Lung T-cell subset composition at the time of surgical resection is a prognostic indicator in non-small cell lung cancer.

NSCLC arises in the complex environment of chronic inflammation. Depending on lung immune polarization, infiltrating immune cells may either promote or suppress tumor growth. Despite the importance of the immune microenvironment, current staging techniques for NSCLC do not take into consideration the immune milieu in which the neoplasms arise. T-cell subset content was compared between paired tumor-bearing and contralateral lungs, patient and control peripheral blood. The relationship between T-cell subset distribution and survival were evaluated. CD4 and CD8+ T cells were subsetted by CD45RA/CD27 and analyzed for expression of activation, adhesion, and homing markers. Strikingly, T-cell content was indistinguishable between lungs. Compared with peripheral blood, naïve CD4 and CD8 T cells were rare in BAL. CD4+ BAL T cells showed increased CD95 (higher apoptotic potential) and CD103 expression (epithelial adhesion), but decreased CD38 (activation) and CCR7 expression (lymph node homing). CD8+ BAL T cells showed increased CD103 expression and decreased CD28 expression (co-stimulation). Differences in CD28, CD95, and CCR7 expression were more pronounced within memory cells, while differences in CD4+ CD103 expression were more prominent in effector/memory cells. Of these populations, the absence of lung CD4 T cells with an effector-like phenotype (CD45RA+/CD27-) emerged as a predictor of favorable outcome. Patients with a low proportion (≤0.44%) had 90% 5-year survival (n = 10, median survival 2,343 days), compared with 0% (n = 9, median survival 516 days) of patients with a higher proportion. Further study is required to confirm this association prospectively and define the function of this subpopulation.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Tumorigenic epithelial stem cells and their normal counterparts.

ABC transporters are highly conserved and represent a major protective mechanism for barrier tissues as well as adult tissue stem cells. Emerging data support the existence of a cancer stem cell that shares features of tissue stem cells, including the ability to self-renew and undergo dysregulated differentiation. Here we show that a rare population of cells coexpressing MDR transporters and stem cell markers is a common feature across therapy-naive epithelial cancers as well as normal epithelial tissue. MDR+ and MDR- candidate tumor stem and progenitor populations were all capable of generating highly anaplastic transplantable human tumors in NOD/SCID. The finding that rare cells bearing stem cell markers and having intrinsic MDR expression and activity are already present within the tumorigenic compartment before treatment with cytotoxic agents is of critical importance to cancer therapy. Just as damaged normal epithelial tissues regenerate after chemotherapy by virtue of highly protected resting tissue stem cells, the existence of malignant counterparts in therapy-naive epithelial cancers suggests a common mechanism by which normal and tumor stem cells protect themselves against toxic injury.

Measurement of basal, substrate induced and total P-glycoprotein activity in bronchoalveolar lavage T-cell subsets.

BACKGROUND: P-glycoprotein (P-gp) is a member of the ABC transporter superfamily. P-gp activity can be detected by measuring efflux of fluorescent substrates such as rhodamine 123 (R123). Our objectives were to evaluate P-gp activity in T cells freshly isolated from bronchoalveolar lavage (BAL) and to develop a strategy to distinguish between basal, in vitro substrate-induced, and total P-gp activities. METHODS: Cells were obtained from blood (n = 44) and BAL (n = 34), stained for expression of CD3, CD4, CD8, and CD14, and incubated with R123 (0.13 microM) +/- cyclosporine (5 microM), a specific P-gp inhibitor. P-gp activity was detected as median fluorescence intensity (MFI) and percentage of cells falling below a pre-established cutoff. RESULTS: BAL T cells displayed significant basal P-gp activity, which was most apparent when measured as percentage below the cutoff. Induced activity (difference between P-gp activity measured after load and efflux) was determined equally well when using the MFI or the percentage below cutoff parameter. Total activity was represented by the efflux parameters (MFI or percentage below cutoff) or by the activity-time area under the curve (AUC) method. The two efflux parameters correlated well but were insensitive to the time-dependent nature of dye efflux. In the AUC method, two samples with identical R123 brightness or percentage below cutoff after dye efflux can have very different total activities, depending on their basal activity. The AUC method was also most sensitive in distinguishing between P-gp activity in peripheral blood and resident lung T cells. Application of this methodology to the comparison of P-gp activity in BAL and peripheral CD8+ T cells best revealed the elevated total P-gp activity in BAL T cells. CONCLUSIONS: We have systematically evaluated several methodologies for analysis of P-gp activity and arrived at a novel and robust strategy amenable to standardization and evaluation of the effects of P-gp modulators in vivo and in vitro.

