Usefulness of cell-mediated immune function in risk stratification for patients with advanced heart failure

    Usefulness of cell-mediated immune function in risk stratification for patients with advanced heart failure Vijaiganesh Nagarajan MD, MRCP, Adrian V. Hernandez MD, PhD, Clay A. Cauthen MD, MPH, Randall C. Starling MD, MPH, W. H. Wilson Tang MD PII: DOI: Reference:

S0002-8703(16)30204-6 doi: 10.1016/j.ahj.2016.09.008 YMHJ 5285

To appear in:

American Heart Journal

Received date: Accepted date:

16 August 2015 6 September 2016

Please cite this article as: Nagarajan Vijaiganesh, Hernandez Adrian V., Cauthen Clay A., Starling Randall C., Tang W. H. Wilson, Usefulness of cell-mediated immune function in risk stratification for patients with advanced heart failure, American Heart Journal (2016), doi: 10.1016/j.ahj.2016.09.008

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ACCEPTED MANUSCRIPT Usefulness of cell-mediated immune function in risk stratification for patients with advanced heart failure

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Vijaiganesh Nagarajan, MD MRCP1, Adrian V. Hernandez, MD PhD2,3, Clay A. Cauthen, MD MPH4, Randall C. Starling, MD MPH4, and W. H. Wilson Tang, MD4 Department of Cardiology, University of Virginia, Charlottesville, 2Department of Quantitative Health Sciences, Lerner Research Institute; and 3Medical School, Faculty of Medicine, Universidad Peruana de Ciencias Aplicadas (UPC), Lima, Peru; and 4 Department of Cardiovascular Medicine, Heart and Vascular Institute, Cleveland Clinic, Cleveland, Ohio

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Journal: American Heart Journal Section: Clinical Investigations

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Running title: Immune function in advanced heart failure

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Funding Support: This research was supported by National Institutes of Health grant RO1 HL103931.

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Conflict of Interest Disclosure: All authors have no relationships to disclose. All authors have approved the final draft of the manuscript. Figures: To be printed in black and white.

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Number of Pages: 21 pages

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Number of words: 3,147 words (including abstract and references) Corresponding Address: W. H. Wilson Tang, MD 9500 Euclid Avenue, Desk J3-4, Cleveland, OH 44195. U.S.A. Phone: (216) 444-2121 / Fax: (216) 445-6165 / E-mail: [email protected]

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ACCEPTED MANUSCRIPT Highlights 1. 11% of stage IV heart failure patients referred for heart transplantation had poor

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immunity, while 18.7% had strong immune function.

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2. The primary outcome of all-cause mortality or cardiac transplantation occurred in 63.4%, 45.3% and 34.8% in the poor immunity, normal immunity and strong immune

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function groups, respectively.

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3. Although the cell mediated immune function was strongly associated with primary outcome of all-cause mortality or cardiac transplantation in univariate analysis, it lost its

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significance in multivariate analysis. But the direction to an increased risk of primary

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outcome was maintained in the poor immune function group.

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ACCEPTED MANUSCRIPT Abstract Background: Although heightened inflammation and autoimmune responses have

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been well-described in patients with heart failure, the role of cell-mediated immune

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function in the pathogenesis and progression of heart failure is unclear. The aim of our study is to evaluate the prognostic role of cell-mediated immune function in patients with

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advanced heart failure.

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Methods: We studied patients with advanced heart failure referred for evaluation of candidacy for advanced heart failure therapies between 2007 and 2010. Cell-mediated

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immune response was categorized into three groups – low or poor immune response (≤ 225 ng/ml), moderate or normal immune response (226 – 524 ng/ml) and strong

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immune response (≥ 525ng/ml) using a phytohemagglutinin-stimulated T-cell response

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assay.

Results: Out of 368 patients, 41 patients (11.1%) had poor immune function, 258

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patients (70.1%) had normal immune function, and 69 patients (18.7%) had strong

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immune function. The primary outcome of all-cause mortality or cardiac transplantation occurred in 63.4%, 45.3% and 34.8% in the poor immunity, normal immunity and strong immune function groups, respectively. In univariate analysis, cell-mediated immune function was strongly associated with the primary outcome (p=0.014). Poor immune function portended worse prognosis (Hazard ratio=2.18, 95%CI 1.01-4.70, p=0.047) and strong immune function was associated with better survival (Hazard ratio = 0.67, 95% CI 0.43-1.04). However, when adjusted for multiple variables in multivariate analysis, immune function status lost its overall significance to predict primary outcome (p=0.11),

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ACCEPTED MANUSCRIPT but the direction to an increased risk of primary outcome was maintained in the poor immune function group.

