T cell response to influenza vaccination remains intact in adults with congenital heart disease who underwent early thymectomy

Abstract

Introduction

T cells developed in the thymus play a key role in vaccine immunity. Thymectomy occurs during infant congenital heart surgery and results in an altered T cell distribution. We investigated if adults with congenital heart disease (ACHD) who underwent early thymectomy have a diminished response to influenza vaccination.

Methods

Blood samples from ACHD with early thymectomy ≤1 year of age (ACHD-ET; n = 12), no thymectomy (ACHD-NT; n = 8), and healthy controls (HC; n = 14) were collected prior to and 4 weeks after influenza vaccination. Flow cytometric analysis of T cell subsets and vaccine-specific cytokine expressing CD4⁺ T cells as well as hemagglutination inhibition (HI) assays were completed.

Results

The mean age of the cohort was 34 ± 10.6 years and similar in all groups. The mean frequencies of naïve CD4⁺ and CD8⁺ T cells were lower in ACHD-ET than in HC (32.7% vs. 46.5%, p = 0.027 and 37.2% vs. 57.4%, p = 0.032, respectively). There was a rise in the frequency of memory CD4⁺ and CD8⁺ T cells in the ACHD-ET group. The ACHD-NT had no statistical difference from either group. The frequencies of influenza-specific memory CD4⁺ T cells expressing IFN-γ and TNF-α were increased after vaccination across all groups (p < 0.05).

Conclusions

ACHD-ET have fewer naïve T cells, suggesting immunosenescence. Despite this, they show an adequate T Cell response to vaccination in young adulthood. Our findings support that routine vaccination is effective in this population, but research into older ACHD is necessary.

Highlights

• •Adults with congenital heart disease and early thymectomy have altered T cells.
• •T cells are necessary for an appropriate response to influenza vaccination.
• •This population is able to produce cytokines following vaccination despite this.
• •They also can produce adequate immunoglobulins following inoculation.
• •Their response was the same as similar aged, healthy controls.

1. Introduction

Removal of the thymus is a routine part of infant congenital heart surgery to obtain access to the heart and great vessels at the time of median sternotomy. Due to the location and size of the thymus in infancy, surgery often results in complete removal of the organ in cases of significant congenital heart disease. The thymus plays an important role in human immunity, particularly in the maturation of T cells. For example, the 22q11.2 deletion syndrome (also known as DiGeorge syndrome) classically causes agenesis of the thymus and results in significant, clinical immunodeficiency [1]. This degree of immune system dysfunction is not seen in neonates who undergo incidental thymectomy. This is mainly due to normal thymic development and function during fetal life [2,3]. Investigations have shown that individuals who undergo thymectomy early in life at the time of cardiac surgery have long-term notable alterations in T cell populations. These include low numbers of CD4⁺ and CD8⁺ T cells with a significantly decreased naïve T cell population (CCR7⁺ CD45RA⁺) [[2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]]. There is then a reciprocal increase in central memory (CM; CCR7⁺ CD45RA⁻), effector memory (EM; CCR7^- CD45RA⁻), and terminal effector memory (TEMRA; CCR7^- CD45RA⁺) T cells [4,6,13,14]. This pattern of T cell distribution signifies terminal differentiation with a decreased ability to respond to new antigens and is comparable to what occurs during age-related immunosenescence [15,16]. It has been shown that adults who had thymectomy in the first month of life had total and naïve CD4⁺ and CD8⁺ cell counts similar to controls who are decades older [13].

Our group has previously described that individuals over 65 years of age have low naïve CD4⁺ T cell and high memory CD4⁺ T cell counts, similar to the pattern described above in those with early thymectomy [15,17,18]. This population also has significantly decreased IgG and memory CD4⁺ T cell responses to influenza vaccine compared to a younger cohort [15,17].

Given the suggestion of premature immunosenescence in subjects who underwent thymectomy early in life, we set out to evaluate if adults with congenital heart disease (ACHD) who are decades removed from their initial heart surgery have an adequate immune response to the influenza vaccine. Our hypothesis is that there would be a blunted T cell mediated response with decreased anti-influenza antibody titers to antigen stimulation as a result of early thymectomy and resultant immunosenescence. To our knowledge, we believe this is first study of its kind to evaluate response to the influenza vaccine in this population of individuals with early thymectomy [3,8,12,19].

