Atrophy of the thymus caused by cytotoxic drugs and glucocorticoid hormones remains a primary obstacle to full immune recovery following chemotherapy, which is dependent on high thymic output of new recent thymic emigrants (RTE) to replenish the naïve T cell pool (1, 2). Failure of restoration of thymus function may lead to increased risks of infections and tumor recurrence, particularly in elderly patients where the thymus is atrophied (1, 3). Enlargement of the thymus above baseline following chemotherapy, known as rebound thymic hyperplasia (TH), sometimes occurs during recovery from chemotherapy (4). TH may be related with robust thymic regeneration and is characterized by an increase in thymic size and density, concurrent with the restoration of thymic output of T cells (5–7). This phenomenon is common in children and adolescents and can occasionally be observed in young adults; however, it is rare in older patients (4, 8–11). Therefore, more effort should be made toward restoring thymic function in post-pubertal patients. However, the factors involved in thymus atrophy and regeneration are not fully understood, and approaches to stimulate rejuvenation of the thymus remain limited (12).

Clinical factors associated with thymic regrowth after chemotherapy have been explored in previous studies. Information on the occurrence of TH following chemotherapy obtained from different age groups revealed that the renewal ability of the thymus may be influenced by host age, and TH after chemotherapy is more common in younger populations with greater amounts of residual thymic tissues and higher thymic activity (4–11). Whereas, thymic regrowth is not likely associated with the tumor types and treatment. It can occur following treatment for various malignancies, of which malignant lymphoma is the most common (8). Besides, TH after cessation of chemotherapy does not appear to be influenced by the degree of lymphocyte depletion, but appears to be a common response to the withdrawal of chemotherapy (4, 6). There is also evidence that TH after chemotherapy maybe associated with endocrine dysfunctions (13). However, the factors mentioned above need to be further validated, since the knowledge of TH after chemotherapy was mainly obtained from analysis of small patient groups, and other potential factors related with TH have not been elicited.

Interleukin-7 (IL-7) acts as a non-redundant cytokine in thymic development (14). Under conditions of lymphopenia, it could optimize the long-term recovery of T cell diversity by restoring thymic function (15, 16). In patients with human immunodeficiency virus (HIV) infection, those with higher baseline levels of IL-7 had a higher incidence of TH after therapy (17). Patients with rheumatoid arthritis and type 1 diabetes demonstrating positive responses to treatment exhibited raised plasma IL-7 levels concurrent with renewed thymopoeisis (18, 19). Thus, IL-7 may be an important factor in thymic regeneration after injury. The responsiveness of IL-7 is dependent on the expression of the IL-7 receptor (IL-7R), which is a heterodimer consisting of the common γ-chain and the α-chain (IL-7Rα) (20, 21). The gene encoding IL-7Rα (IL7R) is polymorphic, and single-nucleotide polymorphisms (SNPs) in IL7R could potentially affect its transcription, thereby influencing IL-7Rα expression levels and signal transduction (22–26). IL7R polymorphisms are likely to modulate the regulation, differentiation, and function of T cell subsets and are associated with the susceptibility to autoimmune diseases, the pathogenesis of graft-versus-host disease after hematopoietic stem cell transplantation (HSCT), and T cell repopulation after lymphocytopenia caused by HIV infection and HSCT (22–32). Moreover, SNPs in IL7R may influence thymic T cell development in patients with multiple sclerosis (MS) (25), indicating a possible role for these SNPs in the process of thymic regeneration after chemotherapy.

Considering these elements, the present study was aimed at examining clinical predictors for the occurrence of TH in a group of adult patients undergoing chemotherapy for lymphoma and exploring the possible contribution of IL7R polymorphisms to thymic renewal capacity by detecting possible links between IL7R SNPs and the recovery of thymic volume and output function after chemotherapy.

