Acute exercise preferentially redeploys NK-cells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells. Part II: impact of latent cytomegalovirus infection and catecholamine sensitivity.

We showed previously that acute exercise is associated with a preferential redeployment of highly-differentiated NK-cells and increased cytotoxicity against HLA-expressing tumor cell lines during exercise recovery. In this part II study, we retrospectively analyzed these findings in the context of latent cytomegalovirus (CMV) infection and performed additional experiments to explore potential mechanisms underpinning the marked reduction in NK-cell redeployment with exercise in CMV-seropositive individuals. We show here that latent CMV infection impairs NK-cell mobilization with exercise, only when the intensity of the exercise bout exceeds the individual blood lactate threshold (BLT). This impaired mobilization is associated with increased proportions of poorly exercise-responsive NK-cell subsets (NKG2C+/KIR-, NKG2C+/NKG2A-, and NKG2C+/CD57+) and decreased NK-cell β(2)-adrenergic receptor (AR) expression in those with CMV. As a result, NK-cell production of cyclic AMP (cAMP) in response to in vitro isoproterenol (synthetic β-agonist) stimulation was drastically lower in those with CMV (6.0 vs. 20.3pmol/mL, p<0.001) and correlated highly with the proportion of NKG2C+/CD57+ NK-cells (R(2)=0.97). Moreover, NK-cell cytotoxic activity (NKCA) against the K562 (36.6% vs. 22.7%, p<0.05), U266 (23.6% vs. 15.9%, p<0.05), and 221.AEH (41.3% vs. 13.3%, p<0.001) cell lines was increased at baseline in those infected with CMV; however, latent CMV infection abated the post-exercise increase in NKCA as a result of decreased NK-cell mobilization. Additionally, NKCA per cell against the U266 (0.24 vs. 0.12, p<0.01), RPMI-8226 (0.17 vs. 0.11, p<0.05), and 221.AEH (0.18 vs. 0.11, p<0.05) cell lines was increased 1h post-exercise (relative to baseline) in CMV-seronegative subjects, but not in those infected with CMV. Collectively, these data indicate that latent CMV infection may compromise NK-cell mediated immunosurveillance after acute exercise due to an increased proportion of "CMV-specific" NK-cell subsets with impaired β-adrenergic receptor signaling pathways.

Bigley AB ，Rezvani K ，Pistillo M ，Reed J ，Agha N ，Kunz H ，O'Connor DP ，Sekine T ，Bollard CM ，Simpson RJ ... -  《-》

Acute exercise preferentially redeploys NK-cells with a highly-differentiated phenotype and augments cytotoxicity against lymphoma and multiple myeloma target cells.

NK-cells undergo a "licensing" process as they develop into fully-functional cells capable of efficiently killing targets. NK-cell differentiation is accompanied by an increased surface expression of inhibitory killer immunoglobulin-like receptor (KIR) molecules, which is positively associated with cytotoxicity against the HLA-deficient K562 cell line. NK-cells are rapidly redeployed between the blood and tissues in response to acute exercise, but it is not known if exercise evokes a preferential trafficking of differentiated NK-cells or impacts NK-cell cytotoxic activity (NKCA) against HLA-expressing target cells. Sixteen healthy cyclists performed three 30-min bouts of cycling exercise at -5%, +5%, and +15% of lactate threshold. Blood samples obtained before, immediately after, and 1h after exercise were used to enumerate NK-cells and their subsets, and determine NKCA and degranulating subsets (CD107+) against cell lines of multiple myeloma (U266 and RPMI-8226), lymphoma (721.221 and 221 AEH), and leukemia (K562) origin by 4 and 10-color flow cytometry, respectively. Exercise evoked a stepwise redeployment of NK-cell subsets in accordance with differentiation status [highly-differentiated (KIR+/NKG2A-) >medium-differentiated (KIR+/NKG2A+)>low-differentiated (KIR-/NKG2A+)] that was consistent across all exercise intensities. NKCA per cell increased ∼1.6-fold against U266 and 221 AEH targets 1h post-exercise and was associated with a decreased proportion of NK-cells expressing the inhibitory receptor CD158b and increased proportion of NK-cells expressing the activating receptor NKG2C, respectively. We conclude that exercise evokes a preferential redeployment of NK-cell subsets with a high differentiation phenotype and augments cytotoxicity against HLA-expressing target cells. Exercise may serve as a simple strategy to enrich the blood compartment of highly cytotoxic NK-cell subsets that can be harvested for clinical use.

