Impact of blood processing variations on natural killer cell frequency, activation, chemokine receptor expression and function

Naranbhai, Vivek; Bartman, Pat.; Ndlovu, Dudu.; Ramkalawon, P; Ndung’u, Thumbi; Wilson, Douglas P.; Altfeld, Marcus; Carr, William H.

doi:10.1016/j.jim.2011.01.001

Cited by 33 publications

(29 citation statements)

References 24 publications

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“…Activation, CD107a expression and intracellular cytokine production were measured in cryopreserved peripheral blood mononuclear cells (PBMC) in batch analyses using optimized flow cytometry methods as previously described [15]. Cryopreserved PBMC were rapidly thawed in warm media (RPMI 1640; Gibco), washed and rested for two hours.…”

Section: Methodsmentioning

confidence: 99%

Changes in Natural Killer Cell Activation and Function during Primary HIV-1 Infection

et al. 2013

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BackgroundRecent reports suggest that Natural Killer (NK) cells may modulate pathogenesis of primary HIV-1 infection. However, HIV dysregulates NK-cell responses. We dissected this bi-directional relationship to understand how HIV impacts NK-cell responses during primary HIV-1 infection.Methodology/Principal FindingsPaired samples from 41 high-risk, initially HIV-uninfected CAPRISA004 participants were analysed prior to HIV acquisition, and during viraemic primary HIV-1 infection. At the time of sampling post-infection five women were seronegative, 11 women were serodiscordant, and 25 women were seropositive by HIV-1 rapid immunoassay. Flow cytometry was used to measure NK and T-cell activation, NK-cell receptor expression, cytotoxic and cytokine-secretory functions, and trafficking marker expression (CCR7, α4β7). Non-parametric statistical tests were used. Both NK cells and T-cells were significantly activated following HIV acquisition (p = 0.03 and p<0.0001, respectively), but correlation between NK-cell and T-cell activation was uncoupled following infection (pre-infection r = 0.68;p<0.0001; post-infection, during primary infection r = 0.074;p = 0.09). Nonetheless, during primary infection NK-cell and T-cell activation correlated with HIV viral load (r = 0.32'p = 0.04 and r = 0.35;p = 0.02, respectively). The frequency of Killer Immunoglobulin-like Receptor-expressing (KIRpos) NK cells increased following HIV acquisition (p = 0.006), and KIRpos NK cells were less activated than KIRneg NK cells amongst individuals sampled while seronegative or serodiscordant (p = 0.001;p<0.0001 respectively). During HIV-1 infection, cytotoxic NK cell responses evaluated after IL-2 stimulation alone, or after co-culture with 721 cells, were impaired (p = 0.006 and p = 0.002, respectively). However, NK-cell IFN-y secretory function was not significantly altered. The frequency of CCR7+ NK cells was elevated during primary infection, particularly at early time-points (p<0.0001).Conclusions/SignificanceAnalyses of immune cells before and after HIV infection revealed an increase in both NK-cell activation and KIR expression, but reduced cytotoxicity during acute infection. The increase in frequency of NK cells able to traffic to lymph nodes following HIV infection suggests that these cells may play a role in events in secondary lymphoid tissue.

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Section: Methodsmentioning

confidence: 99%

Changes in Natural Killer Cell Activation and Function during Primary HIV-1 Infection

et al. 2013

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“…Samples were properly insulated and quality control was maintained to ensure the viability and functional output of innate and adaptive immune cells from all subjects as previously described [27]. Controllers were defined as HIV-seropositive individuals who were not receiving antiretroviral therapy and who had either undetectable HIV-1 RNA levels using conventional assays (<50 copies/mL by Abbott RealTime PCR or <75 copies/mL by bDNA; “ elite controllers ”) or low but detectable levels of viral replication (e.g., 50 to 2000 copies RNA/mL; “ viremic controllers” ) for a minimum of 5 years (Table 1).…”

Section: Methodsmentioning

confidence: 99%

Impact of protective killer inhibitory receptor/human leukocyte antigen genotypes on natural killer cell and T-cell function in HIV-1-infected controllers

