Abstract

Key words : Immunologic monitoring, Lymphocyte count, Opportunistic infections, Renal transplantation, T-lymphocyte subsets

Introduction

The clinical application of potent immunosup-pressive treatment has improved solid-organ graft outcomes. However, this progress has come at a price. Long-term graft and patient survival rates have been affected as a result of impaired immune surveillance, which has increased the risk of infections and neoplasms in patients.¹

Transplant recipients can have serious morbidity and mortality from infectious complications.^2,3 As the North American Pediatric Renal Transplant Cooperative Study has reported, posttransplant infections currently exceed acute rejection as a cause of hospitalization.⁴ Sepsis has been shown to be a major cause of mortality during the first year posttransplant.⁵ Furthermore, viruses not only cause specific diseases but they can also boost the development of graft damage.² Moreover, the immune system plays a critical role in eradication of precancerous lesions. In fact, cancer constitutes one of the major causes of kidney transplant-associated long-term morbidity and mortality.⁶ There is an uncertain balance between overimmunosuppression and underimmunosup-pression. Clinical markers and drug levels are often inaccurate to assess immune status and do not predict complications.

Pediatric kidney transplant recipients (KTRs) usually receive a combination of immunosuppressive drugs that have different targets in the immune system. Anticalcineurin drugs and corticosteroid boluses act on humoral immunity by inhibiting CD4 Th2 T lymphocytes and their cytokines, which are needed for activation and expansion of B lymphocytes.

Mycophenolate mofetil inhibits the de novo synthesis of guanosine nucleotide. It has more potent cytostatic effects on lymphocytes than on other cells, since T and B lymphocytes are decisively dependent on the de novo synthesis of purines for their proliferation. On the other hand, mechanistic target of rapamycin (mTOR) inhibitors, such as sirolimus, act directly on memory B cells by inhibiting their differentiation into plasmatic cells.⁷ The mechanism of action of mTOR inhibitors is based on the inhibition of mTOR kinase activity by binding to the immunophilin FKBP12. This inhibition produced by the sirolimus/FKBP12 complex blocks the activation of protein synthesis and causes an arrest in the G1 phase of the cell cycle. Mycophenolic mofetil and mTOR inhibitors mainly cause a decrease in serum immunoglobulin G (IgG) levels. Therefore, hypogam-maglobulinemia is more frequent in KTRs than in healthy individuals.⁸ On the other hand, the CD4 and CD8 lymphocyte subpopulations in KTRs differ according to the immunosuppressive induction therapy administered.⁹

Quantification of serum immunoglobulin levels, complement fractions, and peripheral blood lymphocyte subpopulations (PBLS) are useful for immune monitoring.^9,10 Some studies have shown that hypogammaglobulinemia^7,11 and low CD4+ and CD8+ T-cell and natural killer cell counts could predict infection risk.^9,12,13 In addition, viral monitoring is useful to identify early stages of virus-related pathology.¹⁴ For this, biomarkers that could reliably assess immunosuppression levels and associated morbidity are needed.

Pediatric KTRs are a particularly vulnerable population within this group of patients. In fact, immunization is partial at younger ages, and they can be highly exposed to multiple pathogens through close contact with other children. To our knowledge, there are few publications that have analyzed secondary immunosuppressive status in pediatric KTRs. Application of strategies for immunological monitoring in KTRs would allow tailoring of immunosuppression and prophylaxis practices according to the patient’s current risk of infection.¹⁰

Therefore, the aim of this study was to describe secondary immune deficiencies related to immuno-suppressant treatment in pediatric KTRs.

Materials and Methods

Ethics approval
This retrospective study involving human participants received approval from the Clinical Research Ethics Committee of Cruces University Hospital (ID: E20/32) before the study began. The protocols conformed to the ethical guidelines of the 1975 Helsinki Declaration, and written informed consent was obtained from study patients or their guardians.

Study design
This retrospective observational and single-center study was conducted in a tertiary hospital (Cruces University Hospital), in cooperation with the Pediatric Nephrology and Immunology Departments. We included 28 pediatric KTRs who were transplanted between 2011 and 2019. We excluded patients with follow-up in other hospitals after transplant surgery and those over 18 years old. All donors were deceased.

We registered demographic (sex and age) and basic clinical variables (transplant date, etiology of kidney failure). We recorded current immunosup-pressive and prophylactic treatment, as well as hospital admissions due to opportunistic infections (OI) and graft rejections. We defined OI as those infections caused by an opportunistic pathogen or by a common pathogen causing unusually severe infections due to secondary immunosuppression.¹⁵

Laboratory tests and reference values
All blood tests were done between December 2019 and January 2020; therefore, time since transplant was variable. We measured immunosuppressive drug levels, viral loads, immunoglobulins, anti-pneumococcal antibodies, leukocytes, and PBLS to investigate immune status in KTRs.

