The impact of latent CMV infection on NK-cell mobilization and expression of KLRG1 and CD57 in response to acute exercise.

Natural killer (NK) cells are cytotoxic effectors of the innate immune system that are able to distinguish healthy autologous cells from tumors and virally infected cells. NK-cells kill the targeted cells by releasing cytotoxic proteins, a process that is governed by inhibitory surface receptors, such as KLRG1. Additionally, activated NK-cells are able to proliferate in response to immunological stimuli, a process that is inhibited in NK-cells expressing the senescence marker CD57. Acute bouts of exercise are known to mobilize NK cells into the blood compartment, which could alter immunity; however, whether or not exercise alters NK-cell KLRG1 and CD57 expression has not been fully elucidated. Furthermore, as latent CMV infection is associated with an increased frequency of inhibitory NK cells, it is not known if CMV status influences NK-cell mobilization in response to acute exercise. PURPOSE: To examine the impact of latent CMV infection on the mobilization of NK-cells and their expression of KLRG1 and CD57 in response to acute exercise. METHODS: Otherwise healthy CMV seropositive (CMV+) and CMV seronegative (CMV-) males (age 23-35 years) completed a 30-min cycling protocol at 85% of maximum power. Lymphocytes isolated from whole blood before, immediately after, and one hour after exercise were surface-stained with monoclonal antibodies against CD3, CD56, KLRG1 and CD57 and analyzed by 4-color flow cytometry. RESULTS: Preliminary analysis of the data show a prodigious increase in the number of CD56 dim (mature, highly cytotoxic subset) NK-cells immediately after exercise in all subjects, which subsequently fell below pre-exercise values 1 hour later. In CMV- subjects, the proportion of CD56 bright (immature, mildly cytotoxic) NK cells was considerably higher 1 hour post-exercise than before exercise, but the number of cells changed very little suggesting that the increased proportion was due merely to the egress of CD56 dim NK cells. Interestingly, CMV seropositivity was associated with a near complete absence of CD56 bright NK cells that was unaffected by exercise. Neither exercise nor CMV status influenced the proportion of NK-cells expressing KLRG1 or CD57. CONCLUSION: Preliminary analysis of this data indicates that acute exercise preferentially mobilizes CD56 dim NK cells without altering KLRG1 and CD57 expression. Latent CMV infection is associated with a lowered proportion of CD56 bright NK-cells; however, the NK-cell response to exercise was not influenced by CMV status. Future work will examine the role of aging on NK-cell response to exercise and CMV status

CMV-specific T-cells Mobilized with Exercise have Broad Epitope Specificity and a High-Differentiated Effector Memory Phenotype.

Introduction: Dynamic exercise evokes a rapid redeployment of cytotoxic T-cell subsets with high surface expression of b2 adrenergic receptors, presumably to enhance immunosurveillance during acute stress. As this response is affected by age and infection history, the main aim of this study was to examine latent CMV infection as a potential confounder to age-related differences in blood CD8+ T-cell responses to exercise. The second aim of this study was to examine the impact of acute exercise on the mobilization of CMV-specific T-cells in the peripheral blood compartment. Methods: Healthy young (n=16) and older (n=16) humans counterbalanced by CMV IgG serostatus (positive or negative) exercised for 30-minutes at ~80% peak cycling power. Isolated blood lymphocytes phenotypes were assessed by flow cytometry and Enzyme-linked immunospot (ELISPOT) analysis was used to determine the frequency and function of T-cells secreting IFN-g in response to CMV antigens. Maximum likelihood linear mixed models (LMM) were used to determine main effects of exercise (pre, post and 1h post-exercise), age (young or old) and CMV status (positive or negative) on total numbers of blood lymphocytes and their subsets. Results: Those with CMV redeployed ~2 times more CD8+ T-cells and ~6-times more KLRG1+/CD28- and CD45RA+/CCR7- CD8+ subsets than non-infected exercisers. Seronegative older exercisers had an impaired redeployment of total CD8+ T-cells, CD45RA+/CCR7+ and (KLRG1-/CD28+) CD8+ subsets. Redeployed CD8+ T-cell numbers were similar between infected young and old. CMVpp65 specific CD8+ cells in HLA/A2* subjects increased ~2.7 fold after exercise, a response that was driven by the KLRG1+/CD28-/CD8+ subset. Stimulating PBMCs before and after exercise with CMVpp65 and CMV IE-1 antigens and overlapping peptide pools revealed a 2.1 and 4.4 fold increases in CMVpp65 and CMV IE-1 IFN-g secreting cells respectively. The breadth of the T cell response was maintained after exercise with the magnitude of the response being amplified across the entire epitope repertoire. Conclusion: We conclude that latent CMV infection overrides age-related impairments in CD8+ T-cell redeployment with exercise. We also show for the first time that many T-cells redeployed with exercise are specific to CMVpp65 and CMV IE-1 antigens, have broad epitope specificity, and are mostly of a high-differentiated effector memory phenotype. We anticipate that these findings may have clinical implications, with acute exercise serving as a simple strategy to increase numbers of available antigen-specific cells in blood that can be harvested for expansion and adoptive T-cell transfer in HSCT recipients