Monitoring the patient off immunosuppression. Conceptual framework for a proposed tolerance assay study in liver transplant recipients.

The mission of the recently established Immune Tolerance Network includes the development of protocols for the induction of transplant tolerance in organ allograft recipients and the development of assays that correlate with and may be predictive of the tolerant state. The state of clinical organ transplant tolerance seems to already exist in a small minority of conventionally immunosuppressed liver and, more rarely, kidney transplant patients. Immunosuppressive drug therapy has been withdrawn from these patients for a variety of reasons, including protocolized weaning for a uniquely large group of liver patients at the University of Pittsburgh. In this study, we propose to evaluate the validity of a variety of in vitro immunologic and molecular biologic tests that may correlate with, and be predictive of, the state of organ transplant tolerance in stable liver patients off immunosuppression. Only peripheral blood will be available for the execution of these tests. Both adult and pediatric liver graft recipients will be studied, in comparison to appropriate controls. We shall examine circulating dendritic cell (DC) subsets [precursor (p) DC1 and p DC2] including cells of donor origin, and assess both the frequency and function of donor-reactive T cells by ELISPOT and by trans-vivo delayed-type hypersensitivity analysis in a surrogate murine model. Cytokine gene polymorphism and alloantibody titers will also be investigated. It is anticipated that the results obtained may provide physicians with a tolerance assay "profile" that may determine those patients from whom immunosuppressive therapy may be safely withdrawn.

P-glycoprotein (P-gp) is upregulated in peripheral T-cell subsets from solid organ transplant recipients.

Immunosuppressive agents such as cyclosporine, tacrolimus, sirolimus, and corticosteroids are substrates for the transmembrane multidrug resistance pump P-glycoprotein (P-gp). Experience in oncologyhas suggested that chronic exposure to P-gp substrates induces upregulation of P-gp activity, which could result in resistance to immunosuppressive drugs. The authors investigated P-gp function in CD4+ and CD8+ T cells from the peripheral blood of solid organ transplant recipients (SOTX). Subjects included 14 stable SOTX (10 liver, 4 lung) and 16 healthy controls. Four-color flow cytometry was used to simultaneously measure intracellular concentration of the fluorescent P-gp substrate Rhodamine 123 (Rh123) and surface expression of CD45RO (nominal memory/effector), CD45RA (naive), and either CD4 or CD8. P-glycoprotein function was measured by a dye efflux assay in which activity was inferred from a decrease in Rh123 fluorescence. CD4+ and CD8+ T cells from patients and control subjects eliminated Rh123, and this activity was inhibited by verapamil, a known P-gp substrate. CD8+ T cells had greater P-gp activity than CD4+ cells, and naive and transitional T cells displayed greater activity than memory T cells. Activity was bimodal in CD8+ CD45RO+ T cells, with a subset of these cells expressing the greatest P-gp activity. Patient CD8+ naive and transitional T cells had upregulated P-gp activity compared to control subjects. We conclude that (1) P-gp activityis significantly upregulated in specific T-cell subsets (CD8+/CD45RA+) in the peripheral blood of SOTX, and (2) the bimodal nature of P-gp response in CD8+ T cells complicates analysis of the effect of chronic administration of P-gp substrates to SOTX.

Rare-event analysis of circulating human dendritic cell subsets and their presumptive mouse counterparts.

