**4. Outcomes of age-related thymic atrophy**

Overt outcomes of age-related thymic atrophy include reduction of functional naïve T cells, which is related to a decline in T cell receptor (TCR) repertoire diversity [8, 55, 83, 84]. However, the atrophied thymus is still functioning, albeit with limitations, in the elderly, continuing to select T cells for the lifetime of the individual. This causes a potential for the atrophied thymus to generate harmful T cells that could increase autoimmune predisposition the elderly [26]. Therefore, we will review recent research progress regarding this area of concern.

#### **4.1 Decreased naïve conventional T cell output**

As stated previously, the most readily observed outcome of age-related thymic involution is the decline in thymic output, which includes reduced naïve conventional T (Tcon) cell output over time [85] and fewer recent thymic emigrants (RTEs) [8]. However, peripheral T cell numbers are not decreased in aged individuals [36, 86, 87]. The actual effect is an overall diminished TCR repertoire diversity observed in the aged peripheral T cell pool [8, 55, 83, 84], which is due to oligoclonal expansion of memory T cells along with insufficient RTE output. This has been suggested to contribute to the decreased capacity for new immune responses to infection and poor vaccination efficacy, which are typical phenotypes of immunosenescence [17–20], observed in the elderly [35].

This phenotype has been recapitulated in FoxN1 cKO mice, which have accelerated aging in the thymus, but maintain a young periphery, as they exhibit impaired peripheral T cell responses in infection with influenza virus [88]. This study also demonstrated a direct role for thymic atrophy in the impairment of T cell function during aging.

#### **4.2 Increased self-reactive conventional T cells due to perturbed negative selection**

In light of the alterations in thymocyte number and diminished naïve T cell output with age-related thymic atrophy, it is of paramount importance to understand the effects of the altered thymic micro-environment on central tolerance establishment of the thymocytes that are still being developed in the atrophied thymus.

Under the current paradigm, negative selection is the process by which thymocytes with high affinity for self-peptides presented by MHC are deleted from the developing thymocyte repertoire via apoptosis [2, 38, 89]. Studies also show that when these high affinity TCRs receive strong signaling, negative selection takes place [90, 91]. However, the TCR signaling strength is not based solely on TCR affinity, but is also influenced by avidity, or the quantity of interactions between self-peptide/MHC (self-pMHC) complexes and the TCR (**Figure 2**). Therefore, if the thymocyte-intrinsic factors (i.e., TCR affinity and number), of self-reactive thymocytes are unchanged, the TCR signaling strength varies based on the ability of effective self-pMHC-II expression. In other words, if self-antigen can be normally presented in the MHC-II groove, the reciprocal TCR signaling should be produced through a strong interaction. We know that MHC-II is expressed on mTECs, however, aging induces mTEC defects (**Figure 1b**), resulting in reduced capacity for self-pMHC-II ligand expression. Therefore, we suggest that a strong signaling strength shifts either to an intermediate strength, which favors CD4SPFoxP3+ tTreg cell generation (**Figure 2**, arrow-a), or to a low strength, which results in the generation of self-reactive thymocytes (**Figure 2**, arrow-b). The selfreactive thymocytes via this pathway are neither depleted nor shifted to Treg cells, but become Tcon cells that are released to the periphery. If they encounter specific self-tissues, they may become effector T (Teff) cells that can attack self-tissues and induce pathological inflammation.

The FoxN1 cKO mouse model is a useful model for studying the capacity for efficient self-pMHC-II ligand expression, because it exhibits a defect in the nonhematopoietic TECs, but maintains intrinsically normal hematopoietic lineage cells and a young periphery. We demonstrated that thymic involution perturbs negative selection, as revealed by the enhanced release of autoreactive interphotoreceptor retinoid-binding protein (IRBP)-specific Tcon cells from the atrophied thymus of FoxN1 cKO mice compared to the thymus from young normal controls [25]. This

**55**

**Figure 2.**

*Age-Related Thymic Atrophy: Mechanisms and Outcomes DOI: http://dx.doi.org/10.5772/intechopen.86412*

result is presumably due to decreased self-pMHC-II expression, confirmed via assessment of a mock self-antigen in normal versus atrophied thymus [92].