Reactive TH after chemotherapy is a well-documented phenomenon in children, adolescents, and young adults, but is rare in older patients, suggesting a high thymic regenerative capacity in the young (4, 5, 8–11). However, by monitoring thymic output, it was determined that substantial output is maintained into late adulthood despite the decline of thymic function with age, and even the aged thymus retains the ability to renew thymopoiesis (34). This study further explored the influence of age on the renewal capacity of the thymus following chemotherapy. In the cohort of adults accepting chemotherapy for lymphoma, TH was observed in 38 (28.4%) cases aged 18–53 years (median 30), who were much younger, with larger thymic volumes and higher thymic output before treatment than those without TH, suggesting a high renewal capacity was retained in the thymus of young adults. In contrast, TH was observed in none of the elderly, possibly due to a decreased regenerative capacity of the thymus with age, which could represent a decline in the intrinsic capacity of lymphopoietic stem cells and/or changes in the thymic microenvironment (7). Besides, by multivariate analysis, patient age under 30 was revealed as an independent predictor for the occurrence of TH, further supporting the conclusion that thymic renewal capacity after chemotherapy is age-dependent. Consistent with this, we have revealed in an earlier study that, the majority (70%) of TH following chemotherapy was found in individuals under 30 years, although it can occasionally be observed in middle-aged patients (10). Furthermore, the renewal of thymopoiesis following chemotherapy was evaluated in the present study. The fast restoration of thymic output within 1 year of follow-up observed in these adults suggested a renewed thymopoiesis. Thymus regeneration, manifesting as partial reversal of thymic atrophy concurrent with a renewed thymopoiesis, in young adults undergoing chemotherapy would contribute to immune recovery, enhancing the clinical relevance of developing strategies to booster thymic activity in this population (2, 5, 12).

Clinical features except for patient age associated with thymic regrowth after chemotherapy have not been studied in-depth. Based on previous observations, it was seemed that, TH is neither tumor nor treatment specific (4, 6, 8). This study revealed that, patients with TH after chemotherapy were more commonly diagnosed with HL than those without TH. While, by multivariate analysis, the diagnosis of HL was not associated with the occurrence of TH independently, it can be conceived that, the lower age of patients with HL may contribute to a higher probability of thymic regrowth. Whether thymic regrowth following chemotherapy is correlated with the biological properties of the tumors remains unknown. It should be reminded that, the influence of rituximab usage on the occurrence of TH post-chemotherapy cannot be excluded, since HL patients were treated with chemotherapy only, in contrast with patients with B cell lymphoma accepted chemotherapy combined with rituximab. Emerging data suggest that rituximab has an inhibitory effect on effector T cells and could induce regulatory T cells expansion, which may be related to B cell deprivation resulting in impaired antigen-presenting cells activity and reduced production of co-stimulatory and immuno-modulatory molecules taking part in T cell activation and interactions; besides, it could exert a direct effect on T cells by binding to a specific T cell subset expressing CD20 (35). A recent study confirmed that, CD20 was also expressed on mature medullary thymocytes (36). Therefore, rituximab may intervene T cell development in the thymus by targeting CD20⁺ thymocytes. Unfortunately, this study design precluded an analysis for the effect of rituximab. Whether the use of rituximab could affect thymic activity and thus delay the regeneration of the thymus in patients with B cell lymphoma deserves further studies. Moreover, TH was demonstrated to be caused by chemotherapy-induced gonadal atrophy, which results in increased luteinizing hormone secretion (13), and sex steroid ablation has been regarded as an important candidate strategy for the improvement of thymic regeneration (12). Thus, the sex-related endocrine status is presumed to influence the renewal capacity of the thymus after chemotherapy. However, sex ratio was comparable in cases with and without TH in this study, excluding sex as a predictor for TH. Measurement of sex-related endocrine function is warranted to clarify this issue. In addition, the occurrence of TH in this cohort of adults we studied was not influenced by the size of peripheral CD4⁺ T cell pool as expected, since renewed thymopoiesis correlated with TH play a limited role in the early repopulation of peripheral CD4⁺ T cells following chemotherapy in adults (10).

The mechanisms for the reconstitution of thymic cellularity, accompanied by the restoration of thymic T cell development, after the removal of chemo-therapeutic toxicity is complex (37, 38). Interleukin-7, a growth and anti-apoptotic factor for T cells, was found to be associated with thymus enlargement in HIV patients undergoing antiretroviral therapy, suggesting an important role of IL-7 in thymus regrowth (17). Consistent with this findings, administration of recombinant human IL-7 was shown to increase the numbers of RTEs and levels of TRECs and results in a broadening of the T-cell receptor repertoire, indicating increased thymopoiesis (39). The response of thymocytes to IL-7 correlates with IL-7Rα expression. Accordingly, this study focused on four specific SNPs within the IL7R locus, known to influence the IL-7Rα expression on T cells (23–25), and explored their potential contributions to the thymic regeneration after chemotherapy in adults with lymphoma. It was found that the frequencies of the minor allele T and the TT + GT genotype of rs7718919, located in the promoter region of IL7R, were higher in patients with TH compared with those without TH. These results provided a possible link between rs7718919 and thymus enlargement after chemotherapy. Considering rs7718919 could potentially influence the expression of IL-7Rα as a transcription factor binding site (30), it is conceivable that it may affect IL-7-mediated regulation of thymocytes, and thus be implicated in thymic regrowth. However, this association between rs7718919 and the occurrence of TH was not confirmed by multivariate analysis. Moreover, we did not observe an accelerated recovery of thymic output after chemotherapy in patients with TT + GT genotype compared with GG genotype carriers, suggesting that the presence of the rs7718919 T allele had limited effects on the restoration of thymopoiesis. Consistent with these results, Shamim et al. (31) were unable to demonstrate any association between IL7R polymorphisms and TREC levels before or after HSCT in a Danish cohort. Nevertheless, the influence of rs7718919 polymorphisms on the renewal of thymopoiesis should be carefully evaluated. As IL-7Rα expression is finely tuned and differentially regulated during thymocyte development (21), it is important to better understand which thymocyte subset could be influenced by the altered IL-7 signaling associated with rs7718919 and to what extent this could affect thymic T cell development.

