The lymphoid tissue-specific proteome
The lymphoid tissues play an important role in the body's defence against exogenous pathogens, in the production and development of lymphocytes as well as in the transportation of interstitial fluids. The lymphoid tissues consist of primary lymphoid tissues and secondary lymphoid tissues. The primary tissues include bone marrow and thymus, while the secondary tissues include tonsils, lymph nodes, spleen and appendix. Transcriptome analysis shows that 76% (n=15405) of all human proteins (n=20162) are expressed in the lymphoid tissue and 1470 of these genes show an elevated expression in the lymphoid tissue compared to other tissue types.
The lymphoid tissue transcriptome
Transcriptome analysis of the lymphoid tissue can be visualized with regard to the specificity and distribution of transcribed mRNA molecules (Figure 1). Specificity illustrates the number of genes with elevated or non-elevated expression in the lymphoid tissue compared to other tissues. Elevated expression includes three subcategory types of elevated expression:
Distribution, on the other hand, visualizes how many genes have, or do not have, detectable levels (nTPM≥1) of transcribed mRNA molecules in the lymphoid tissue compared to other tissues. As evident in Table 1, all genes elevated in lymphoid tissue are categorized as:
Figure 1. (A) The distribution of all genes across the five categories based on transcript specificity in lymphoid tissue as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (nTPM≥1) in lymphoid tissue as well as in all other tissues.
As shown in Figure 1, 1470 genes show some level of elevated expression in the lymphoid tissue compared to other tissues. The three categories of genes with elevated expression in lymphoid tissue compared to other organs are shown in Table 1. In Table 2, the 12 genes with the highest enrichment in lymphoid tissue are defined.
Table 1. The number of genes in the subdivided categories of elevated expression in lymphoid tissue.
Protein expression of genes elevated in primary lymphoid tissues
The lymphoid tissues are categorized into primary and secondary lymphoid tissues. The primary tissue consists of the bone marrow and the thymus gland and it is here the T-cells and B cells are created and matured. For more information about the bone marrow, see separate bone marrow chapter.
The thymus gland is primarily a lymphoid tissue where the maturation of T-cells occurs, but it also produces several hormones. The thymus is active in children, but after puberty it undergoes involution, which involves the replacement of the gland by adipose tissue and a decrease in lymphocytes.
The thymus is located beneath the sternum and consists of two lobes surrounded by a fibrous capsule. The lobes are divided by a fine septa into many lobules, with an outer cortex with high cellular density, and an inner medulla with lower cellular density. Hassall’s corpuscles are structures only found in the thymic medulla which increase in number throughout life. These are non-secreting flattened thymic epithelial cells in a whorl-like formation arranged in concentric layers.
T-cells are a type of lymphocyte that is a part of the adaptive immune system together with B-cells. T-cells originate from hematopoietic cells in the bone marrow, which develop into immature thymocytes in the thymus. The thymocytes differentiate into several types of mature T-cells; T helper cells, cytotoxic T-cells, memory T-cells, regulatory T-cells and natural killer T-cells. During maturation, T-cells undergo β-selection and positive selection in the thymic cortex and negative selection in the thymic medulla. β-selection ultimately produces a functional αβ T-cell receptor by rearranging the β-chain and pairing it with a constant α-chain. Before maturation, thymocytes do not express CD4 or CD8. The expression of CD4 or CD8 occurs during the β-selection. After β-selection, thymocytes go through positive selection, where cells that are able to bind to MHC presented by thymic epithelial cells, are selected for. During this process, thymocytes binding to MHC class II, using CD4 as a coreceptor, become CD4⁺ T-cells (helper T-cells), and thymocytes binding to MHC class I, using CD8 as a coreceptor, become CD8⁺ T-cells (cytotoxic T-cells). The final step of T-cell maturation is negative selection, where autoreactive thymocytes are eliminated. T-cells that bind too strongly to self-antigens presented on MHC complex of thymic epithelial cells receive an apoptotic signal. Remaining, now mature T-cells, enter the bloodstream as naïve T-cells.
