Inverted CD4:CD8 ratio is not associated with three-year mortality in a sample of community-dwelling oldest old: The octabaix immune study

Abstract

Background

The presence of an immune risk phenotype (IRP) has been correlated with survival rates in elderly people.

Objective

To determine whether an inverted CD4:CD8 ratio might be a marker of IRP in a sample of oldest old by assessing its relationship with mortality.

Design

Prospective cohort study.

Setting

Community-based survey study of seven primary healthcare centres.

Participants

328 people born in 1924 and registered with primary healthcare centres.

Measurements

Chronic drug prescription, functional status (Barthel and Lawton indexes) and cognitive status (Spanish version of the Mini-Mental State Examination) were recorded. CD4:CD8 ratios were determined, with a ratio of 1.00 or less being used to define IRP.

Results

The CD4:CD8 ratio was 1.00 or less in 47 subjects (15.6%). After three years, 51 subjects had died (16.3%); 9 were from among the 47 (19.1%) with an inverted CD4:CD8 ratio and 42 (15.8%) from the remainder (P=0.52). Multivariate analysis identified two significant clinical variables (Lawton Index scores and the number of chronic drugs prescribed) as being independent predictors of three-year mortality risk in this cohort of octogenarians. This risk profile did not change when introducing the CD4:CD8 ratio into the calculation.

Conclusion

In this communitydwelling population of oldest old (85 years old at baseline) an inverted CD4:CD8 ratio was not associated with three-year mortality.

Introduction

Age-related changes in the immune system include alteration in T cell phenotype and effector functions and a reduced ability of B cells to produce high affinity antibodies. An immune risk phenotype (IRP) consisting of low CD4+ T cells and high CD8+ T cells and poor proliferative response to concanavalin A (con A) has been described in research using cluster analysis (1). An inverted CD4:CD8 ratio has been reported as being useful as a marker of IRP 1, 2, and its prevalence increases from around 8% in the 20-59 age range to 18% in the 60-94 range (2). In a previous study of the Octabaix cohort (3), 15% of individuals had an inverted CD4:CD8 ratio at baseline, substantially lower than the figure of 31% reported in the oldest patients (mean age 92 years) in our area (4) and very similar to the 16-20% reported in the Swedish NONA immune studies 5, 6.

An inverted CD4: CD8 ratio has been correlated with survival rates in elderly people 7, 8. We recently conducted a prospective follow-up of Octabaix subjects, aged 85 years old at baseline, and assessed mortality over a three-year period (9). The results showed that an objective evaluation based on comprehensive geriatric assessment tools might be useful to predict mortality risk when performed in the oldest-old, community-dwelling subjects. In particular, the ability to perform instrumental activities of daily living and taking fewer prescription drugs seem to predict a better chance of survival in this population.

The primary aim of the present study was to determine whether an inverted CD4:CD8 ratio is associated with three-year mortality in this same population group. A second objective was to assess whether addition of the inverted CD4:CD8 ratio to the variables previously identified as being of prognostic value would change the mortality risk profile.

Methods

This study forms part of the OCTABAIX project, a prospective, population-based study of 328 community-dwelling inhabitants born in 1924 (85 years old at the time of inclusion) and assigned to one of seven primary care teams in the geographical area of Baix Llobregat (Barcelona, Spain). The sample is described in more detail elsewhere 3, 9, 10. All subjects in the OCTABAIX project who gave their consent to participate were enrolled in the immunity study. Exclusion criteria for this sub-study were being under chemotherapy for active neoplasms or metastatic dissemination at the time of assessment, a diagnosis of AIDS, or a drug regime that might affect the immune response. No other exclusion criteria related to compromised health were applied. Given that morbidity does not seem to influence significantly the T-cell immune risk profile in the elderly (11), we decided, when designing this sub-study of certain aspects of IRP prevalence in old people, not to adopt the broad exclusion criteria recommended by the SENIEUR protocol (12). This approach meant that most of the 85-year-olds included in the initial OCTABAIX study (10) (95%) could be included in this immunity sub-study.