*Signaling strength decides self-reactive CD4SP T clone fates. Interaction between self-pMHC on mTEC and self-reactive TCR on CD4SP thymocyte produces thress types of signaling strength: a strong signal leads to negative selection, resulting in depletion, an intermediate signal leads to tTreg generation, and a weak signal results in thymocyte survival to differentiate into Tcon. Thymic aging (green arrow—a) shifts signaling strength from strong to intermediate and relatively enhances polyclonal tTreg generation; while some antigen-specific interactions exhibit an extremely weak signal, resulting in diminished antigen-specific tTreg cells and increased* 

As mentioned earlier, central tolerance establishment encompasses two mechanisms. The first mechanism, negative selection, is not entirely perfect [5] resulting in some self-reactive T clones being released into the periphery as Tcon cells. The

cell-mediated autoimmune suppression. It is believed that 80–95% of pTreg cells are generated within the thymus, as thymic-derived T regulatory (tTreg) cells [93–95]. Under the current paradigm, the processes of both negative selection and tTreg generation in the thymus utilize the same set of agonist self-peptides [93, 96]. Whether self-reactive thymocytes developing in the thymus are negatively selected or develop into tTreg cells depends on TCR signaling strength, or the sum of TCR affinity and avidity, (or the number of TCR interactions with self-peptide/MHC) when all other variables, such as IL-2, etc., are fixed. Put simply, strong signaling induces the apoptosis of self-reactive thymocytes, intermediate signaling leads to tTreg generation, and weak signaling results in the survival of thymocytes that differentiate into Tcon cells (**Figure 2**). This paradigm implies that depletion or survival for thymocytes is dependent on overall TCR signaling strength [38, 93]. Although there are cell extrinsic factors that can impact thymocyte development, such as the thymic cytokine milieu (IL-2 [97, 98], TGF-β [98, 99], etc.), we propose that there are two cell types that directly regulate TCR signaling strength. One is intrinsic to thymocytes and the other is intrinsic to TECs. When the TCR binds to self-peptide/MHC on an antigen presenting cell, the immunoreceptor tyrosine-based activation motifs (ITAMs) are activated and the Zap70 kinase is subsequently phosphorylated. A mouse model with a knock-in allele of TCR zeta (ζ) chain gene with tyrosine-to-phenylalanine mutations in 6 out of 10 ITAMs led to a 60% decrease in TCR signaling potential [100]. This mouse model exhibited a

peripheral Treg (pTreg)

**4.3 Changes in thymic-derived regulatory T cell generation**

second defense against self-reactivity is CD4SPFoxP3+

*antigen-specific Tcon cells (green arrow—b).*

#### **Figure 2.**

*Thymus*

during aging.

CD4SPFoxP3+

induce pathological inflammation.

**selection**

**4.1 Decreased naïve conventional T cell output**

nosenescence [17–20], observed in the elderly [35].

As stated previously, the most readily observed outcome of age-related thymic

This phenotype has been recapitulated in FoxN1 cKO mice, which have accelerated aging in the thymus, but maintain a young periphery, as they exhibit impaired peripheral T cell responses in infection with influenza virus [88]. This study also demonstrated a direct role for thymic atrophy in the impairment of T cell function