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Conclusion: Poor cell-mediated immune function measured by a clinically available

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assay could be associated with more adverse long-term prognosis in patients with

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advanced heart failure.

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Keywords: cell-mediated immune function; heart failure; mortality; heart transplantation

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ACCEPTED MANUSCRIPT Introduction Heart failure is recognized as a complex clinical syndrome as it involves

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endocrine, molecular and neurohormonal axes. Although the activation of renin-

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angiotensin system and sympathetic system plays a major role in the pathogenesis of heart failure, there is growing recent evidence to suggest involvement of immune

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system in its development and progression. Early reports suggesting elevation of serum

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tumor necrosis factor (TNF) level in heart failure patients started coming more than two decades ago.(1) Since then, multiple cytokines were noted to be elevated in patients

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with heart failure like interleukin-1 (IL-1), IL-6 and IL-10.(2) (3) (4) Whether activation of immune system is a cause or consequence to myocyte injury is still controversial.

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Measurement of cell-mediated immunity is frequently employed in the post-transplant

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patients receiving immunosuppressants as a measure of immune response.(5) The aim of our study is to evaluate the role of immune function testing in patients with pre-

Methods

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transplant stage D heart failure patients.

This study was approved by the Cleveland Clinic Institutional Review Board. No extramural funding was used to support this work. We evaluated cell-mediated immune function in a large database of more than 500 consecutive patients with advanced end stage heart failure referred for cardiac transplant evaluation and other advanced heart failure therapies to a tertiary level reference center between 2007 and 2010. Out of these, 368 patients had performed testing of cell-mediated immune function to assess the underlying immune status prior to immunosuppressive therapy. Patients underwent

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ACCEPTED MANUSCRIPT comprehensive evaluation either in the hospital or in our advanced heart failure clinic, including laboratory investigations, echocardiogram, metabolic stress test and cardiac reviewed

characteristics,

which

electronic

included

medical

demographic

records

to

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information,

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catheterization.

collect baseline

baseline clinical

characteristics, past medical history, history of infection and immunodeficient states,

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medications (including active antibiotics use at the time of testing), laboratory test

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results, and procedures performed including echocardiographic data. Participation in this retrospective study did not require any alteration to the medical management of

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patients, and the database was managed by independent researchers not responsible for care of these patients.

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We utilize a cell-mediated immune function assay (Immuknow, Cylex Inc,

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Columbia MD, USA) to estimate the degree of cell-mediated immunity. Briefly, patient’s whole blood is incubated for 15- 18 hours with and without the stimulant

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phytohemagglutinin (PHA). PHA increases adenosine triphosphate (ATP) synthesis

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within the cells. Magnetic beads coated with anti-CD4 monoclonal antibodies are added to both samples. Then the samples are washed over a strong magnet and a lysis reagent is added. This releases intracellular ATP which is measured using a luminometer. Luciferin, a luminescence reagent is used for this step. The results of the immune function assay were reported in ng/mL of ATP. Based on previous studies and laboratory data, immune response was categorized into three groups (5) (6) (7) – low or poor immune response (≤225 ng/ml), moderate or normal immune response (226–524 ng/ml) and strong immune response (≥525ng/ml). The primary outcome of interest was the composite of all-cause mortality or cardiac transplantation. We used medical

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ACCEPTED MANUSCRIPT records and the hospital transplantation index to identify both patients and date of cardiac transplantation. Since infection or other mediators of immune response can

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affect post-transplant outcomes, the cases were censored upon the occurrence of

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transplant as reaching the primary endpoint.

Dichotomous categorical data were reported as percentages and the chi-square

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test was used to detect differences. Continuous variables were described as mean and

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standard deviation; comparisons between 2 groups were done with the t-test or Wilcoxon rank sum text Because we were expecting non-normal distribution for some

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continuous variables across the three immune response groups due to their small sample sizes, description of these continuous outcomes were with median and

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interquartile range (IQR); comparisons among the three groups were done with the

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Kruskal-Wallis test. Time to event analysis was performed using Kaplan-Meier method, and differences between curves were calculated using the log rank test. Univariate

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analyses for association between immune function groups, important clinical and

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laboratory variables and the primary outcome were performed using Cox proportional hazards models. Variables with a p<0.2 were included for multivariate Cox models. The associations were provided as hazard ratios (HR) and their 95% confidence intervals (CI). Differences with p<0.05 were considered statistically significant in multivariate analyses. We also performed univariate and multivariate logistic regression analysis to evaluate the association between clinical and laboratory variables and poor immune function (<225 ng/mL). The authors are solely responsible for the design and conduct of this study, all study analyses, the drafting and editing of the paper and its final contents.

