Association of T Cell Subsets and Platelet/Lymphocyte Ratio with Long-Term Complications in Kidney Transplant Recipients

Introduction

In light of the continuous improvement of the technical and perioperative management of kidney transplantation and the application of new immunosuppressants, kidney transplantation has become the best kidney replacement therapy for patients with end-stage renal disease, and has greatly improved the survival of patients with uremia [1]. However, infection and chronic rejection remain major issues for kidney transplant recipients (KTRs) and nephrologists. At present, most studies still focus on early infection and acute rejection in the perioperative period of kidney transplantation [2–4], and few studies exist on long-term complications of KTRs, such as predictors of long-term infection or chronic renal failure. As markers of circulating immune system components, T cell subsets can reflect the immune status of KTRs. A large number of studies have shown that the levels of CD4+ T cells and CD8+ T cells play important roles in infection and acute rejection in the perioperative period of kidney transplantation. This period has always been characterized by difficulty, and is a hotspot of organ transplantation research [5–8]. The derivative indexes platelet/lymphocyte ratio (PLR) and neutrophil/lymphocyte ratio (NLR) in the peripheral blood cell count, which can reflect the immune-inflammatory state of patients with chronic kidney disease, have been reported in multiple studies [9–14], but have been reported less often as markers in long-term chronic renal insufficiency in KTRs.

Therefore, the purpose of the present study was to investigate the relationship between T cell subsets and PLR in long-term infection and chronic renal insufficiency in KTRs. By providing new clinical evidence that could be used for the prevention and treatment of long-term complications in KTRs, the lifespan of a transplanted kidney could be extended, improving the quality of life for the patients.

Material and Methods

STUDY POPULATION:

The clinical data for kidney transplantation patients who were hospitalized in Huazhong University of Science and Technology Union Shenzhen Hospital (Nanshan Hospital) from June 2014 to December 2021 for more than half a year were analyzed retrospectively. The glomerular filtration rate (GFR) was calculated from the formula provided by the C16 000 biochemical instrument (Abbott, USA). The patients were divided into an infected group and a non-infected group, according to whether they were diagnosed with an infection during hospitalization. In the present study, long-term infection refers to acute infections diagnosed through medical records, clinical evaluations, and antibiotic prescriptions, excluding chronic infections such as hepatitis B, hepatitis C, and tuberculosis. Kidney insufficiency is defined as chronic kidney disease (CKD) stage 3 that is evident 6 months after transplantation, regardless of renal imaging changes. Inclusion criteria were: 1) patients who received kidney transplantation over 6 months and for whom the concentration of anti-rejection drugs was stable and within the standard range [15]; 2) adults (over 18 years old) with complete clinical data. Exclusion criteria were: patients with malignant tumors, severe hematological diseases, severe immune deficiency diseases, severe liver diseases, thyroid diseases, major trauma or surgery, and patients who underwent dialysis again after kidney transplantation. All the patients signed informed consent forms. This study has been approved by the hospital ethics committee [Application ID: KY-2021-066-01].

DETECTION INDEXES AND METHODS: COLLECTION OF CLINICAL DATA:

By querying the KTRs who met the standards and were hospitalized in our hospital from June 2014 to December 2021, the general clinical data and laboratory test indexes were obtained. These included sex, age, time of kidney transplantation, NLR, PLR, hemoglobin (HB), CRP, erythrocyte sedimentation rate (ESR), uric acid, creatinine, GFR, serum albumin (ALB), CD4+ T cells, CD8+ T cells, and CD4+/CD8+ ratio.

DETECTION INDEXES AND METHODS: DETECTION OF SERUM INDEXES:

Upon admittance, a complete blood count, which included evaluation of leukocytes, neutrophils, hemoglobin, lymphocytes, and platelets, and biochemical indexes such as creatinine, uric acid, GFR, and ALB were detected using venous blood. The NLR was calculated as the ratio of neutrophils to lymphocytes and the PLR was calculated as the ratio of platelets to lymphocytes. The percentage of T cell subsets such as CD4+ T cells and CD8+ T cells and the ratio of CD4+ T cells to CD8+ T cells were detected by flow cytometry.