Geriatric assessment

Functional status was measured by the Barthel Index (BI) (13) for basic activities of daily living (ADL) and the Lawton Index (LI) (14) for instrumental ADL. Cognitive status was assessed using the validated Spanish version of the Mini-Mental State Examination, known as the MEC (15). The MEC assesses orientation, memory, concentration, language and praxis, and it provides a score of up to 35 (a score of 23 or below indicates cognitive impairment).

Laboratory methods

Blood samples were drawn between 9:00 a.m. and 10:00 a.m. Total leukocytes and lymphocytes were measured on an automated counter. T-lymphocyte subsets were determined by flow cytometry. Aliquots from peripheral blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies in double combinations: anti-CD4-FITC (clone SK3) and anti-CD8-PE (clone SK1), both purchased from Becton Dickinson (San Jose, CA, USA). A minimum of 4000 events per sample were acquired on a FACScalibur flow cytometer (Becton Dickinson). Forward light scatter (FSC) and side light scatter (SSC) parameters were used to gate viable lymphocytes. These gated lymphocytes were then analysed by two-colour immunofluorescence with CellQuest software (Becton Dickinson).

CD4:CD8 ratio

A CD4:CD8 ratio (numbers of CD4 and CD8 cells) of 1.00 or less was used to identify individuals with a possible IRP. The use of CD4 and CD8 percentages resulted in identical CD4:CD8 ratio categorization.

Outcome events

Since mortality, measured as time-to-event data, was the main outcome variable of this study, the vital status of participants was assessed every year during the three years of follow-up.

Data analysis

Normally distributed continuous variables are reported as mean ± standard deviation (SD), while categorical variables are reported as proportions. The normality of continuous variables was assessed using the Kolmogorov-Smirnov test. The Student's t test was used to compare continuous variables, with a previous Levene test for equality of variances, while either the chi-square statistic or Fisher's exact test was used to compare categorical or dichotomous variables. Correlation analysis was based on Pearson's correlation coefficients. All statistical tests were two-tailed and the level of statistical significance was set at p=0.05. Proportional hazards and linearity assumptions (for continuous variables) were evaluated for Cox models in order to determine the variables associated with three-year mortality. The variables entered into the model using a backward stepwise approach were those previously shown to have a significant association in the total cohort, namely the LI score and number of chronic drug prescriptions, with gender and BI and MEC scores also being introduced. We then repeated the model, adding the inverted CD4:CD8 ratio as both a categorical score and a continuous variable (mean values). An adjusted hazard ratio (HR) with a 95% confidence interval (CI) was used. The results were considered significant when p < 0.05. All analyses were performed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA).

Results

A total of 312 subjects were finally included in the OCTABAIX immune study, 190 women (60.9%) and 122 men. In the geriatric assessment the mean LI score was 5.4 ± 2.5, the mean BI score was 88.1 ± 18 and the mean MEC score was 26.9 ± 6.6.

CD4:CD8 status

The laboratory tests yielded the following mean values: leukocytes 6757 ± 1882, lymphocytes 1873 ± 910, T-CD4 42.9% ± 10, T-CD8 24.9% ± 10, and CD4:CD8 ratio 2.2 ± 1.5. The CD4:CD8 ratio was 1.00 or less in 47 subjects (15.6%). Inverted CD4:CD8 ratio was more frequent (p>0.01) in men (55.3%) than females (44.7%).