Examples of genes expressed in CD8⁺ T-cells or during the maturation of CD8⁺ T-cells include SATB1, PSMB11 and CD8B. SATB1 modulates genes that are essential in the maturation of CD8⁺ T-cells. PSMB11 generates peptides that are presented by MHC class I molecules during the maturation of CD8⁺ T-cells in the thymic cortex. CD8B is the beta chain of the cell surface glycoprotein CD8 and is an important molecule mediating cell-cell interactions in lymphoid tissues. By acting as a co-receptor to the T-cell receptor on the T-cell, CD8 recognizes MHC class I molecules displayed by an antigen-presenting cell.
The THEMIS gene encodes a protein involved in the late phases of T-cell development. It is necessary for lineage commitment and functions through T-cell antigen receptor signaling. The gene UHRF1 encodes a member of a subfamily E3 ubiquitin ligases. The protein regulates gene expression by recruiting a histone deacetylase. It is involved in the phases of the cell cycle, and plays a role in the p53-dependent DNA damage checkpoint.
The gene CCR8 encodes a member of the beta chemokine receptor family and a transmembrane protein. Chemokines are of importance for recruitment to inflammatory processes. I-309, thymus activation-regulated cytokine (TARC) and macrophage inflammatory protein-1 beta (MIP-1 beta) have been identified as ligands of this receptor. It is believed that this protein is involved in the regulation of monocyte chemotaxis and thymic cell apoptosis. This receptor may contribute to the proper positioning of activated T-cells within lymphoid tissues. SLAMF1 belongs to the self-ligand receptor of the signaling lymphocytic activation molecule (SLAM) family. SLAM receptors trigger interactions that affect the activation and differentiation of several immune cell types. The IHC staining shows strong cytoplasmic expression in the medullary cells in the thymus.
RAD51 is involved with homologous strand exchange in DNA repair. This protein is also found to interact with BRCA1 and BRCA2, which may be important for the cellular response to DNA damage. This protein is highly expressed in the thymus, however it has been detected with IHC staining in other immune tissues as well as in testis.
Protein expression of genes elevated in secondary lymphoid tissues
The secondary lymphoid tissues that have been evaluated on the Human Protein Atlas include spleen, tonsil, lymph node and appendix. These tissues filter lymphatic fluids, tissue fluid and blood, and are involved in the production of antibodies, the detection of antigens, as well as clonal expansion and affinity maturation of residing lymphocytes.
The spleen is divided into two main compartments, the red pulp and the white pulp, and is surrounded by a dense fibrous covering called the splenic capsule. The main functions (in the adult) can be described as follows:
The spleen consists of the red pulp and white pulp within a meshwork of reticular fibers enclosed by a dense connective tissue capsule. The white pulp consists of lymphatic tissue and monitors the incoming blood for harmful substances. Aggregations of mainly T-cells envelop the central arteries in a periarterial lymphatic sheath (PALS). At some places, the white pulp expands into greater spherical aggregations to form splenic nodules containing a light germinal center, consisting of proliferating B-cells. Surrounding the B-cells is a darker stained mantel zone, and peripheral to this a lighter stained marginal zone that marks the border to the red pulp. The splenic nodules have an appearance similar to lymph follicles, with the exception of a central located artery.
The red pulp filters blood to detect damaged and old red blood cells and platelets. Cells that are selected for breakdown are phagocytized by splenic macrophages. The red pulp consists of splenic cords and splenic sinuses. A meshwork of reticular cells and fibers, together with dendritic cells, macrophages, lymphatic cells, and red blood cells constitute the splenic cords. Branches of the central arteries penetrate into the red pulp where they further branch into smaller macrophage sheathed capillaries. Within the splenic cords, the red blood cells are exposed to the macrophages and can be selected for breakdown.