On the day of admission, 3 mL of venous blood was taken from each patient. The supernatant was tested after centrifugation. Measurement of neutrophils, platelets, and lymphocytes was conducted with a fully automated blood cell analyzer [Heizenmekang XN9000, Japan], and the NLR and PLR ratios were calculated. CRP concentration in the serum was determined by a specific protein analyzer [CSC Bio Aristo, China]. An automatic chemiluminescence detector [New industry MAGLUMI 4000 PLus, China] was used to determine the procalcitonin concentration level. An automatic biochemical analyzer [Abbott C16 000, USA] was used to detect renal liver function and biochemical indexes.

Detection Indexes and Methods: Stages of Chronic Kidney Disease [16–17]

The estimated glomerular filtration rate (eGFR) was computed utilizing the creatinine equation from the Chronic Kidney Disease Epidemiology Collaboration. The patients were classified into 5 stages of CKD based on their eGFR: CKD stage1, GFR ≥90 mL/min; CKD stage 2, 90 mL/min >GFR ≥60 mL/min; CKD stage 3, 60 mL/min >GFR ≥30 mL/min; CKD stage 4, 30 mL/min >GFR ≥150 mL/min, CKD stage 5, GFR<15 mL/min.

STATISTICAL ANALYSIS:

SPSS 20.0 software (Chicago, USA) was used for statistical analysis. The normal measurement data were expressed by mean±standard deviation (χ̄±s), using an independent sample test, and skewed data were expressed as median and quartile, and analyzed by rank U test. Quantitative data were expressed as the number of cases and percentage, and were compared using a χ² test. Spearman correlation analysis was conducted for the correlation of circulating immune-inflammatory indexes to long-term infection and CKD in KTRs. Survival analysis and survival curves were used for long-term complications in KTRs. P<0.05 was considered as statistically significant.

Results

COMPARISON OF GENERAL CLINICAL DATA FOR KTRS:

A total of 271 hospitalized KTRs were included in this study, of which 164 KTRs had long-term infection (60.52%). There were 94 male patients (57.32%), which was a significantly higher number than female patients (P=0.015). However, no statistically significant difference was detected for age, kidney transplantation time, hemoglobin, ALB, serum creatinine, and GFR between the 2 groups (P>0.05). Demographic data for the 2 studied groups are shown in Table 1.

COMPARISON OF CIRCULATING IMMUNE-INFLAMMATORY INDEXES BETWEEN THE 2 KTR GROUPS:

Univariate analysis showed that there were significant differences in general immune-inflammatory indexes of CRP, ESR, procalcitonin, NLR, PLR, CD4+, CD8+, and CD4+/CD8+ ratio between the 2 groups (all P<0.05). The levels of CRP, ESR, procalcitonin, NLR, PLR, and CD8+ cell count were higher in the infected group than in the non-infected group. However, the CD4+ count and the CD4+/CD8+ ratio were lower in the infected group than in the non-infected group. The results of the laboratory tests are shown in Table 2.

Spearman Correlation Analysis of Circulating Immune-Inflammatory Indexes and Long-Term Infection and Chronic Kidney Insufficiency in KTRs

CRP, procalcitonin, ESR, NLR, and PLR were positively correlated with long-term infection in KTRs (P < 0.05), while blood uric acid and CD4+/CD8+ ratio were negatively correlated with long-term infection in KTRs (P<0.05). The results also showed that long-term renal insufficiency was positively correlated with ESR, uric acid, and PLR, and negatively correlated with CRP and CD4+/CD8+ ratio (P<0.05). PLR was positively correlated with CRP, procalcitonin, ESR, and NLR; and negatively correlated with CD4+/CD8+ ratio (P<0.05). CD4+/CD8+ ratio was negatively correlated with CRP, NLR, and PLR (P<0.05). All of these results are depicted in Table 3.

ADJUSTED COX PROPORTIONAL HAZARDS MODEL OF LONG-TERM INFECTION AND CHRONIC KIDNEY INSUFFICIENCY IN KTRS:

Predictive factors for long-term infection and chronic kidney insufficiency were evaluated using a Cox proportional hazards model, and the results are shown in Tables 4 and 5. Predictive factors associated with both long-term infection and chronic kidney insufficiency risk in KTRs included age, PLR, and CD4+/CD8+ ratio (all P<0.05).

