Post‐hospitalization rehabilitation alleviates long‐term immune repertoire alteration in COVID‐19 convalescent patients

Abstract The global pandemic of Coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is an once‐in‐a‐lifetime public health crisis. Among hundreds of millions of people who have contracted with or are being infected with COVID‐19, the question of whether COVID‐19 infection may cause long‐term health concern, even being completely recovered from the disease clinically, especially immune system damage, needs to be addressed. Here, we performed seven‐chain adaptome immune repertoire analyses on convalescent COVID‐19 patients who have been discharged from hospitals for at least 6 months. Surprisingly, we discovered lymphopenia, reduced number of unique CDR3s, and reduced diversity of the TCR/BCR immune repertoire in convalescent COVID‐19 patients. In addition, the BCR repertoire appears to be activated, which is consistent with the protective antibody titres, but serological experiments reveal significantly lower IL‐4 and IL‐7 levels in convalescent patients compared to those in healthy controls. Finally, in comparison with convalescent patients who did not receive post‐hospitalization rehabilitation, the convalescent patients who received post‐hospitalization rehabilitation had attenuated immune repertoire abnormality, almost back to the level of healthy control, despite no detectable clinic demographic difference. Overall, we report the potential long‐term immunological impairment for COVID‐19 infection, and correction of this impairment via post‐hospitalization rehabilitation may offer a new prospect for COVID‐19 recovery strategy.

back to the level of healthy control, despite no detectable clinic demographic difference.Overall, we report the potential long-term immunological impairment for COVID-19 infection, and correction of this impairment via post-hospitalization rehabilitation may offer a new prospect for COVID-19 recovery strategy.

| INTRODUCTION
The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the most severe public health emergency event for decades. 1 As of January 2023, SARS-CoV-2 had infected more than 660 million people and caused more than 6 million deaths worldwide.Although the epidemiological and clinical features, pathogenesis, and complications of patients infected with SARS-CoV-2 in the acute phase have been well explored and documented, studies on the clinical and scientific features of convalescent COVID-19 patients remain limited. 2,3Tremendous efforts have been made to develop therapeutic strategies and vaccines for COVID-19 infection, yet the efficacy and specificity of various treatments needs to be further evaluated and tested. 4In addition, the long-term effects of COVID-19 infection on the immune system of convalescent COVID-19 patients have been neglected.Therefore, it is an urgent need to explore the response of the immune system to SARS-CoV-2 infection in convalescent COVID-19 patients.Moreover, evaluating and understanding the immune system change in convalescent COVID-19 patients will also have great clinical, scientific and sociological significance.
Adaptive immunity confers a major immune response to viral infection, 5 including COVID-19 infection. 6,7The adaptive immune system is mainly comprised of lymphocytes, of which T cells and B cells execute their cellular and humoral immune responses, respectively, via specific binding of pathogens with T cell receptor (TCR) and B cell receptor (BCR) 8,9 and the aggregates of the entire TCR/BCR repertoire termed adaptome. 10,113][14] The CDR3 (complementaritydetermining region 3) region is located at the junction of VDJ genes and provides the basis of immune repertoire diversity and antigenbinding specificity of TCR and BCR. 13,15,16This random VDJ recombination bestows the extraordinary diversity of TCR/BCR immune repertoire, ranging from 10 15 to 10 25 for each chain, that is IgH (all isotypes), IgK, IgL, TCR-Alpha, Beta chains, and TCR-Gamma, Delta chains. 17The TCR and BCR diversity provides extensive protection against various pathogens and viruses. 18rious studies have profiled the TCR 19 and BCR 20,21 immune repertoire separately and holistically 22,23 on COVID-19 patients.Consistently, the TCR/BCR diversity is significantly decreased during the acute stage of COVID-19 infection 19,24 in different disease cohorts.In addition, certain dynamic V/J preference, IgM to IgG class switch, B cell hypermutation, and clonotype expansion were also reported, [25][26][27] describing the characteristic pattern of COVID-19 infection.
As estimated, hundreds of millions, even billions of people have or will be infected with COVID-19, it is of great importance to evaluate the clinical and immunologic characteristics of convalescent patients.Serologic analysis discovered that protective adaptive immune responses of anti-SARS-CoV-2 antibodies and specific memory B and T cell responses caused by natural SARS-CoV-2 infection may last for at least 6-8 months after the onset of symptoms, 28 and the diversity of the immune repertoire in convalescent patients with COVID-19 is associated with outcomes. 29though for most of the people infected with COVID-19, the symptoms are mild, even asymptomatic, 30,31 the long-term effect of COVID-19 infection on the immune system still attracts tremendous public attention.Here, we discovered characteristic pan-immune repertoire alterations, like lymphopenia, reduced unique CDR3 number and reduced diversity, together with the

