**4. Surgical thymic ablation**

Thymus anatomically locates in the anterior mediastinum, just behind the sternum, facing the heart and the great vessels. This location places the thymus in the surgical field of critical open heart surgery. Since short-time consequences of either children or adult thymectomy have not been reported, thymus is partially or completely removed during this surgical procedure. However, recent studies suggest that individuals thymectomized during early childhood could have an immunological misbalance later on adulthood. Despite still a controversial topic, a continuously increasing body of knowledge supports the choice of preserving, as far as possible, thymus integrity.

#### **4.1 Neonatal thymectomy during congenital heart disease correction**

Congenital heart disease (CHD) comprises aberrant embryonic development or failure to progress beyond some early stage during fetal development of any cardiac structure. Septal or cyanotic defects, defects causing obstruction in either the heart or blood vessels and complex abnormalities are encompassed into CHD. Complex multifactorial genetic and environmental causes, rather chromosomal aberrations or single gene mutation (less than

mortality rates (Nahmias et al., 1998) when compared with children with preserved thymic

Atrophy implies thymic function reduction and it has been long time assumed that TL repertory was fixed during childhood and the thymus was no longer needed during adult life. However, different studies showed that adult thymus not only is functional but it can even booster its function to expedite immune reconstitution in different scenarios as chemotherapy (Mackall et al., 1995) or HIV-infected patients under highly active

During elderly, thymus shows even more reduced functionality. However, despite TES reduction and architectural changes, elderly thymus is able to maintain a certain degree of thymopoiesis (Jamieson et al., 1999). Thymopoietic degree is widely heterogeneous among individuals (Figure 3). In addition, thymic function in elderly people has an active role in the peripheral immune system rejuvenation (Ferrando-Martínez et al., 2009). Higher thymic function in elderly human is associated with a better preservation of the immune system. To add "insult to injury", thymic failure in healthy elderly leads to a discrete lymphopenia and naive T cell drop that eventually allows non-antigen driven homeostatic proliferation (Ferrando-Martinez et al., 2011). Homeostatic proliferation correlates with naive TL gathering age-related defects leading to irresponsiveness. Despite causality still needs to be assessed, altogether these results strongly suggest that elderly thymic function preservation or rejuvenation could be of great importance to ameliorate the age-related immune system deterioration. In fact, recent results show that thymic function, accurately measured with the sj/β-TREC ratio quantification method, in healthy elderly subjects (aged over 65) is independently associated with two-years all-cause mortality (Ferrando-Martínez et al., 2011b).Thus, elderly thymic function still has an active role in the maintenance of the immune system and this information should be taken into account to design innovative therapies capable of improve elderly quality of life through the immune system

Thymus anatomically locates in the anterior mediastinum, just behind the sternum, facing the heart and the great vessels. This location places the thymus in the surgical field of critical open heart surgery. Since short-time consequences of either children or adult thymectomy have not been reported, thymus is partially or completely removed during this surgical procedure. However, recent studies suggest that individuals thymectomized during early childhood could have an immunological misbalance later on adulthood. Despite still a controversial topic, a continuously increasing body of knowledge supports the choice of

Congenital heart disease (CHD) comprises aberrant embryonic development or failure to progress beyond some early stage during fetal development of any cardiac structure. Septal or cyanotic defects, defects causing obstruction in either the heart or blood vessels and complex abnormalities are encompassed into CHD. Complex multifactorial genetic and environmental causes, rather chromosomal aberrations or single gene mutation (less than

function (usually infected at the birth canal).

rejuvenation.

**4. Surgical thymic ablation** 

preserving, as far as possible, thymus integrity.

**4.1 Neonatal thymectomy during congenital heart disease correction** 

**3.2 Thymic failure during adulthood and elderly** 

antiretroviral therapy (HAART) (de la Rosa and Leal, 2003).

Fig. 3. Heterogeneity of the thymic function. Functionality was measured by ex-vivo quantification of DP thymocytes. Green arrows show high thymic function among elderly individuals (older than 65) while the red arrow show lack of functionality in adult individuals (50 to 60 years old). Modified from Ferrando-Martinez et al., 2009.

10% of diagnosed CHD), are the origin this disease. Prevalence in general population is estimated in 1% of all live deliveries. However, frequency rises up to 4% for childbirths of women which have been diagnosed with CHD during childhood. Disorders showing mild seriousness usually need little or no medical treatment lifetime. However, CHD can be a life-threatening condition. Since CHD is a progressive disease from prenatal to adulthood, severe conditions can be lethal immediately after birth and also during child/adulthood. Deathly forms of CHD usually need invasive surgery to guarantee the patient's survival. All forms together, more than 85% of diagnosed newborns reach adulthood thanks to positive adaptation to their condition (non-severe forms) or to successful medical and surgical interventions (lethal forms). Over the last 30 years, neonatal surgery to correct serious CHD has become usual and, since surgical access is obstructed by the thymus, thymectomy is a common practice.

