The developmental diversion of thymocytes

I like reports that make me learn something new and appreciate novel developments leading to more integral view of immunological concepts. My understanding of the thymic central tolerance process was that thymocytes receiving strong signals from tissue antigens through TCR undergo invariably the clonal deletion. But it looks like the clonal deletion of cells that can develop into potentially autoreactive T lymphocytes is not the only way which exists for such population in the thymus. I have read the paper that suggests that there may be actually two outcomes for thymocytes with self-reactive TCR – first is the clonal deletion whereas second the developmental diversion.

The link: http://www.nature.com/ni/journal/v13/n6/abs/ni.2292.html

What is the developmental diversion, though? According to authors it is a process that happens when a thymocyte gets the signal through its autoreactive TCR but is not able to receive the costimulation with CD28 molecule. In such case it can enter a pool of DN cells (double negative for CD4 and CD8) and turn up in the intestinal epithelium where it re-expresses CD8 (in its αα form). Cells derived from the developmental diversion are anergic and when the clonal deletion is impaired (as for example in CD28 knockout mice) the efficiency of central tolerance is not reduced because autoreactive thymocytes have an substitute pathway that sequesters them from harmful and self-reactive mature population.

How the the developmental diversion was detected? The publication contains a lot of data, so I will focus on most crucial evidence. The initial observation made by investigators was that CD28 knockout mice (and also B7 double knockout with no CD80 and CD86 which are CD28 ligands) has unusually numerous population of DN thymocytes that express TCRαβ. In normal mice DN thymocytes are in their majority TCRαβ-negative. The DN population from mice deleted for CD28 contains also the similar proportion of autoreactive TCRs as pre-selection DP (double positive) thymocytes but mature C4 or CD8 T cells from the same strain are mostly deprived of self-reactive rearrangement. Therefore authors conclude that the clonal deletion of autoreactive thymocytes requires CD28 costimulation and in its absence such cells are diverted into the alternative developmental way.

Investigators follow this phenomenon by studying at what exact stage of thymocyte development the diversion may occur and what happens with diverted thymocytes once they leave the thymus (they end up in the intestinal epithelium as already has been remarked). The most interesting thing, however, is that the developmental diversion seems to take place in normal mice as well as TCRαβ+CD8ααintraepithelial lymphocytes from the wild type strain are enriched for autoreactive specificities. I definitely need to start following this story.

Pobezinsky LA, Angelov GS, Tai X, Jeurling S, Van Laethem F, Feigenbaum L, Park JH, & Singer A (2012). Clonal deletion and the fate of autoreactive thymocytes that survive negative selection. Nature immunology, 13 (6), 569-78 PMID: 22544394

Tonsils and T cell development process

I have found yet another interesting publication that covers the thymocyte stage of T cell life. The understanding of this paper may require some extra effort to become acquainted with a number of cellular populations that are encountered along the stepwise T cell development process (unless you’re an expert on thymocyte maturation). But the conclusions are quite intriguing because according to authors the thymus may not be the only anatomical place which supports the organized production of mature T cells. This research is performed on human tonsilar samples that have been collected during routine tonsillectomies. Most presented data comprise the meticulous use of flow cytometry which is supported by real-time PCR and immunohistochemical staining.

The link: http://www.jci.org/articles/view/46125

Authors track the presence of putative T cell progenitors in human tonsils using markers associated with thymocyte development. These markers include CD34 (which is expressed on bone marrow-derived stem cells and lost along T cell differentiation), CD38 (whose increased expression denotes commitment to the T cell lineage) and CD1a (another surface protein associated with the stem cell to thymocyte transition). Initially investigators try to make sure if tonsils may be seeded by stem cells that are converting to thymocytes. It turns out that tonsilar CD34-positive and lineage negative population contains two subpopulations – CD38dim and CD38brigth. Another experiment identifies CD34+CD1a+ population which is divided into two distinct subsets: CD11c+CD10 cells (that are dendritic cells) and CD11cCD10+ cells (that may be putative thymocytes). Collectively, the first part of paper provides the evidence that human tonsils hold lineage negative populations whose surface marker expression is remarkably similar to developing thymocytes: CD34+CD38dim, CD34+CD38bright and CD34+CD1a+CD11c.

Another characteristic feature of thymocyte is the double positive stage when developing T lymphocytes express both CD4 and CD8 co-receptors and loose stem cell marker CD34. At this stage some thymocytes start also expressing the additional component of TCR complex – CD3. Authors compare CD34-negative DP cells from thymus, tonsils and blood to find that thymic DP population is exclusively CD1a-positive. Blood has only CD1a-negative DP cells (they are probably memory cells as joint CD4/CD8 expression is not exclusive for thymocytes) and tonsils hold a mixture of both. Remarkably, the tonsilar CD1a-positive DP population is identical to thymic DP cells as it is comprised of two subsets that are CD3 and CD3+. It is therefore concluded that human tonsils contain two additional thymocyte-like populations: CD34CD1a+CD4+CD8+CD3 and CD34CD1a+CD4+CD8+CD3+.

