Detailed endometrial immune assessment of both normal and adverse reproductive outcome populations.

PURPOSE: Using a comprehensive flow cytometric panel, do endometrial immune profiles in adverse reproductive outcomes such as repeat implantation failure (RIF) and repeat pregnancy loss (RPL) differ from each other and male-factor controls? METHODS: Six-hundred and twelve patients had an endometrial biopsy to assess the immunophenotype. History on presentation was used to subdivide the population into recurrent implantation failure (RIF) [n = 178], recurrent pregnancy loss (RPL) [n = 155], primary infertility [n = 130] and secondary infertility [n = 114]. A control group was utilised for comparative purposes [n = 35] and lymphocyte subpopulations were described. RESULTS: Distinct lymphocyte percentage differences were noted across the populations. Relative to controls and RPL, patients with a history of RIF had significantly raised uterine NKs (53.2 vs 45.2 & 42.9%, p < 0.0001). All sub-fertile populations had increased percentage peripheral type NKs (p = 0.001), and exhibited increased CD69+ activation (p = 0.005), higher levels of B cells (p < 0.001), elevated CD4:CD8 ratio (p < 0.0001), lower T-regs (p = 0.034) and a higher proportion of Th1+ CD4s (p = 0.001). Patient aetiology confers some distinct findings, RPL; pNK, Bcells and CD4 elevated; RIF; uNK and CD56 raised while CD-8 and NK-T lowered. CONCLUSIONS: Flow cytometric endometrial evaluation has the ability to provide a rapid and objective analysis of lymphocyte subpopulations. The findings show significant variations in cellular proportions of immune cells across the patient categories relative to control tissue. The cell types involved suggest that a potential differential pro-inflammatory bias may exist in patients with a history of adverse reproductive outcomes. Immunological assessment in appropriate populations may provide insight into the underlying aetiology of some cases of reproductive failure.

The assessment of host and bacterial proteins in sputum from active pulmonary tuberculosis.

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL-17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase-1 in sputum. Results of detection of IL-10, IFN-γ, perforin-1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acid-fast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.

Rapid assessment of in vitro expanded human regulatory T cell function.

Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127(lo/⁻) and FoxP3+. CD45RA can be used to further differentiate the population into naïve (CD45RA(+)) and induced (CD45RA⁻) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3-5day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127(lo/⁻)CD45RA+ cells with a BD FACSAria™ II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.

Preliminary evaluation of whole-blood gamma interferon release for clinical assessment of cellular immunity in patients with active coccidioidomycosis.

Assessment of the cellular immune response in coccidioidomycosis has epidemiologic and prognostic importance. Measurement of delayed-type hypersensitivity to skin testing has been used in the past to determine cellular immunity in coccidioidomycosis. However, no skin tests are currently available in the United States. Assay of gamma interferon (IFN-gamma) release in whole blood in response to incubation with antigen has been used to assess cellular immunity in tuberculosis. We used a similar assay using the coccidioidal antigen preparation T27K to measure the in vitro cellular immune responses among a cohort of 69 subjects with active coccidioidomycosis. IFN-gamma release was bimodal, with concentrations above and below 5 IU/ml. Using multivariate logistic regression, underlying disease and disseminated or chronic pulmonary coccidioidomycosis was significantly associated with the release of IFN-gamma at a concentration of <5 IU/ml (P = 0.02 or 0.05, respectively). In addition, the release IFN-gamma concentration was <5 IU/ml in all subjects with a clinical severity score of > or =6 (P = 0.02). The release IFN-gamma concentration correlated with expression of CD69 on T lymphocytes in an in vitro assay using T27K as the antigen (Spearman's rho = 0.59; P < 0.01). These results suggest that the IFN-gamma release assay with T27K as the antigen may be a useful clinical test for assessing cellular immunity in patients with active coccidioidomycosis.

Deceptive multilineage reconstitution analysis of mice transplanted with hemopoietic stem cells, and implications for assessment of stem cell numbers and lineage potentials.

Hemopoietic stem cells (HSC) are identified through their unique ability, at the single cell level, to long-term reconstitute all blood cell lineages. Sustained myeloid reconstitution is considered the hallmark of HSC, because myeloid progenitors and their progeny have very short half-lives. Here we demonstrate that the established practice of relying on RB6-8C5 as a myeloid specific Ab can result in overestimation of HSC frequencies because the RB6-8C5 Ab also detects Ags expressed on a sizeable population of CD3(+)CD8(+) T cells, constitutively as well as following transplantation. Likewise, a high fraction of mice transplanted with limiting numbers of ex vivo expanded Lin(-)Sca(+)kit(+)CD34(-) HSC show long-term RB6-8C5(+)CD3(+) (lymphoid) but no RB6-8C5(+)CD3(-) (myeloid) reconstitution. Most noteworthy, the use of RB6-8C5 as a myeloid specific Ab can be deceptive by implicating the existence of lineage-restricted HSC capable of long-term reconstituting the myeloid and T, but not B, cell lineage. Because cross-lineage expression of "lineage-specific" markers is unlikely to be unique to the blood system, claims of unexpected cell fates should be substantiated not only by acquisition of lineage-specific markers, but also absence of markers of other lineages normally derived from the investigated stem cells.