We quantified total blood levels of tacrolimus in all patients. We established the therapeutic levels as between 10 and 12 ng/mL during the first year after transplant and between 5 and 10 ng/mL after the first year posttransplant.¹⁶

We measured serum cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV) viremias by polymerase chain reaction. We considered a negative result when there was no detectable viremia. Viral loads were measured periodically according to the department’s protocol in asymptomatic patients or when there was clinical suspicion of viral infection. All patients received CMV prophylaxis during the first 6 months after transplant, unless both the donor and recipient had negative CMV serology.

We quantified serum immunoglobulins (IgA, IgM, and IgG) by the nephelometric method. We used reference values from Bayram and associates, which considered a value below the limit of normality when it was below -2 SD for age.¹⁷

We also measured specific serum IgG antibodies against pneumococcal capsular polysaccharide by the enzyme-linked immunosorbent assay method. The kit that we used (VaccZyme) was designed to measure antibody responses to pneumococcal vaccines incorporating 23 polysaccharides isolated from Streptococcus pneumoniae. We considered an adequate postimmunization response when the result was higher than 80 mg/dL, based on previous results.¹⁸

T-lymphocyte subpopulations were measured by automated flow cytometer (AQUIOS CL; Beckman Coulter). Reference values for absolute counts and percent leukocytes and specific lymphocytic populations (CD3, CD4, CD8, and CD4/CD8 ratio) in healthy children were provided by Garcia-Prat and colleagues.¹⁹ We defined a normal range based on the median and the tenth and ninetieth percentiles according to a variable age range in a reference population similar to ours.

Statistical analyses
The statistical analysis was performed with IBM SPSS version 23. Categorical variables are reported as numbers and percentages and analyzed with the Fisher exact test. Continuous variables are reported as mean and standard deviation (mean ± SD) when normally distributed or as median and interquartile range (IQR) when nonnormally distributed. Variables were analyzed with t test for normal distributions or the Mann-Whitney U test for nonnormal distributions. P < .05 was considered statistically significant.

Results

Patient characteristics, immunosuppressants, and prophylactic treatments
We included 28 pediatric KTRs in our study. Table 1 lists information about the clinical characteristics of these patients as well as the immunosuppressive and prophylactic treatments that they received.

Viral infections
Isolated CMV, EBV, or BKV viremia was detected in 17 patients (60.7%) during posttransplant follow-up; 6 patients had 2 or more viruses detected.

Cytomegalovirus serology was positive in 14 recipients and 18 donors prior to transplant. There were only 2 cases where serology was negative in both the recipient and donor. Twenty-two patients (78.6%) received prophylaxis with valganciclovir for 5.95 ± 1 months. Fifteen patients (53.6%) showed CMV viremia during the first year posttransplant (5.77 ± 3.51 mo), which was beyond the prophylaxis period. Seven patients had primary infection, and 3 patients had CMV disease.

BK virus infection occurred in 6 patients (21.4%) at a mean time after transplant of 3.76 ± 3.31 months. Two of these patients developed BKV disease, 1 with acute kidney injury and 1 with hemorrhagic cystitis.

Epstein-Barr virus serology was positive in 17 recipients prior to transplant. After transplant, 5 patients (17.9%) had detectable EBV viremia, mainly after the first year posttransplant at a median time of 38.4 months (IQR, 11.5-56.3 mo).

Two patients had detectable CMV viremia at the time of the study, with 1 being associated with BKV. Both patients had lymphopenia (less than tenth percentile for age range) at time of diagnosis of CMV infection, showing 1260 cells/μL and 350 cells/μL, respectively.

Hospitalizations related to opportunistic infections
Sixteen patients (57.1%) had an OI that required admission, with a total of 30 hospitalizations. Table 2 details the microbiological results and clinical manifestations. These hospitalizations occurred at a median time from transplant of 1.02 years (IQR, 0.46-2.07 y). Most hospitalizations (73.3%) happened during the first 2 years.

Patients who were transplanted before 6 years of age were more likely to be hospitalized more than once due to OIs (odds ratio of 4.6; 95% CI, 2.1-9.9; P = .003). No significant difference was observed between male and female KTRs in the number of admissions.

Humoral and cellular immunity
Table 3 shows detailed results of the blood analyses. Patients with low total antipneumococcal antibody levels had lower IgG and IgM values compared with the rest of the sample (962.2 vs 1140.6 mg/dL [P = .08] and 58.5 vs 105.6 mg/dL [P = .02], respectively).

Most patients (85.7%) had been vaccinated with at least 1 dose of each type of pneumococcal vaccine, 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine. Regarding time from kidney transplant, patients transplanted less than 2 years previously had lower values of total antipneumococcal antibodies com-pared with the rest of the sample (53.9 ± 47.8 vs 132.9 ± 114.8 mg/L; P = .049). We found no significant differences in PBLS and immunoglobulin levels between patients transplanted less than 2 years previously and the remaining sample.

Patients with positive viral loads during follow-up also had significantly higher total IgG levels than those without (1227.1 ± 450.5 vs 850.8 ± 329.5 mg/dL; P = .04). We found no significant differences in laboratory results between those patients who required admission and those who did not (Table 4).

