Chen et al. BMC Pediatrics(2024) 24:587
https://doi.org/10.1186/s12887-024-05051-z

BMC Pediatrics

CASE REPORTOpen Access

Changes of CD8+CD28- Tregs and GammaDelta-T-cells in a Neonate with Intrauterine
Cytomegalovirus Infection: A Case Report
Xian Chen1†, Zhenchao Jin2†, Ping Zhou2, Tingyan Xie3, Fan Jiang1 and Quan Tang3*
Abstract
Background Congenital cytomegalovirus (cCMV) infection can lead to a range of adverse outcomes. The majority ofcCMV neonates with clinical symptoms are infected postnatally; however, established cases of intrauterine infection
are uncommon, resulting in a paucity of reports on clinical findings and lymphocytes expression in CMV-infectedneonates.
Case presentation We followed a neonate with cCMV infection from the onset of hospitalization to several monthsof follow-up. This infant was intrauterine CMV-positive in the amniotic fluid of the mother at 21 weeks’ gestation
and received intravenous ganciclovir infusion and sequential oral valganciclovir after birth. The typical clinical signs
manifested in the nervous system, liver, and peripheral blood and were documented during the hospitalizaion period
and up to the follow-up visit. Flow cytometry was employed to examine the expression of T cells, their subsets, andthe associated cytokines in peripheral blood samples at various time points. The flow data for the cCMV neonate werecompared with those of the controls at each time point. Following treatment, clinical symptoms improved and theinfant became CMV negative. However, developmental delays occurred later in life. The proportion of CD8+CD28−
Tregs in the peripheral blood of the neonate with congenital CMV infection was higher than that in the controls at
the three time points. The expression levels of perforin and granzyme B secreted by γδ T cells (Vδ1 and Vδ2 T cells),
increased during the course of hospitalization until follow-up and were higher than those in the controls at the threetime points.
Conclusions Despite the alleviation of clinical symptoms, developmental delay in later life remains inevitable in thisintrauterine cCMV neonate. CD8+CD28− Tregs and Vδ1 and Vδ2 T cells secreting perforin and granzyme B may be
involved in congenital CMV infection, although this hypothesis requires validation in a larger study. This report may
contribute to our understanding of the effect of current treatment and the immune status of intrauterine cCMVinfected neonates.
Keywords Congenital CMV infection, Neonate, CD8+CD28− Tregs, Γδ T cells, Vδ1 and Vδ2 T cells†

Xian Chen and Zhenchao Jin contributed equally to this work.

Xian Chen and Zhenchao Jin are co-first authors.
*Correspondence:
Quan Tangtangquan1987@126.com

1

Department of Laboratory Medicine, Shenzhen Baoan Women’s andChildren’s Hospital, Shenzhen, China
2
Department of Neonatology, Shenzhen Baoan Women’s and Children’s
Hospital, Shenzhen, China
3
Research Laboratory, Shenzhen Baoan Women’s and Children’s Hospital,
Shenzhen, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Chen et al. BMC Pediatrics(2024) 24:587