Lymphocytes and monocytes egress peripheral blood within minutes after cessation of steady state exercise: A detailed temporal analysis of leukocyte extravasation

Acute exercise evokes an almost instantaneous lymphocytosis, followed by sustained lymphopenia that occurs within just 30–60 min after exercise cessation. The aim of this study was to characterize the immediate (order of minutes) post-exercise kinetics of lymphocyte and monocyte egress, and to determine whether this egress is associated with heart rate recovery following a single bout of steady state dynamic exercise. Eleven healthy subjects cycled for 30-min at ~70% of their estimated peak power. Blood samples were collected from an intravenous catheter before exercise, during exercise (E) at +15 and +30 min, and during passive recovery (R) at exactly +1, +2, +3, +4, +5 and +10 min after exercise cessation. Complete blood counts and flow cytometry were used to enumerate total monocytes, lymphocytes: CD3+ T-cells, CD4+ T-cells, CD8+ T-cells, NK-cells and γδ T-cells in whole blood. Both lymphocytes and monocytes displayed rapid egress kinetics, by R+3 the total numbers of all cell types examined were significantly lower than E+30. NK-cells egressed more rapidly than other lymphocyte subtypes, followed by CD8+, γδ, and then CD4+ T-cells. Further, the egress of NK-cells, CD4+, and CD8+ T-cells positively correlated with heart rate recovery after exercise cessation. In conclusion, lymphocyte and monocyte egress is rapid and occurs within minutes of exercise recovery, underscoring both the importance of collection time for post exercise blood samples, and the use of intravenous catheters to capture peak cell mobilization. The rate of egress may be dependent on how quickly hemodynamic equilibrium is restored on cessation of exercise and is, therefore, likely to be influenced by individual fitness levels

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The Effect of Acute Exercise and Latent Cytomegalovirus Infection on Nk-Cell Redeployment and Anti-Tumor Cytotoxicity in Healthy Adults