In light of the alterations in thymocyte number and diminished naïve T cell output with age-related thymic atrophy, it is of paramount importance to understand the effects of the altered thymic micro-environment on central tolerance establishment of the thymocytes that are still being developed in the atrophied thymus. Under the current paradigm, negative selection is the process by which thymocytes with high affinity for self-peptides presented by MHC are deleted from the developing thymocyte repertoire via apoptosis [2, 38, 89]. Studies also show that when these high affinity TCRs receive strong signaling, negative selection takes place [90, 91]. However, the TCR signaling strength is not based solely on TCR affinity, but is also influenced by avidity, or the quantity of interactions between self-peptide/MHC (self-pMHC) complexes and the TCR (**Figure 2**). Therefore, if the thymocyte-intrinsic factors (i.e., TCR affinity and number), of self-reactive thymocytes are unchanged, the TCR signaling strength varies based on the ability of effective self-pMHC-II expression. In other words, if self-antigen can be normally presented in the MHC-II groove, the reciprocal TCR signaling should be produced through a strong interaction. We know that MHC-II is expressed on mTECs, however, aging induces mTEC defects (**Figure 1b**), resulting in reduced capacity for self-pMHC-II ligand expression. Therefore, we suggest that a strong signaling strength shifts either to an intermediate strength, which favors

tTreg cell generation (**Figure 2**, arrow-a), or to a low strength, which

results in the generation of self-reactive thymocytes (**Figure 2**, arrow-b). The selfreactive thymocytes via this pathway are neither depleted nor shifted to Treg cells, but become Tcon cells that are released to the periphery. If they encounter specific self-tissues, they may become effector T (Teff) cells that can attack self-tissues and

The FoxN1 cKO mouse model is a useful model for studying the capacity for efficient self-pMHC-II ligand expression, because it exhibits a defect in the nonhematopoietic TECs, but maintains intrinsically normal hematopoietic lineage cells and a young periphery. We demonstrated that thymic involution perturbs negative selection, as revealed by the enhanced release of autoreactive interphotoreceptor retinoid-binding protein (IRBP)-specific Tcon cells from the atrophied thymus of FoxN1 cKO mice compared to the thymus from young normal controls [25]. This

**4.2 Increased self-reactive conventional T cells due to perturbed negative** 

involution is the decline in thymic output, which includes reduced naïve conventional T (Tcon) cell output over time [85] and fewer recent thymic emigrants (RTEs) [8]. However, peripheral T cell numbers are not decreased in aged individuals [36, 86, 87]. The actual effect is an overall diminished TCR repertoire diversity observed in the aged peripheral T cell pool [8, 55, 83, 84], which is due to oligoclonal expansion of memory T cells along with insufficient RTE output. This has been suggested to contribute to the decreased capacity for new immune responses to infection and poor vaccination efficacy, which are typical phenotypes of immu-

**54**

*Signaling strength decides self-reactive CD4SP T clone fates. Interaction between self-pMHC on mTEC and self-reactive TCR on CD4SP thymocyte produces thress types of signaling strength: a strong signal leads to negative selection, resulting in depletion, an intermediate signal leads to tTreg generation, and a weak signal results in thymocyte survival to differentiate into Tcon. Thymic aging (green arrow—a) shifts signaling strength from strong to intermediate and relatively enhances polyclonal tTreg generation; while some antigen-specific interactions exhibit an extremely weak signal, resulting in diminished antigen-specific tTreg cells and increased antigen-specific Tcon cells (green arrow—b).*

result is presumably due to decreased self-pMHC-II expression, confirmed via assessment of a mock self-antigen in normal versus atrophied thymus [92].

#### **4.3 Changes in thymic-derived regulatory T cell generation**

As mentioned earlier, central tolerance establishment encompasses two mechanisms. The first mechanism, negative selection, is not entirely perfect [5] resulting in some self-reactive T clones being released into the periphery as Tcon cells. The second defense against self-reactivity is CD4SPFoxP3+ peripheral Treg (pTreg) cell-mediated autoimmune suppression. It is believed that 80–95% of pTreg cells are generated within the thymus, as thymic-derived T regulatory (tTreg) cells [93–95]. Under the current paradigm, the processes of both negative selection and tTreg generation in the thymus utilize the same set of agonist self-peptides [93, 96]. Whether self-reactive thymocytes developing in the thymus are negatively selected or develop into tTreg cells depends on TCR signaling strength, or the sum of TCR affinity and avidity, (or the number of TCR interactions with self-peptide/MHC) when all other variables, such as IL-2, etc., are fixed. Put simply, strong signaling induces the apoptosis of self-reactive thymocytes, intermediate signaling leads to tTreg generation, and weak signaling results in the survival of thymocytes that differentiate into Tcon cells (**Figure 2**). This paradigm implies that depletion or survival for thymocytes is dependent on overall TCR signaling strength [38, 93].

