The lymph node-specific proteome
Lymph nodes constitute bean-shaped masses of lymphoid tissue enclosed by capsules of connective tissue found in association with lymphatic vessels. As part of the lymphatic system, lymph nodes provide specialized tissues where foreign antigens are trapped and exposed to cells of the immune system for destruction. Lymph nodes are spread out throughout the body, typically found in the mediastinum, the retroperitoneum adjacent to large vessels, and near junctions of the lymphatic vessels, most prominently in the neck, groin, and armpits. The transcriptome analysis shows that 67% (n=13141) of all human proteins (n=19613) are expressed in the lymph node and 279 of these genes show an elevated expression in lymph node compared to other tissue types.
An analysis of the genes with elevated expression in the lymph node, with regards to subcellular localization, reveals that corresponding proteins are predominantly located in the cytoplasmic membrane.
- 45 genes defined as group enriched in lymph node
- Most group enriched genes share expression with tonsil
- 279 genes defined as elevated in lymph node
- Most of the elevated genes in lymph node encode proteins involved in immune system processes
Figure 1. The distribution of all genes across the five categories based on transcript abundance in lymph node as well as in all other tissues.
279 genes show some level of elevated expression in the lymph node compared to other tissues. The three categories of genes with elevated expression in lymph node compared to other organs are shown in Table 1.
Table 1. Number of genes in the subdivided categories of elevated expression in lymph node.
||Number of genes
||At least five-fold higher mRNA levels in a particular tissue as compared to all other tissues
||At least five-fold higher mRNA levels in a group of 2-7 tissues
||At least five-fold higher mRNA levels in a particular tissue as compared to average levels in all tissues
||Total number of elevated genes in lymph node
There were no genes in the category of genes with tissue enriched expression in the lymph node. This is not unexpected as the included cell types, function and morphological features of the lymph node are highly similar to that of other related tissue types, such as the appendix, tonsil and spleen. Genes that specifically signify these types of tissues will thus be categorized as group enriched genes (see below).
The lymph node transcriptome
An analysis of the expression level of each gene made it possible to calculate the relative mRNA pool for each of the categories. The analysis shows that 89% of the mRNA molecules in the lymph node correspond to housekeeping genes and only 3% of the mRNA pool corresponds to genes categorized to be either lymph node enriched, group enriched, or enhanced. Thus, most of the transcriptional activity in the lymph node relates to proteins with presumed housekeeping functions as they are found in all tissues and cells analyzed.
Protein expression of genes elevated in the lymph node
The list of elevated genes (n=279)
are well in line with the function of the lymph node, as it includes an overrepresentation of proteins associated with immune
system process. Many proteins are involved in the regulation of lymphocyte activity (e.g.
TRAT1 and CXCL9).
Proteins specifically expressed in chemotaxis
CCL21 is the gene with the highest TPM-value among the elevated genes for lymph node. CCL21 is expressed in secondary lymphoid organs (e.g. lymph nodes) and is known to attract T- and B-cells and dendritic cells via their chemokine receptor. Our RNA-seq data shows a moderately enriched expression in lymph node, which is in concordance to the IHC staining.
Proteins specifically expressed in T-cell activity
TCL1A is expressed at high levels in lymph node. IHC staining supports the RNA-data, showing staining of lymphocytes mainly in lymph node and the other secondary lymphoid organs. Dysregulation of TCL1A leading to overexpression of the protein is associated with T-cell leukemia.
Proteins specifically expressed in B-cell immune response
CD20 (MS4A1) is increasingly expressed on the surface of B-cells during maturation, however it is absent in early pro-B-cells and the fully differentiated plasma cells. The expression is maintained in neoplasms of B-cell origin, and CD20 is used as a diagnostic biomarker to distinguish B-cell lymphomas and leukemias from histologically similar T-cell neoplasms. CD20 constitutes the target for monoclonal antibodies Rituximab, Ibritumomab tiuxetan, and Tositumomab, that are used in the treatment of B-cell lymphomas and leukemias.
