. Importantly, herpes simplex virus-stimulated CD123++ pre-DCs, but not CD11c+ pre-DCs, secreted interferon-alpha (IFN-alpha). Corresponding DC subsets were identified by flow analysis and immunohistochemistry in lymph nodes wherein both populations were increased 2- to 3-fold by Flt3L administration. CD123+ pre-DCs produced IFN-alpha in response to in vivo viral infection. Thus, rhesus monkeys exhibit 2 distinct DC precursor populations that closely resemble those of humans. Both are mobilized into blood and lymphoid tissue by Flt3L, offering potential for their further characterization and possible therapeutic application.

Animals↗

PubMed2003

Rapid turnover of the CD8(+)CD28(-) T-cell subset of effector cells in the circulation of patients with head and neck cancer.

CD8(+) T cells in the circulation of patients with head and neck cancer (HNC) were previously shown to be significantly more sensitive to, and preferentially targeted for, apoptosis than CD4(+) T cells (Hoffmann et al., Clin Cancer Res, 8:2553-2562, 2002). To distinguish global from CD8(+) subset-specific apoptosis, we studied Annexin-binding to naïve, memory, and effector subsets of CD8(+) cells by multicolor flow cytometry. Age-related changes in naïve and effector CD8(+) cell subsets were observed in patients and normal controls (NC). The frequencies of naïve (CD28(+)CD45RO(-)) CD8(+) T cells were lower and those of memory (CD28(+)CD45RO(+)) and effector (CD28(-)) CD8(+) T cells significantly higher in the circulation of HNC patients relative to age-matched NC. Among CD8(+) T cells, the CD28(-) effector cell subset contained the highest proportion of Annexin-binding cells, while the naïve CD28(+)CD45RO(-) subset contained the lowest. This suggested a high turnover rate of the CD8(+)CD28(-) effector cell subset in patients with HNC, which was being compensated by a rapid transition of naïve CD8(+) T cells to the effector cell pool. Following tumor resection, the frequency of CD8(+)CD28(-) T cells normalized in the patients, an indication that the presence of tumor had an influence on the size of CD8(+)CD28(-) T-cell pool. Ex vivo, in mixed lymphocyte-tumor cultures (MLTC) with semiallogeneic T cells as responders, CD8(+)CD28(-) T cells could be generated from CD8(+)CD28(+) cells by repeated stimulations with tumor cells. These CD8(+)CD28(-) effector cells lysed the tumor, produced IFN-gamma in response to the tumor, and strongly expressed granzyme B. Thus, the high rate of their apoptosis in the circulation of patients with HNC might be expected to contribute to tumor progression. However, the ex vivo generation of this cell subset was suppressed by strong CD28/B7 ligation or by overexpresson of MHC molecules on tumor cells, suggesting that adequate costimulation is necessary for protection from apoptosis. It appears that interactions of immune and tumor cells might determine the fate of this terminally differentiated effector cell subset.

Adult↗

PubMed2003

Direct visualization of antigen-specific T cells using peptide-MHC-class I tetrameric complexes.

Peptide-major histocompatibility complexes (MHC) class I tetrameric complexes ("tetramers") are proving invaluable as fluorescent reagents for enumeration, characterization, and isolation of peptide-specific T cells and have afforded many advantages over previous techniques, particularly the ability to directly quantify and phenotype antigen-specific T cells with minimal in vitro manipulation. Recently, various technical peculiarities of tetramers have led to modifications within this important immunoassay. The current manuscript details potential pitfalls and provides guidance for interpretation of results.

Epitopes, T-Lymphocyte↗

PubMed2002

Frequencies of tetramer+ T cells specific for the wild-type sequence p53(264-272) peptide in the circulation of patients with head and neck cancer.

Immunization with wild-type sequence (wt) p53 epitopes represents a novel therapeutic strategy for cancer patients with tumors accumulating mutant p53. To evaluate usefulness of p53-derived peptides as future cancer vaccines, frequencies of wt p53(264-272) peptide-specific CD8+ T cells were determined in the peripheral circulation of patients with squamous cell carcinoma of the head and neck (SCCHN). T cells of 30 HLA-A2.1+ patients and 31 HLA-A2.1+ healthy individuals were evaluated by multicolor flow cytometry analysis using peptide-HLA-A2.1 complexes (tetramers). T cells specific for an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were studied in parallel. Patients with SCCHN had a significantly higher mean frequency of CD8+ T cells specific for wt p53(264-272) than normal donors (P = 0.0041). Surprisingly, the frequency of epitope-specific T cells in the circulation of patients did not correlate with p53 accumulation in the tumor. In patients whose tumors had normal p53 expression or had p53 gene mutations preventing presentation of this epitope, high frequencies of wt p53(264-272)-specific CD8+ T cells were found, of which many were memory T cells. In contrast, patients whose tumors accumulated p53 had low frequencies of wt p53(264-272)-specific CD8+ T cells, which predominantly had a naive phenotype and were unable to proliferate ex vivo in response to the epitope, as reported by us previously (T. K. Hoffmann, J. Immunol., 165: 5938-5944, 2000). This seemingly contradictory relationship between the high frequency of epitope-specific T cells and wt p53 expression in the tumor suggests that other factors may contribute to the observed anti-p53 responses. Human papillomavirus-16 E6/E7 expression is common in SCCHN, and E6 is known to promote presentation of wt p53 epitopes. Although human papillomavirus-16 E6/E7 expression was detected in 46% of the tumors, it did not correlate with the frequency of wt p53(264-272)-specific CD8+ T cells or with p53 expression in the tumor. These findings emphasize the complexity of interactions between the tumor and the host immune system, and, thus, have particularly important implications for future p53-based immunization strategies.

Antibodies, Neoplasm↗

PubMed2002

Strategies for preclinical evaluation of dendritic cell subsets for promotion of transplant tolerance in the nonhuman primate.

A role for dendritic cells (DC) as critical regulators of immune reactivity has become increasingly recognized. There is evidence in rodent models that donor-derived DC, particularly in the immature state, can prolong organ allograft survival and even induce donor-specific tolerance. To allow the potential tolerogenic properties of these cells to be evaluated more fully with a view to clinical testing, it is necessary to identify DC subsets in nonhuman primates. We have identified the putative rhesus monkey equivalents of circulating human DC subset precursors as lineage(-), HLA-DR(+), CD123(lo),CD11c(hi)(pDC1) and lineage(-), HLA-DR(+), CD123(hi),CD11c(lo)(pDC2). Testing of these DC populations both in vitro and in vivo, as well as in transplant models in combination with conventional or experimental immunosuppressive reagents, will aid the development of novel strategies for the promotion of allo-antigen specific tolerance in transplantation.

Animals↗