This study also investigated rs6897932, a missense polymorphism located in exon 6 of IL7R, which encodes the trans-membrane region of IL-7Rα. The C allele of rs6897932 is associated with increased skipping of exon 6, resulting in a higher ratio of membrane-bound vs. soluble receptor, which could cause reduced IL-7Rα expression on T cells (26). In a Danish cohort, homozygosity for the rs6897932 T-allele was associated with more rapid CD4⁺ T-cell recovery after the initiation of antiretroviral therapy (28). Unfortunately, the design of that study did not allow evaluation of whether the increase in CD4⁺ T cells was caused by increased thymopoiesis or expansion of the peripheral T-cell pool. Our study revealed no significant association of rs6897932 polymorphisms with either thymic enlargement or the restoration of thymic output, indicating a limited role for rs6897932 variants in thymopoiesis recovery after chemotherapy. This is consistent with the report by Broux et al. (25) of no influence of rs6897932 polymorphisms on thymopoiesis, as measured by the frequency of CD31⁺ RTEs in MS patients and healthy controls. Further studies investigating the potential effects of rs6897932 on the expression of IL-7Rα and its possible influence on the affinity and/or signaling of IL-7Rα in various thymocyte subsets are warranted to elucidate its role on thymopoietic activity. In addition, rs11567685, located in the promoter region of IL7R, was included in this study. It could potentially influence thymic development and peripheral homeostasis of T cells by altering the expression of IL-7Rα on CD4⁺ T cells in MS patients (25). Unfortunately, the effect of rs11567685 on the recovery of thymic cellularity and function after chemotherapy in this study cannot be evaluated, due to its low minor allele frequency.

This study has some limitations. Our observations were based on relatively few cases, and this may have masked significant effects due to lack of statistical power. In addition, thymic output measured by determination of sjTREC and CD31⁺ RTE may be influenced by the proliferation of peripheral T cells, potentially leading to an underestimation of thymic activity (40, 41). Hence, future studies with larger sample sizes and more accurate tools for thymic output measurement are necessary to further define the factors associated with thymic regrowth after chemotherapy. Moreover, evaluating the effect of rituximab usage on thymus regeneration following chemotherapy by comparing the occurrence of TH and the recovery of thymic output in two groups with comparable clinical features, including age, sex, disease type and stage, thymic size, and output function at baseline, is warranted in future studies.

To conclude, these findings suggest that patient age is an independent predictor for thymic regrowth after chemotherapy, which should promote awareness among physicians to make a timely diagnosis of TH in young adults and help physicians to prioritize intervention strategies for thymus rejuvenation after chemotherapy in this population. Besides, the potential link between the IL7R SNP and thymic regrowth after chemotherapy warrant further investigations, given the key roles of IL-7 and IL-7Rα in thymic development. These results will contribute to an improved understanding of the mechanisms involved in thymic regeneration.

Ethics approval and consent to participate was obtained. In accordance with the 1975 guidelines of the Declaration of Helsinki and after obtaining approval for the study from the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, the medical staff of each participating center informed their patients of the potential risks and benefits of the planned treatment and the need for periodical clinical and laboratory checkups in detail. Informed consent was obtained from all the subjects involved in this study.

WX and J-YL conceived and designed the study; D-PS, LW, C-YD, and J-HL contributed to the experimental design and provided intellectual input; D-PS and Y-JW performed experiments; LW, H-YZ, and LF contributed patients and provided clinical data; D-PS and LW analyzed data and wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.