Background
Congenital cytomegalovirus (cCMV) infection can resultin a range of adverse outcomes, including stillbirth, neonatal death, neurological deafness, neurodevelopmental
disorders, and liver complications [1]. It is estimated thatbetween 35 and 40% of pregnant women with a primary
CMV infection transmit the virus to their fetuses, withapproximately 10% of infected fetuses exhibiting symptoms at birth. Furthermore, while some newborns may
not display overt ymptoms of CMV infection, they may
still develop neural deafness caused by CMV infection
later in life [2].
Studies in different regions of China revealed a prevalence of cCMV between 0.23 and 0.7%, with 10 to 15%
of cases resulting in symptomatic births [3, 4]. Cases
presenting with multiple-organ damage at birth are lesscommon, and cases with severe neurological lesions areeven rarer. A review of the cases at our hospital over the
past three years reveals that there were 60,000 deliveries,
with nearly 10,000 cases discharged from the neonatal
department. However, only four cases were diagnosed
with multisystem damage due to cCMV and administered antiviral drugs, of which two cases exhibited neurological lesions.
This neonate represents a rare case due to the presenceof multi organ dysfunction (liver, haematological system,
and lungs) and neurological lesions at birth. Secondly, theneonate was confirmed to have an intrauterine congenital infection with a clear route of intrauterine transmission. This was evidenced by the positive quantification of
CMV DNA in the maternal amniotic fluid at mid-pregnancy and in the neonate’s blood and urine. However,it was not possible to ascertain with certainty whether
the other cases were perinatal or postpartum infections.
Furthermore, the neonate was followed from the outsetof the hospitalization until the follow-up visit. The clinical findings and laboratory tests at different time points
were recorded and analyzed. Additionally, it proved challenging to obtain fresh peripheral blood samples fromthe neonate at different time points and corresponding
controls for flow cytometry. The timeline is presented inFig. 1A.
Case presentation
Patient and treatment

The infant was delivered spontaneously at home on theevening of 23 April 2022, at a gestational age of 35 + 6
weeks. Consequently, the amniotic fluid, umbilical cord,
placenta, and Apgar scores were unavailable at the timeof birth. The infant was observed to cry immediately
after birth, as described by the father. The infant was
subsequently transferred to the local hospital one hour
after birth by an emergency medical service. The infant’s
weight and length were recorded as 1570 g and 41 cm,

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respectively. The initial blood test of the neonate at the
local hospital demonstrated a diminished platelet count(59*10^9/L), elevated direct bilirubin (33.8 µmol/L) andtotal bilirubin (89.8 µmol/L), a normal leukocyte count,
C-reactive protein (CRP), and alanine aminotransferase(ALT) levels. On day 5, four days later, the platelet countdecreased to 44*10^9/L, while the direct bilirubin level
increased to 78.7 µmol/L and the total bilirubin reached129.7 µmol/L. The initial quantification of CMV DNA in
urine was 1.41e + 7 copies/mL (see Supplementary Table
1). Due to the limited resources available at the local hospital, the infant was transferred to our tertiary neonatal
intensive care unit (NICU) on 29 April 2022 (day 6) toreceive more specialized care. The neonate was initiallydiagnosed with congenital CMV infection, preterm low
birth weight, small for gestational age, thrombocytopenia, and neonatal cholestasis. In accordance with theestablished guidelines, ganciclovir (6 mg/kg, q12 h) was
administered intravenously from day 7 until discharge,
and oral ursodeoxycholic acid (10 mg/kg/day) was initiated on day 9 (Fig. 1A). The patient was treated withintravenous ganciclovir for 3 weeks in our hospital, andthen switched to oral valganciclovir for a further 3 weeks(there was no oral valganciclovir in our hospital, and theneonate was taken to the other hospital for valganciclovir
and re-evaluated). Following a 6-week course of antiviraltreatment, the neonate was CMV negative. The physician
at the hosptital providing valganciclovir decided to discontinue valganciclovir (the treatment at the other hospital was described by the family members, more specificreasons unknown).
Diagnosis

The obstetric examination of the mother of the newborn
suggested a high risk of trisomy 21 with intrauterine
growth retardation. The antenatal ultrasound examination revealed a significantly reduced fetal head circumference in comparison to the gestational age. Additionally,
the fetal head exhibited sonographic alterations, and thepossibility of intracranial infection could not be excluded.
Amniocentesis was performed at 21 weeks of gestation in
our hospital. The results of the karyotype and microarray analysis were negative. However, the concentrationof CMV nucleic acid in the amniotic fluid was markedly
elevated at 2.76e + 7 copies/mL. The diagnosis of congenital CMV infection was established, and the routeof intrauterine transmission was identified. The infant
exhibited typical multi-organ dysfunction (central nervous system, liver, haematological system, and lungs)
at birth, with positive quantification of CMV DNA inmaternal amniotic fluid of the mother at mid-pregnancy
and in neonatal blood and urine. Polymerase chain reaction (PCR) was employed to examine the cerebrospinal
fluid, serum, and urine for the presence of CMV nucleic

