The trend of Epstein-Barr virus DNA loads and CD8⁺ T lymphocyte numbers can predict the prognosis of pediatric liver transplant recipients with PTLD

Introduction

Epstein-Barr virus (EBV) is a herpesvirus in which over 90 % of the population worldwide has been infected. It has a double-stranded, linear DNA genome enclosed by a protein capsid. The virus transmits primarily through saliva, and transmission via blood and droplets also occurs [1], [2], [3]. The incidence of EBV infection in children is increasing with each passing year, it is characterized by high fever, sore throat, lymphadenopathy, hepatitis, eyelid edema and other inflammatory symptoms accompanied by an increase of atypical lymphocytes (ALY) [4], 5]. Individuals respond differently to EBV infection due to different immune status [6]. Infectious mononucleosis (IM) is an acute infectious disease caused by EBV infection, which is a kind of self-limited disease in patients with normal immune function, and the virus will be cleaned in a few weeks. However, individuals with immunodeficiency may develop lymphoproliferative disorders and malignancies, the virus will be persistently positive for a long time, and it is difficult to be cleared. The development of post-transplantation lymphoproliferative disease (PTLD) is linked to a deficient cellular immune response due to immunosuppression administered to prevent graft rejection.

At present, the pathogenesis of EBV infection is not completely clear, but the host immune system exerts a key function in the recognition and elimination of EBV infection. Both innate and adaptive immune responses are initiated to clear the virus. The Toll-like receptors (TLRs) of immune cells are activated, triggering an interferon-mediated innate immune response. B lymphocytes express TLR7, dendritic cells express TLR9, and epithelial cells, as well as monocytes, express TLR2. EBV activates the TLRs of immune cells, mediating the expression of downstream factors, stimulating the proliferation of monocytes, macrophages, neutrophils, dendritic cells, and natural killer (NK) cells. At the same time, humoral and cellular immune responses in the adaptive immune response are activated. EBV primarily targets B lymphocytes and causes changes in the surface antigen of these cells, which in turn triggers CD3⁺ T lymphocytes’ defense response. CD3⁺CD4⁺ lymphocyte is a type of helper T lymphocyte, where the intracellular molecules are phosphorylated to activate the signal transduction process and assist in the activation of B lymphocytes, cytotoxic T cells, and natural killer cells by secreting cytokines. CD3⁺CD8⁺ T lymphocyte is a cytotoxic type of T-lymphocyte with a killing effect that directly destroys target cells or induces the apoptosis of target cells [7], 8]. Therefore, EBV infection will affect the absolute count and the percentage of peripheral lymphocyte subsets (PLS), and PLS may conversely influence the clearance of virus.

Trends of the PLS and EBV-DNA loads as well as their interaction in EBV-infected patients are still unknown. In this study, we performed dynamic monitoring of PLS and EBV-DNA loads in pediatric liver transplant recipients with PTLD and IM children, to investigate the effect of immune response on the elimination of EBV.

Materials and methods

In a period of 24 months (from January 2021 to December 2022), a total of 63 patients were enrolled in this mono-center retrospective study, including 17 pediatric liver transplant recipients with PTLD (PTLD group) and 46 patients diagnosed as IM (IM group). Based on the response to treatment, PTLD group were divided into PTLD – stable disease (PTLD – SD) group and PTLD – partial response (PTLD-PR) group.

The diagnostic criteria of PTLD followed the Clinical guidelines on Epstein-Barr virus infection in solid organ transplantation recipients and post-transplant lymphoproliferative disease (2019 edition) [9]. The criteria of PTLD-SD group was that after treatment, the symptoms of PTLD disappeared, and the EBV loads in PBMC declined below the detection limit. The concept of PTLD-PR group was that after treatment, the symptoms of PTLD alleviated. The EBV loads decreased, but they have not declined below the detection limit. The IM was diagnosed following Experts’ consensus on diagnosis and treatment of Epstein-Barr virus infection-related diseases in children [10], 11].

According to our Internal Review Board guidelines, all participants provided full informed consent to participate in the study.

Blood samples for examination of PLS and EBV-DNA loads were collected right after the diagnosis and subsequent management of EBV infection. Blood samples were taken on the 1st, 7th, 14th, 21st, 28th, 35th, 42nd, 60th, 90th, 180th, 270th, and 360th day for PTLD group as well as on the 1st day for IM group.

Results

Peripheral lymphocyte subsets in the IM group, PTLD-SD group and PTLD-PR group

As can be seen from Tables 2-1, 2-2 and Figure 1, there were significant differences in CD3⁺, CD3⁺CD8⁺, CD16⁺CD56⁺ lymphocyte numbers and CD4⁺/CD8⁺ ratio among the IM group, PTLD-SD group and PTLD-PR group, p<0.05. The CD3⁺CD8⁺ lymphocyte numbers in the IM group and PTLD-SD group were obviously higher than that in the PTLD-PR group, which led to the increased number of CD3⁺ lymphocytes and the decreased CD4⁺/CD8⁺ ratio, all p<0.05. What is more, CD3⁺CD8⁺ lymphocyte numbers in the IM group were remarkably higher than that in the PTLD-SD group, p<0.05. Besides, the number of CD16⁺CD56⁺ lymphocytes in the IM group is higher than that in the PTLD group.