Although there are cell extrinsic factors that can impact thymocyte development, such as the thymic cytokine milieu (IL-2 [97, 98], TGF-β [98, 99], etc.), we propose that there are two cell types that directly regulate TCR signaling strength. One is intrinsic to thymocytes and the other is intrinsic to TECs. When the TCR binds to self-peptide/MHC on an antigen presenting cell, the immunoreceptor tyrosine-based activation motifs (ITAMs) are activated and the Zap70 kinase is subsequently phosphorylated. A mouse model with a knock-in allele of TCR zeta (ζ) chain gene with tyrosine-to-phenylalanine mutations in 6 out of 10 ITAMs led to a 60% decrease in TCR signaling potential [100]. This mouse model exhibited a defect in negative selection, but an increase in tTreg generation [100]. The second variable is the relative expression level of self-peptide/MHC on TECs. Transgenic expression of a microRNA targeting the MHC class-II transactivator (CIITA) resulted in reduced MHC-II on mTECs [37], leading to insufficient mTEC presentation of self-peptides thus reducing the overall avidity of the TCR interaction with self-peptide/MHC. This also resulted in the enhancement of tTreg generation at the expense of negative selection [37].

In the aged thymus, as we mentioned earlier, mTECs are flawed and self-antigen cannot be normally presented in the MHC-II groove, which results in a diminished interaction with TCRs on developing thymocytes. This is similar to the second scenario described above, in which a defect exists in the TEC compartment causing reduced TCR signaling strength. We observed a relatively enhanced tTreg generation in the atrophied thymus, exhibiting no change in overall tTreg numbers, but an increased ratio of tTreg to tTcon cells in the aged, atrophied thymus compared to young controls [92]. This is probably a demonstration of the atrophied thymus attempting to compensate for defective negative selection [25] in order to maintain central T cell tolerance in the elderly.

If self-reactive TCR signaling strength is too low these thymocytes may neither be depleted nor form tTreg cells, but rather may directly differentiate into selfreactive Tcon cells. As an artifact of impaired promiscuous self-antigen expression in mTECs through an autoimmune regulator (*Aire*) knock-out model, the *Aire*dependent TCAF3 epitope of prostate antigen cannot be promiscuously expressed on mTECs [101]. This resulted in prostate-specific thymocytes, which should be negatively selected, but in contrast were redirected into prostate-reactive Tcon cells. The authors observed loss of prostate-specific tTreg cells for this same epitope, and heightened prostate-reactive Tcon cells that infiltrated the prostate of these mice causing auto-inflammatory lesions [102, 103]. Defects in self-peptide expression on mTECs due to protein knock-out [104], are beginning to suggest that some of the same impairments exhibited by the atrophied thymus, may impact antigen-specific (monoclonal) tTreg generation, meanwhile increasing this same self-antigen specific Tcon generation, despite an unchanged or increased total (polyclonal) tTreg population [105]. It will be interesting to see what further subtle implications the aging thymus has on central tolerance establishment via potentially altering certain self-tissue specific tTreg populations and altering the overall aged Treg TCR repertoire, in spite of a relatively increased aged polyclonal Treg population [92].

