**2.2 T Lymphocyte (TL) maturation**

Thymus is the major site of maturation of TL. Bone marrow-derived lymphoid precursor entering the thymus commit to the T lineage. Maturation processes begin in the cortex and thymocytes move toward the medulla as they progress in the tuition program. Thymus is not only the provider of new mature naive TL to preserve the immune system integrity but also ensures that all departing cells are functional and non-self-reactive.

As shown in Figure 2 most immature cells of the T lineage enter the thymic cortex through the blood vessels. After a proliferation step Pro-T thymocytes, CD4-CD8- double negative (DN) irresponsive cells that still do not express the T cell receptor (TCR), start expressing the terminal deoxynucleotidyl transferase (TdT) enzyme, which adds random nucleotides to the TCR genomic sequence to increase lymphocyte diversity. Expression of the Rag-1 and Rag-2 proteins, necessary for the TCR rearrangement, marks the pre-T cell stage. Pre-T cells are still DN thymocytes. Beta chain of the TCR is rearranged at this stage. Β-chain rearrangement success is tested in the cellular membrane using a pre-T α-chain. Expression of a functional β-chain triggers another proliferation round and the entry into the CD4+CD8+ double positive (DP) stage. Thymus atrophy diminishes TES lymphoid components by impairing this proliferation step. As a consequence, DP numbers, or DN to DP proliferation rates, are the best indicative of thymic functionality. DP thymocytes rearrange then the α-chain of the TCR. Once a functional αβ TCR protein is expressed, DP thymocytes start a strict scrutiny program. TCR affinity and avidity is checked from every thymocyte. Positive selection ensures that only cells presenting a functional TCR reach the next step. Thymocytes that do not receive a positive signaling thru a successful TCR

recognition go to programmed cell death. Afterwards, a negative selection warrants that survivors do not have self-reactivity. Thymocytes with strong TCR signaling due to selfantigen recognition will be also directed to cell death. A small percentage of DP thymocytes, approximately 1%, qualify to further maturation. Thymocytes migrate to the medullar space expressing a fully functional non-self-reactive TCR. CD4 and CD8 co-receptor expression is modulated in this stage and mature thymocyte are CD4 or CD8 single positive (SP) cells. Thymocyte maturation and selection need the stimuli provided by the thymic microenvironment. TECs, bone marrow-derived macrophages and dendritic cells locate in the pathways of thymocyte migration, allowing physical interactions necessary for the maturation process. MHC class I and class II molecules (expressed by epithelial and

dendritic cells); cytokines and chemokines (secreted by thymic stromal cells) provide signaling for selection process, stimulate thymocyte proliferation and coordinate the cortical to medullary transit. Therefore, non-lymphoid thymic cells play a crucial role in the immune system generation.

#### **2.3 Thymic function-related markers**

Study of thymic function is of great interest either to determine immune system impairment in lymphopenia scenarios or to monitor immune reconstitution. However, despite the necessity, lack of accurate measurement tools has hampered thymic output assessment. Due to its anatomical situation (in the anterior mediastinum, above the heart and ahead the great vessels) thymic biopsies are formally contraindicated and indirect measurements are needed. Thymic volume determination by thoracic computed tomography (CT) or magnetic resonance imaging (MRI) is an accurate indirect approximation and has been very useful to determine the role of thymic function in HIV-infected patients' immune reconstitution

recognition go to programmed cell death. Afterwards, a negative selection warrants that survivors do not have self-reactivity. Thymocytes with strong TCR signaling due to selfantigen recognition will be also directed to cell death. A small percentage of DP thymocytes, approximately 1%, qualify to further maturation. Thymocytes migrate to the medullar space expressing a fully functional non-self-reactive TCR. CD4 and CD8 co-receptor expression is modulated in this stage and mature thymocyte are CD4 or CD8 single positive (SP) cells. Thymocyte maturation and selection need the stimuli provided by the thymic microenvironment. TECs, bone marrow-derived macrophages and dendritic cells locate in the pathways of thymocyte migration, allowing physical interactions necessary for the maturation process. MHC class I and class II molecules (expressed by epithelial and dendritic cells); cytokines and chemokines (secreted by thymic stromal cells) provide signaling for selection process, stimulate thymocyte proliferation and coordinate the cortical to medullary transit. Therefore, non-lymphoid thymic cells play a crucial role in the immune