2. Materials and methods

2.1. Subjects

This study was a prospective, case-control evaluation. We recruited adults aged 18–65 years old. Subjects were screened during their routine visits in the Yale Adult Congenital Heart Program. Patients (n = 12) who had an early thymectomy at the time of median sternotomy within the first year of life (ACHD-ET) were recruited. A second group of adults (n = 8) with congenital heart disease who did not require surgery through a midline incision or underwent sternotomy later than 1 year of age (ACHD-NT) were also enrolled. A third group (n = 14) was recruited as healthy controls (HC) and consisted primarily of volunteers from the hospital and university employee pool. Care was taken to recruit similar aged subjects across groups. Two subjects were lost to follow up and did not return for their second blood draw (one in the ACHD-ET and one in the ACHD-NT). Their first visit data were included in analysis.

Demographic data along with medical and surgical history were obtained via subject interview and review of the electronic medical record (Table 1). Where available, operative reports were reviewed, but description of thymectomy and remote records from previous institutions were not always available. The long-term, institutional preference was to perform total thymectomy at time of median sternotomy.

Table 1.

Subject demographics and heart disease.

	HC	ACHD-NT	ACHD-ET	p-value
n	14	8	12
Age (years)	36.6 ± 11.1	31.6 ± 5.8	35.3 ± 12.6	0.58
Female n (%)	10 (71.4%)	3 (37.5%)	7 (58.3%)	0.30
Age at sternotomy (months)	N/A	N/A	4 ± 4
Congenital Heart Defect
		Sub-Aortic Stenosis	Multiple VSD's
		Coarctation	AVSD
		TOF (Thoracotomy)	Truncus Arteriosus
		Coarctation	DORV/TOF
		Aortic Stenosis	DORV/D-TGA
		Tricuspid Atresia (Thoracotomy)	D-TGA
		Sub-Aortic Stenosis	AVSD/Heterotaxy
		Tricuspid Dysplasia	TOF
			L-TGA/Tricuspid Atresia
			D-TGA
			Truncus Arteriosus
			Large VSD

Age is presented as mean ± standard deviation. P-value determined by One-way ANOVA analysis. The range for age at sternotomy was 0–10 months. Difference amongst groups for sex were determined by Chi Square Test. P < 0.05 was considered as statistically significant. List of congenital heart defects for each individual subject is listed for the adults with congenital heart disease with early thymectomy (ACHD-ET) and no thymectomy (ACHD-NT). Two subjects in the ACHD-NT had surgery via a lateral thoracotomy in the first years and identified above. TOF = Tetralogy of Fallot, VSD = Ventricular Septal Defect, DORV = Double Outlet Right Ventricle, D-TGA = D-Transposition of the Great Arteries, L-TGA = L-Transposition of the great arteries, AVSD = Atrioventricular Septal Defect.

Subjects from all three groups were excluded from consideration if they met any of the following criteria: use of steroids, antibiotics, or antivirals in the past two weeks; acute infection; history of cancer; severe reaction to the influenza vaccine (such as rash, hives, anaphylaxis); current pregnancy; organ, bone marrow, or stem cell transplant; decompensated heart failure; liver cirrhosis; end stage kidney disease; HIV/AIDs; hepatitis B or C; auto-immune conditions; diabetes; or coronary artery disease. In addition, subjects were excluded if they had any diagnosed genetic condition or syndrome (e.g., Trisomy 21 or 21q11.2 deletion syndrome).

All subjects signed informed consent. Study protocol was reviewed and approved by the Yale University Institutional Review Board (HIC#2000028115).

2.2. Influenza virus vaccination

All subjects were identified to be receiving the influenza vaccination as part of their routine medical care; the vaccine was not administered as part of the study. Subjects recorded the date of their vaccination, and this was confirmed through the electronic medical record or contact with commercial pharmacies. All subjects were verified to have received the only commercially available quadrivalent influenza vaccine available during the 2020–2021 season [A/Guangdong-Maonan/SWL1536/2019 (H1N1), A/Hong Kong/2671/2019 (H3N2), B/Washington/02/2019 (B/Victoria lineage), B/Phuket/3073/2013 (Yamagata lineage)].