#### **4.4 Overall contribution to inflammaging**

Inflammaging or the age-related, persistent increase in basal pro-inflammatory phenotype, has long been thought to be primarily a result of senescent somatic cells exhibiting senescence-associated secretory phenotype (SASP) [30, 31, 34, 106]. However, it is has come to be appreciated that chronic immune activation in the elderly contributes to a pro-inflammatory secretory milieu. This activation in the elderly includes chronic innate immune activation, which may result from immunosenescence related to accumulation of memory T cells. Chronic innate immune activation is also attributed to long-term virus, such as cytomegalovirus (CMV) [29, 107, 108], infection; or a degeneration-associated autotoxic reaction [109]. However, age-related autoimmune predisposition (an adaptive immune activation), induced by adaptive immune reaction to self-tissues by self-reactive T cells, has recently been recognized as a potential factor and/or synergistic cause of chronic inflammation in the elderly [25, 34]. Therefore, the role of the adaptive immune system in mediating inflammaging, as a result of self-reactive T cell immune

**57**

*Age-Related Thymic Atrophy: Mechanisms and Outcomes DOI: http://dx.doi.org/10.5772/intechopen.86412*

thymus [25, 34].

serum) [121].

was restored to normal levels [81].

responses to self-tissue that increases with age, is directly related to the atrophied

Since it has recently been confirmed that the involuted thymus releases selfreactive Tcon cells as a result of perturbed negative selection, the direct implications of age-related thymic atrophy on the risks of inflammaging and the associated subclinical increase in the pro-inflammatory milieu has become more clear [25]. Subsequent alterations in tTreg development may also play an unappreciated role in the increased self-reactivity associated with aging, as changes in the tTreg repertoire may in fact impair sufficient suppression of appropriate self-reactivity in

Rejuvenation of aged thymic function is one of the strategies to reduce inflammaging because it can reduce self-reactive Tcon cell release and potentially readjust tTreg cell function so that the adaptive immune aspects of inflammaging may be ameliorated. Several strategies to rejuvenate the atrophied thymus have been reported, including: (1) TEC stem cell-based strategies, including utilization of human embryonic/pluripotent stem cells [110–112], FoxN1eGFP/+ knock-in epithelial

cells [113], young TEC-based [114] or inducible TEC-based [115] strategies;

(2) cytokine-to-TEC based therapy, such as keratinocyte growth factor (KGF) [116, 117] and IL-22 [118–120]; (3) genetically-based methods (enhancement of exogenous FoxN1 expression with FoxN1 cDNA plasmid and FoxN1 transgene) [79–81], and (4) epigenetically-based methods (via exosomes extracted from young healthy

As to the genetic rejuvenation strategy via exogenous FoxN1, intrathymic injection of plasmid vectors carrying FoxN1-cDNA into middle-aged and aged mice was able to partially rescue thymic atrophy and function. The investigators observed increased thymic size and thymocyte number in the treated group compared to mice receiving empty vector [79]. Another group utilized an inducible FoxN1 overexpression reporter gene system, and it was demonstrated that *in vivo* upregulation of FoxN1 expression in middle-aged and aged mice resulted in increased thymic size and thymocyte numbers as well as increased numbers of early thymic progenitor cells [81]. Additionally, the ratio of mTECs to cTECs, which is normally declined,

As to cell-based therapy, this has also been investigated as a potential source of thymic rejuvenation via the use of exogenous TECs from newborn thymi. The investigators, after observing that circulating factors alone (via a heterochronic parabiosis model, in which young and aged mice are surgically joined resulting in mutual influence of blood-borne factors [122–130]) did not rejuvenate the aged thymus, utilized a model of direct transplantation of TECs from newborn mice intrathymically into middle-aged recipients [114]. This group observed renewed

Other groups are investigating the use of reprogrammed mouse embryonic fibroblasts (MEF), as sources of exogenous FoxN1, as a means of generating *de novo* ectopic thymus. One such group generated induced TECs (iTECs) from MEF cells by initiating FoxN1 expression that converted MEF cells into epithelial-like cells *in vitro* [115]. Then, these iTECs, after some testing, were re-aggregated and grafted under the kidney capsule of syngenic adult mice to evaluate the ability of these iTECs to develop into a functional thymus-like organ. Interestingly, the grafts were seeded by host T cell progenitors and reflected thymocyte distributions associated

growth of the thymus as well as enhanced T cell generation [114].

the periphery, however, this still remains largely uninvestigated.

**5. Trends in rejuvenation of age-related thymic atrophy**

*Thymus*

expense of negative selection [37].

central T cell tolerance in the elderly.