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ACCEPTED MANUSCRIPT All statistics were performed using SPSS version 20.0 (IBM, Armonk NY) and R 2.15.1

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(www.r-project.org).

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Results

Among 500 consecutive subjects in the database, 368 subjects were included for

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analysis with cell-mediated immune function assay performed. None of the subjects

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experienced active infection or received antibiotics at the time of testing by chart review. In this study cohort, 258 patients (70.1%) had normal immunity, while 41 patients

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(11.1%) had poor immunity and 69 patients (18.7%) had strong immunity. The distribution of the cell-mediated immune function measurement is illustrated in Figure 1,

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and baseline characteristics of all the three groups are illustrated in Table 1.

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The primary outcome occurred in 63.4%, 45.3% and 34.8% in the poor immunity, normal immunity and strong immunity group, respectively. Patients with strong immunity

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had better survival when compared to other two groups and patients with poor immunity

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had the worst chance of survival in Kaplan-Meier analysis (log rank test p = 0.012) (Figure 1). In univariate analysis, immune function was strongly associated with the primary outcome of all-cause mortality or cardiac transplantation (p=0.014). In comparison to the normal immune group, the poor immune function seems to increase the risk of the primary outcome (Hazard ratio 1.53, 95% CI 1.00-2.34); the strong immune function seems to protect against the occurrence of the primary outcome (Hazard ratio 0.67, CI 0.43-1.04) (Table 2). Other variables which had some association with the primary outcome (p<0.2) were race, NYHA class, diabetes, use of betablockers and ACE inhibitors, serum sodium, blood urea nitrogen, albumin, bilirubin, B-

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ACCEPTED MANUSCRIPT type natriuretic peptide, and hemoglobin. When all these variables were included in multivariate analysis, immune function test lost its overall significance to predict primary

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outcome (p=0.11). Although the effect estimate was larger for the poor immune function

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group, the direction to an increased risk of the primary outcome was maintained (Hazard ratio 2.18, 95% CI 1.01-4.71). In contrast, the borderline protective effect of the

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strong immune function disappeared in multivariate analyses (Hazard ratio 0.84, 95% CI

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0.39-1.78) (Table 2).

We also looked for associations between patient variables and immune function.

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Comparing between the three groups, patients with the weakest cell-mediated immune function has higher B-type natriuretic peptide and blood urea nitrogen levels (Table 1).

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In univariate and multivariate analyses, none of the patient variables had significant

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association with immune function when analyzed as continuous variable. But when variables with p<0.2 were included for multivariate analysis, only history of ventricular

Discussion

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tachycardia (VT) reached borderline significance (OR 0.35, 95% CI, 0.13, 0.96; p=0.04).

In patients with advanced, "stage D" heart failure, we observed that stronger cellmediated immunity was associated with better survival free of transplantation. However, stronger cell-mediated immune function was not associated with lower all-cause mortality or cardiac transplantation when adjusted for other clinical variables. Also, we did not find any significant association between immune function and individual patient variables. Taken together, diminished cell-mediated immunity tracks with disease progression in advanced heart failure but did not provide independent prognostic value.

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ACCEPTED MANUSCRIPT Immune system plays an important, but complex role in the pathogenesis of heart failure. Overall levels of multiple cytokines have been elevated in patients with

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heart failure, but in most cases they are highly variable and may not provide the best

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discrimination in patients with versus without adverse consequences for reliable clinical use. The most studied cytokine is tumor necrosis factor alpha (TNFα). The circulating

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level of this cytokine was increased in patients with cachexia. (8) Subsequently TNF-α

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was labeled as a major pathogenic mediator of heart failure progression. However, even after extensive investigations, the role of modulating immune system in heart failure

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remains elusive. The largest of such anti-TNFα trial was the RENEWAL trial.(9) However, this trial was terminated prematurely as etanercept did not have any clinically