There were no significant differences in im-munoglobulin values, leukocyte counts, neutrophil counts, and PBLS counts between patients with 2 or 3 immunosuppressant drugs at the time of the study.

Three patients had graft rejection, diagnosed by kidney biopsy, during the study period. One patient developed chronic humoral rejection 6 years after transplant and was successfully treated with gammaglobulin and rituximab. The second patient developed acute cellular rejection 6 months after transplant and was successfully treated with methylprednisolone pulse therapy and intravenous thymoglobulin. The third patient had severe cellular and humoral rejection 2 weeks after transplant with no response to initial treatment (methylprednisolone pulse therapy and thymoglobulin) and graft loss 2 weeks later, despite treatment with plasmapheresis and intravenous gammaglobulin. None of the 3 patients had underlying infections.

Discussion

Pediatric patients are a particularly vulnerable population within KTRs. This study analyzed the prevalence and characteristics of secondary immune deficiencies related to immunosuppressant treatment in pediatric KTRs.

More than one-half of the patients in our sample were hospitalized for OI. The leading causes of admission were similar to those published previously.^2,3,20 Patients transplanted at younger ages and those transplanted less than 2 years previously were more likely to be admitted for OIs. This could be due to an immature immune system in younger patients and the high levels of immunosuppression required in the early years after transplant. One in 10 patients in our sample was admitted for sepsis; this rate is higher than that in a healthy pediatric popula-tion but similar to what has been described previously in pediatric KTRs.^13,21 Therefore, KTRs require a low threshold of suspicion regarding infections.

Infections related to CMV, EBV, and BKV were frequent during follow-up. Both patients in our sample who had positive viral loads for CMV when the study was performed presented with lymphopenia. In fact, the presence of lymphopenia, when treatment for CMV is completed, can be a strong and independent predictor of recurrent disease.⁴ Thus, the detection of lymphopenia could be useful for identifying patients at higher risk of reinfection and to provide them optimized management.

Secondary hypogammaglobulinemia was frequent in our sample. Some recent publications have highlighted the importance of recognizing secondary antibody deficiency since it is related to higher risk of infection.^11,22 Prevention and treatment strategies for secondary antibody deficiency include monitoring risk parameters, antibiotic strategies, immunoglob-ulin replacement therapy, and vaccination.^11,23

Although pneumococcal vaccination is highly recommended for KTRs, its effectiveness in this population remains largely unknown.^24-26 Although most patients were correctly vaccinated, almost half of them had low anti-pneumococcal antibody levels. In addition, patients who had recent transplant procedures had lower anti-pneumococcal antibody levels. For this reason, most publications have encouraged pretransplant correct immunization in order to maintain adequate posttransplant antibodies levels.²⁷ Furthermore, the response to the pneu-mococcal vaccine should be monitored because antibodies return to baseline over time.²⁸ Anti-pneumococcal antibody monitoring may be useful for tailoring empirical antibiotic treatment in patients with fever and low antibody levels. Nevertheless, in our sample, no pneumococcal infection was evidenced after transplant.

Lymphopenia was frequent in our sample. One-third of the patients had low PBLS for age. Although OIs have been linked to lower T-lymphocyte CD4 and CD8 absolute counts,^12,29 we did not find a significant relation between PBLS and the number of admissions for OI in our patient sample. This was probably because blood tests were not performed at the time of admission. In addition, the absolute counts of lymphocytes are variable over time and do not accurately predict the risk of infection. However, CD4 T-lymphocyte levels below 200 cells/μL imply the need for prophylactic antibiotics.¹²

Most patients had tacrolimus levels in the thera-peutic range. Nonetheless, response to immunozsup-pressant treatment was different in each patient. Immunologic status was not related to tacrolimus levels or corticosteroid treatment in our sample. Thus, immune monitoring might be helpful in combination with immunosuppressant levels to assess immunosuppression status. Moreover, we found no differences in PBLS between patients transplanted more than 2 years previously and those who had transplants within 2 years. This may justify the need to monitor immune status even after the first few years posttransplant.

This study had some limitations. Blood samples for PBLS and immunoglobulin testing were taken only once after transplant. Therefore, we do not have posttransplant follow-up or data on the baseline immune status of patients prior to transplant. Studies in bigger samples are needed to identify analytical examinations that might be more useful for immune monitoring. In addition, the association between immunological status and the risk of infection needs to be assessed.

Nonetheless, this study provided additional information about the importance of periodic immune monitoring in pediatric KTRs. Furthermore, the tests we have used for immune monitoring are inexpensive and available in most laboratories. We suggest that immunological status could be checked before transplant in order to adequately manage these patients. These may include monitoring of PBLS, immunoglobulin levels, and vaccine responses.

Conclusions

Pediatric KTRs require a low threshold of suspicion regarding infection and require accurate preventive measures. Immune monitoring in pediatric KTRs under immunosuppressant treatment could help to recognize overimmunosuppression. Additionally, it could lead as a guide to establishment of individu-alized preventive measures.

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