Bigley AB ，Rezvani K ，Chew C ，Sekine T ，Pistillo M ，Crucian B ，Bollard CM ，Simpson RJ ... -  《-》

Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans.

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK cells, which are potent killers of infected cells expressing human leucocyte antigen (HLA)-E. As HLA-E is also over-expressed in several haematological malignancies and CMV has been linked to a reduced risk of leukaemic relapse, we determined the impact of latent CMV infection on NK cell cytotoxicity against four tumour target cell lines with varying levels of HLA-E expression. NK cell cytotoxicity against K562 (leukaemia origin) and U266 (multiple myeloma origin) target cells was strikingly greater in healthy CMV-seropositive donors than seronegative donors and was associated strongly with target cell HLA-E and NK cell NKG2C expression. NK cell cytotoxicity against HLA-E transfected lymphoma target cells (221.AEH) was ∼threefold higher with CMV, while NK cell cytotoxicity against non-transfected 721.221 cells was identical between the CMV groups. NK cell degranulation (CD107a(+) ) and interferon (IFN)-γ production to 221.AEH cells was localized almost exclusively to the NKG2C subset, and antibody blocking of NKG2C completely eliminated the effect of CMV on NK cell cytotoxicity against 221.AEH cells. Moreover, 221.AEH feeder cells and interleukin (IL)-15 were found to expand NKG2C(+) /NKG2A(-) NK cells preferentially from CMV-seronegative donors and increase NK cell cytotoxicity against HLA-E(+) tumour cell lines. We conclude that latent CMV infection enhances NK cell cytotoxicity through accumulation of NKG2C(+) NK cells, which may be beneficial in preventing the initiation and progression of haematological malignancies characterized by high HLA-E expression.

Bigley AB ，Rezvani K ，Shah N ，Sekine T ，Balneger N ，Pistillo M ，Agha N ，Kunz H ，O'Connor DP ，Bollard CM ，Simpson RJ ... -  《-》

NK-cells have an impaired response to acute exercise and a lower expression of the inhibitory receptors KLRG1 and CD158a in humans with latent cytomegalovirus infection.

NK-cells and γδ T-cells are cytotoxic effectors of the immune system that are preferentially mobilized into the blood compartment in response to acute stress and exercise. While infection history is known to alter the phenotype and exercise-responsiveness of CD8+ T-cells, the influence of latent cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections on the phenotypes and exercise-responsiveness of NK-cells and γδ T-cells are unknown. Twenty healthy males (age: 28.4±5.4 years) cycled for 30 min at 85% peak power. Blood lymphocytes isolated before, immediately after, and 1 h after exercise were surface stained for CD3, CD4, CD8, CD56, CD57, CD158a, KLRG1, and γδ-TCR antigens by four-color flow cytometry. CMV and EBV serostatus (pos/neg) was determined by ELISA. CMVpos had lower proportions of NK-cells expressing inhibitory receptors (KLRG1+ and CD158a+) and higher proportions of terminally differentiated NK-cells (KLRG1-/CD57+) compared to CMVneg. CMVpos mobilized far fewer (132 cells/μL vs. 245 cells/μL) NK-cells in response to exercise despite having similar baseline NK-cell counts and physiological responses to exercise as CMVneg, although terminally differentiated NK-cells were equally responsive to exercise regardless of CMV serostatus (p=0.658). EBVpos had higher proportions of CD8+ NK-cells, but cellular responses to exercise were not influenced by EBV. The frequency and exercise-responsiveness of γδ T-cells was not affected by CMV or EBV serostatus (p>0.05). In conclusion, latent CMV infection is associated with lowered numbers of NK-cells expressing inhibitory receptors and a blunted mobilization of NK-cells in response to acute exercise. This may indicate a compromised immune response to "fight-or-flight" situations in those infected with CMV.

Bigley AB ，Lowder TW ，Spielmann G ，Rector JL ，Pircher H ，Woods JA ，Simpson RJ ... -  《-》

The Effects of Age and Latent Cytomegalovirus Infection on NK-Cell Phenotype and Exercise Responsiveness in Man.

Bigley AB ，Spielmann G ，Agha N ，Simpson RJ ... -  《-》