| Sample collection
Blood samples were collected from patients with informed consent.
Whole blood samples were collected via venipuncture and the sera were separated by centrifugation at 3000 Â g for 15 min within 8 h after collection, and then the supernatant was collected.The sera were inactivated at 56 C for 30 min, and then stored at À80 C.
The peripheral blood samples for the immune repertoire sequence were collected via venipuncture using the PAXgene Blood RNA Tube.Then the samples were placed at room temperature for 4 h, and then transferred to À80 C until total RNA was extracted.

| Cytokine and chemokine measurement
Cytokine and chemokine were measured using the Human Cytokine HSTCMAG-28SK-21 and TGFBMAG-64K-03 Assays panels, and the MILLIPLEX ® MAGPIX system (Thermo Fisher, USA) according to the manufacturer's instructions.

| Detection of immune repertoire of T cell and B cell
Extracted total RNA was amplified using a commercially available iR-RepSeq-plus 7-Chain Cassette (iRepertoire Inc, US) covering the human TCR and BCR all seven chains using a strategy that allows the incorporation of unique molecular identifiers during the reverse transcription (RT) step.One disposable cassette is for one sample's library

| Statistical analyses
Statistical analyses were performed using SPSS version 20 (SPSS, Chicago, IL).Statistical significance between two groups was determined using Mann-Whitney U test, and between multiple groups by Kruskal-Wallis test.

| Altered adaptome in convalescent COVID-19 patients
To discover the potential long-term effect of COVID-19 infection on convalescent patients, we collected 80 peripheral blood mononuclear     32,33 number and reduced expression of TCR and BCR. 19For convalescent patients, who were discharged from the hospital for at least 180 days, unique TCR reads remained significantly lower than healthy controls (Figure 1A), which is consistent with the results of the FACS analysis (Figure 5A).Adaptome immune repertoire consists of TCR chains (TCR-alpha, TRA; TCR-beta, TRB; A unique CDR3 (uCDR3) sequence is also defined as a clonotype, which targets a particular antigen, therefore, the uCDR3 may reflect the total adaptive immunity. 34,35Compared to healthy control, convalescent patients displayed a reduced uCDR3 count (Figure 1B), of which the average uCDR3 count for each chain was all reduced.Principal coordinate analysis (PCoA) was performed on the uCDR3 frequency profile of the adaptome immune repertoire.Significant differences were discovered between healthy controls and convalescent patient groups in TCR and BCR clusters were separated in PCoA plots (Figure 1C).
Taken together, we discovered reduced lymphocyte numbers as well as unique CDR3 numbers in the convalescent patients, which may suggest impaired long-term immune repertoire following COVID-19 infection.Moreover, overall TCR and BCR features were also altered represented by PCoA.
In summary, we discovered the diminished TCR/BCR repertoire diversity in convalescent patients, further implying the long-term effect of COVID-19 infection on the adaptome.

| Skewed TRBV/J and IGHV/J gene usage in convalescent COVID-19 patients
To further evaluate whether there is a preferential use of V and J  On the other hand, we discovered no substantial differences in CDR3 length and amino acid composition between healthy control and convalescent patients (Figure S2).