Chen et al. BMC Pediatrics(2024) 24:587

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Fig. 1 Timeline of this study and clinical manifestations of the cCMV neonate. (A) The long arrow represents the timeline of peripheral blood sampling
for flow cytometry and treatments. (B) Calcifications in the brain. Calcifications scattered in bilateral cerebral hemispheres (left) and lateral paraventricular(right). White dots indicate calcifications. (C) The viral load of CMV was determined in urine, serum, and cerebrospinal fluid (CSF) at various time points.
The cut-off is indicated by the dashed line; a value below 500 indicates a negative CMV stauts. (D) DBIL (direct bilirubin) was tested in serum at different time points. The dashed line indicates the normal level of DBIL, and the value higher than 17.1 indicates pathological elevation. (E) Platelet count inperipheral blood was tested at different time points. The dashed lines incidate the reference range (183–614), and the value lower than 183 indicatesthrombocytopenia

acid, in acccordance with the instructions provided by
the manufacturer [5] (Human Cytomegalovirus Nucleic
Acid Quantification Test kit, DAAN GENE, Guangzhou,
China).
Clinical findings

Typical calcifications were observed in both cerebral
hemispheres, the lateral ventricles, and the right cerebellar hemisphere, with CT values ranging from 125 to 230
HU. The bilateral ventricles exhibited asymmetry andslight dilation, while the bilateral anterior ventricularangle distance was widened (Fig. 1B). On the third day ofadmission, the infant exhibited dyspnoea and pulmonary
rales, leading to a diagnosis of CMV-associated pneumonia. The infant was treated with nCPAP-assisted ventilation for one day and high-flow oxygen for five days. The
levels of CMV DNA in serum and urine were quantified
by PCR at various time points, demonstrating a declining trend throughout the course of antiviral therapy. The
patient’s cerebrospinal fluid was CMV negative (Fig. 1C).
The direct bilirubin level exhibited a decline throughout the course of therapy (Fig. 1D). The platelet countChen et al. BMC Pediatrics(2024) 24:587

demonstrated an increase from the time of admission to
the follow-up assessment on day 46 (Fig. 1E). The trends
in key blood counts and significant liver function tests
are illustrated in Supplementary Figs. 1 and 2. The brainstem auditory evoked potentials were within the normal
range. Following a 26-day hospitalization period, thepatient was discharged and continued to receive valganciclovir and ursodeoxycholic acid.
Subsequent to discharge, a series of assessments wereconducted at our institution and at the hospital where theoral medications were dispensed. These included evaluations of the patient’s blood count, liver function, regularvirological nucleic acid monitoring, and neurodevelopmental follow-up. The neonate exhibited the following
clinical manifestations: (1) The imaging results revealed
multiple cerebellar gyrus, giant gyrus, and abnormal
white matter signalling, as well as post-cerebellar injury
changes and bilateral ventricular dilatation. These findings were observed through nuclear magnetic resonanceimaging (MRI). (2) The infant exhibited mild hearing
abnormalities, an abnormal electroencephalogram (EEG),
and microcephaly. Following a 20-day course of treatment, the CMV test yielded a negative result, prompting
the discontinuation of valganciclovir. At six and sevenand a half months of age, the neonate was admitted to the
paediatric rehabilitation unit of our hospital for a periodof six weeks, during which time he was diagnosed with“brain injury syndrome”. Eight months after birth, theneonate was evaluated as an inpatient in the paediatricrehabilitation unit, and the following observations were
made: (1) Gross motor: head stable, can raise the headabout 90º with elbow support; no delay in pulling up; canroll from supine to lateral position, but can’t complete therollover; when no hand support in prone position, can sit
with both hands to support leaning for a few moments
and lift the armpit can support part of the body weight;
pointy feet. (2) Fine motor: both hands clench fists, have
hand-mouth movement, can hold hands together, canhold flower bell stick, cannot grasp objects; (3) Speech:
can pronounce single rhyming sounds; can laugh, but is
not very responsive to sound; does not vocalise to people
or objects; (4) Social adaptation: can smile, look at people’s faces, respond to teasing, cannot turn head to look
for sound source; (5) Others: increased muscle tone in all
four limbs, ATNR (±), standing drape (+).
Flow cytometry analysis