Tomescu

Duh

Hoh

et al. 2012

Aids

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Objective Both protective T cell genotypes and NK cell genotypes have been associated with delayed progression to AIDS and shown to be co-inherited in HIV-1 infected subjects who limit viral replication in absence of antiretroviral therapy (“controllers”). However, a comparative analysis of the genotype and function of the innate and adaptive immune compartments in HIV-1 infected controller subjects has been understudied to date. Design Here, we simultaneously tested NK and T cell function in controllers to investigate the mechanism(s) that might account for host-immune control over viral replication. Methods We measured CD8 T cell responses against HIV-1 utilizing overlapping 15-mer peptides spanning the HIV-1 Consensus Clade B Gag protein and tested NK cell degranulation and cytokine secretion against tumor target cells following IFN-alpha stimulation. Results Among a cohort of 37 controllers, the presence of protective MHC-Class I HLA alleles (such as HLA-B*57) was not correlated with HIV-specific CD8 responses. In contrast, the inheritance of a protective KIR3DL1*h/*y receptor genotype along with the corresponding HLA-Bw4*80I ligand was associated with significantly heightened target cell-induced NK degranulation and cytokine secretion following IFN-alpha stimulation (p=0.0201, n=13). Interestingly, we observed a significant inverse association between the IFN-alpha stimulated NK response to K562 cells and the HIV-specific CD8 T cell response to Gag among elite controllers. (rho=−0.8321, p=0.0010, n=12). Conclusions Together, these results suggest that heightened NK responses can be evidenced independently of HIV-specific T cell responses in HIV-1 infected elite controllers.

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“…This is critical since the efficacy of autologous BMNC transplantation is likely dose-dependent [20], and little data is available on a possible asymmetry of the cell loss [21], [22]. Recently, it was described that Ficoll DGC even depleted cells with a high regenerative potential, such as MSC [23] and VSEL [24], and irreversibly impaired cell function by decreasing expression of chemokines receptors [25], [26]. Accordingly, the objective of this study was to determine whether and how Ficoll DGC affects the yield and composition of the cell graft compared to alternative methods such as adjusted Percoll DGC [27] and immunomagnetic bead separation of granulocytes [28].…”

Section: Introductionmentioning

confidence: 99%

Density Gradient Centrifugation Compromises Bone Marrow Mononuclear Cell Yield

et al. 2012

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Bone marrow mononuclear cells (BMNCs) are widely used in regenerative medicine, but recent data suggests that the isolation of BMNCs by commonly used Ficoll-Paque density gradient centrifugation (DGC) causes significant cell loss and influences graft function. The objective of this study was to determine in an animal study whether and how Ficoll-Paque DGC affects the yield and composition of BMNCs compared to alternative isolation methods such as adjusted Percoll DGC or immunomagnetic separation of polymorphonuclear cells (PMNs). Each isolation procedure was confounded by a significant loss of BMNCs that was maximal after Ficoll-Paque DGC, moderate after adjusted Percoll DGC and least after immunomagnetic PMN depletion (25.6±5.8%, 51.5±2.3 and 72.3±6.7% recovery of total BMNCs in lysed bone marrow). Interestingly, proportions of BMNC subpopulations resembled those of lysed bone marrow indicating symmetric BMNC loss independent from the isolation protocol. Hematopoietic stem cell (HSC) content, determined by colony-forming units for granulocytes-macrophages (CFU-GM), was significantly reduced after Ficoll-Paque DGC compared to Percoll DGC and immunomagnetic PMN depletion. Finally, in a proof-of-concept study, we successfully applied the protocol for BMNC isolation by immunodepletion to fresh human bone marrow aspirates. Our findings indicate that the common method to isolate BMNCs in both preclinical and clinical research can be considerably improved by replacing Ficoll-Paque DGC with adapted Percoll DGC, or particularly by immunodepletion of PMNs.

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Impact of blood processing variations on natural killer cell frequency, activation, chemokine receptor expression and function

Cited by 33 publications

References 24 publications

Changes in Natural Killer Cell Activation and Function during Primary HIV-1 Infection

Changes in Natural Killer Cell Activation and Function during Primary HIV-1 Infection

Impact of protective killer inhibitory receptor/human leukocyte antigen genotypes on natural killer cell and T-cell function in HIV-1-infected controllers

Density Gradient Centrifugation Compromises Bone Marrow Mononuclear Cell Yield

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