| BCR repertoire display activation pattern
Previous study has described the characteristic dynamics of the B cell repertoire after COVID-19 infection, yet the study scope was limited to 1 month after infection. 19Therefore, in order to follow up on the feature of the B-cell repertoire over time, we further explored the B-cell repertoire in our study cohort.Utilizing the algorithm described by Bashford-rogers et al., 36

| Serological analysis of convalescent COVID-19 patients
Cytokines produced by COVID-19 infection play a crucial role in the aetiology and pathophysiology of the disease, especially the overproduction of pro-inflammatory cytokines such as IL-1, IL-6, IL-12, IFN-γ and TNF-α, [37][38][39] and the levels of these cytokines correlate with disease prognosis. 40,41However, the serological cytokine profile of convalescent COVID-19 patients remains unclear.Therefore, we first performed immunoglobulin isotypes of IgA, IgM, IgG and neutralizing antibody analysis (Figure 5B).Moreover, we conducted cytokine profiling study on our serum samples (Figure 5C, Figure S5).
Of the 24 cytokines measured, only IL-4 and IL-7 were significantly decreased in convalescent patients when compared to healthy controls.IL-4 has anti-inflammatory properties and elevated IL-4 levels have been reported as part of a cytokine storm associated with severe respiratory symptoms 42,43 ; IL-7 can activate T cells, and its levels are elevated in COVID-19 patients, directly related to disease severity. 44,45Here, our finding demonstrated the abnormal level of certain cytokines in convalescent COVID-19 patients, indicating residual long-term effect of the infection., and reduced IL-7 level may correlate with diminished T cell number and function.

| Post-hospitalization rehabilitation enhances immune repertoire normalization in convalescent patients
Of 80 convalescent patients, 56 of them performed posthospitalization rehabilitation (PHR) after being discharged from hospitals (Table 2).Rehabilitation includes sports training, breath training, and traditional Chinese medicine intervention.etc. (Table 3).We found no significant difference in CT, respiratory function, and blood biochemical examination between the 56 PHR patients and the 24 non-PHR patients (Table 2).
We further analysed the adaptome immune repertoire of PHR, non-PHR and healthy control groups.The unique CDR3 count, reflecting the total adaptive immunity, of PHR patients is significantly higher than that of non-PHR patients.Meanwhile, the PHR uCDR3 count recovered to the level of healthy control (Figure 6A,B, Figure S6), indicating PHR markedly improved adaptive immunity in convalescent patients.In addition, the diversity of the adaptome, defined by the Shannon index, is used to evaluate the potential of the adaptive immune system.Here, we found a significant improvement in the diversity of TRA, TRB, IgH, IgK and IgL for PHR patients compared to non-PHR patients (Figure 7A).Moreover, the diversity of TRA, TRB, TRG, IgH, IgK and IgL of PHR patients is indistinguishable from healthy controls (Figure 7A).
Meanwhile, the clusters of PHR patients were significantly closer to healthy control clusters compared to non-PHR patients in TCR (TRA, TRB, TRD and TRG) and BCR (IGH, IGL and IGK) (Figure 6C) in PCoA plots.Furthermore, compared with non-PHR patients, clonal diversification of IGH, IGHD, IGHM and IGHG were increased in PHR patients (Figure 7B).
In addition, TRBV5-5_J2-1, TRBV9_J2-7 and IGHV3-21_J6 pairs were significantly down-regulated in non-PHR patients compared to healthy controls.Meanwhile, the levels of these pairs were indistinguishable between healthy controls and PHR patients (Figure 8), indicating PHR may correct some skewed V_J pairs caused by COVID-19 infection.What is more, there was no significant difference in serum cytokine levels between PHR patients, non-PHR patients and healthy controls (Figure S7).
Hence, although the potency of the adaptive immune system is jeopardized in convalescent patients, PHR can significantly normalize adaptive immune function to a normal state, which validates the necessity of PHR.

| DISCUSSION
We are almost 3 years into COVID-19 pandemic, different variants of SARS-COV-2 has appeared all over the world.Meanwhile, the number of infected subjects might have been largely underestimated, due to the large number of asymptomatic infection and limited testing resources in many areas. 30,31Although millions have succumbed to the illness, billions who survived this pandemic have been getting back to normal life.Therefore, it is of great interest and importance to evaluate the potential impact and possible sequela of infection in convalescent COVID-19 patients.8][49] Therefore, although the major target of SARS-CoV-2 infection is lung, [50][51][52] and multi-organ damages had been reported upon infection, 53,54 evaluating long-term adaptive immune system changes in convalescent COVID-19 patients remains of great significance.In our study, TCR/BCR immune repertoire profiling reveals the residual alterations in the adaptive immune system of convalescent patients recovering from COVID-19 6 months after infection, including lymphopenia, reduced diversity, skewed V/J gene usage and B cell repertoire activation.Interestingly, when patients underwent post-hospitalization rehabilitation after being discharged from hospitals, all of these abnormal patterns could be significantly corrected, which proves the validity and necessity of posthospitalization rehabilitation.
Deep sequencing of TCR/BCR adaptome has become a powerful strategy for profiling immune repertoires and reveals clonal selection, expansion and evolution pattern of SARS-CoV-2 infection on adaptive immune system. 55Seven-chain immune repertoire profiling was first conducted in a longitudinal holistic for COVID-19 infection, and demonstrated dynamic changes over the disease course in first month upon infection. 22Here, we used the same technology platform to profile the panoramic immune repertoire of long-term convalescent COVID-19 patients.Surprisingly, lymphopenia, reduced TCR/BCR diversity remained even six-month after infection, indicating aberrant immune repertoire after recovery.
A large-scale, multi-centre study reported decreased TRB repertoire diversity and skewed TRB V/J utilization in COVID-19 patients. 56Meanwhile, a small-scale longitudinal study measuring IgH pattern upon COVID-19 infection yielded no common tendencies among patients, including uCDR3 number, diversity, high-frequency clone number etc., yet multiple V gene usage preference were discovered in COVID-19 patients. 57In our study, using the holistic immune repertoire technology, we found reduced uCDR3 numbers, reduced repertoire diversity in all seven TCR/BCR chains and skewed TRB and IgH V/J usage in convalescent COVID-19 patients.In addition, reduced diversity of BCR and TCR in COVID-19 patients (non-ICU and ICU) and recovered in convalescent patient was reported. 19This finding appears to be not consistent with ours that showing reduced diversity of TCR and BCR, and this discrepancy may cause by the sample number difference.Due to the larger sample size (82 vs. 12) of convalescent COVID-19 patients in our study, our finding may have more immunological significance.
BCR repertoire is the key factor defining humoral immunity against viral infection, and its feature may serve as a viable predictive biomarker for particular vaccination or actual infection. 58Various studies have reported the distinct B cell repertoire pattern in COVID-19 patients: including distinctive IGHV rearrangements and CDR3H length for B cell heavy chain, 59 transient IgA surge and characteristic IgM to IgG isotype switch, as well as reduced BCR repertoire diversity during early onset of infection. 22Previously, the concept of immune repertoire convergence was used to measure immunogenicity, especially in tumour. 60 Multiple evidences are supporting residual multi-system symptoms and complications after the initial stage of acute infection. 62systematic review showed that at 6 months after infected SARS-CoV-2, 54% of COVID-19 survivors suffered at least one long-term symptom.63 Fatigue, muscle weakness, sleep difficulties, difficulty with memory and concentration were the most commonly persistent symptoms in convalescent COVID-19 patients.63,64 The latest research data suggests that COVID-19 will enhance the incidence rate of type 2 diabetes and the risk and 1-year burden of cardiovascular disease.[65][66][67] Hence, more and more governments and experts begin to pay attention to long-term symptom of COVID-19 survivals.The National Institute for Health and Care Excellence (NICE) published a clinical guideline for managing long-term symptoms of COVID-19 patients.68 The guideline points out that multidisciplinary rehabilitation is critical for managing hospitalized COVID-19 patients, but when and how to administrate rehabilitation remains to be further investigation. Threfore, establishing an objective standard is crucial for evaluating the effect of rehabilitation and formulating rehabilitation strategies for convalescent COVID-19 patients.
Various studies have reported that early rehabilitation is essential for minimizing the risk of developing long-term symptoms and promoting functional restoration for patients, and rehabilitation should be started as early as possible. 69,70The early rehabilitation may be personalized and focus on addressing patient-specific problems, targeting such as pulmonary and cardiac function, etc.A randomized controlled study showed that a 6-week respiratory rehabilitation can improve respiratory functions, quality of life, and anxiety in convalescent COVID-19 patients. 71In addition, individuals who suffered from COVID-19 with continuous respiratory symptoms should be given rehabilitation. 70Regular exercise training programs have immuneregulatory and anti-inflammatory effects. 72For example, aerobic exercise not only can relieve anxiety and depression, but also can enhance aerobic capacity, thus improving short-term immunity, including increasing immune cell function, immunoglobulins level and improving respiratory function in COVID-19 patients. 73A six-month study for pulmonary rehabilitation among patients hospitalized with COVID-19 showed that pulmonary rehabilitation can increase exercise capacity, without altering immunity variables. 74Our current study discovered that opportune rehabilitation may normalize the impaired immune functions, such as lymphopenia, TCR/BCR diversity, etc.This crucial finding may change our strategy for post-hospitalization patients.In addition, evaluating adaptive immune system function using immune repertoire sequencing technology may be an objective way to evaluate the efficacy of rehabilitation strategy from molecular immunology prospect, and more data is needed to establish the standard.Therefore, this study will initiate a trend of biomarker discovery for COVID-19 recovery, and direct further way of COVID-19 rehabilitation.