Peripheral blood samples were obtained from the cCMV
neonate on day 23 (during treatment), day 30–32 (discharge), and day 46 (follow-up) for laboratory analysis.
Flow cytometry was employed to ascertain the proportion of circulating T cells (CD4+ and CD8+ T cells), B
cells, natural killer (NK) cells, CD8+CD28− T regulatory cells (Tregs), and γδ T cells (Vδ1 and Vδ2 T cells).

Page 4 of 9

The objective was to determine the expression levels of
tumour necrosis factor (TNF)-α, interferon (IFN)-γ,interleukin (IL)-17, perforin, and granzyme B secreted byγδ T cells in the peripheral blood of the cCMV neonate
at the three time points. At each time point, the flow dataof the cCMV neonate were compared with those of thecontrols. The demographic data of the case and controls
are presented in Supplementary Table 2. The peripheral blood of the healthy neonates of the same age as the
CMV-infected neonate served as controls. A total of 29
healthy neonates from Shenzhen Baoan Women’s andChildren’s Hospital were included in this study with theconsent of their guardians.
For surface marker staining, freshly isolated PBMCs(1 × 106/mL) were washed and incubated with monoclonal antibodies. For TBNK staining, a commerciallyavailable kit (Mandary, China) was utilised, containing
two panels (T cell panel: Percp-anti-CD45, FITC-antiCD3, APC-anti-CD4, PE-anti-CD8; B-NK panel:
Percp-anti-CD45, FITC-anti-CD3, APC-anti-CD19,
and PE-anti-CD16+CD56−); for staining of CD8+CD28−
Tregs, the antibodies used were FITC-anti-CD8 and
APC-anti-CD28.
For perforin and granzyme B staining, isolated PBMCs
were washed and stained with Alexa Fluor 700-antiCD3, Brilliant Violet 421-anti-TCRγδ, PE/Cy7-anti-Vδ2(all from BioLegend, San Jose, CA, USA), and FITCanti-Vδ1 (Genetex, Germany). Subsequently, the cells
were fixed, permeabilized, and stained with Alexa Fluor647-anti-perforin and Brilliant Violet 510-anti-granzyme
B (BD Bioscience) in accordance with the manufacturers’
insturctions.
Prior to staining for intracellular cytokines secretedby γδ T cells, isolated PBMCs were suspended in RPMI
1640 medium supplemented with 10% FBS and stimulated with phorbol-12-myristate 13-acetate (PMA) (50ng/mL), ionomycin (1 µg/mL), and brefeldin (1 µg/mL)(all from Biogems, Rocky Hill, NJ, USA) for 4 h in 5%
CO2 atmosphere at 37 ℃. Subsequently, the cells were
washed and stained with Alexa Fluor 700-anti-CD3 andBrilliant Violet 421-anti-TCRγδ in the dark at room temperature for 20 min. Following these staining, the cells
were fixed, permeabilized, and stained with Alexa Fluor
647-anti-IL17A, PE-anti-TNFα, Brilliant Violet 605-antiINFγ (BioLegend) according to the manufacturers’ protocol. The panels and antibodies utilized in the study aredetailed in Supplementary Tables 3 and 4.
The labeled cells were quantified using the BD Fortessa
system (BD Bioscience) and the Bricyte E6 (Mandary,
China), and the data were analyzed using Flowjo software(Tree Star). The gating strategy of different lymphocytes
is illustrated in Supplementary Figs. 3–6.