Several studies, with case-control designs, assessed the potential immune failure of thymectomyzed children. Despite data are scarce and methodology heterogeneous, some conclusion can be already extracted. When short-term consequences are analyzed (less than one year after surgery) low peripheral lymphocyte counts are already reported (Wells et al., 1998; Turan et al., 2004). From the lymphocyte populations, T cells are preferentially decreased. Normal responsiveness to mitogens and none clinical consequence have been reported despite the described lymphopenia. Other studies focused in children that underwent surgery one to five years before, in order to analyze medium-term consequences of thymectomy. The fast drop of the lymphocyte populations was still observed, suggesting that peripheral homeostasis is not enough to maintain normal lymphocyte numbers during childhood (Halnon et al., 2005; Mancebo et al., 2006). In addition, low sj-TREC levels are also associated with thymectomy. Low levels of sj-TREC per million of cells in the thymectomyzed group has been associated with insufficient thymic function. Therefore, despite thymectomy is only partial in some individuals and thymic regeneration could be observed in some reoperated children, the general appreciation is that remaining thymopoietic degree will never reach normal levels in early thymectomyzed individuals. However it should be noted that sj-TREC counts are not a good measure of thymic function, especially when high peripheral proliferation is involved. On account of thymectomyzed individuals evidence low lymphocyte counts early after the surgical procedure, it can be assumed that homeostatic-driven proliferation will try to adjust lymphopenia to normal levels. High proliferation rates will dilute the sj-TREC content, disregarding changes of thymic function. Consequently, even if inefficient thymic function is indisputable due to persistently low lymphocyte counts, thymic function dynamics need to be further analyzed. It is noteworthy that medium-time costs of thymectomy still do not involve any clinical consequence.

In addition, long-term consequences should be evaluated. Interestingly, long-term studies also report persistent lymphopenia (preferentially T lymphocyte drops, as expected) and maintained low sj-TREC levels. However, when 20 to 30 years old individuals are analyzed, a trend to normalization in naive TL is observed, despite not in all studies. On the other hand Eysteinsdottir et al., 2004 reported new immune defects, as impaired Th2 T cell response despite normal Th1 response. In this study, thymectomyzed subjects also present higher numbers of neutrophils and low platelet counts but normal B lymphocyte and natural killer (NK) cell numbers. Low naive T cell counts, high proliferation rates among the naive subset and increased IL-7 bioavailability have been also observed (Prelog et al., 2009). This later result is especially interesting, since this naive T lymphocytes alteration is also a hallmark feature of immune senescence on healthy aging (Ferrando-Martínez et al., 2011). Whether this non-antigen driven proliferation is also leading to a naive T cell pool exhaustion (described in age-related changes of naive TL) needs to be explored.

Accordingly, more recent works specifically focused on premature aging. Immune senescence, or *immunosenescence*, comprises the age-related changes of the immune system that gather phenotypic and functional defects in both, the innate and adaptive immune response. It is generally accepted that thymic atrophy, as the first age-related change, triggers the accumulation of lymphocyte frailty. Despite causality needs to be established, the prevalence and severity of infectious diseases, probable direct immunosenescence consequence, discloses de outstanding importance of the age-related immune exhaustion. Immune senescence has also been linked to lack of vaccination response, autoimmunity and increase in oncological pathology in elderly subjects. Other clinical situations involving immune weakness (as HIV-infection or immune reconstitution after extensive chemotherapy) have been analyzed under this point of view, interestingly finding that TL which are forced to replenish a strongly damaged immune space usually show premature immunosenescence-related defects. Thymic function defects in early childhood can be an added model of premature immunosenescence. In this regard, thymectomyzed children show several features usually reported in chronological immunosenescence. Naive TL have increased proliferation rates, strong oligoclonal CMV-specific responses (that probably can accumulate because thymus is not able to replenish the immune space with

that peripheral homeostasis is not enough to maintain normal lymphocyte numbers during childhood (Halnon et al., 2005; Mancebo et al., 2006). In addition, low sj-TREC levels are also associated with thymectomy. Low levels of sj-TREC per million of cells in the thymectomyzed group has been associated with insufficient thymic function. Therefore, despite thymectomy is only partial in some individuals and thymic regeneration could be observed in some reoperated children, the general appreciation is that remaining thymopoietic degree will never reach normal levels in early thymectomyzed individuals. However it should be noted that sj-TREC counts are not a good measure of thymic function, especially when high peripheral proliferation is involved. On account of thymectomyzed individuals evidence low lymphocyte counts early after the surgical procedure, it can be assumed that homeostatic-driven proliferation will try to adjust lymphopenia to normal levels. High proliferation rates will dilute the sj-TREC content, disregarding changes of thymic function. Consequently, even if inefficient thymic function is indisputable due to persistently low lymphocyte counts, thymic function dynamics need to be further analyzed. It is noteworthy that medium-time costs of thymectomy still do not involve any clinical

In addition, long-term consequences should be evaluated. Interestingly, long-term studies also report persistent lymphopenia (preferentially T lymphocyte drops, as expected) and maintained low sj-TREC levels. However, when 20 to 30 years old individuals are analyzed, a trend to normalization in naive TL is observed, despite not in all studies. On the other hand Eysteinsdottir et al., 2004 reported new immune defects, as impaired Th2 T cell response despite normal Th1 response. In this study, thymectomyzed subjects also present higher numbers of neutrophils and low platelet counts but normal B lymphocyte and natural killer (NK) cell numbers. Low naive T cell counts, high proliferation rates among the naive subset and increased IL-7 bioavailability have been also observed (Prelog et al., 2009). This later result is especially interesting, since this naive T lymphocytes alteration is also a hallmark feature of immune senescence on healthy aging (Ferrando-Martínez et al., 2011). Whether this non-antigen driven proliferation is also leading to a naive T cell pool

exhaustion (described in age-related changes of naive TL) needs to be explored.