CD5L encodes a protein that is involved in lipid synthesis and is expressed by macrophages. It is mainly expressed in lymphoid tissues and involved in inflammatory responses from infections or atherosclerosis. CD5L is believed to induce lipolysis during obesity and inflammation in adipose tissue. This in turn can lead to insulin resistance and other metabolic diseases. CD5L works as a metabolic switch in T helper 17 cells by regulating the transcription of nuclear receptors ROR-gamma (RORC) that will have negative effects on downstream metabolism.
The gene NOS3 encodes for nitric oxide synthase 3, an enzyme that produces nitric oxide, a reactive free radical that acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. IHC shows cytoplasmic expression in endothelial cells, most abundant in the spleen.
Tonsils consist of partly encapsulated aggregations of lymphoid tissue. They are located in the epipharynx and mesopharynx where they serve as a defense against pathogens from the air we breathe. The tonsils are covered by a stratified squamous epithelium that forms deep irregular invaginations into the tonsils. Underlying the epithelium numerous lymph follicles are present. Lymph follicles are spherical aggregations of lymphocytes. Primary lymph follicles appear as homogeneous aggregations of small lymphocytes. Secondary lymph follicles have a lighter germinal center, representing proliferating B-cells. A typical feature of tonsils is the presence of lymphocytes that infiltrate into the squamous epithelium of crypts and the mucosal surface.
The SP140 gene encodes a member of the SP100 family of proteins. The encoded protein is interferon-inducible and is expressed at high levels in the nuclei of leukocytes. Variants of this gene have been associated with multiple sclerosis, Crohn's disease, and chronic lymphocytic leukemia. Alternative splicing results in multiple variants. IHC staining shows nuclear positivity in tonsil and lymph node.
Lymph nodes are comprised of small, bean-shaped organs in the lymphoid tissues, which filters lymph entering the lymph nodes via lymph vessels. Each lymph node is surrounded by a fibrous capsule, and the inside consists of thin reticular fibers and elastin which form a supporting meshwork called reticular network (RN). Within the RN primarily lymphocytes are tightly packed in follicles (B-cells) and within the cortex (mainly T-cells). Lymph entering via afferent lymphatic vessels is drained just beneath the capsule. During its course through the cortex, the lymph is slowly filtered and immunogenic peptides thereby encounter lymphocytes and macrophages, which leads to elimination and/or activation of an adaptive immune response. The filtered lymph ultimately reaches the medulla and exits via efferent lymph vessels towards the lymphatic ducts.
The main functions can be categorized as follows:
CCL21 is a gene among the elevated genes in lymphoid tissues. CCL21 is expressed in secondary lymphoid organs and is involved in chemotaxis as well as having an inhibiting effect on hematopoiesis. It is a ligand for chemokine receptor 7 that is expressed on T- and B-cells. It is known to attract T-, B-, and dendritic cells via their chemokine receptor. Our IHC shows a moderately enriched expression in lymph nodes and can also be observed in the tonsil.
IRAG2 is a gene which codes for a membranous and cytoplasmic protein and can be observed in IHC. The full name is Lymphoid-restricted membrane protein and is believed to play a role in the delivery of peptides to MHC-1 molecules. However, the function of this protein is not well investigated.
The appendix is located in the lower right of the GI-tract close to the pelvic bone and can vary in size, however on average it measures 9 centimeters in length. It is well accepted that the immune tissue called gut-associated lymphoid tissue (GALT) is important for fighting pathogens passing through the glandular epithelium of the gut and believed to be involved in regulating the gut microbiota. However, the function of the appendix is widely debated due to the apparent lack of importance, as judged by an absence of side effects following an appendectomy. One hypothesis is that the appendix constitutes a vestigial remnant of a once larger cecum, while another hypothesis suggests that it acts as storage for beneficial bacteria during times of illness.