BACKGROUND: Considerable interest has focused recently on murine CD8alpha- and CD8alpha+ dendritic cell (DC) subsets, because of their roles in initiating and regulating immune responses. Attention has also centered on their presumed human counterparts, DC1 and DC2, respectively, and their precursors. Identification and quantification of these subsets in the blood may be crucial to understanding and monitoring of their immunologic significance, particularly in humans, where blood may be the only tissue readily or routinely available. METHODS: Leukocytes were isolated from anticoagulated human or mouse (C57BL/10J) blood using conventional procedures. Four-color, rare-event, flow cytometric analysis was used to identify DC1 precursors (pDC1; lineage [lin]- CD4+ CD11c+ HLA-DR+) or DC2 precursors (pDC2; lin- CD4+ CD11c- CD123(hi) [IL-3Ralpha(hi)] HLA-DR+) in normal humans. In mice, CD8alpha+ (CD11b(lo), CD11c+) and CD8alpha- (CD11b(hi), CD11c+) DC subsets were identified both in normal animals and after administration of the potent DC growth factor, fms-like tyrosine kinase 3 ligand (Flt3L). RESULTS: All human subjects examined had discrete populations of pDC1 and pDC2 comprising approximately 0.6% and 0.1% of blood mononuclear cells. CD8alpha- and CD8alpha+ DC constituted approximately 0.75% and 0.2%, respectively, of blood mononuclear cells in normal mice, and 12% and 0.5%, respectively, in Flt3L-treated animals. Flt3L administration substantially increased the absolute numbers of circulating CD11c+ DC by approximately 200-fold. CONCLUSIONS: In addition to pDC1 and CD8alpha- DC, pDC2 and CD8alpha+ DC can be identified in normal human or mouse blood, respectively. Monitoring and isolation or characterization of these cells may provide novel insights into their functional significance in transplantation and other clinical conditions.

Competition of peptide-MHC class I tetrameric complexes with anti-CD3 provides evidence for specificity of peptide binding to the TCR complex.

BACKGROUND: Major histocompatibility complex (MHC)-peptide tetrameric complexes (tetramers) are valuable tools for detecting and characterizing peptide-specific T cells. Because the frequency of these cells is generally very low, it may be difficult to discriminate between nonspecific and specific tetramer binding. METHODS: A four-color flow cytometric assay that simultaneously measures tetramer, CD3, CD8, and CD14 was used to investigate the sensitivity and specificity of MHC class I tetramer staining. This was accomplished by using the influenza virus matrix protein peptide, GILGFVFTL (FLU), as a model recall antigen and the human immunodeficiency virus (HIV) reverse transcriptase peptide, ILKEPVHGV (HIV), as a model novel antigen. Peripheral blood mononuclear cells (PBMC) from 31 HLA-A2.1(+) and 10 HLA-A2.1(-) healthy individuals were stained with the tetramers. RESULTS: The lower limit of detection was established at approximately 1/8,000. In HLA-A2(+) PMBC, frequencies of tetramer-positive CD8(+) T cells were log normally distributed and were high for FLU (1/910) but low for HIV (1/6,067). A novel competition assay, in which tetramer binding was shown to diminish subsequent staining with anti-CD3 antibody, was used to confirm the specificity of tetramer binding to the T-cell receptor (TCR) complex. The competition assay was validated by evaluating several anti-CD3 antibodies and showing that in PBMC from HLA-A2(-) subjects, spurious tetramer-positive events (1/20,000) failed to compete with CD3 binding. For the "recall" FLU tetramer, the degree of competition was proportional to the frequency, suggesting a selection of high avidity cells. Although CD3 competition was also highly correlated with the intensity of tetramer staining, competition allowed the identification of false positive cases with relatively high tetramer staining intensity. CONCLUSION: The data indicate that competition of CD3 binding allows confirmation of the specificity of tetramer binding to the TCR, extending the usefulness of tetramers in the frequency analysis of peptide-specific T lymphocytes.