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significant benefit either on death or heart failure hospitalization. Multiple hypotheses

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had been used to explain this phenomenon. Possible cardioprotective effect of TNFα was one of them.(10) Studies involving another anti-TNF inhibitor, thalidomide again

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showed contradictory results.(11) (12)

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Monitoring of cell-mediated immune function has been used in the posttransplant setting in patients on immunosuppression. Poor cell-mediated immune function measured by this clinically available assay was associated with increased infection risk in 296 heart transplant patients, when the predictive ability to detect rejections was inconclusive.(13) Cell-mediated immune function assay was helpful in assessing the overall immune function in lung transplant patients, but it was not a reliable predictor of rejections or infections as the test had poor performance characteristics in receiver operating characteristic curve analysis.(14) Similarly, in 83 pediatric heart transplant patients, cell-mediated immune function assay was not

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ACCEPTED MANUSCRIPT predictive of infections or rejections and hence was not recommended for routine monitoring of such patients.(15) In 504 solid organ transplant patients, a higher risk of

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rejection was associated with stronger immune response whereas higher risk of

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infection with weaker immune response.(16) Another meta-analysis showed a high specificity, but low sensitivity in identifying patients at risk of rejection. Summary ROC

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curve showed an area under the curve (AUC) of 0.51 for rejection confirming poor

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overall accuracy. Similarly, this analysis produced inconsistent results in identifying patients at risk of infection as well with low sensitivity and low specificity.(17) Recent

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studies evaluating the use of cell-mediated immune function assay in non-transplant patient population like inflammatory bowel disease and rheumatoid arthritis are

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emerging.(18) (19) Although the immune assay did not show any correlation with

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disease activity, it was helpful in identifying patients at increased risk of infection in these studies. Specifically, the immune assay level was significantly lower in patients

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with infection when compared to patients without.

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To our knowledge, there are no studies looking into the role of immune function assay in advanced, "stage D" heart failure patients. Our initial hypothesis before this study, based on the available literature was that the higher the activity of cytokines (hence the better the immune function), patients are likely to have poorer prognosis. In contrast, the main finding that patients with stronger cell-mediated immune function portend better prognosis in univariate analysis and in Kaplan-Meier analysis, yet lost its significance in multivariate analysis suggested that disease severity may play a contributing role to these findings. We also noted the poor immune function group was at increased risk of primary outcome compared to the group with normal immunity. This

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ACCEPTED MANUSCRIPT effect was attenuated after adjusting for other variables in multivariable analysis, but the direction was maintained. Although limited by a retrospective chart review, we have

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carefully considered concomitant factors that may have influenced the outcome of

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interest secondary to ongoing infections or other immunodeficiency states, issues that were unikely encountered in this setting as these would be obvious contraindications for

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cardiac transplantation. It is conceivable that as patients progressed to more advanced

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stage of heart failure, their end-organ perfusion towards hematologic, hepatic, and immune systems maybe more markedly altered. Hence, these adverse functional and

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long-term consequences that were detectable by a cell-mediated immunity function assay likely represent underlying disease severity. Naturally, immunosuppressive

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therapy may have a difficult time balancing potential risks and benefits if underlying

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immune dysfunction is underappreciated.(20),(21),(22),(23) Indeed, our findings are also consistent with prior research on the prognostic role of lymphopenia in advanced heart

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stratification.(25)

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failure,(24) which is one variable utilized in the Seattle Heart Failure Model for risk

This is primarily a hypothesis-generating study, but sheds some light on the complex interaction between immune system and heart failure. It may be reasonable to obtain pre-transplant immune function testing as it may provide some prognostic information although there is no clear way to modify this risk at the present time. This may help to tailor the post-transplant immunosuppressive therapy. If further studies show a similar relationship, it may be time to think about novel therapies to enhance immunity. Nevertheless, our hypothesis-generating study has several limitations. Our study is a retrospective study and was not designed primarily to evaluate the role of cell-

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ACCEPTED MANUSCRIPT mediated immunity in heart failure. We did not measure any cytokine level in any patients, nor do we have any information regarding infections and other coexisting

In a population with no immunosuppressive therapy, the proportion of

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ineligible.

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immunodeficiencies, although these patients are often excluded as being transplant

individuals with poor immune function was relatively low (11%), and we only performed

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a one-time measurement as part of pre-transplant evaluation.