Finally, despite these promising results, some questions remain to be addressed, especially from the micro-prospect of the immune repertoire, that is clone level analysis need to be further performed.
SARS-CoV-2 associated/specific clones have been identified in TCRβ 56 and IgH. 75 imbalance of V/J usage even in convalescent patients at least 6 months after hospitalization, indicating long-term immune system marks upon COVID-19 infection.Interestingly, convalescent patients with severe symptoms and underwent posthospitalization rehabilitation, have markedly improved immune repertoire profile compared to the convalescent patients without rehabilitation, justifying the necessity of rehabilitation upon COVID-19 recovery.2 | MATERIALS AND METHODS 2.1 | Sample collection Screening of COVID-19 convalescent patient donors and sample collection were conducted at Hubei Provincial Hospital of Traditional Chinese & Western Medicine.The study protocols were approved by the Ethical Committee of Guangdong Provincial The diversity of adaptome in PBMCs of convalescent COVID-19 patients.The diversity of was demonstrated shannon-index at the level of unique uCDR3 clones, as shown of TRA, TRB, TCD, TRG, IgH, IgK and IgL (A).Immune repertoire diversity is shown by tree maps in two representative individual samples (B), in which each round rectangle represents a unique uCDR3, and the size of each rectangle denotes the relative frequency of that uCDR3 clone.*p < 0.05, **p < 0.01.preparation;all necessary reagents for amplification and purification are preloaded into the cassette.For the construction of human TCR and BCR libraries, RT is performed using Qiagen OneStep RT PCR mix (Qiagen).First-strand cDNA is selected and unused primer is removed by SPRIselect bead selection (Beckman Coulter), followed by the second round of binding and extension with the V-gene primer mix.After binding and extension, SPRIselect beads are used to purify the first and second strand synthesis products.Library amplification isF I G U R E 3 V/Jpreference usage of convalescent COVID-19 patients.Mann-Whitney U test was used to statistically define the significant difference, and data are presented as the mean ± SD values.All of the TRBV-TRBJs conjunctions (A) and IGHV-IGHJ conjunctions (B) with p value <0.05, between control and convalescent COVID-19 patients are presented.Heat maps of V gene usage in conjunction with J usage in convalescent COVID-19 patients showing preferred TRBV-TRBJ pairs (C) and IGHV-IGHJ pairs (D).performed with a pair of primers that are specific for communal sites engineered onto the 5 0 end of the C-and V-primers used in the first and second strand synthesis.The final constructed library includes Illumina dual index sequencing adapters, a 10-nucleotide unique molecular identifier region, and an 8-nucleotide internal barcode associated with the C-gene primer.Amplified libraries were multiplexed and pooled for sequencing on the Illumina MiSeq platform using a 600-cycle kit and were sequenced as 250 paired-end reads.The output of the immune receptor sequence covers the second framework region through the beginning of the constant region including CDR2 and CDR3.Sequencing raw data were analysed using the previously described iRmap program (Wang et al., PNAS, 2010; Yang et al., Elife, 2015).Briefly, sequence reads were demultiplexed according to both Illumina dual indices incorporated during the amplification process and barcode sequences at the 5 0 end of reads from the constant region.Reads were then trimmed according to their base qualities with a 2-base sliding window.If either quality value in this window is lower than 20, this sequence stretches from the window to 3 0 end is trimmed out from the original read.Trimmed pair-end reads were joined together through overlapping alignment with a modified Needleman-Wunsch algorithm.If paired forward and reverse reads in the overlapping region were not perfectly matched, both forward and reverse reads were thrown out without further consideration.The merged reads were mapped using a Smith-Waterman algorithm to germline V, D, J and C reference sequences using an IMGT reference library.To define the complementary determining regions three (CDR3s) region, the position of CDR3 boundaries of reference sequences from the IMGT database was migrated onto reads through mapping results, and the resulting CDR3 regions were extracted and translated into amino acids.
repertoire clonality and clonal network parameters of convalescent COVID-19 patients.Clonal expansion index (A) and clonal diversification index of BCRs (B) for BCR repertoires in PBMCs.Between healthy control and convalescent COVID-19 patients, IGH mean cluster size (C) and IGH largest clusters (D) were shown, as well as the percentage expression of the largest clone for IGHM out of the total expression of all sequences belonging to IGHM (E), the percentage expression of the top 10 largest clones for IGHM out of the total expression of all sequences belonging to IGHM (F), the percentage of reads that for IGHM of the top 100 unique clones of the sample (G) and the percentage of reads for IGHM out of the top 50% of reads in the repertoire (H).B cell hypermutation is shown in two representative individual samples (I), in which each dot represents a unique B cell uCDR3, and the size of each dot denotes the relative frequency of that uCDR3 clone, and the dots connected by line represent hypermutation.cell (PBMC) samples from convalescent COVID-19 patients and 12 healthy donors as normal control.The demographic and clinical characteristics of the patients are shown in