One of the elevated genes in the appendix is FPR1. FPR1 encodes a G protein-coupled receptor protein expressed by e.g. neutrophils, and plays a role in chemotaxis, phagocytosis and generation of reactive oxygen species. This protein is important in host defense and inflammation. The IHC shows strong positivity in a cell population indicative of phagocytes in bone marrow, appendix, as well as in many other tissues.
Another gene prevalent in appendix is the LEF1 gene. This gene encodes a transcription factor protein that binds to a T-cell receptor-alpha enhancer. It is believed to be involved in the Wnt signalling pathway and may regulate cell differentiation. Below, we can observe the protein in the appendix.
Gene expression shared between lymphoid tissues and other tissues
There are 307 group enriched genes expressed in lymphoid tissue. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, including lymphoid tissue, compared to all other tissues.
To illustrate the relation of lymphoid tissue tissue to other tissue types, a network plot was generated, displaying the number of genes with a shared expression between different tissue types.
Figure 2. An interactive network plot of the lymphoid tissue enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of lymphoid tissue enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 5 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.
The lymphoid tissues have most group enriched gene expression in common with bone marrow and intestine. Below are some proteins expressed in the lymphoid tissues together with other tissue types.
Lymphocyte cytosolic protein 2 (LCP2) and FGR proto-oncogene (FGR) are two proteins that are group enriched in lymphoid tissue and bone marrow. Both are downstream signaling elements to immune cell activating ligands. LCP2 is a downstream element to the T-cell receptor and associated with T-cell development and activation. FGR is a downstream element to receptors that bind the Fc region of antibodies and contributes to the regulation of immune responses involvning neutrophils, monocytes, macrophages and mast cells, and in addition to bone marrow, it is also enriched in lung.
The gene CD1D is group enriched in intestine, liver and lymphoid tissue and the encoded protein have been found to be expressed on the surface of antigen-presenting cells in order to bind and present self and non-self glycolipids to innate immunity-associated natural killer T (NKT)-cells. RAS protein activator like 3 (RASAL3) is group enriched in bone marrow, intestine, lung and lymphoid tissue and have been found to play an important role in the function and expansion of NKT-cells by negative regulation of RAS/ERK signaling activity.
The G protein-coupled receptor GPR182 is fairly uncharacterized, however the expression of GPR182 shows enhanced elevation in lymphoid tissue and testis, while IHC shows some expression in testis and strong staining in spleen and lymph nodes.
The lymphoid tissues in the human body constitute an important part of the adaptive immune system and it is in the primary lymphoid tissues B cells and T-cells are created. Primary lymphoid tissues consist of the bone marrow and thymus glands. The bone marrow is the major producer of all the cells in the lymphoid tissues and in charge of thematuration of B cells. Immature T-cells created in the bone marrow are transported to the thymus gland where they are developed into more specific T-cells. Antigen-presenting cells (APCs), dendritic cells, are transported to the lymph nodes where an immune response is provoked and antibodies and cytokines can be recruited to fight off pathogens. Secondary lymphoid tissues can be found at different locations in the body; lymph nodes, tonsils, appendix and the spleen. These sites are connected through lymph vessels, which are responsible for regulating bodily fluids and controlling fat absorption through its intricate network of lymph vessels, also known as the lymphoid circulatory system. Here, it absorbs lipids from the gut before they are transported to the blood. The fluid in these vessels is called lymph.
Here, the protein-coding genes expressed in lymphoid tissue are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in lymphoid tissue.
Transcript profiling was based on a combination of two transcriptomics datasets (HPA and GTEx), corresponding to a total of 14590 samples from 50 different human normal tissue types. The final consensus normalized expression (nTPM) value for each tissue type was used for the classification of all genes according to the tissue-specific expression into two different categories, based on specificity or distribution.
Relevant links and publications
Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
The histology of the human lymphoid tissues with detailed information can be viewed in the Protein Atlas Histology Dictionary