Predictors of mortality

After three years, 51 subjects (16.3%) had died; 9 were from among the 47 (19.1%) with an inverted CD4:CD8 ratio and 42 (15.8%) from the remainder (P=0.52). The bivariate analysis of baseline variables associated with three-year survival is shown in Table 1. Multivariate analysis identified two significant clinical variables (LI scores and the number of chronic drugs prescribed) as being independent predictors of three-year mortality risk in this cohort of octogenarians. However, the prognostic value of this risk profile (LI: hazard ratio 0.804, 95% CI 0.723-0.893; chronic drug prescription: hazard ratio 1.108, 95% CI 1.021-1.202) was not improved by introducing the inverted CD4:CD8 ratio into the calculation, neither as a categorical variable nor as a continuous variable (mean value).

Table 1.

Comparison of variables associated with three-year survival. Proportions and mean (standard deviation) are shown

	Survivors (n=261)	Non-Survivors (n=51)	P
Gender
Female	165 (63.2%)	25 (49%)	0.06
Male	96 (36.8%)	26 (51%)
Lawton Index	5.7 ± 2.4	3.8 ± 2.7	0.0001
Barthel Index	89.5 ± 17	80.8 ± 22	0.002
MEC1	27.1 ± 6.3	25.5 ± 7.8	0.09
Chronic drug prescriptions	5.8 ± 3.2	7.6 ± 3.1	0.0001
Leukocytes	6715 ± 1826	6971 ± 2159	0.37
Lymphocytes	1898±931	1743 ± 787	0.27
T-CD4	42.9 ± 10	42.9 ± 12	0.97
T-CD8	24.6 ± 10	25.8 ± 11	0.48
CD4:CD8 ratio	2.1 ± 1.4	2.2 ± 2.1	0.65
Inverted CD4:CD8 ratio	38 (14.6%)	9 (17.6%)	0.57

¹MEC = Spanish version of the Mini-Mental State Examination

Discussion

The results of the OCTABAIX immunity study, involving a Mediterranean population of oldest old subjects, did not confirm the hypothesis that an inverted CD4:CD8 ratio, as a possible IRP, would be associated with increased mortality. This contrasts with previous findings reported in very elderly Swedish subjects 6, 7, even when broader samples (individuals aged 60 years or older) were assessed (2). In a sample that formed part of the Healthy Ageing Study (16), which examined 2041 elderly community residents with a follow-up that varied from 3 to 115 months, inverted CD4:CD8 ratio was associated with an increased risk of death (hazard ratio 1.56, 95% CI 1.09–2.22). However, as in the present study these results became non-significant when adjusting for sex (1.37, 95% CI 0.96–1.97).

Poor overall health, defined in terms of physical function, and/or the presence of cognitive impairment is associated with worse prognosis and increased mortality in older people (17). The lack of association between an inverted CD4:CD8 ratio and poorer health status which was already reported in the initial evaluation of this sample (3) may partly explain the lack of association now observed with respect to mortality. In this regard, it is also important to take into account the small percentage of patients who had an inverted CD4:CD8 ratio at baseline in the Octabaix study, as well as the relatively modest mortality rate after three years of follow-up.

Despite the strengths of this study, particularly the inclusion of geriatric assessment alongside the analysis of the inverted CD4:CD8 ratio in a large sample of oldest old subjects aged 85 years at baseline, an important limitation needs to be acknowledged when interpreting the findings, namely that we did not assess the presence or absence of chronic cytomegalovirus (CMV) infection at baseline. Assessing this aspect can be important for a better understanding of immune status in older subjects.

To conclude, in this study of a community-dwelling, single birth year cohort the inverted CD4: CD8 ratio was not associated with higher mortality after three years of follow-up. A longer follow-up study would be of interest in order to determine whether mortality and disability (in terms of physical function and cognition) increases in the group of subjects with an inverted CD4:CD8 ratio.

Key points

• •Limited data are available with regard to immune risk phenotype and mortality in oldest old patients, particularly those in the Mediterranean area.
• •A low percentage of subjects who were 85 years old at baseline had an inverted CD4:CD8 ratio.
• •The presence of an inverted CD4: CD8 ratio was not associated with mortality in oldest old subjects after three years of follow-up