Chen et al. BMC Pediatrics(2024) 24:587

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Table 1 Comparison of TBNK lymphocytes between the cCMV
neonate and controls
Lymphocytes
parameters

Day24
cCMV
neonateLymphocytes
numbers (/µl)
Total T cellnumbers (/µl)
Total T (%)
CD4+T cells
numbers (/µl)
CD4+T cell (%)
CD8+T cells
numbers (/µl)
CD8+T cell (%)
CD4/CD82635

B cell numbers(/µl)
B cell (%)
NK cell numbers (/µl)
NK cell (%)

745
28.88
294

M(P25 ~ P75)
2266
(1932 ~ 2630)
1642(1547 ~ 2108)
79 (75 ~ 81)
1211(1098 ~ 1247)
53 (50 ~ 58)426(354 ~ 882)
20 (17 ~ 25)
2.61(2.11 ~ 3.34)
287(230 ~ 367)
12 (11 ~ 15)
131 (90 ~ 240)

Day30-32
cCMV
Controls
neonateM(P25 ~ P75)
1645
1960
(1645 ~ 2255)
748
1487(1257 ~ 1765)
60.23
79 (73 ~ 82)598
1029(923 ~ 1219)
40.87
56 (50 ~ 61)
218
447(310 ~ 492)
18.0
20 (16 ~ 24.5)
2.34
2.91
(2.16 ~ 3.47)350
365(170 ~ 464)
20.85
11 (9 ~ 15)264
97 (64 ~ 192)

11.4

8 (5 ~ 13)

17.20

1498
56.86
1025
38.90
462
17.52
2.22

Controls

3 (3 ~ 5)

Data of the cCMV neonate was presented. Data of healthy controls waspresented as median (the figure outside the brackets) and quartile with percent
25 ~ percent 75 (the figures in the brackets)

Flow cytometry results

The proportion of T cells in the lymphocytes of the cCMV
neonate was lower than that of the controls at both day
24 (58.86% vs. 79%, interquartile range (IQR) = 75–81)
and day 30–32 (60.23% vs. 79%, IQR = 73–82) (Table 1).
Additionally, the proportions and numbers of other lymphocytes are presented in Table 1.
The subsets of T cells are then examined. CD8+CD28−
Tregs play a suppressive regulatory role in several diseasestates [6, 7]. The proportion of CD8+CD28− Tregs among
the CD8+ T cells demonstrated a declining trend fromday 24 to day 30–32, followed by an increase until day 46.
In comparison to the corresponding controls, the proportion of CD8+CD28− Tregs was higher in the cCMV
neonate at day 24, day 30–32, and day 46, respectively(Table 2; Fig. 2A).
Given the crucial role of γδ T cells in the immune
response to CMV infection [8], we further investigatedthe production of TNF-α, IFN-γ, and IL-17 A by γδ T
cells in the cCMV neonate. The expression levels of TNFα, IFN-γ, and IL-17 A were higher on day 24 than on day
30–32. The expression levels of TNF-α and IFN-γ at bothtime points in the cCMV neonate were higher than thosein the corresponding controls, whereas the expression
levels of IL-17 were lower than those in the controls on
day 30–32 (Table 2).
Vδ1 and Vδ2 T cells represent two major subsets of γδ
T cells that play pivotal roles in CMV infection [8]. The
proportion of Vδ1 T cells in CD3 exhibited a decline
from day 24 to day 30–32, followed by an increase until
discharge. In contrast, the proportion of Vδ2 T cells
demonstrated a similar pattern from day 24 to day 30–32,
before undergoing a decline from day 30–32 to day 46.
(Table 2). It is noteworthy that both perforin and granzyme B, secreted by Vδ1 and Vδ2 T cells, demonstrated
an increasing trend from the onset of hospitalization todischarge. Moreover, the expression levels of perforin
and granzyme B secreted by Vδ1 and Vδ2 T cells werehigher than those of the controls at all three time points(Table 2; Fig. 2B, C, D, E).