2.3. Cells and flow cytometry

Between October and February during the 2020–2021 influenza season, peripheral blood samples were collected before vaccination and at a mean of 32 days (range: 27–64 days) after vaccination. Peripheral blood mononuclear cells (PBMCs) were prepared from blood on FicollPAQUE gradients. To identify the distribution of T cell subsets, the cells were stained with antibodies to BUV395-CD3, Alexa Fluor 700-CD4, Pacific Blue-CD8, PE-Cy7-CCR7, and PE-Cy5-CD45RA (all from BD Biosciences, San Jose, CA) or isotype antibodies. For intracellular cytokine staining, PBMCs were incubated for 6 h with or without a combination of Quadrivalent Influenza Vaccine (Seqirus, Parkville, Australia) and anti-CD28/49d costimulatory antibodies (BD Biosciences) in the presence of Golgiplug (BD Biosciences). Incubated cells were stained with antibodies to BUV395-CD3 and Alexa Fluor 700-CD4 (all from BD Biosciences). Cells were fixed and permeabilized using Foxp3/transcription factor staining buffer set according to the manufacturer's protocol (Invitrogen, Waltham, MA) and stained with antibodies to APC-Cy7- IFN-γ and APC-TNF-α (Biolegend, San Diego, CA). Cells were analyzed using an LSRII® flow cytometer (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR).

2.4. Anti-influenza viral antibody titers

Hemagglutination inhibition (HI) assays on pre- and post-vaccine serum samples were performed to determine antibody titers against each influenza strain included in the 2020–2021 vaccine as described previously [20]. The titer was considered to be the highest dilution at which inhibition of hemagglutination occurred. Seroconversion to a strain in the vaccine was defined as a 4-fold or greater increase in antibody titer between pre- and post-vaccine serum samples and a seroprotective antibody titer was considered to be ≥ 1:40 [15,[21], [22], [23]].

2.5. Statistical analysis

The paired t-test, one-way ANOVA, and Chi-square tests (for hemagglutination testing) were used for statistical analyses as appropriate using GraphPad Prism 9 software. P values of less than 0.05 were considered statistically significant. Data are shown as mean ± SEM, unless otherwise specified.

3. Results

The mean ages (SD) for the ACHD-ET, ACHD-NT, and HC groups were 35.5 years (12.6), 31.6 years (5.8), and 36.6 (11.1), respectively. There was no statistical difference in age between the three groups (p = 0.58).

3.1. Adults with congenital heart disease and early thymectomy (ACHD-ET) have an altered frequency of peripheral T cell subsets

In this evaluation, the ACHD-ET group exhibited a decreased frequency of naïve CD4⁺ T cells (32.7% ± 10.7) and an increased frequency of total memory CD4⁺ T cells (67.3% ± 10.7) compared to HC (46.5% ± 9.4 and 53.5% ± 9.4, respectively; p = 0.027). However, the frequency of effector memory CD4⁺ T cells was comparable between the groups (Fig. 1A). The ACHD-NT group did not show any differences in the frequency of naïve (38.0% ± 14.3) or memory (62.0% ± 14.3) CD4⁺ T cells compared to either HC or ACHD-ET. A similar alteration was also observed in naïve and total memory CD8⁺ T cells across the groups. The ACHD-ET group had a decreased frequency of naïve CD8⁺ T cells compared to the HC group (37.2% ± 17.9 vs. 57.4% ± 12.8, p = 0.032; Fig. 1B) while the frequency of memory CD8⁺ T cells was higher in the former than in the latter (62.8% ± 17.9 vs. 42.6% ± 12.8, p = 0.032; Fig. 1B). There was no statistical difference in the same measurements between the ACHD-NT and other groups.

Fig. 1.

Adults with congenital heart disease and early thymectomy (ACHD-ET) have an altered frequency of peripheral T cell subsets. The basal frequency of T cell subsets in peripheral blood mononuclear cells (PBMCs) collected from healthy controls (HC), adults with congenital heart disease and no thymectomy (ACHD-NT), and adults with congenital heart disease with early thymectomy (ACHD-ET) before vaccination were analyzed by flow cytometry. (A) Naïve (CCR7⁺CD45RA⁺) and total memory (central and effector memory) T cells, central memory (CCR7⁺CD45RA⁻) and effector memory (CCR7⁻CD45RA⁻) T cells were identified based on the surface marker expression gated on CD3⁺CD4⁺ T cells. (B) As shown in panel A, naïve (CCR7⁺CD45RA⁺) and total memory (central, CD45RA⁻ effector memory, and CD45RA⁺ effector memory or TEMRA) T cells, central memory (CCR7⁺CD45RA⁻), effector memory (CCR7⁻CD45RA⁻), and TEMRA (CCR7⁻CD45RA⁺) CD8⁺ T cells were identified with the surface marker expression gated on CD3⁺CD8⁺ T cells. One-way ANOVA analysis was performed to assess the statistical significance and P-values were corrected for multiple comparisons. P < 0.05 was considered as statistically significant.

