Analysis of the differentially expressed gene content in human mTECs

3. Results

3.1. Expression patterns and evolutionary conservation of promiscuous gene

3.1.5. Analysis of gene expression between immature and mature mTECs in the thymus

3.1.5.5. Analysis of the differentially expressed gene content in human mTECs

The immature and mature mTECs (MHCII^loand MHCII^hirespectively) from 11 different individuals ranging from 3 days to 3 years of age were isolated from their thymus. The mTECs were enriched using three different methods (Density gradients, depletion of CD45⁺ cells using either Dynal beads or Miltenyi beads) as described earlier (Section 2.2.3.3). Gene array results showed substantial variations in the number of differentially expressed gene between the different individuals. The number of differentially expressed genes ranged from ~700 to 6000 (Table 3.5A). Differences were observed irrespective of the different isolation methods used.

Hence, it can be excluded that the variation arose due to the isolation methods used.

Seeking for a common set of differentially expressed genes between individuals we found a negative correlation between the number of individuals and the number of common/overlapping genes. The intersection interval which is the extreme point in the comparison containing genes that are differentially expressed in all 11 individuals showed only 24 common differentially expressed genes, 18 genes were up-regulated and 6 genes were down-regulated (Table 3.5B).

AIRE can be considered as a measure for the degree of enrichment of MHCII^himTECs of the individual samples. Since, AIRE is the driving force for many of the promiscuously expressed genes in the mature mTEC population, the correlation between the number of differentially expressed genes and fold change in AIRE expression was determined. There does exist a correlation between the fold change in AIRE expression (between MHCII^loand MHCII^hi mTECs) and the number of differentially expressed genes, (correlation coefficient, r = 0.5), though two individual samples were outliers (Table 3.5C and Figure 26).

This is the first time that such striking inter-individual difference in pGE is found, though Taubert et al. also reported considerable variability in the expression of several target auto-antigens in mTECs (Taubert et al., 2007). These variations could result from differences in the genetic background, environmental factors, disease/health condition of the individuals and pre-medication. The observed variations in antigen expression are likely to influence T-cell repertoire selection and thus tolerance thresholds in human provided they reflect steady state values.

Taken together, the results in the three species show that promiscuously expressed genes are enriched in TRAs that are partitioned into clusters and this phenomenon is conserved between species. Clusters harbor both TRAs and non-TRAs that are interspersed between each other.

TRAs are preferentially regulated during mTEC differentiation. Moreover, genes within a particular gene cluster are subject to partial co-regulation. The bulk of the TRA clusters contain structurally unrelated genes. Based on these data, we propose these clusters to be the

“operational genomic unit” of pGE in the thymus.

Different methods of separation used to isolate human mTECs Dynal BeadMiltenyi beads (MACS)Density Gradients IndividualsInd. 14Ind. 24Ind. 32Ind. 72Ind. 74Ind. 77Ind. 89Ind. 92Ind. 53Ind. 55Ind. 59 Differentially expressed genes15602153211469710037061097167458856383984 Up‐regulated genes10811557130642665739147297229422937706 Down‐regulated genes47959680827134631562570229433410278

In at least 3 individuals In at least 4 individuals In at least 5 individuals In at least 6 individuals In at least 7 individuals In at least 8 individuals

UnionIn at least 2 individuals In at least 3 individuals In at least 4 individuals In at least 5 individuals In at least 6 individuals In at least 7 individuals In at least 8 individuals

In at least 9 individualsIn at least 10 individualsIntersection Differentially expressed genes1393744002096125787861642031219910824 Up‐regulated genes6805292113317985583942691941286718 Down‐regulated genes7132147975645932022215111871416 Different methods of separation used to isolate human mTECs Dynal BeadMiltenyi beads (MACS)Density Gradients IndividualsInd. 14Ind. 24Ind. 32Ind. 72Ind. 74Ind. 77Ind. 89Ind. 92Ind. 53Ind. 55Ind. 59 AIRE fold change MHCIIhivs. MHCIIlo mTECs23.722.550.123.221.014.04.536.228.769.546.0

Different methods of separation used to isolate human mTECs Dynal BeadMiltenyi beads (MACS)Density Gradients IndividualsInd. 14Ind. 24Ind. 32Ind. 72Ind. 74Ind. 77Ind. 89Ind. 92Ind. 53Ind. 55Ind. 59 AIRE fold change MHCIIhivs. MHCIIlo mTECs23.722.550.123.221.014.04.536.228.769.546.0

A B C

Table 3.5

Representation of gene expression data from different individual thymi

Human mTECs (MHCII^loand MHCII^hi) were isolated from thymus samples for 11 different individuals ranging from 3 days to 3 years of age. The mTECs were enriched using three different methods (Density gradients, Dynal beads and Miltenyi beads) as described earlier (Section 2.2.3.3).

(A) Comparison of differentially-, up- and down-regulated genes between MHCII^lo and MHCII^himTECs from each individual thymus.

(B) Number of differentially-, up- and down-regulated genes expressed in at least “n” individuals, n = 2 to 10.

Union and intersection intervals are the extreme points in the comparison: where union means all genes that are found to be differentially expressed in at least one individual; intersection contains genes that are differentially expressed in all 11 individuals.

Fold Change in AIRE (mature to immature mTECs)

Number of Differentially Expressed Genes

Fold Change in AIRE (mature to immature mTECs)

Number of Differentially Expressed Genes

Figure 26

Correlation between number of differentially expressed genes and fold change in AIRE expression in the different individual thymi

Correlation plot of the number of differentially expressed genes and fold change in AIRE expression between MHCII^hiand MHCII^lo mTECs in the 11 individual samples, (correlation coefficient, r = 0.5). Two individual samples were outliers.

3.2. “Holes” in the thymic antigen repertoire: implications for central tolerance and autoimmunity

Genome-wide studies of pGE in mTECs showed a respectful representation of the genome in the thymic antigen repertoire, but this is not a complete representation. “Holes” or absence in expression of certain genes in the thymus, should lead to lack of central tolerance for particular antigens, therefore it would be a preferential target for autoimmune disease. An important question in organ-specific autoimmune diseases is: why the loss of self-tolerance is so selective that only certain tissues and auto-antigens are targeted?

Two prominent examples of target antigens in autoimmune diseases are: glutamic acid decarboxylase (GAD) involved in insulin-dependent diabetes (IDD) (Tisch, 1996; Yoon et al., 2000; Lernmark, 2002) and cardiac myosin heavy chain (MyHC) involved in myocarditis (Wolfgram et al., 1985; Neu et al., 1987; Rose, 2000). Both cases involve two isoforms in which the expression of either one of the two is lacking in the thymus. This lack of expression may represent a “pitfall” of pGE. It is possible that the non-expressed isoform is embedded in a silent chromosomal context i.e. “region of closed chromatin’’. We started to investigate this possibility by analyzing the thymic expression of the flaking regions of these gene loci.

In document Dissertation submitted to the Combined Faculties for Natural Sciences and Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the (Page 97-100)