Discussion and conclusions
We followed a cCMV neonate with typical symptoms
and a clear route of intrauterine transmission from the
beginning of hospitalization to the follow-up visit, which
is a rare occurrence. The prevalence of cCMV in China
has been estimated to be between 0.23% and 0.7% [3, 4],while in developed countries, it is thought to be between
0.14% and 0.7% [9]. However, the majority of cCMV

Table 2 The fraction of T cell subsets of the cCMV neonate and controls at day 24, day 30–32, and day 46
T cell subsets

CD8+CD28−(%)
TCRγδ+TNFα+(%)
TCRγδ+IFNγ+(%)
TCRγδ+IL17A+(%)
CD3+Vδ1+(%)
CD3+Vδ1+perforin+(%)
CD3+Vδ1+granzymeB+(%)
CD3+Vδ2+(%)
CD3+Vδ2+perforin+(%)
CD3+Vδ2+granzymeB+(%)
NA : not available

Day 24cCMV neonate
15.9
82.3
68.4
0.84
1.0
2.28
3.37
2.34
25.5
24.8

Controls
M(P25 ~ P75)
4.05 [2.69, 5.48]
45.30 [39.95, 46.85]
11.50 [9.77, 19.20]
0.44 [0.40, 0.70]
0.75 [0.46, 0.97]
0.40 [0.18, 0.78]
1.08 [0.58, 1.52]
1.16 [0.73, 1.95]
1.10 [0.67, 1.63]
5.55 [3.88, 6.96]

Day 30–32
cCMV neonate
12.54
50.1
30.6
0.38
0.55
12.8
14.1
2.39
52.3
55.5

Controls
M(P25 ~ P75)
2.33 [1.71, 4.35]
28.90 [17.80, 30.20]
6.43 [3.14, 10.66]
0.82 [0.53, 1.05]
0.50 [0.41, 0.80]
2.05 [1.40, 3.23]
2.10 [1.40, 3.11]
1.24 [0.70, 3.00]
1.78 [0.60, 2.68]
9.80 [5.85, 14.70]

Day 46cCMV neonate
15.2
NA
NA
NA
0.95
13.9
18.1
1.49
70.9
78.3

Controls
M(P25 ~ P75)
2.42 [1.80, 4.34]
NANANA
0.60 [0.24, 0.84]
2.69 [1.40, 3.55]
2.64 [1.48, 3.26]
3.33 [2.03, 3.88]
5.19 [1.78, 11.36]
21.60 [6.90, 35.90]

Chen et al. BMC Pediatrics(2024) 24:587

Fig. 2 (See legend on next page.)

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Chen et al. BMC Pediatrics(2024) 24:587