Authors follow up with thorough comparison of thymocyte-like populations from tonsils to corresponding cells collected from thymus by flow cytometry and observe general (but not absolute) similarities in surface marker expression in all studied subsets. It is also detected that tonsilar putative thymocytes match their thymus counterparts in gene expression patterns. Apart from that investigators employ immunohistochemistry staining to show that thymocyte-like cells localize to the discreet anatomical region of tonsil – fibrous scaffold. Finally, in a series of ex vivo experiments they demonstrate that tonsilar thymocytes have T cell differentiation potential (CD34+ thymocytes are apparently able to give rise not only to T cells but also to NK population).

The description of what looks like the complete T cell differentiation pathway outside the thymus is very intriguing. Authors collected their samples from children that have undergone tonsillectomy which means that they must have suffered numerous onsets of tonsil inflammation. May repetitive inflammatory events contribute to that unusual extrathymic production of T cells? And what can you think about their functional characteristics? Are tonsil-derived T cells subject to the central tolerance process?

McClory S, Hughes T, Freud AG, Briercheck EL, Martin C, Trimboli AJ, Yu J, Zhang X, Leone G, Nuovo G, & Caligiuri MA (2012). Evidence for a stepwise program of extrathymic T cell development within the human tonsil. The Journal of clinical investigation, 122 (4), 1403-15 PMID: 22378041

Plasmacytoid dendritic cells may have the role in central tolerance

The principle of central tolerance implies that developing T cells be exposed to tissue antigens and become deleted if they display auto-reactivity features. However, the understanding of gene expression control has engendered the problem of how peripheral tissue-specific antigens reach the anatomic organ where the central tolerance process takes place – the thymus. Among mechanisms that provide explanation for this problem are those that either circumvent the gene expression aspect (passive diffusion of antigens through the blood) or embrace it (promiscuous intrathymic gene expression). There are also data that indicate the tolerogenic role for dendritic cells residing in thymic compartments. These dendritic cells have been suggested to be able to collect peripheral antigens in tissues and supply them to the thymus. In this line I have found very interesting publication that describes plasmacytoid dendritic cells as potential players in the central tolerance induction.

The link: http://www.cell.com/immunity/retrieve/pii/S1074761312000908

Authors show that CCR9-deficient mice have decreased numbers of thymic plasmacytoid dendritic cells. Following this initial finding it is concluded that WT pDCs possess the advantage over CCR9-deficient pDCs in homing ability to the thymus and additionally to the lamina propria and the intestinal intraepithelium. To prove such point investigators have used diverse techniques that included parabiosis experiments (animals of different genetic background that are surgically joined to enable blood cells exchange), generation of bone marrow chimaeras combined with adoptive transfer methodology and in vivo enrichment of dendritic cell populations by grafting mice with tumors that express Flt3L.

To address the possible role of plasmacytoid dendritic cells in the central tolerance authors examine if pDCs may be able to transport OVA antigen to the thymus in order to induce the deletion of OVA-specific CD4 T cells there. Following the generation of bone marrow chimaeras that enable monitoring fate of transgenic OT-II thymocytes and the intravenous injection of pDCs loaded with OVA antigen it is demonstrated that this is indeed the case. However, the tolerogenic activity of pDCs is abrogated when these cells become activated with TLR9 ligand. For the full ability to delete CD4 T cells pDCs need to express CCR9 – as this molecule directs them to the thymus but is not involved in the proper deletion mechanism. The publication also contains clever visualization scheme devised to prove that pDCs are actually capable to collect tissue antigens and shuttle them to the thymus.

So, it seems like plasmacytoid dendritic cells beyond their known participation in viral defense and peripheral Treg induction may have an additional function. The deletion data obtained with OVA transgenic system is very convincing and entice to seek some further evidences supporting this novel physiological role of pDCs. For example, it might be interesting to look for a link between the ability of pDCs to transport peripheral antigens to the thymus and tolerance to intestinal commensal flora. It is well established that postpartum bacterial colonization has profound effects on the functionality of immune system. May it also influence the central tolerance process? I don’t know the answer, but I think there are tools like gnotobiotic mono-colonized mice and TCR transgenic systems that may enable us to get some more information.

Hadeiba H, Lahl K, Edalati A, Oderup C, Habtezion A, Pachynski R, Nguyen L, Ghodsi A, Adler S, & Butcher EC (2012). Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promote central tolerance. Immunity, 36 (3), 438-50 PMID: 22444632