Kaplan-Meier Survival Analysis and Survival Curves Analyzing Associations Between CD4+/CD8+ Ratio, PLR, Long-Term Infection, and Chronic Kidney Insufficiency in KTRs

Kaplan-Meier survival curves estimating the risks of long-term infection and chronic kidney insufficiency in kidney transplant recipients are presented in Figures 1–4. The curves are stratified by median values of the CD4+/CD8+ ratio and PLR groups. Notably, the higher CD4+/CD8+ ratio and lower PLR groups demonstrated a significantly increased cumulative risk of long-term infection (all P<0.05).

Discussion

Due to the implantation of grafts and the use of immunosuppressants, even a half year after receiving renal transplantation and successfully transitioning to the stable stage, KTRs are still at risk of immune state imbalance [18]. At the same time, long-term infection and chronic rejection are the main long-term complications for KTRs [19–21]. Our retrospective observational study aimed to evaluate the role and clinical significance of CD4+/CD8+ ratio and PLR in the process of immune-inflammatory activation in KTRs.

T lymphocytes, including CD4+ T cells and CD8+ T cells, mainly exist in peripheral lymphoid tissue and blood. The ratio of the 2 cell types is about 2: 1, and CD4+ cells are integral in regulating the immune state in the human body [22]. Because immunosuppressants are used after kidney transplantation to prevent rejection, the occurrence of infection caused by bacteria, viruses, and eukaryotic pathogens after transplantation is greatly increased. This is often accompanied by changes in the levels of lymphocyte subsets, and these changes can be valuable for evaluating prognosis in KTRs [7,23,24]. In general, the CD4+/CD8+ T cell ratio contains information on CD4+ T cells and CD8+ T cells. Our study indicated that the ratio of CD4+/CD8+ T cells was negatively correlated with long-term infection and chronic kidney insufficiency in KTRs, and this correlation was statistically significant (respectively, r=−0.451, P<0.05; r=−0.392, P<0.05).

As markers of new circulating blood cells, PLR and NLR include a variety of blood cell information, such as platelets, lymphocytes, and neutrophils. They have important clinical value in evaluating infection and inflammatory response. At the same time, they have a certain predictive value for the loss of kidney function, particularly in CKD patients treated with non-renal replacement therapy [10,12]. Our study showed that NLR had a strong correlation with long-term infection in KTRs (r=0.740, P<0.05), which was consistent with previous studies, but NLR had no significant correlation with CKD progression in KTRs (P>0.05), which may be due to the negative effect of long-term use of immunosuppressive agents on leukocytes in KTRs. However, in our study, we also found that PLR was positively correlated with long-term infection and CKD in KTRs (respectively, r=0.357, P<0.05; r=0.249, P<0.05).

In summary, it can be seen that CD4+/CD8+ ratio and PLR are related to long-term infection events and chronic decline of renal function after kidney transplantation. The present study also used survival analysis and survival curves to demonstrate that PLR is strongly correlated with long-term infection in KTRs (P<0.05), compared with the index of CRP. Compared with serum uric acid, the ratio of CD4+/CD8+ cells was negatively correlated with the long-term progression of CKD after kidney transplantation (P<0.05).

Conclusions

Our study demonstrated that CD4+/CD8+ ratio and PLR were important risk factors for long-term infection in KTRs. They were also related to the progress of CKD in KTRs. Finally, they were also important for the prediction of long-term immune status in KTRs. Therefore, this study could have clinical application value in preventing long-term complications, and thereby improving the lifespan of transplanted kidneys and delaying the decline of transplanted kidney function. It also provided a certain reference value for the monitoring of immune status and long-term health education in KTRs.

There are various forms of long-term complications for KTRs, such as chronic kidney transplant failure and immune disorders caused by the use of immunosuppressants, including chronic rejection after kidney transplantation, chronic inflammatory infections, tumors, hematopoietic dysfunction, and others. In the follow-up to this study, multiple patients with tumors and polycythemia have been found. However, more research about the long-term complications in KTRs would be beneficial for patients and society.