Low-cost CD4 enumeration in HIV-infected patients in Thailand.

In Thailand, over one million people have been infected with HIV since the beginning of the epidemic. This has created a great burden on the country's limited health care budget. Monitoring CD4+ T-lymphocytes is important to determine the success of any antiretroviral therapy as well as HIV vaccine trials. However, the high cost of CD4 counts makes monitoring of every HIV-infected patient impossible in Thailand. Therefore, the development of affordable strategies is necessary in order to allow more HIV infected persons to access CD4 testing to control the disease. The current standard methods for enumeration of CD4+ T-lymphocytes are performed on whole blood by flow cytometric immunophenotyping using the 6-tube 2-color and 3-tube 3-color panels recommended by the Centers for Diseases Control (CDC). In this study, percentage CD4+ T-lymphocyte values (from 142 HIV-seropositive patients and 26 anti-HIV negative adult blood donors) generated by the use of just 2 reagents (CD45/CD4) in a 1-tube 2-color panel employing side scatter/CD45 morphospectral gating were compared to those obtained by state of the art methods. We also compared the use of generic monoclonal antibody reagents with commercial reagents and found the results to be comparable with an overall correlation coefficient (r) of more than 0.95 for both CD4+ and CD8+ T-lymphocytes. Bland-Altman analysis of the mean CD4 values plotted against the difference in values between the generic reagents and the commercial reagents showed no bias. The 1-tube 2-color method using generic monoclonal antibody reagents potentially permits more affordable but reliable CD4 testing and therefore could increase access for more HIV-infected patients in resource-poor countries.

Individual variability in cyclosporin A sensitivity: the assessment of functional measures on CD28-mediated costimulation of human whole blood T lymphocytes.

The quantitative analysis of cyclosporin A (CsA) effects might be helpful for optimizing immunosuppressive treatment after allogeneic organ transplantation in individual patients, as rejection can occur despite the existence of CsA blood levels within therapeutic ranges. Previous investigations found that costimulation of the CD28 pathway generally mediates CsA-resistant proliferation of T cell receptor (TCR)-activated T lymphocytes. However, here we describe considerable interindividual variation regarding the immunosuppressive effects of CsA (1000 microg/L) on anti-CD3/CD28 T cell costimulation in a human whole blood assay. In the in vitro study, we found a significant reduction of T cell proliferation, activation marker expression (CD25, CD69) on the T cell surface, and interleukin-2 (IL-2) protein expression in whole blood samples of all healthy subjects (n = 11). However, the investigation of cytokine mRNA profiles revealed variable results of in vitro CsA sensitivity. Whole blood samples of 3 of 11 healthy individuals demonstrated a marked suppression of IL-2 mRNA expression (>50%) and a partial inhibition of IL-4, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) mRNA expression on addition of CsA. In contrast, the remaining 8 healthy individuals had cytokine mRNA expression levels that were unaffected or even increased when CsA was administered in vitro. In patients undergoing CsA monotherapy (ex vivo study, n = 9), we found a significant suppression of IL-2 mRNA levels in 4 of 9 patients ex vivo. Thus, we cannot confirm a universal CsA resistance of T cells on anti-CD3/CD28 costimulation. Instead, our results suggest an individual degree of CsA sensitivity that might be more consistent with clinical experience. Prospective studies are necessary to determine if individual degrees of CsA sensitivity correlate with clinical events and are associated with a low or high risk of transplant rejection.

Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines.