Accordingly, more recent works specifically focused on premature aging. Immune senescence, or *immunosenescence*, comprises the age-related changes of the immune system that gather phenotypic and functional defects in both, the innate and adaptive immune response. It is generally accepted that thymic atrophy, as the first age-related change, triggers the accumulation of lymphocyte frailty. Despite causality needs to be established, the prevalence and severity of infectious diseases, probable direct immunosenescence consequence, discloses de outstanding importance of the age-related immune exhaustion. Immune senescence has also been linked to lack of vaccination response, autoimmunity and increase in oncological pathology in elderly subjects. Other clinical situations involving immune weakness (as HIV-infection or immune reconstitution after extensive chemotherapy) have been analyzed under this point of view, interestingly finding that TL which are forced to replenish a strongly damaged immune space usually show premature immunosenescence-related defects. Thymic function defects in early childhood can be an added model of premature immunosenescence. In this regard, thymectomyzed children show several features usually reported in chronological immunosenescence. Naive TL have increased proliferation rates, strong oligoclonal CMV-specific responses (that probably can accumulate because thymus is not able to replenish the immune space with

consequence.

naive TL), CD57-expressing exhausted cells or systemic inflammation biomarkers (Sauce et al., 2009). Lack of vaccine response, as elderly individuals, seems to focus on new antigens, despite normal memory responses (Zlamy et al., 2010). As a difference, thymectomyzed children have delayed vaccine response, rather that complete lack of immunization.

Finally, some studies report about normal TL counts after 20 – 30 years post-thymectomy, suggesting a reestablishment of thymic function later on life. Van Gent et al. (2011) report normal naive T cell numbers, adjusted proliferation rates and sj-TRECs numbers, concluding that thymectomization has an early impact on the LT compartment that will be restored later on life. Thymic regeneration (as measure by image techniques and inferred by sj-TREC dynamics) is proposed as major restoration mechanism. The reason why some cohorts can observe thymic and LT renewal (van Gen et al., 2011) while other do not (Prelog et al., 2009), remains a matter of debate. Partial results and animal models point to the possibility that younger thymus (months after birth) could have higher epithelial precursor content and, then, higher regeneration potential. However, further studies are needed to better clarify whether thymus actually have an age-related drop of regeneration capability. The lack of clinical consequences reported by all studies could be explained by thymic renewal and immune system restoration during adulthood. Despite all these results are very reassuring, it should be note that thymectomyzed individuals grow up with a frail immune system the first five to ten years of life and premature immunosenescence features have been reported in young adulthood (20 – 30 years old), suggesting that early thymectomy have immune consequences that, despite not being overwhelming, could have clinical consequences once premature immunosenescence joins the age-related exhaustion of the immune system.

#### **4.2 Adult thymectomy: Open heart surgery**

Valve replacement and ischemic cardiopathy are common open heart surgical procedures in adulthood / elderly subjects. Despite neonatal thymus size is larger than the atrophied adult thymus is; thymectomy to gain unrestricted view of the operation site is performed at any age. Moreover, the widespread belief that thymus from adult individuals completely lacks of functionality makes it usually despised. Anyhow, several factors should be pondered. First of all, despite the atrophy, elderly thymus still impacts on the peripheral T cell pool rejuvenation (Ferrando-Martínez et al., 2009). The more important, thymic function failure, in subjects over 65, predicts two-year all-cause mortality (Figure 4) (Ferrando-Martínez et al., 2011b). Moreover, myasthenia gravis patients (neuromuscular autoimmune disease where thymectomy is associated with better prognosis by unknown mechanisms) benefit of complete thymectomy even if thymus is already atrophied (Chen et al., 2011). All results together strongly suggest that atrophied thymus is active in different ways rather than an inactive surplus.

In addition, immune system of adult and elderly subjects undergoing open heart surgery already present exhaustion and senescence features. Thus, short- and medium-time defects induced by thymectomy (clearly present in children up to five to ten years old) will be acting on a previously damaged system. Clinical consequences of this damage have not been studied yet. Associated comorbidities of an elderly cohort with major cardiac illness is not easy, but further studies are needed to evaluate the potential effect of thymus removal on survival rates.

Fig. 4. Relationship between thymic function and two-year all-cause mortality in healthy elderly. Modified from Ferrando-Martínez et al., 2011b.