F I G U R E 5
The lymphocyte, cytokines and SARS-CoV-2 specific immunoglobulin in peripheral blood of convalescent COVID-19 patients.T cell, B cell and NK cell count were measured using flow cytometry (A).Levels of SARS-CoV-2 specific immunoglobulin isotypes of IgA, IgM, IgG and neutralizing antibody (B) were measured by ELISA, between control and convalescent COVID-19 patients.Plasma cytokine IL-4 and IL-7 were measured using Luminex, resulting in the mean of plate-detrended median fluorescence intensity values of each cytokine per sample (C).Mann-Whitney U test were performed for each cytokine, comparing healthy controls with convalescent COVID-19 patients.
the B cell clones are measured by size (clonal expansion), which is characterized as clone expansion index (CEI), and diversification (somatic hypermutation and isotype switching), which is characterized as clone diversification index (CDI).CEI is used to indicate the unevenness of the number of RNA molecules per VDJ region sequences, which is T A B L E 3 Sequelae symptom of the convalescent COVID-19 individuals.
defined by the Gini index, and CDI is used to indicate the unevenness of unique VDJ region sequences per clone, which is defined by Renyi entropy.In our study, clonal diversification (Figure 4A) was decreased in convalescent patients.This trend of clone diversification, a comprehensive measure of B cell clone diversity, hypermutation and isotype switching, was discovered in all IgH isotypes, including IgHA, IgHD, IgHE, IgHG and IgHM (Figure S3).Moreover, larger mean IgHM cluster sizes, largest IgHM clones (Figure 4C,D), larger mean IgHD cluster sizes, and largest IgHD clones were discovered in convalescent patients (Figures S3 and S4).Overall, we found the B cell repertoire remained activated in convalescent patients.F I G U R E 6 Immune repertoire profile of post-hospitalization rehabilitation (PHR) and non-post-hospitalization rehabilitation (non-PHR) convalescent COVID-19 patients.The proportion of reads for overall adaptome immune repertoire and seven-chain adaptome immune repertoire (A) in healthy control, PHR and non-PHR subjects.The abundance of immune repertoire measured by the number of unique CDR3 (uCDR3) in the overall adaptome and seven-chain adaptome (B).Principle of component analysis (PoCA) based on the abundance of TRA, TRB, TRD, TRG, IGH, IGK and IGL clones and the distance between the dots indicates the degree of dissimilarity between samples (C).*p < 0.05, **p < 0.01.