**4.4 Overall contribution to inflammaging**

defect in negative selection, but an increase in tTreg generation [100]. The second variable is the relative expression level of self-peptide/MHC on TECs. Transgenic expression of a microRNA targeting the MHC class-II transactivator (CIITA) resulted in reduced MHC-II on mTECs [37], leading to insufficient mTEC presentation of self-peptides thus reducing the overall avidity of the TCR interaction with self-peptide/MHC. This also resulted in the enhancement of tTreg generation at the

In the aged thymus, as we mentioned earlier, mTECs are flawed and self-antigen cannot be normally presented in the MHC-II groove, which results in a diminished interaction with TCRs on developing thymocytes. This is similar to the second scenario described above, in which a defect exists in the TEC compartment causing reduced TCR signaling strength. We observed a relatively enhanced tTreg generation in the atrophied thymus, exhibiting no change in overall tTreg numbers, but an increased ratio of tTreg to tTcon cells in the aged, atrophied thymus compared to young controls [92]. This is probably a demonstration of the atrophied thymus attempting to compensate for defective negative selection [25] in order to maintain

If self-reactive TCR signaling strength is too low these thymocytes may neither be depleted nor form tTreg cells, but rather may directly differentiate into selfreactive Tcon cells. As an artifact of impaired promiscuous self-antigen expression in mTECs through an autoimmune regulator (*Aire*) knock-out model, the *Aire*dependent TCAF3 epitope of prostate antigen cannot be promiscuously expressed on mTECs [101]. This resulted in prostate-specific thymocytes, which should be negatively selected, but in contrast were redirected into prostate-reactive Tcon cells. The authors observed loss of prostate-specific tTreg cells for this same epitope, and heightened prostate-reactive Tcon cells that infiltrated the prostate of these mice causing auto-inflammatory lesions [102, 103]. Defects in self-peptide expression on mTECs due to protein knock-out [104], are beginning to suggest that some of the same impairments exhibited by the atrophied thymus, may impact antigen-specific (monoclonal) tTreg generation, meanwhile increasing this same self-antigen specific Tcon generation, despite an unchanged or increased total (polyclonal) tTreg population [105]. It will be interesting to see what further subtle implications the aging thymus has on central tolerance establishment via potentially altering certain self-tissue specific tTreg populations and altering the overall aged Treg TCR repertoire, in spite of a relatively increased aged polyclonal Treg population [92].

Inflammaging or the age-related, persistent increase in basal pro-inflammatory phenotype, has long been thought to be primarily a result of senescent somatic cells exhibiting senescence-associated secretory phenotype (SASP) [30, 31, 34, 106]. However, it is has come to be appreciated that chronic immune activation in the elderly contributes to a pro-inflammatory secretory milieu. This activation in the elderly includes chronic innate immune activation, which may result from immunosenescence related to accumulation of memory T cells. Chronic innate immune activation is also attributed to long-term virus, such as cytomegalovirus (CMV) [29, 107, 108], infection; or a degeneration-associated autotoxic reaction [109]. However, age-related autoimmune predisposition (an adaptive immune activation), induced by adaptive immune reaction to self-tissues by self-reactive T cells, has recently been recognized as a potential factor and/or synergistic cause of chronic inflammation in the elderly [25, 34]. Therefore, the role of the adaptive immune system in mediating inflammaging, as a result of self-reactive T cell immune

**56**

responses to self-tissue that increases with age, is directly related to the atrophied thymus [25, 34].

Since it has recently been confirmed that the involuted thymus releases selfreactive Tcon cells as a result of perturbed negative selection, the direct implications of age-related thymic atrophy on the risks of inflammaging and the associated subclinical increase in the pro-inflammatory milieu has become more clear [25].

Subsequent alterations in tTreg development may also play an unappreciated role in the increased self-reactivity associated with aging, as changes in the tTreg repertoire may in fact impair sufficient suppression of appropriate self-reactivity in the periphery, however, this still remains largely uninvestigated.