Proteins specifically expressed in development of lymphocytes
LRMP, lymphocyte-restricted membrane protein (Jaw1), displays an enhanced expression in lymph node and tonsil. This gene was first described for T- and B-cell lineages (Behrens et al., 1994). The gene encodes a protein associated with the endoplasmatic reticulum, and is involved in the delivery of peptides to the MHC class I molecules (Snyder et al., 1997). IHC supports RNA-seq data, displaying ER-positivity in lymphocytes.
Previously uncharacterized genes
Also elevated in lymph node was the gene SP140, about very little is known. SP140 is a nuclear body protein, and immunohistochemical stainings reveal nuclear staining in lymphoid cells in lymph node, tonsil, spleen and appendix.
Genes shared between lymph node and other tissues
There are 45 group-enriched genes expressed in the spleen. Group enriched genes are defined as genes showing a 5-fold higher average level of mRNA expression in a group of 2-7 tissues, including lymph node, compared to all other tissues.
In order to illustrate the relation of lymph node tissue to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types.
Figure 2. An interactive network plot of the lymph node enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of lymph node 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 4 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.
The network plot shows that most genes are shared with the other three organs with a major component of lymphoid cells, namely spleen, tonsil and appendix.
One such gene is MS4A1, which encodes CD20, an activated-glycosylated phosphoprotein expressed on the surface of B-cells beginning
at the pro-B phase with progressively increasing concentrations until maturity.
Lymph node function
Lymph nodes are comprised of small, bean-shaped organs in the lymphatic system, which filters lymph entering the lymph nodes via
lymph vessels. Each lymph node is surrounded by a fibrous capsule, and inside thin reticular fibers and elastin
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:
- Filtration of lymph. Detection and elimination of foreign antigens, primarily involving macrophages.
- Activation of adaptive immune response, i.e. proliferation and maturation of lymphocytes.
- Production of antibodies. In response to the antigens, the lymphocytes in the lymph node produce antibodies which exit from the lymph node and enter the circulation, to seek and target antigens produced by pathogens and thus leading to the destruction of pathogens.
The histology of human lymph node including detailed images and information about the different cell types can be viewed in the Protein Atlas Histology Dictionary.
Here, the protein-coding genes expressed in the lymph node are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize protein expression patterns of proteins that correspond to genes with elevated expression in the lymph node.
Transcript profiling and RNA-data analyses based on normal human tissues have been described previously (Fagerberg et al., 2013). Analyses of mRNA expression including over 99% of all human protein-coding genes was performed using deep RNA sequencing of 172 individual samples corresponding to 37 different human normal tissue types. RNA sequencing results of 5 fresh frozen tissues representing normal lymph node was compared to 167 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in lymph node. A tissue-specific score, defined as the ratio between mRNA levels in lymph node compared to the mRNA levels in all other tissues, was used to divide the genes into different categories of expression.
These categories include: genes with elevated expression in lymph node, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in lymph node, and genes not expressed in any tissue. Genes with elevated expression in lymph node were further sub-categorized as i) genes with enriched expression in lymph node, ii) genes with group enriched expression including lymph node and iii) genes with enhanced expression in lymph node.
Human tissue samples used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation and obtained from the Department of Pathology, Uppsala University Hospital, Uppsala, Sweden as part of the sample collection governed by the Uppsala Biobank. All human tissue samples used in the present study were anonymized in accordance with approval and advisory report from the Uppsala Ethical Review Board.
Relevant links and publications
Uhlén M et al, 2015. Tissue-based map of the human proteome. Science
PubMed: 25613900 DOI: 10.1126/science.1260419
Yu NY et al, 2015. Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res.
PubMed: 26117540 DOI: 10.1093/nar/gkv608
Fagerberg L et al, 2014. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics.
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600
Andersson S et al, 2014. The transcriptomic and proteomic landscapes of bone marrow and secondary lymphoid tissues. PLoS One.
PubMed: 25541736 DOI: 10.1371/journal.pone.0115911
Behrens TW et al, 1994. Jaw1, A lymphoid-restricted membrane protein localized to the endoplasmic reticulum. J Immunol.
Snyder HL et al, 1997. Two novel routes of transporter associated with antigen processing (TAP)-independent major histocompatibility complex class I antigen processing. J Exp Med.
Histology dictionary - lymph node