Nevertheless, these

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findings provide insights into the need to better understand how the immune system

approach to immune modulation.

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Funding

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may affect different individuals with advanced heart failure, rather than a one-size-fits-all

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There is no extramural funding for this project. Dr. Tang is funded by grants from the

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Disclosure

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National Institutes of Health (R01HL103931).

No relationships to disclose.

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ACCEPTED MANUSCRIPT 3. Francis SE, Holden H, Holt CM, et al. Interleukin-1 in myocardium and coronary

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arteries of patients with dilated cardiomyopathy. J Mol Cell Cardiol. 1998;30:215-23.

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4. Amir O, Rogowski O, David M, et al. Circulating interleukin-10: Association with higher mortality in systolic heart failure patients with elevated tumor necrosis factor-

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alpha. Isr Med Assoc J. 2010;12:158-62.

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5. Kowalski R, Post D, Schneider MC, et al. Immune cell function testing: An adjunct to therapeutic drug monitoring in transplant patient management. Clin Transplant.

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2003;17:77-88.

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6. Husain S, Raza K, Pilewski JM, et al. Experience with immune monitoring in lung transplant recipients: Correlation of low immune function with infection. Transplantation.

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2009;87:1852-7.

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7. Kowalski RJ, Post DR, Mannon RB, et al. Assessing relative risks of infection and

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rejection: A meta-analysis using an immune function assay. Transplantation. 2006;82:663-8.

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9. Mann DL, McMurray JJ, Packer M, et al. Targeted anticytokine therapy in patients with chronic heart failure: Results of the randomized etanercept worldwide evaluation (RENEWAL). Circulation. 2004;109:1594-602.

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ACCEPTED MANUSCRIPT 10. Kurrelmeyer KM, Michael LH, Baumgarten G, et al. Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a

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12. Orea-Tejeda A, Arrieta-Rodriguez O, Castillo-Martinez L, et al. Effects of

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thalidomide treatment in heart failure patients. Cardiology. 2007;108:237-42.

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transplant patients using ATP production in activated lymphocytes. J Heart Lung

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14. Shino MY, Weigt SS, Saggar R, et al. Usefulness of immune monitoring in lung transplantation using adenosine triphosphate production in activated lymphocytes. J Heart Lung Transplant. 2012;31:996-1002.

15. Rossano JW, Denfield SW, Kim JJ, et al. Assessment of the cylex ImmuKnow cell function assay in pediatric heart transplant patients. J Heart Lung Transplant. 2009;28:26-31.

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ACCEPTED MANUSCRIPT 16. Kowalski RJ, Post DR, Mannon RB, et al. Assessing relative risks of infection and rejection: A meta-analysis using an immune function assay. Transplantation.

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2006;82:663-8.

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18. Brandhorst G, Weigand S, Eberle C, et al. CD4+ immune response as a potential biomarker of patient reported inflammatory bowel disease (IBD) activity. Clin Chim Acta.

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19. Akimoto M, Yunoue S, Otsubo H, et al. Assessment of peripheral blood CD4+ adenosine triphosphate activity in patients with rheumatoid arthritis. Mod Rheumatol.

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20. Mann DL, McMurray JJ, Packer M, et al. Targeted anticytokine therapy in patients

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with chronic heart failure: Results of the randomized etanercept worldwide evaluation (RENEWAL). Circulation. 2004;109:1594-602.

21. Chung ES, Packer M, Lo KH, et al. Randomized, double-blind, placebo-controlled, pilot trial of infliximab, a chimeric monoclonal antibody to tumor necrosis factor-alpha, in patients with moderate-to-severe heart failure: Results of the anti-TNF therapy against congestive heart failure (ATTACH) trial. Circulation. 2003;107:3133-40.

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ACCEPTED MANUSCRIPT 22. Torre-Amione G, Anker SD, Bourge RC, et al. Results of a non-specific immunomodulation therapy in chronic heart failure (ACCLAIM trial): A placebo-

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controlled randomised trial. Lancet. 2008;371:228-36.

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24. Ommen SR, Hodge DO, Rodeheffer RJ, et al. Predictive power of the relative lymphocyte concentration in patients with advanced heart failure. Circulation.

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1998;97:19-22.

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25. Levy WC, Mozaffarian D, Linker DT, et al. The seattle heart failure model: Prediction

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of survival in heart failure. Circulation. 2006;113:1424-33.