3.2. Adults with congenital heart Disease and early thymectomy (ACHD-ET) can mount antigen specific CD4⁺ T cell responses to influenza vaccine

Next, we examined whether early thymectomy could affect CD4⁺ T cell responses to the influenza vaccine by analyzing cytokine expressing CD4⁺ T cells in ACHD-ET, ACHD-NT, and healthy controls. Four weeks following vaccination, all three groups had an increase in the frequency of influenza virus-specific memory CD4⁺ T cells expressing IFN-γ and TNF-α (Fig. 2A, C). The levels of changes between pre and post vaccination were also similar in the three groups (Fig. 2B and D).

Fig. 2.

Adults with congenital heart disease and early thymectomy (ACHD-ET) can mount antigen specific CD4⁺T cell responses to influenza vaccine. Influenza virus-specific CD4⁺ T cells expressing IFN-γ and TNF-α were analyzed in peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), adults with congenital heart disease and no thymectomy (ACHD-NT), and adults with congenital heart disease with early thymectomy (ACHD-ET) by flow cytometric intracellular cytokine analysis. PBMCs were stimulated for 6 h with influenza viral antigens in the presence of Golgi-plug followed by intracellular cytokine staining. The frequency of memory CD4⁺ T cells expressing (A) IFN-γ and (C) TNF-α pre- and post-vaccination. The difference in frequency of (B) IFN-γ and (D) TNF-α cytokine expressing memory CD4⁺ T cells between post- and pre-vaccination was examined. Statistical comparisons were performed using paired Student's t-test and p < 0.05 was considered as statistically significant.

3.3. Adults with congenital heart disease and early thymectomy (ACHD-ET) have hemagglutination inhibition titers comparable to ACHD with no thymectomy (ACHD-NT) and healthy controls

All three study groups had an increase in HI titers after vaccination. However, the number of individuals who met the criteria for seroconversion, consisting of a 4-fold increase in titers, was relatively low in all three groups. Seroconversion rates to at least one strain tested were 3 (21%), 5 (71%), and 5 (45%) in the HC, ACHD-NT, and ACHD-ET groups, respectively (p = 0.082; Fig. 3A). The second characteristic evaluated with the HI assay was seroprotection. This is defined as a 1:40 or greater titer response. Prior to vaccination, the frequency of seroprotection to at least one strain in the HC, ACHD-NT, and ACHD-ET groups were 6 (43%), 2 (29%), and 8 (73%), respectively. Following vaccination, this rose to 8 (57%), 6 (86%), and 11 (100%), respectively (p = 0.031; Fig. 3B). Collectively, the ACHD-ET group had normal and similar antibody titers compared to other control groups.

Fig. 3.

Adults with congenital heart disease and early thymectomy (ACHD-ET) have hemagglutination inhibition titers comparable to adults with congenital heart disease with no thymectomy (ACHD-NT) and healthy controls. Seroconversion and seroprotection were assessed using the hemagglutination inhibition (HI) assay. The sera were obtained from peripheral blood of HC, ACHD-NT, and ACHD-ET pre- and post-influenza vaccination. (A) Individuals with a 4-fold or higher increase in the HI titer from pre- to post-vaccination sera were considered seroconversion (responders). (B) Seroprotection was defined by HAI titers ≥1:40. Statistically significant differences were evaluated using the Chi-square test and P < 0.05 was considered as statistically significant.

4. Discussion

This study is part of the ongoing investigation to understand some of the long-lasting effects of congenital heart surgery at a young age. While it has long been thought that there is little downside to the removal of the thymus, there is emerging evidence that this does result in some long-term alterations to the immune system. There are newer longitudinal studies that have also shown an increased incidence in carcinomas and the suggestion of higher rates of autoimmune conditions [[24], [25], [26]]. Although there are multiple factors that contribute to the development of these conditions, changes to the immune system may play a role and would require further study [27].