Table 2-1:

Peripheral lymphocyte subsets among the IM group, PTLD-SD group and PTLD-PR group.

Number of lymphocytes, ×109/L	IM group	PTLD-SD group	PTLD-PR group	F-Value	p-Value
CD3+	6.62 ± 3.92	4.17 ± 1.00	2.11 ± 0.57	19.676	0.000a
CD3+CD4+	1.40 ± 0.71	1.59 ± 0.48	1.19 ± 0.36	2.799	0.066
CD3+CD8+	4.71 ± 2.93	2.48 ± 0.81	0.86 ± 0.24	26.223	0.000a
CD4+/CD8+	0.35 ± 0.19	0.69 ± 0.24	1.42 ± 0.37	129.148	0.000a
CD16+CD56+	0.93 ± 0.52	0.58 ± 0.26	0.49 ± 0.24	10.724	0.000a
CD3−CD19+	0.30 ± 0.19	0.37 ± 0.17	0.30 ± 0.14	1.4	0.252

1. IM, infectious mononucleosis; PTLD-SD, post-transplantation lymphoproliferative disease-stable disease; PTLD-PR, post-transplantation lymphoproliferative disease-partial response. ^ap<0.01.

Table 2-2:

Peripheral lymphocyte subsets between two groups.

LSD-t, p	IM group vs. PTLD-PR group	PTLD-SD group vs. PTLD-PR group	IM group vs. PTLD-SD group
CD3+	4.50, 0.000b	2.06, 0.016a	2.44, 0.001b
CD3+CD4+	0.22, 0.146	0.41, 0.02a	0.19, 0.21
CD3+CD8+	3.85, 0.000b	1.63, 0.011a	2.22, 0.000b
CD4+/CD8+	−1.06, 0.000b	−0.73, 0.000b	−0.33, 0.000b
CD16+CD56+	0.44, 0.000b	0.10, 0.443	0.35, 0.001b
CD3−CD19+	0.002, 0.962	0.07, 0.173	−0.07, 0.122

1. IM, infectious mononucleosis; PTLD-SD, post-transplantation lymphoproliferative disease-stable disease; PTLD-PR, post-transplantation lymphoproliferative disease-partial response. ^ap<0.05, ^bp<0.01.

Figure 1:

Peripheral lymphocyte subsets in the IM group, PTLD-SD group and PTLD-PR group. *p<0.05, **p<0.01. IM, infectious mononucleosis; PTLD-SD, post-transplantation lymphoproliferative disease-stable disease; PTLD-PR, post-transplantation lymphoproliferative disease-partial response.

The trend of EBV-DNA loads and the percentage of CD3⁺CD8⁺ T lymphocytes among pediatric liver transplant recipients in the PTLD-SD group and PTLD-PR group

As can be seen from Table 3 and Figure 2, the trend of EBV-DNA loads and the percentage of CD3⁺CD8⁺ T lymphocytes in patients with stable disease differed from those with partial response. In the PTLD-SD group, the percentage of CD3⁺CD8⁺ T lymphocytes increased beyond the normal range with the ascending of EBV-DNA loads. After a period (usually ranging from 42 to 360 days), the percentage of CD3⁺CD8⁺ T lymphocytes decreased to the normal range accompanied by the clearance of EBV. However, in the PTLD-PR group, the percentage of CD3⁺CD8⁺ T lymphocytes kept in the normal range, which was too low to clear the virus. Therefore, the EBV kept on replicating. As for patient No. 17, both the EBV-DNA loads and the percentage of CD3⁺CD8⁺ T lymphocytes kept on replicating at a high level due to hemophagocytic syndrome.

Table 3:

The EBV-DNA loads and percentage of CD3⁺CD8⁺ T lymphocytes among pediatric liver transplant recipients in PTLD-SD group and PTLD-PR group.

	PTLD-SD group		PTLD-PR group
	EBV-DNA loads (copies/mL, Log10)	Percentage of CD3+CD8+ T lymphocytes, %	EBV-DNA loads (copies/mL, Log10)	Percentage of CD3+CD8+ T lymphocytes, %
1st day	6.02 [4.23, 6.48]	0.42 ± 0.12	3.76 [3.63, 4.90]	0.32 ± 0.07
7th day	5.15 [4.31, 7.17]	0.42 ± 0.10	4.58 [4.52, 4.74]	0.33 ± 0.06
21st day	5.84 [5.48, 6.10]	0.46 ± 0.09	4.90 [4.76, 5.64]	0.34 ± 0.06
28th day	5.51 [4.96, 5.92]	0.49 ± 0.09	4.58 [4.46, 5.61]	0.34 ± 0.06
35th day	4.41 [3.48, 5.31]	0.50 ± 0.06	5.39 [4.53, 6.26]	0.33 ± 0.07
42nd day	3.85 [1.52, 5.31]	0.51 ± 0.06	5.03 [4.48, 5.95]	0.30 ± 0.03
60th day	3.01 [0, 5.29]	0.49 ± 0.06	4.79 [4.50, 5.60]	0.30 ± 0.04
90th day	0 [0, 4.67]	0.48 ± 0.09	4.44 [4.32, 4.74]	0.29 ± 0.04
180th day	0 [0, 1.78]	0.43 ± 0.07	4.26 [4.22, 4.44]	0.28 ± 0.04
270th day	0 [0, 1.46]	0.38 ± 0.06	3.6 [3.03, 4.53]	0.28 ± 0.04
360th day	0 [0, 0]	0.36 ± 0.04	3.98 [3.19, 4.38]	0.29 ± 0.04