Dendritic cells (DCs) are potent antigen-presenting cells and play a pivotal role in T cell-mediated immunity. DCs have been shown to induce strong antitumor immune responses in vitro and in vivo, and their efficacy is being investigated in clinical trials. Compared with vaccination strategies directed against a single tumor antigen, tumor-cell lysate as the source of antigen offers the potential advantage of inducing a broad T-cell response against multiple known, as well as unknown, tumor-associated antigens expressed by the individual tumor. We used pancreatic carcinoma cell lines to develop an in vitro model for monitoring T-cell responses induced by lysate-pulsed DCs. Monocyte-derived DCs of HLA-A2(+) donors were pulsed with lysate generated from the HLA-A2(+) pancreatic carcinoma cell line Panc-1. In some experiments, the immunogenic protein keyhole limpet hemocyanin (KLH) was added to the lysate. Subsequently, the antigen-loaded DCs were activated with tumor necrosis factor-alpha and prostaglandin E(2). Autologous mononuclear cells were cocultured with DCs in the presence of low-dose interleukin (IL)-2 and IL-7 and were restimulated weekly with new DCs. High levels of IL-12 and IFN-gamma could be detected in the supernatants, indicating a T-helper type 1-type immune response. This cytokine profile was associated with the expression of the activation marker CD69 on both T helper and CTLs and with an antigen-induced proliferative T-cell response. After 4 weeks, CTL-mediated cytotoxicity was assessed. Tumor cell lysis was specific for Panc-1 tumor cells and was MHC class I-restricted. Cytokine secretion, CD69 expression of T cells, and antigen-induced T-cell proliferation correlated with the cytotoxic activity and were more pronounced when KLH was added to the lysate. This is the first study to show that T cells specific for pancreatic carcinoma cells can be generated in vitro by lysate-pulsed DCs and that the T-cell response can be enhanced by KLH. This in vitro model can be applied to compare different strategies in the development of DC-based tumor vaccines.

A comparative assessment of the roles of CD8 and CD2 in the functions of activated murine CD8+ T lymphocytes.

Both CD8 and CD2 are T cell surface receptors involved in physical cell interaction and in transmembrane signalling. The present paper addresses their role in the induction of two different functions of the cloned murine cytotoxic T cell C196: target cell lysis and IFN-gamma production. These functions were induced in C196 either by stimulation with the specific stimulator/target cell P815 or, bypassing specific recognition, by the aCD3 hybridoma 145-2C11 or by solid phase aTCR antibodies. These responses were tested for their susceptibility to inhibition/enhancement by a panel of aCD8 and aCD2 mAb. In addition, CD8 deficient and CD8/CD2 double-deficient variants of C196 were transfected with the CD8 and CD2 genes and the resulting cell lines were analysed for their functional capacities. The following results were obtained: (i) CD8 is primarily important in the specific recognition process of activated CTL; (ii) transmembrane signalling of activated CTL through the TCR does not require CD8, nor is it sensitive to modification through CD8; (iii) CTL can nevertheless be directly activated through CD8; however, this is restricted to induction of cytotoxicity but does not result in IFN-gamma production; (iv) CD2 does not seem to be important in any of these responses.

Comprehensive quality assessment approach for flow cytometric immunophenotyping of human lymphocytes.

Flow cytometric immunophenotypic (IPT) evaluation has become an important adjunct to clinical patient management and epidemiological studies. This has precipitated a need for stringent quality assessment (QA) procedures to ascertain data integrity. We evaluated a QA approach to monitor all elements of the immunophenotyping process, inclusive of blood collection and processing procedures as well as of staining reagent and instrument performance. Central to our approach was preparation each day, in parallel with clinical analytes, of lymphocytes from healthy donors, selected from a 15 donor panel. IPT parameters evaluated over a 19 month period included frequencies of CD3+, CD4+, CD8+, and CD20+ lymphocytes and the ratio of CD4+ to CD8+ lymphocytes. The sensitivity for analytical error detection was reflected by median coefficients of variation of these parameters within individual panel donors, which were 4.1%, 4.5%, 3.9%, 8.2%, and 10.1%, respectively. IPT parameter values were determined each day for two of the panel donors, then averaged and standardized to obtain a quality or Q variate, which was the basis of QA. Error detection sensitivity decreased 0.6-1.7% and the number of false rejections increased 1.2-3.3% when one panel donor rather than two was used daily for QA. This study also illuminated important aspects of what constitutes the norm for longitudinal IPT parameter variation in healthy individuals including: 1) a generally low degree of temporal parameter variation within individual donors, but 2) significant differences between donors with respect to variance estimates for CD3+ and CD8+ lymphocyte frequencies and CD4+/CD8+ lymphocyte ratios, and 3) an apparent seasonal pattern of variation in CD4+ T-cell frequencies.

Quantitative assessment of interleukin-2-producing alloreactive human T cells by limiting dilution analysis.

A limiting dilution (LD) culture system was developed to estimate the frequency of IL-2 producing alloantigen-reactive human helper T lymphocytes (HTL). E rosette-purified T cells or cell sorter-separated CD4+ and CD8+ subsets were co-cultured under LD conditions with irradiated allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) in the absence of additional growth factors. IL-2 in microculture supernatants was detected in a bioassay combined with rapid colorimetric visualization (cleavage of MTT). Under these conditions, one out of 250-800 E+, 180-280 CD4+, and 440-1000 CD8+ T cells produced IL-2 after a culture period of 3 days. This quantitative analysis of alloantigen-specific human HTL is complete within 4 days and thus provides a rapid method with potential applications in various clinical situations, e.g., transplantation medicine.