Akin to past evaluations of similar populations, the ACHD group who underwent early thymectomy demonstrated a shift in the T cell population that is consistent with “immunological aging.” This includes a decrease in naïve CD4⁺ and CD8⁺ T cells and an increase in memory CD4⁺ and CD8⁺ T cells [3,8,12,19]. These changes are similar to those who are over age 65 with a decreased frequency of naïve T cells. These older individuals have been shown to have a reduced response to the influenza vaccine that is, in part, mediated by T cells [15,17]. However, randomized control trials in this older population have shown that these individuals can overcome this diminished response and have an improvement in seroprotection and a reduction in clinical influenza infection with use of high dose influenza vaccination [28].

We hypothesized that the observed change in T cell subsets would lead to immunosenescence and reduced T cell response to influenza vaccination. Fortunately, this was not observed. The ACHD-ET, ACHD-NT, and HC groups had comparable levels of T cell activation in response to influenza vaccine as determined by measuring the frequency of influenza antigen-specific CD4⁺ T cells expressing IFN-γ and TNF-α following in vitro stimulation. The seroprotective titer response to the vaccine as defined by the HI titers greater than 1:40 was found in all subjects of the ACHD-ET group. It is unclear why the HC group did not seem to have a significant response to the vaccine by HI alone. This group may not be completely representative of the general population, as it consists of hospital and university workers who are required to obtain the influenza vaccine yearly for employment. It is possible that this factor could alter the titer response. In addition, there has been a concern for using HI alone as a measure of vaccine efficacy due to the lack of correlation with breakthrough cases and inconsistencies with measurement between laboratories [29,30].

Overall, these findings support that the immune system of ACHD-ET was able to compensate for the long-term alteration to T cell populations and mount an appropriate response to influenza vaccination. The results of our study provide reassurance for ACHD, particularly in primary prevention of influenza infections. However, this research raises further questions that need to be addressed. The group of recruited individuals was relatively young with a mean age of 37 years. As the number of emerging adults with congenital heart disease has continued to increase due to advances in medical and surgical therapies, the average age of these individuals is also becoming older. There are significant changes seen in T cells populations that start as little as 1 year following thymectomy. This raises the question of how this population in their 6th or 7th decade of life will respond to the influenza or other protective vaccines. This response has never been studied and will need to be the topic of future investigations. In addition, it will be critical to understand how ACHD respond to other vaccine types such as the messenger RNA vaccines recently developed for SARS-CoV-2 or conjugate vaccines used for pneumococcus.

There may be a role in high dose influenza vaccination in adults with congenital heart disease at a younger age than 65 years; however, there is no evidence to support this as of now. The INfluenza Vaccine to Effectively Stop Cardio Thoracic Events and Decompensated heart failure (INVESTED) trial was designed to compare high dose influenza vaccination in patients with heart failure due to their high risk of morbidity and mortality following influenza infection [31]. This was a large, multicentered, double-blinded, randomized control trial that recruited over 5000 patients with heart failure. In addition, the protocol was designed to include the recruitment of 500 ACHD. However, the trial was stopped early due to lack of difference in the primary endpoint in the treatment arm [32]. Because of the early termination and low number of ACHD recruited, analysis will not be performed on this subgroup, including antibody testing.

5. Limitations

This study has limitations that include a small sample size, particularly in the ACHD-NT group. Thus, there may be some limitations to extrapolating these data to the ACHD population at large. In addition, it took place a single center. Due to the sample size and very low incidence of influenza during the 2020–2021 influenza season in the United States, no clinical data regarding influenza infection was able to be obtained [33]. Finally, in the ACHD-ET group, presence of early thymectomy was dependent on the available clinical records and operative reports. These reports did not always mention if thymectomy was performed or to what degree (total, sub-total, etc.). Thus, in some cases, thymectomy was assumed based on the operative approach.

6. Conclusion

As more adults are surviving with congenital heart disease there is a greater population of individuals who underwent infant thymectomy. This results in an altered T cell population in adulthood that is similar to that seen in immunosenescence. Despite this, young adults with congenital heart disease and early thymectomy are able to mount an appropriate T cell-mediated immune response following influenza virus vaccination which is comparable to that of ACHD-NT and healthy controls. Further investigations with older survivors of congenital heart disease and other vaccine types are needed.