Increased T cell immunosenescence and accelerated maturation phenotypes in older kidney transplant recipients.

Older kidney transplant recipients experience increased rates of infection and death, and less rejection, compared with younger patients. However, little is known about immune dysfunction in older compared with younger kidney transplant recipients and whether it is associated with infection. We evaluated T cell phenotypes including maturation, immune senescence, and exhaustion in a novel investigation into differences in older compared with younger patients receiving identical immune suppression regimens. We evaluated PBMC from 60 kidney transplant recipients (23 older and 37 matched younger patients) by multiparameter immune phenotyping. Older kidney transplant recipients demonstrated decreased frequency of naïve CD4+ and CD8+ T cells, and increased frequency of terminally differentiated, immune senescent, and NK T cells expressing KLRG1. There was a trend towards increased frequency of T cell immune senescence in patients experiencing infection in the first year after transplantation, which reached statistical significance in a multivariate analysis. This pilot study reveals immune dysfunction in older compared with younger transplant recipients, and suggests a likely mechanism for increased vulnerability to infection. The ability to assess T cell maturation and immune senescence in transplant recipients offers the potential for risk stratification and customization of immune suppression to prevent infection and rejection after transplantation.

Standardization and cross validation of alloreactive IFNγ ELISPOT assays within the clinical trials in organ transplantation consortium.

Emerging evidence indicates memory donor-reactive T cells are detrimental to transplant outcome and that quantifying the frequency of IFNγ-producing, donor-reactive PBMCs by ELISPOT has potential utility as an immune monitoring tool. Nonetheless, differences in assay performance among laboratories limit the ability to compare results. In an effort to standardize assays, we prepared a panel of common cellular reagent standards, developed and cross validated a standard operating procedure (SOP) for alloreactive IFNγ ELISPOT assays in several research laboratories supported by the NIH-funded Clinical Trials in Organ Transplantation (CTOT) Consortium. We demonstrate that strict adherence to the SOP and centralized data analysis results in high reproducibility with a coefficient of variance (CV) of ≈ 30%. This standardization of IFNγ ELISPOT assay will facilitate interpretation of data from multicenter transplantation research studies and provide the foundation for developing clinical laboratory testing strategies to guide therapeutic decision-making in transplant patients.

HIV-1 actively replicates in naive CD4(+) T cells residing within human lymphoid tissues.

Although HIV-1 gene expression is detected in naive, resting T cells in vivo, such cells are resistant to productive infection in vitro. However, we found that the endogenous microenvironment of human lymphoid tissues supports de novo infection and depletion of this population. Cell cycle analysis and DNA labeling experiments established that these cells were definitively quiescent and thus infected de novo. Quantitation of the "burst size" within naive cells further demonstrated that these cells were productively infected and contributed to the local viral burden. These findings demonstrate that lymphoid tissues support active HIV-1 replication in resting, naive T cells. Moreover, these cells are not solely reservoirs of latent virus but are permissive hosts for viral replication that likely targets them for elimination.

Detection of cell cycle subcompartments by flow cytometric estimation of DNA-RNA content in combination with dual-color immunofluorescence.

BACKGROUND: Correlated flow cytometric measurements of phenotype and DNA-RNA content offer detailed information on cell cycle status of subpopulations in heterogeneous cell preparations in response to stimulation. We have developed a method for flow cytometric analysis of DNA-RNA content that has been optimized for simultaneous measurement of dual-color immunofluorescence. METHODS: Nucleic acid staining was performed at low pH in the presence of saponin. DNA was stained with 7-aminoactinomycin D (7-AAD) and RNA with pyronin Y(G) (PY); both dyes were used at low concentrations, and 7-AAD was exchanged with nonfluorescent actinomycin D after DNA staining to minimize fluorochrome-fluorochrome interactions. For cell surface antigen staining, allophycocyanin was combined with pH-independent Alexa488 instead of fluorescein-isothiocyanate (FITC) because FITC is pH sensitive. RESULTS: This method identified cell cycle subcompartments in CEM cells comparable to published results on cell lines using other dyes and staining methods. Measurement of DNA-RNA content in CD8 lymphocyte subsets of human peripheral blood mononuclear cells costimulated with CD3/CD28.2 showed that, after 48 h of stimulation, 80% of CD8(+) T cells were in the proliferative state, whereas 86% of CD8(+) non-T cells remained in G(0). CONCLUSIONS: This technique permits the clear identification of cellular subpopulations by phenotype and assessment of their cell cycle status.

Nonproductive human immunodeficiency virus type 1 infection in nucleoside-treated G0 lymphocytes.

Productive infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile, reverse transcripts. We have previously identified G1b as the cell cycle stage required for the optimal completion of the reverse transcription process in T lymphocytes. However, the mechanism(s) involved in the blockage of reverse transcription remains undefined. In this study we investigated whether nucleotide levels influence viral reverse transcription in G0 cells. For this purpose the role of the enzyme ribonucleotide reductase was bypassed, by adding exogenous deoxyribonucleosides to highly purified T cells in the G0 or the G1a phase of the cell cycle. Our data showed a significant increase in the efficiency of the reverse transcription process following the addition of the deoxyribonucleosides. To define the stability and functionality of these full reverse transcripts, we used an HIV-1 reporter virus that expresses the murine heat-stable antigen on the surfaces of infected cells. Following activation of infected quiescent cells treated with exogenous nucleosides, no increased rescue of productive infection was seen. Thus, in addition to failure to complete reverse transcription, there was an additional nonreversible blockage of productive infection in quiescent T cells. These experiments have important relevance in the gene therapy arena, in terms of improving the ability of lentivirus vectors to enter metabolically inactive cells, such as hematopoietic stem cells.

Costimulation of naive CD8(+) lymphocytes induces CD4 expression and allows human immunodeficiency virus type 1 infection.

Human immunodeficiency virus type 1 (HIV-1) infection requires cell surface expression of CD4. Costimulation of CD8(+)/CD4(-) T lymphocytes by anti-CD3 and anti-CD28 antibodies or by allogeneic dendritic cells induced expression of CD4 and rendered these CD8 cells susceptible to HIV-1 infection. Naive CD45RA+ cells responded with greater expression of CD4 than did CD45RO+ cells. CD8(+) lymphocytes derived from fetal or newborn sources exhibited a greater tendency to express CD4, consistent with their naive states. This mechanism of infection suggests HIV-induced perturbation of the CD8 arm of the immune response and could explain the generally rapid disease progression seen in HIV-infected children.

Norepinephrine accelerates HIV replication via protein kinase A-dependent effects on cytokine production.

To explore the role of sympathetic nervous system activation in HIV pathogenesis, we examined the effect of the neuroeffector molecule norepinephrine (NE) on HIV-1 replication in quiescently infected PBMCs that were subsequently activated with Abs to CD3 and CD28. NE accelerated HIV-1 replication at concentrations ranging from 10(-8) to 10(-5) M. This effect could be mimicked by protein kinase A (PKA) activators (forskolin or dibutyryl-cAMP) and abrogated by beta-adrenoreceptor antagonists or the PKA inhibitor rp-cAMP, indicating transduction via the adrenoreceptor signaling pathway. NE reduced cellular activation and altered the production of several HIV-modulating cytokines: IL-10 and IFN-gamma were markedly suppressed; TNF-alpha, IL-1beta, IL-2, IL-4, and IL-6 were mildly suppressed; and levels of IL-12 were not significantly altered. The addition of either exogenous IFN-gamma or IL-10 abrogated the effect of NE on virus production. Thus PKA-dependent suppression of cytokine production appears to mediate the enhancement of HIV-1 replication by NE.

Progression to the G1b phase of the cell cycle is required for completion of human immunodeficiency virus type 1 reverse transcription in T cells.

Successful infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile, reverse transcripts. In the present study, we isolated highly purified quiescent T cells and utilized the CD3/CD28 activation pathways as well as cell cycle inhibitors to further define the role of costimulation and cell cycle progression in HIV-1 reverse transcription. Activation with alphaCD3 alone resulted in cell cycle progression into only G1a and incomplete HIV-1 reverse transcription. Costimulation through the CD28 receptor and transition into G1b was required to efficiently complete the reverse transcription process. These findings have relevance to immune activation in vivo, since lymphocytes rendered anergic by a single activation signal would be nonpermissive for productive infection with HIV-1. Importantly, these data also suggest that HIV vector-based genetic transduction strategies might be successful only in target cells that transition into the G1b phase of the cell cycle.

Role of Vif in human immunodeficiency virus type 1 reverse transcription.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown that vif mutant HIV-1 virions are defective in their ability to synthesize proviral DNA in vivo. Here, we examine the role of Vif in viral DNA synthesis in the endogenous reverse transcriptase (RT) reaction, an in vitro assay in which virions synthesize viral DNA by using endogenous viral RNA as a template. vif mutant virions showed a significant reduction in endogenous RT activity despite similar levels of exogenous RT activity. Analysis of the viral DNA products on agarose gels demonstrated that this reflects reduced synthesis of short minus- and plus-strand DNA products in addition to those of full genomic length. Quantitative PCR analysis of endogenous reverse transcription provided further evidence for reduced formation of both initial and completed reverse transcripts. Vif had no effect on genomic RNA dimerization or the stability of the RNA dimer linkage. These results suggest that Vif is important for an early event after virus entry but preceding or during the early stages of viral DNA synthesis. This may be due to an intrinsic effect on reverse transcription or a preceding postentry event(s), such as virion uncoating or disassembly of the virion core. Drugs targeted to Vif function may provide a new therapeutic approach to inhibiting HIV-1 reverse transcription.

Performance of chronic schizophrenics on matched word and design recall tasks.

Matched word and design recall tasks were constructed and used to assess the performance of chronic schizophrenics on neuroleptics alone and on both neuroleptic and anticholinergic drugs. The two groups of patients performed at an equally low level on both tasks, without evidence for a differential deficit. The tasks did, however, evoke a differential deficit in clustering performance for designs as opposed to words. Although clustering, which is based on recall, is not necessarily as well matched for words and designs as recall itself, this result suggests a lateralized dysfunction in brain structures related to use of mnemonic organization at retrieval.

Verbal and non-verbal recall by depressed and euthymic affective patients.

This study uses matched-tasks methodology in order to test memory function in depressed and euthymic patients with major affective disorder. Neither drug-free depressed patients nor lithium-treated euthymic patients show a differential deficit in verbal versus non-verbal recall. However, while euthymic patients show no memory impairment, drug-free depressives do show poor memory functioning. The results support the view that memory deficits observed in affective patients in the depressed state are transient, secondary manifestations of depression and are neither indicative of underlying organic pathology, nor of abnormal hemispheric laterality. This suggests that memory impairment in depression can be treated by treating depressive symptoms, both chemically and behaviourally. The results also support the view that prophylactic lithium treatment has no adverse effects on these memory tasks.