1. IM, infectious mononucleosis; PTLD-SD, post-transplantation lymphoproliferative disease-stable disease; PTLD-PR, post-transplantation lymphoproliferative disease-partial response.

Figure 2:

The trends of EBV-DNA loads and the percentage of CD3⁺CD8⁺ T lymphocytes among patients in PTLD-SD group and PTLD-PR group. (A and B) Show the trend of EBV-DNA loads and the percentage of CD3⁺CD8⁺ T lymphocytes of patients in PTLD-SD group. (C and D) Show the trend of EBV-DNA loads and the percentage of CD3⁺CD8⁺ T lymphocytes of patients in PTLD-PR group.

Discussion

Liver transplantation has become a standard procedure for children with end-stage liver disease, but immunosuppressive drugs have serious side effects in both short and long perspective. Complications caused by EBV infection are frequent, which show as a disease spectrum ranging from asymptomatic EBV infection to PTLD, and sometimes develop into malignant lymphomas with high mortality. Successful transplantation of liver transplantation requires effective control of EBV infection. Previous studies have given evidence that molecular EBV monitoring is effective in finding patients with a high risk of developing PTLD. However, it has become also evident that molecular EBV monitoring alone is not sufficient to achieve complete prediction and hence prevention of PTLD [12]. Some researchers propose that monitoring of humoral immune parameters, including serum immunoglobulin concentrations and mono-/oligoclonal gammopathy, in combination with EBV monitoring, is valuable in identifying high-risk patients [13], 14].

EBV infection will bring a series of pathophysiological changes, such as activation of immune cells, secretion of cytokines, production of EBV-specific antibodies, and even tissue damage and inflammation. EBV initially attacks B lymphocytes, the viral envelope glycoprotein 350 (GP350) binds to the B lymphocyte membrane receptor CD21 for latent infection and then promotes EBV replication, which triggers CD3⁺ T lymphocytes’ defense response [15], 16]. Based on the antigen-dependent and cytokine-dependent manner, CD3⁺CD4⁺ lymphocytes differentiate into functional helper T cell subset, which plays a key role in initiating and strengthening immune response to promote humoral and innate immune response. Besides, it can regulate the biological activity of other immune cells in inflammation and infection. CD3⁺CD8⁺ T lymphocyte is a cytotoxic subtype of T cell, which can directly destroy target cells or induce the apoptosis of target cells by secreting perforin interferon-gamma, as well as granzyme. Some studies have indicated that, in IM patients, CD3⁺CD4⁺ lymphocytes were highly activated, which could produce functional cytokines and help CD3⁺CD8⁺ T cells to recognize and kill EBV-infected cells [17], 18].

In the present study, the expression of lymphocyte markers was analyzed in pediatric liver transplant recipients with PTLD. The absolute number of CD3⁺CD8⁺ T lymphocytes in the PTLD-SD group was higher than that in the PTLD-PR group, whereas lower than that of IM children, which is in accordance with previous studies [19]. As can be seen from the two patterns of the trends between EBV-DNA loads and the percentage of CD3⁺CD8⁺ T lymphocytes, if the CD3⁺CD8⁺ T lymphocytes increased beyond the normal range following the infection of EBV, the virus would be cleared in a few months. However, the depletion of CD8⁺ T cells results in increased viral load and persistent viremia in pediatric liver transplant recipients might reflect a weak EBV-specific immune response resulting from a poor induction of EBV-specific CD3⁺CD8⁺ lymphocytes when primary EBV infection is acquired during immunosuppressive therapy. Therefore, immunomodulation is more important than antivirus therapy in immunodeficiency patients [20].

The increase of CD3⁺CD8⁺ T lymphocyte numbers as well as the decrease in CD4/CD8 ratio was considered to reflect the recovery of T lymphocyte functions because these changes occurred in parallel with the decline in EBV-DNA loads. CD3⁺CD8⁺ T lymphocytes are the most useful index in estimating the host capacity of immuno-surveillance against EBV because its increase was more prominent in those patients whose EBV-DNA loads decreased to an undetectable level than whose EBV-DNA loads retained high viral load.

Our limitation lies in the fact that test blood samples were obtained after the initiation of management. Prospective serial monitoring of PLS from the time of diagnosis through the course of management in a large cohort of children is necessary to confirm these observations. Despite this limitation, we believe that the results are important, this is the first observation between EBV-DNA loads and PLS in the long-term EBV viremia of children with different immunity. In conclusion, PLS, the easily available laboratory test, may be used as a potential surrogate measure of disease progression and treatment effects.