Hawley Kunz

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.

Latent CMV infection enhances anti‐tumor 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.

The effects of age and viral serology on γδ T-cell numbers and exercise responsiveness in humans

γδ T-cells are cytotoxic effector cells that preferentially migrate to peripheral tissues and recognize many types of antigen. We examined the effects of age and viral serology on the exercise responsiveness of γδ T-cells. Blood was collected from 17 younger (age: 23-35yrs) and 17 older (50-64yrs) healthy males matched for cytomegalovirus (CMV), Epstein-Barr virus, herpes simplex virus-1 and Parvovirus B19 serologic status before and after a single bout of cycling exercise. Older had lower numbers and proportions of γδ T-cells than younger, while CMV was associated with increased numbers and proportions of γδ T-cells in younger but not older. Exercise evoked a ∼2-fold increase in circulating γδ T-cell numbers. The magnitude of this response was 3-times greater in younger compared to older, and 1.6-times greater in younger CMV-infected compared to younger non CMV-infected. To conclude, γδ T-cell numbers and exercise responsiveness decreases with age and may contribute to impaired immunosurveillance after acute acute physical stress.

Regulatory T Cells in Asthma and Airway Hyperresponsiveness

Regulatory T cells, or Tregs, have been shown to play a major role in reducing Th2 cell proliferation, potentially reducing (often significantly) airway-associated inflammation seen in airway diseases, such as asthma. These cells are characterized as a sub-population of T cells that maintain peripheral tolerance through a variety of biological mechanisms. Although Tregs make up only 5-10% of peripheral CD4 +

A single exercise bout enhances the manufacture of viral-specific T-cells from healthy donors: implications for allogeneic adoptive transfer immunotherapy

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). The adoptive transfer of donor-derived viral-specific cytotoxic T-cells (VSTs) is an effective treatment for controlling CMV and EBV infections after HSCT; however, new practical methods are required to augment the ex vivo manufacture of multi-VSTs from healthy donors. This study investigated the effects of a single exercise bout on the ex vivo manufacture of multi-VSTs. PBMCs isolated from healthy CMV/EBV seropositive participants before (PRE) and immediately after (POST) 30-minutes of cycling exercise were stimulated with CMV (pp65 and IE1) and EBV (LMP2A and BMLF1) peptides and expanded over 8 days. The number (fold difference from PRE) of T-cells specific for CMV pp65 (2.6), EBV LMP2A (2.5), and EBV BMLF1 (4.4) was greater among the VSTs expanded POST. VSTs expanded PRE and POST had similar phenotype characteristics and were equally capable of MHC-restricted killing of autologous target cells. We conclude that a single exercise bout enhances the manufacture of multi-VSTs from healthy donors without altering their phenotype or function and may serve as a simple and economical adjuvant to boost the production of multi-VSTs for allogeneic adoptive transfer immunotherapy.

Vigorous exercise mobilizes CD34+ hematopoietic stem cells to peripheral blood via the β2-adrenergic receptor

Acute dynamic exercise mobilizes CD34+ hematopoietic stem cells (HSCs) to the bloodstream, potentially serving as an economical adjuvant to boost the collection of HSCs from stem cell transplant donors. The mechanisms responsible for HSC mobilization with exercise are unknown but are likely due to hemodynamic perturbations, endogenous granulocyte-colony stimulating factor (G-CSF), and/or β2-adrenergic receptor (β2-AR) signaling. We characterized the temporal response of HSC mobilization and plasma G-CSF following exercise, and determined the impact of in vivo β-AR blockade on the exercise-induced mobilization of HSCs. Healthy runners (n = 15) completed, in balanced order, two single bouts of steady state treadmill running exercise at moderate (lasting 90-min) or vigorous (lasting 30-min) intensity. A separate cohort of healthy cyclists (n = 12) completed three 30-min cycling ergometer trials at vigorous intensity after ingesting: (i) 10 mg bisoprolol (β1-AR antagonist); (ii) 80 mg nadolol (β1 + β2-AR antagonist); or (iii) placebo, in balanced order with a double-blind design. Blood samples collected before, during (runners only), immediately after, and at several points during exercise recovery were used to determine circulating G-CSF levels (runners only) and enumerate CD34+ HSCs by flow cytometry (runners and cyclists). Steady state vigorous but not moderate intensity exercise mobilized HSCs, increasing the total blood CD34+ count by ∼4.15 ± 1.62 Δcells/µl (+202 ± 92%) compared to resting conditions. Plasma G-CSF increased in response to moderate but not vigorous exercise. Relative to placebo, nadolol and bisoprolol lowered exercising heart rate and blood pressure to comparable levels. The number of CD34+ HSCs increased with exercise after the placebo and bisoprolol trials, but not the nadolol trial, suggesting β2-AR signaling mediated the mobilization of CD34+ cells [Placebo: 2.10 ± 1.16 (207 ± 69.2%), Bisoprolol 1.66 ± 0.79 (+163 ± 29%), Nadolol: 0.68 ± 0.54 (+143 ± 36%) Δcells/µL]. We conclude that the mobilization of CD34+ HSCs with exercise is not dependent on circulating G-CSF and is likely due to the combined actions of β2-AR signaling and hemodynamic shear stress.

T-cell redeployment and intracellular cytokine expression following exercise: effects of exercise intensity and cytomegalovirus infection.