F I G U R E 7
The diversity of adaptome and B cell repertoire clonality in convalescent COVID-19 patients with or without post-hospitalization rehabilitation (PHR).The diversity of was demonstrated shannon-index at the level of unique uCDR3 clones, as shown of TRA, TRB, TCD, TRG, IgH, IgK and IgL (A).Clonal expansion index (CDI) of IGH and IGH isotype (IgHA, IgHD, IgHE, IgHG12, IgHG34 and IgHM) (B).
preference usage of post-hospitalization rehabilitation (PHR) and non-post-hospitalization rehabilitation (non-PHR) convalescent COVID-19 patients.Kruskal-Wallis test was used to statistically define the significant difference, and data are presented as the mean ± SD values.All of the TRBV-TRBJs conjunctions (A) and IGHV-IGHJ conjunctions (B) with p value <0.05, between control, PHR and non-PHR are presented.Heat maps of V gene usage in conjunction with J usage in control, PHR and non-PHR showing preferred TRBV-TRBJ pairs (C) and IGHV-IGHJ pairs (D).
Galson et al. report a stereotypical naive immune response of BCR heavy chain across their study cohort, with clonal expansion and strong convergent signature. 61Here, we measured these COVID-19 specific BCR signature in the convalescent patients, and discovered individual clone expansion and diversification, including somatic hypermutation and class switch.This information supports the potential of BCR signature as a biomarker to measure the recovery of COVID-19 infection.Similar to severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS) and influenza, T and B lymphocytes also plays the key role in defending against SARS-CoV-2 infections.46,47Moreover, the full recovery of adaptive immunity may take more than 2 years in convalescent SARS patients.48Therefore, exploring the immunological characteristics of convalescent COVID-19 patients will help to understand the impact of SARS-CoV-2 infections on adaptive immune responses more comprehensively, and the diversity of TCR and BCR will provide valuable insights into the dynamic change of immune function in convalescent COVID-19 patients.In our study, we found that the seropositivity rate of IgA, IgM, IgG and neutralizing antibodies of convalescent COVID-19 patients were 53.75%, 25%, 92.5% and 92.5%, respectively.However, the IgA, IgM, IgG and neutralizing antibodies of 12 healthy volunteers are negative.In addition, decreased T and B cell repertoire diversity also indicates a dysfunctional immune response in convalescent COVID-19 patients.Taken together, this evidence suggests that convalescent COVID-19 patients may take a long time to fully recover adaptive immunity, even more than 6 months.Therefore, exploring the dynamic change of immune function in convalescent COVID-19 patients will be a very worthwhile subject in further.
It would be enlightening to perform data mining comparing our data set to the existing COVID-19 specific clones, and find out the T cell and B cell signatures of COVID-19 infection recovery.In summary, our study demonstrated a holistic manifestation of crucial adaptive immune system alteration in long-term convalescent COVID-19 patients, involving both T cell and B cell repertoire.Moreover, we discovered that post-hospitalization rehabilitation markedly improved adaptive immune system function in convalescent COVID-19 patients, which may help avoid the occurrence of immune diseases.Finally, our findings offer a new strategic avenue for guiding COVID-19 patients back to normal life.

1
Summary of demographic and clinical characteristics in convalescent COVID-19 individuals and healthy individuals.