Page 7 of 9

(See figure on previous page.)
Fig. 2 The proportion of CD8+CD28− Tregs and the expression of Vδ1 and Vδ2 T cells secreting perforin and granzymeB in the cCMV neonate. (A) The
changes in the proportion of CD8+CD28- Tregs out of CD8+ T cells during the antiviral treatment of the cCMV neonate. The comparison of proportion of
CD8+CD28− Tregs out of CD8+ T cells between the cCMV neonate and the controls at day 24 (a), day 30–32 (b), and day 46 (c). The changes in Vδ1 T cells
secreting (B) perforin and (C) granzyme B during antiviral therapy of the cCMV neonate. A comparison of Vδ1 T cells secreting perforin and granzyme B
between the cCMV neonate and controls at day 24 (a), day 30–32 (b), and day 46 (c). The changes in Vδ2 T cells secreting (D) perforin and (E) granzyme
B during the antiviral therapy of the cCMV neonate. A comparison of Vδ2 T cells secreting perforin and granzyme B between the cCMV neonate andcontrols at day 24 (a), day 30–32 (b), and day 46 (c)neonates are born asymptomatic. Furthermore, there areeven fewer cases of multiple organ dysfunction at birth,and even fewer cases of severe neurological lesions. A
search for cases of CMV diagnosed in our hospital in thelast 3 years revealed that the total number of deliveriesin our hospital was 60,000, with nearly 10,000 cases weredischarged from the neonatal department. Nevertheless,only four cases were diagnosed with multi-system damage due to cCMV and treated with antiviral drugs, two of
which exhibited neurological lesions. Our case presented
with the typical multi-organ dysfunction and neurological lesions observed in such instances. It is noteworthy
that only our case was confirmed as an intrauterine infection, whereas the other cases were perinatal or postpartum infections. In addition, the acquisition of fresh
peripheral blood samples from the neonate at varying
time points, along with corresponding controls for flow
cytometry, is an uncommon occurrence.
The patient was administered intravenous ganciclovir for a period exceeding three weeks, followed by oralvalganciclovir to maintain antiviral therapy. The clinicalpresentation, blood test, and CMV DNA quantification
demonstrated favourable treatment outcomes and thepresence of leukopenia, which did not necessitate intervention [10]. Despite the absence of CMV DNA in the
cerebrospinal fluid, the presence of typical foci of calcification in the brain provides evidence of CMV infectionof the central nervous system (CNS) during early or midpregnancy. The negative result may be atrributed to theactivation of the immune system, which effectively eliminates the virus following intrauterine neurological CMV
infection. However, the presence of multiple abnormal
neurological manifestations and poor prognosis (as evidenced by abnormal EEG, brainstem evoked potentials,
GM and cranial imaging during hospitalization and follow-up, as well as significant microcephaly and developmental delay) indicates a high incidence of long-termsequelae following CMV CNS infection. Consequently,
regular follow-up is essential to assess hearing and mental development.
CD8+CD28− Tregs have been associated with the presence of CMV infection [11]. Compared to the corresponding controls, the proportion of CD8+CD28− Tregs
was higher in the cCMV neonate at the three time points,
which is in consistent with the findings of other reports.
In patients with rheumatoid arthritis, the proportion of
CD8+CD28− Tregs is higher in those with CMV-positive

rheumatoid arthritis than in those with CMV-negative
rheumatoid arthritis [12]. Additionally, CMV infection
induced an elevation in CD8+CD28− Tregs in immunosenescent patients [13]. These findings suggest that
CD8+CD28− Tregs are not only implicated in diverse disease states but also exhibit an increasing prevalence during CMV infection.
γδ T cells secrete substantial quantities of TNF-α andIFN-γ when cultured with CMV-infected cells, suggesting a potential correaltion between inflammation
and CMV infection [14, 15]. Additionally, our findings
revealed elevated levels of TNF-α and IFN-γ secretionby γδ T cells in the cCMV neonate. Vδ1 and Vδ2 T cells
represent the predominant subsets of γδ T cells [16]. In
the peripheral blood of the cCMV neonate, the proportion of Vδ1 and Vδ2 T cells fluctuated at different time
points. However, the number of Vδ1 and Vδ2 T cells that
secreted perforin and granzyme B increased. These datasuggest that perforin and granzyme B produced by Vδ1
and Vδ2 T cells may be involved in the control of cCMV
infection.
In a study of 182 cCMV neonates, Soriano-Ramos etal. employed flow cytometry to assess the role of T cell
immune responses and their potential association withthe development of long-term sequelae [9]. They foundthat the number of CD4+ T cells at birth was significantly
lower in infants who subsequently developed sequelae
compared to those who did not. In our study, the CD4+
T cells count of the neonate with long-term sequelae
was lower than that of the healthy controls at two time
points. It is important to note that the control groups andclinical status of the neonates in the two studies are notdirectly comparable. However, both studies demonstrate
the significance and potential of the T cell response in
cCMV neonates.
It is challenging to ascertain the impact of antiviral therapy or the congenital infection itself on the flow
cytometry results, as evidenced by our findings. The
neonate was administered antiviral therapy upon her
admission to our hospital. It is possible that the higher
expression of CD8+CD28− Tregs in cCMV may inhibit
the killing function of other effector cells, thereby contribute to the latency of the virus. The expression of
perforin and granzyme B continued to increase after
treatment, which may be a potential reason for the reduction in CMV and improvement in clinical symptoms.