Clinical significance of cytomegalovirus-specific T helper responses and cytokine production in lung transplant recipients.

Cytomegalovirus (CMV) disease continues to be a major problem for lung transplant recipients. In CMV-seropositive individuals, we detected two types of CMV-specific responses: a self-restricted response stimulated by soluble CMV antigen (sCMV-Ag) and a non-self-restricted response induced by CMV-infected cells (cCMV-Ag). Lung transplant recipients who develop the CMV-specific self-restricted T helper response have a low risk of recurrent CMV disease. In contrast, during CMV disease, lung transplant recipients exhibit only the non-self-restricted T helper responses. We characterized the T cell activation and the kinetics of cytokine production of sorted CD4+ and CD8+ T cells from PBLs of CMV seropositive donors. The two types of CMV antigens induced cytokine production in both T cell subsets. We also performed competitive RT-PCR for Granzyme B (GB) in BAL cells of lung transplant recipients prior to, during and following CMV disease. CMV disease was associated with increase in GB gene expression when was accompanied by acute cellular rejection while it remained low in patients with CMV disease that did not have a complicated course. In summary, CMV-activated T cells within the allograft may produce inflammatory cytokines and effector molecules that may promote allograft rejection.

Lymphopoiesis, apoptosis, and immune amnesia.

Bone marrow transplant recipients have a functional T-cell deficit long after T-cell counts have returned to normal levels. Early after BMT, T-cell phenotype is predominantly CD45RO+/CD29high/HLA-DR+/CD38high. This profile is associated with activated memory cells in healthy subjects, but also appears on the earliest mature naive T-cells in times of lymphopoietic stress. Most of these cells apoptose in short-term unstimulated culture, suggesting that they would have had a similar fate in vivo. Twelve to 24 months after BMT, CD45RA+/CD29low/HLA-DR-/CD38low T cells increase, apoptosis decreases, and T-cell function normalizes. We hypothesize that in the adult, mature memory T cells regulate their own replacement by rescuing a proportion of newly generated naive cells from apoptosis. Ablation of memory cells consequent to high dose therapy disrupts this process, resulting in a protracted period of high lymphocyte turnover with few cells surviving to make the antigen-driven transition to memory cells. Infection with HIV-1 also eventuates in immune deficiency associated with a loss in CD4+ T cells and dominance of the phenotypic/apoptotic profile which we have associated with lymphopoietic stress. Recent data independently confirm that T-cell turnover is greatly elevated in HIV infection. Catastrophic or chronic depletion of memory T cells due to marrow ablative therapy or HIV-1 infection interferes with memory replacement, substituting short-lived hypofunctional naive T cells which characterize the state of immune amnesia.

Apoptosis parallels lymphopoiesis in bone marrow transplantation and HIV disease.

Apoptosis has been implicated in a variety of physiological processes ranging from tissue modeling to deletion of autoreactive T lymphocytes during thymic development. The recent finding that a large proportion of peripheral T cells from HIV-infected subjects (corrected from subjects) apoptose in culture raises an important issue: does this represent a pathologic mechanism by which the virus disrupts the immune system, or a normal physiologic response to virus-mediated T-cell loss? To study the potential relationship between apoptosis and lymphopoiesis, we compared apoptosis rates in unstimulated lymphocyte cultures from healthy subjects, HIV+ gay men, and bone marrow transplant (BMT) recipients undergoing immune reconstruction. BMT recipients were chosen because they undergo massive regeneration of lymphocytes following marrow ablation and graft infusion. The data obtained in BMT recipients suggests that elevated apoptosis accompanies, and is the consequence of, elevated lymphopoiesis. We also found a strong inverse relationship between in vitro T-cell apoptosis rates and peripheral T-cell counts. These results provide new interpretation for elevated apoptosis observed in HIV-infected individuals--that it reflects increased T-cell turnover consequent to virus-mediated destruction of CD4+ T-cells.