The magnitude of lymphocytosis following exercise is directly related to exercise intensity. Infection with cytomegalovirus (CMV) also augments lymphocytosis after exercise. It is not known if the enhanced T‐cell response to exercise due to CMV depends on exercise intensity. Furthermore, exercise‐induced changes in T‐cell expression of type I and type II cytokines are thought to be intensity dependent, but direct comparisons are lacking. The aim of this experiment was to determine if CMV affects the exercise‐induced redistribution of T‐cell subsets at varying intensities, and determine the effect of exercise intensity on CD8+ T‐cell cytokine expression. Seventeen cyclists (nine CMV seropositive; CMV+) completed three 30 min cycling trials at −5, +5, and +15% of blood lactate threshold (LT). T‐cell subsets in blood and intracellular expression of type I (IL‐2, interferon(IFN)‐γ) and type II (IL‐4, IL‐10) cytokines by CD8+ T cells pre, post, and 1‐h post‐exercise were assessed by flow cytometry. Independently of CMV, T‐cell subset redistribution was greater after +15%LT compared to −5%LT (P < 0.05). Independently of intensity, CMV− mobilized more low‐ (CD27+ CD28+) and medium‐ (CD27+ CD28−) differentiated T cells than CMV+, whereas CMV+ mobilized more high (CD27− CD28−) differentiated T cells. The numbers of IL‐2+, IFN‐γ+, IL‐4+, and IL‐10+ CD8+ T cells increased after exercise above LT. Only type I cytokine expression was influenced by exercise intensity (P < 0.05). In conclusion, T‐cell redeployment by exercise is directly related to exercise intensity, as are changes in the number of CD8+ T‐cells expressing type I cytokines. Although CMV+ mobilized more high‐differentiated T cells than CMV−, this occurred at all intensities. Therefore, the augmenting effect of CMV on T‐cell mobilization is independent of exercise intensity.

Rapid morphological characterization of isolated mitochondria using Brownian motion.

Mitochondrial morphology has been associated with numerous pathologies including cancer, diabetes, obesity and heart disease. However, the connection is poorly understood-in part due to the difficulty of characterizing the morphology. This impedes the use of morphology as a tool for disease detection/monitoring. Here, we use the Brownian motion of isolated mitochondria to characterize their size and shape in a high throughput fashion. By using treadmill exercise training, mitochondria from heart and gastrocnemius of Balb/c mice were modulated in size and used to investigate the protocol. Consistent with previous reports, the heart mitochondria of untrained mice increased 5% in diameter immediately after a single bout of moderate exercise (1.091 ± 0.004 μm) as compared to completely sedentary controls (1.040 ± 0.022 μm). In addition, no change was observed in the size of gastrocnemius mitochondria (1.025 ± 0.018 μm), which was also in agreement with previous studies. The method was also successfully applied to smaller Saccharomyces cerevisiae mitochondria.

A single exercise bout augments adenovirus-specific T-cell mobilization and function

Adoptive transfer of virus-specific T-cells (VSTs) effectively treats viral infections following allogeneic hematopoietic stem cell transplantation (alloHSCT), but logistical difficulties have limited widespread availability of VSTs as a post-transplant therapeutic. A single exercise bout mobilizes VSTs specific for latent herpesviruses (i.e. CMV and EBV) to peripheral blood and augments their ex vivo expansion. We investigated whether exercise exerts similar effects on T-cells specific for a NON-latent virus such as adenovirus, which is a major contributor to infection-related morbidity and mortality after alloHSCT. Thirty minutes of cycling exercise increased circulating adenovirus-specific T-cells 2.0-fold and augmented their ex vivo expansion by ~33% compared to rest without altering antigen and MHC-specific autologous target cell killing capabilities. We conclude that exercise is a simple and economical adjuvant to boost the isolation and manufacture of therapeutic VSTs specific to latent and non-latent viruses from healthy donors.

153. Latent cytomegalovirus infection enhances baseline anti-tumor cytotoxicity but impairs NK-cell responses to acute exercise through preferential expansion of NKG2C+ NK-cells in healthy humans

Cytomegalovirus (CMV) infection markedly expands NKG2C+/NKG2A- NK-cells, which are potent killers of infected cells expressing the non-classical MHC molecule HLA-E. As HLA-E is overexpressed in several bloodborne malignancies, we examined the impact of latent CMV infection and acute exercise on NK-cell cytotoxic activity (NKCA) against a wide range of tumor target cells with varying levels of HLA-E expression. In resting blood, the expansion of NKG2C+ NK-cells with CMV was associated with strong anti-leukemia (K562) and anti-myeloma (U266) NKCA that was proportionate to the level of target cell HLA-E expression. NKCA against transfected (HLA-E-bright) 221.AEH cells was 3-times higher in those with CMV, but no effect of CMV was found on NKCA against HLA-E-negative 721.221 cells. Blocking the interaction between NKG2C and HLA-E completely eliminated the effect of CMV on NKCA against 221.AEH cells. Although NK-cells were redeployed in large numbers with exercise, NKG2C+ NK-cells were among the poorest responders of the NK-cell subsets to 30-min of cycling exercise. This contributed to a blunted exercise-induced redeployment of total NK-cells in those with CMV. Moreover, NKCA against U266 and 221.AEH target cells was enhanced during exercise recovery, but not in those with CMV. In conclusion, latent CMV infection enhances NKCA through preferential expansion of NKG2C+ NK-cells; however, these cells respond poorly to a single bout of exercise indicating that CMV may impair acute stress-induced immunosurveillance.