Chen et al. BMC Pediatrics(2024) 24:587

Nevertheless, further investigation is required to elucidate the underlying mechanisms.
Furthermore, the analysis is constrained by the fact that
only a single case of intrauterine cCMV was available for
examination. This is due to the rarity of specific cCMV
in clinical practice. Despite the fact that our hospital isa maternity hospital with the largest number of deliveries in Shenzhen city and a level 3 neonatal intensive careunit, we have only encountered one case of cCMV with aclear intrauterine infection route in recent years. Obtaining blood samples at three time points for our case waschallenging, resulting in the availability of flow data for
only two time points for certain lymphocytes. Determining the changes in CD8+CD28− Tregs and γδ T cellsby flow cytometry is a potential tool for physicians tomonitor the immune status of patients. However, further
investigation is required to ascertain the suitability of thismethod for cases diagnosed with cCMV infection. Plans
exist to increase the sample size and to collect more clinical neonatal CMV cases, including both congenital andnon-congenital CMV, for testing and analysis. This will
facilitate future investigations to ascertain whether comparable findings are present.
In conclusion, we have investigated the changes in clinical manifestations and T-cell subsets at different time
points in a typical intrauterine cCMV-infected neonate.
It was observed that while CMV can become negative
after treatment, developmental delay can occur in theinfant in later life. The involvement of CD8+CD28− Tregs
and Vδ1 and Vδ2 T cells secreting perforin and granzyme B in congenital CMV infection is a hypothesis thatmerits further investigation in a larger study.
List of abbreviations
cCMV	Congenital cytomegalovirus
CMV	Cytomegalovirus
Tregs	T regulatory cells
TNF	Tumor necrosis factor
IFN	InterferonIL	Interleukin
CRP	C-reactive protein
ALT	Alanine aminotransferase
IQR	Interquartile range
CNS	Central nervous system

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12887-024-05051-z.
Supplementary Material 1
Author contributions
XC and ZJ contributed equally to this work. XC and ZJ performed theexperiments, analyzed data; PZ collected the clinical data; FJ and TX collected
the laboratory data; QT designed and supervised the study, and wrote themanuscript. All authors reviewed the manuscript.

Page 8 of 9

Funding
This study was supported by the Science and Technology Innovation
Commission of Shenzhen (JCYJ20210324130005013), the National Natural
Science Foundation of China (No.82201897), and the Baoan District of Basic
Research project of Shenzhen (No.2020JD462). The publication fee is from
Shenzhen Baoan Women’s and Children’s Hospital.
Data availability
All data generated or analyzed during this study are included in this publishedarticle and its supplementary information files. The datasets used and/or
analyzed during the current study are available from the corresponding
author on reasonable request.

Declarations
Ethics approval and consent to participate
We confirm that all experimental protocols were approved by the ethics
committee of Shenzhen Baoan Women’s and Children’s Hospital and thecommittee’s reference number is LLSC-2022-02-07-29-KS.
We confirm that all methods were performed in accordance with the ethicalstandards as laid down in the Declaration of Helsinki and its later amendments
or comparable ethical standards.
We confirm that written informed consent was obtained from the guardiansof all participants.
Consent for publication
We confirm that the consent for publication was obtained from the guardiansof all participants.
We confirm that the “written informed consent” was given from the guardiansof all participants/patients for their personal or clinical details along with any
identifying images to be published in this study.
Competing interests
The authors declare no competing interests.
Received: 20 March 2024 / Accepted: 3 September 2024

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