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ACCEPTED MANUSCRIPT Table.1 Baseline Characteristics Normal Cell-

Strong Cell-

Mediated

Mediated

Mediated

Immune

Immune

function (n=41)

function

57 (48-64)

(IQR) 28 (70)

BMI (kg/m2), median

27.8 (22.5-31.4)

0.4

48 (70)

0.9

27.5 (23.7-31.2)

28.3 (25.1-32.7)

0.3

187 (73)

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(IQR)

function (n=69)

53 (45-62)

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Male, (%)

Immune

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57 (48-63)

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Age (years), median

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(n=258)

p-value

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Poor Cell-

11 (28)

57 (22)

19 (28)

0.5

Hypertension, (%)

22 (55)

97 (38)

24 (39)

0.1

18 (45)

108 (42)

27 (39)

0.8

6 (15)

20 (8)

4 (6)

0.2

18 (45)

110 (43)

25 (36)

0.6

CVA/TIA, (%)

4 (10)

26 (10)

7 (10)

1.0

Smoking, (%)

11 (27)

57 (22)

12 (17)

0.5

Atrial Fibrillation, (%)

16 (40)

92 (36)

20 (29)

0.5

Systolic BP (mmHg),

102 (89-117)

100 (91-111)

101 (90-114)

0.8

61 (56-67)

62 (57-70)

61 (53-70)

0.7

Aspirin, (%)

21 (53)

148 (57)

42 (61)

0.7

Clopidogrel, (%)

6 (15)

37 (14)

12 (17)

0.8

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Diabetes, (%)

COPD, (%)

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CAD, (%)

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Dyslipidemia, (%)

median (IQR) Diastolic BP (mmHg), median (IQR)

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ACCEPTED MANUSCRIPT 25 (63)

178 (69)

48 (70)

0.7

ACE-Inhibitors, (%)

16 (40)

129 (50)

36 (52)

0.4

Spironolactone, (%)

16 (40)

125 (48)

36 (52)

0.5

Hydralazine, (%)

9 (23)

62 (24)

Nitrates, (%)

10 (25)

70 (27)

ARB, (%)

3 (8)

39 (15)

Sodium (mmol/L),

135 (133-138)

136 (133-139)

0.5

16 (23)

0.8

5 (7)

0.1

136 (133-138)

0.7

1.1 (0.9-1.4)

0.2

3.9 (3.5-4.3)

4.1 (3.4-4.4)

0.2

0.12 (0.03-1.46)

0.17 (0.05-0.62)

0.8

522 (258-1317)

461 (213-916)

500 (172-798)

0.02

32 (19-46)

23 (16-31)

23 (17-32)

0.03

16.3 (15.3-17.7)

15.4 (14.1-16.9)

15.7 (13.7-16.7)

0.2

3.6 (2.9-4.2)

median (IQR)

0.18 (0.07-0.72)

median (IQR)

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BNP (pg/mL), median

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Troponin (ng/mL),

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(IQR)

Blood Urea Nitrogen

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Albumin (g/dL),

1.1 (0.9-1.5)

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1.3 (1.0-1.8)

median (IQR)

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12 (17)

median (IQR) Creatinine (mg/dL),

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Beta-blockers, (%)

(mg/dL), median (IQR) RDWCV, median (IQR)

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ACCEPTED MANUSCRIPT Table 2 Unadjusted and adjusted associations between cell-mediated immune function groups and all-cause mortality or cardiac transplantation HR (95%CI)

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Immune function group Poor

1.53 (1.00-2.34)

analyses

Normal

0.014

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Unadjusted

P value

1 (reference)

0.67 (0.43-1.04)

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Strong Poor

2.18 (1.01-4.71)

analyses*

Normal

0.11

1 (reference) 0.84 (0.39-1.78)

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Strong

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Adjusted

* Adjusted for race, NYHA class, diabetes, chronic kidney disease, beta-blocker, ACE

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inhibitor, sodium, blood urea nitrogen, albumin, bilirubin, troponin, hemoglobin and BNP.

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ACCEPTED MANUSCRIPT Figure Legends Figure 1: Distribution of cell-mediated immune function in our study cohort.

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Figure 2: Kaplan–Meier curves showing better survival of patients with stronger

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immune function compared to normal and poor immune function (Log rank test

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p=0.012).

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ACCEPTED MANUSCRIPT

Figure 1

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Figure 2

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ACCEPTED MANUSCRIPT

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