Study of thymic function is of great interest either to determine immune system impairment in lymphopenia scenarios or to monitor immune reconstitution. However, despite the necessity, lack of accurate measurement tools has hampered thymic output assessment. Due to its anatomical situation (in the anterior mediastinum, above the heart and ahead the great vessels) thymic biopsies are formally contraindicated and indirect measurements are needed. Thymic volume determination by thoracic computed tomography (CT) or magnetic resonance imaging (MRI) is an accurate indirect approximation and has been very useful to determine the role of thymic function in HIV-infected patients' immune reconstitution

Fig. 2. Thymocyte maturation process.

**2.3 Thymic function-related markers** 

system generation.

(Ruiz-Mateos et al. 2004). Nevertheless, both are expensive techniques where thymic inference needs specifically trained radiologists. Moreover, thoracic CT generates high levels of radiation. Thus, thymic function-related markers that could be determined from peripheral blood samples have usually been first choice for thymic output determination.

Naive TL quantification was the first proposed related-marker and is still being used. Controversy exist regarding the surface markers election that best discriminate the naive subset by flow cytometry. High expression of CD45RA, CCR7 and CD27 are commonly used to categorize this TL subset, and CD45RA+CD27+ or CD45RA+CCR7+ phenotypes seem to both be accurate enough to identify naive T cells (Ferrando-Martínez et al., 2010). However, naive TL are defined as mature lymphocytes that still have not find their specific antigen (non-experienced lymphocytes), disregarding whether they are recent thymic emigrants (RTEs) or long-lived cells. As a consequence, surface markers that could truthfully identify RTEs have been long chased. CD31-positive naive TL have very short proliferative history and are a RTE-enriched population (Junge et al., 2007). Moreover, protein tyrosine kinase 7 (PTK7)-expressing lymphcytes are proposed as precursor cells for later differentiation into mature naive TL (Haines et al., 2009). However, a marker that specifically characterizes mature thymocytes leaving the thymus is still missing.

T cell receptor excision circles (TREC) description was a milestone in thymic function quantification. TRECs are circular episomal DNA discarded through TCR rearrangement processes that can only be thymically produced and lack of replication origin (they cannot be copied in periphery). Signal-joint (sj)-TREC quantification, discarded product of the αchain rearrangement at DP stage, was described as a new thymic output-related marker (Douek et al., 1998). However, TREC counts are deeply affected by peripheral proliferation (Hazenberg et al., 2000), increasing the difficulty of interpretation (Harris et al., 2005). Moreover, subsequent mathematical models showed that, even in the steady state, the sj-TREC content is not a good measurement of thymic function (Ribeiro and Perelson, 2007). To overcome these limitations, Dion et al. (2004) proposed an elegant technique to quantify the ratio between sj-TREC and the β-TRECs, discarded during the β-chain rearrangement at a more immature stage (see Figure 2). A proliferation step, directly related to thymic function, occurs between the β- and the α-chain rearrangement. Thus, the sj/β-TREC ratio is an indirect measure of this intrathymic proliferation step and a thymic function-related marker. In addition, since the proportion between both TREC types is evaluated, rather than TREC numbers, the results are not affected by peripheral proliferation, even if absolute TREC numbers are. Recently, we have described a simplified version of this technique that allows an accurate and time- and cost-effective quantification of human thymic function from peripheral blood samples (Ferrando-Martínez et al., 2010b). Table 1 summarizes different techniques that have been used to determine thymic function together with their advantages and disadvantages.