Allogeneic adoptive transfer of NK-cells from healthy donors to cancer patients has shown promise as a means of controlling or reversing the spread of multiple human malignancies including multiple myeloma and acute myeloid leukemia. However, multiple issues remain that undermine the efficacy of long-term cancer treatment using adoptive transfer of NK-cells including loss of activating receptors and cytotoxic potential in transferred NK-cells. We sought to improve the clinical usefulness of NK-cells by using either acute exercise or cytomegalovirus (CMV)-driven expansion of NKG2C+ NK-cells as an adjuvant. Acute exercise mobilizes NK-cells, while latent CMV infection is associated with impaired mobilization of NK-cells and expansion of NK-cells expressing the activating receptor NKG2C. Thus, we hypothesized that a single bout of exercise or latent CMV infection would be associated with enhanced anti-tumor cytotoxicity, and that latent CMV infection would be associated with a blunted increase in NK-cell cytotoxicity with exercise. This was divided into three specific hypotheses: 1) Exercise-induced changes in the composition of NK-cell subsets will increase anti-tumor cytotoxicity; 2) CMV-driven NKG2C+ NK-cell expansion will increase anti-tumor cytotoxicity; and 3) latent CMV infection will impair the exercise-induced mobilization of NK-cells through decreased β-AR sensitivity. To test hypothesis 1, sixteen healthy cyclists performed three 30-minute bouts of cycling exercise at -5%, +5%, and +15% of blood lactate threshold. Blood samples were 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. To test hypothesis 2, 30 young healthy subjects donated blood samples to enumerate NK-cell subsets, NKCA (against U266, 721.221, 221 AEH, and K562 cells), and degranulating subsets in response to 221 AEH cells in the context of latent CMV infection. To test hypothesis 3, the same subjects from aim 1 were used and NK-cell responsiveness to exercise and β-AR stimulation were explored in the context of latent CMV infection. In support of hypothesis 1, we found a 1h post-exercise increase in NK-cell anti-tumor cytotoxicity in association with a proportional increase in NK-cells lacking inhibitory KIR for classical HLA molecules and expressing activating receptor for HLA-E (NKG2C). In support of hypothesis 2, we found that latent CMV-infection was associated with a marked, NKG2C-dependent increase in NK-cell activity against tumor cell lines expressing HLA-E (U266, K562, and 221 AEH). In support of hypothesis 3, we found that latent CMV infection was associated with a blunted NK-cell redeployment and 1h post-exercise increase in NK-cell activity following exercise above the blood lactate threshold. This blunted NK-cell mobilization was associated with decreased β2-AR expression and impaired β-AR sensitivity. We conclude that exercise and CMV are both capable of enhancing NK-cell killing efficiency through distinct mechanisms; however, these effects are not additive as CMV infection is associated with an impaired exercise response. This works opens the door to using either exercise or in vitro expansion of NKG2C+ NK-cells as a simple strategy for enhancing the anti-tumor cytotoxicity of NK-cells for immunotherapy.Health and Human Performance, Department o

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

The redeployment of NK-cells in response to an acute bout of exercise is thought to be an integral component of the “fight-or-flight” response, preparing the body for potential injury or infection. We showed previously that CMV seropositivity impairs the redeployment of NK-cells with exercise in the young. In the current study, we examined the effect of aging on the redeployment of NK-cells with exercise in the context of CMV. We show here that CMV blunts the exercise-induced redeployment of NK-cells in both younger (23–39 yrs) and older (50–64 yrs) subjects with older CMVneg subjects showing the largest postexercise mobilization and 1 h postexercise egress of NK-cells. The blunted exercise response in CMVpos individuals was associated with a decreased relative redeployment of the CD158a+ and CD57+ NK-cell subsets in younger and older individuals. In addition, we show that aging is associated with a CMV-independent increase in the proportion of NK-cells expressing the terminal differentiation marker CD57, while CMV is associated with an age-dependent decrease in the proportion of NK-cells expressing the inhibitory receptors KLRG1 (in the younger group) and CD158a (in the older group). Collectively, these data suggest that CMV may decrease NK-cell mediated immunosurveillance after exercise in both younger and older individuals

Systemic β-adrenergic receptor activation augments the ex vivo expansion and anti-tumor activity of Vγ9Vδ2 T-cells

TCR-gamma delta (γδ) T-cells are considered important players in the graft-versus-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic β-adrenergic receptor (β-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic -AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the -AR subtypes involved, by administering a preferential β¬1-AR antagonist (bisoprolol) and a non-preferential β¬1 + β2-AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their ex vivo expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L-, CD158a/b/e+ and NKG2A- cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) in vitro by 40-60%. Blocking NKG2D on TCR-γδ cells in vitro eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a β1 + β2-AR (nadolol), but not a β1-AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and ex vivo expansion. Nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic β-AR activation in healthy donors markedly augments the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells and some of these effects are due to β2-AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight β-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics