Infectivity in chimpanzees (Pan troglodytes) of plasma collected before HCV RNA detectability by FDA-licensed assays: implications for transfusion safety and HCV infection outcomes.

Serial plasma aliquots (50 mL) obtained from 10 commercial donors who converted from hepatitis C virus (HCV) RNA negative to positive were transfused into 2 chimpanzees to assess infectivity during early HCV infection. Plasma, obtained 4 days before HCV RNA detectability by licensed assays, transmitted HCV infection to chimpanzee X355. The infectious PCR-negative plasma was subsequently shown to be positive in 2 of 23 replicates using a sensitive transcription-mediated amplification (TMA) assay, and estimated to contain 1.2 HCV RNA copies/mL (60 copies/50 mL transfused). Plasma units obtained up to 8 weeks earlier were not infectious in a second susceptible chimp, even when from donors with low-level, intermittent HCV RNA detection. Chimp x355 developed acute viremia with subsequent seroconversion, but cleared both virus and Ab in 17 weeks. When rechallenged 38 months later with 6000 RNA copies/mL from the same donor, X355 was transiently reinfected and again rapidly lost all HCV markers. We conclude that: (1) transfusions can transmit HCV infection before RNA detection, but the interval of test-negative infectivity is very brief; (2) early "blips" of HCV RNA appear noninfectious and can be ignored when calculating residual transfusion risk; and (3) markers of HCV infection can be lost rapidly after exposure to low-dose inocula.

The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells.

Foxp3 is a key marker for CD4(+) regulatory T cells (T(regs)) and was used in developing a multiparameter flow cytometric panel to identify T(regs). Achieving reproducible staining and analysis first required optimization of Foxp3 staining. We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti-human-Foxp3 antibodies used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of peripheral blood mononuclear cells from 10 individuals, and then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3(+) from Foxp3(-) events. Foxp3 gates were set using two gating strategies based on CD127(+)CD25(-) "non-T(regs)" or based on isotype controls. For Foxp3 staining, the best conditions for fixation/permeabilization were obtained using the eBioscience Foxp3, Imgenex, BioLegend, and BD Foxp3 buffers. Comparing results from 10 subjects, 259D/C7, PCH101, 236A/E7, and 206D antibodies yielded statistically higher levels of Foxp3 cells than those by 150D and 3G3 antibodies (mean = 6.9, 5.1, 4.7, and 3.7% compared with 1.7, and 0.3% of CD25(+)Foxp3(+) events within CD4(+) cells, respectively). Importantly, the "nonspecificity" of some antibodies observed with a Foxp3 gate based on isotype controls could be eliminated by setting the Foxp3 gate on "non-T(regs)". Better separation of Foxp3(+) and Foxp3(-) populations was observed using the PCH101 clone coupled to Alexa647 compared with FITC or the 259D/C7 clone coupled to PE compared with Alexa488 fluorochrome. Foxp3 staining can be highly variable and depends on the choice of antibody/buffer pair and the fluorochrome used. Selecting the correct population for setting the Foxp3 gate is critical to avoid including non-T(regs) in the Foxp3(+) gate. The experiments presented here will aid in optimization of flow cytometry staining panels to quantify T(reg) frequencies in humans.

Loss of T cell responses following long-term cryopreservation.

Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-gamma responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4(+) T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8(+) T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8(+) T cell responses to peptides and peptide pools were well preserved. Loss of both CD4(+) and CD8(+) T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4(+) T cell responses to peptide stimulation and partially restored T cell IFN-gamma responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-gamma responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4(+) T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4(+) and CD8(+) T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4(+) T cell responses and mild skewing of T cell phenotypic marker expression.

Antagonism of HIV-specific CD4+ T cells by C-terminal truncation of a minimum epitope.

Antagonism of T cell responses by variants of the cognate peptide is a potential mechanism of viral escape from immune responses and may play a role in the ability of HIV to evade immune control. We show here a rarely described mechanism of antagonism by a peptide shorter than the minimum length epitope for an HIV p24-specific CD4+ T cell clone. The shorter antagonist peptide-MHC complex bound the T cell receptor (TCR), albeit with lower affinity than the full-length agonist peptide. Prior work showing the crystal structure of the peptide-MHC complex revealed a unique glycine hinge near the C-terminus of the agonist peptide, allowing the generation of full-length antagonist peptide lacking the hinge. These results confirm the dependence of productive TCR engagement on residues spilling out from the C-terminus of the MHC binding groove and show that partial engagement of the TCR with a truncated, low-affinity ligand can result in T cell antagonism.

Effects of blood sample age at time of separation on measured cytokine concentrations in human plasma.

Measurement of peripheral blood cytokines and other immunomodulatory proteins is a useful and popular tool for assessing human immune responses to a wide range of assaults. A common challenge in this work is obtaining fresh, high-quality samples and limiting the time between blood collection and the separation of plasma or serum from cells. In this study we sought to determine the effect of sample age at the time of processing on the measured levels of 41 soluble immune mediators. Two cohorts were examined: healthy lab donors and trauma patients, who have significant immune perturbation. Whole-blood samples were aliquoted, and plasma was isolated, at days 0, 1, 2, and 3 after collection. Multiplexing techniques were used to measure protein concentrations, and general estimating equations were used to determine if there was a significant change over time. Over the 3-day period examined, only 15 of the 41 proteins showed no significant change in either cohort. Among the remaining proteins both increases and decreases were observed, with changes ranging from 2.4% per day to 325% per day. Proteins with significant changes in one cohort did not always show significant changes in the other group. These results support the need to separate plasma or serum from whole blood as quickly as possible and/or to standardize the length of time to processing within a given study of peripheral blood protein concentrations. When this is not possible, care should be taken to account for differences due to sample age.

Human T cell leukemia virus type 1 infection drives spontaneous proliferation of natural killer cells.

Most human T cell leukemia virus type 1 (HTLV-1) infected subjects remain asymptomatic throughout their lives, with a few individuals developing HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukemia. Lymphocytes from about half of HTLV-1 infected subjects spontaneously proliferate in vitro, and how this phenomenon relates to symptomatic disease outcome and viral burden is poorly understood. Spontaneous proliferation was measured in lymphocyte subsets, and these findings were correlated with HTLV-1 proviral load and Tax expression in PBMCs. We found that in addition to previously described vigorous CD8+ T cell spontaneous proliferation, natural killer (NK) cells spontaneously proliferated to a similar high level, resulting in expansion of CD56-expressing NK cells. Spontaneous NK cell proliferation positively correlated with HTLV-1 proviral load but not with Tax expression or the presence of HAM/TSP. The strongest correlate with clinical outcome in this cohort was the ability of cells to express Tax, while HTLV-1 proviral load was more closely related to spontaneous NK cell proliferation. These results demonstrate that spontaneous proliferation, Tax expression, and proviral load are inter-related but not equivalent, and that spontaneous lymphocyte proliferation is not restricted to T cells, the targets of HTLV-1 infection.

HIV+ elite controllers have low HIV-specific T-cell activation yet maintain strong, polyfunctional T-cell responses.

OBJECTIVE: HIV elite controllers are a unique group of rare individuals who maintain undetectable viral loads in the absence of antiretroviral therapy. We studied immune responses in these individuals to inform vaccine development, with the goal of identifying the immune correlates of protection from HIV. METHODS: We compared markers of cellular activation, HIV-specific immune responses and regulatory T (Treg) cell frequencies in four groups of individuals: HIV-negative healthy controls, elite controllers (HIV RNA level <75 copies/ml), individuals on HAART and individuals with HIV RNA level more than 10,000 copies/ml (noncontrollers). RESULTS: Elite controllers possessed significantly lower levels of activated HIV-specific CD8 T cells and of recently divided HIV-specific CD4 T cells than noncontrollers, whereas these differences were not seen in the respective cytomegalovirus-specific T-cell populations. Elite controllers also mounted a stronger and broader cytokine and chemokine response following HIV-specific stimulation than individuals on HAART and noncontrollers. Finally, we found that HAART-suppressed individuals had elevated Treg cell frequencies, whereas elite controllers and noncontrollers maintained normal percentages of Treg cells. CONCLUSION: Elite controllers maintain high levels of HIV-specific immune responses with low levels of HIV-specific T-cell activation and do not have elevated Treg cell levels. Based on these data an ideal HIV vaccine would induce strong HIV-specific immune responses whereas minimizing HIV-specific T-cell activation.

Tregs control the development of symptomatic West Nile virus infection in humans and mice.

West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4+ Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA+) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans.