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

Germinal centers in pathogenic SIV infection

The characteristic feature of HIV and some SIV infections is the chronic immune activation that probably drives the continuous CD4 T cell depletion and progression towards AIDS. What exactly causes this aberrant activation is not clear; however, traits inherent to the immune response seem to be more responsible than the virus itself. For example, there are data showing that the same strain of virus (SIV) can induce lasting but non-pathogenic infection in one primate species (sooty mangabeys) and devastating disease which resembles AIDS in the other (rhesus macaques). It may be therefore instructive to study details of immune response against SIV in pathogenic conditions. B lymphocytes are not spared from overall activation during HIV/SIV infection – in fact, sick individuals display hypergammaglobulinemia, or elevated levels of IgG antibodies. Switched antibodies are generally produced by cells that derive from the germinal center reaction where B lymphocytes interact with the unique subset of CD4 T cells called follicular helpers (TFH). Follicular helpers can be distinguished from other CD4 T cell subsets by their expression of CXCR5, ICOS, PD-1 and the transcription factor Bcl-6 as well as the high level of IL-21 cytokine secretion. TFH have been shown to be crucial for the successful generation of plasma cell and memory compartments. The recent paper takes a look at germinal centers during SIV infection in monkeys that develop AIDS-like disease.

The link: http://www.jimmunol.org/content/188/7/3247.abstract

Authors use immunohistological staining and flow cytometry to prove that PD-1 positive CD4 T cells (according to what we know TFH population) accumulate in germinal centers along disease progression in rhesus macaques infected with SIVmac239. They also correlate the enhanced PD-1 expression on CD4 T cells with remodeling of B cell populations that reside in lymph nodes (the trend is towards reducing naïve subset and enriching memory subsets) and the increased IgG secretion. Additionally, it is demonstrated that CD8 T cells are excluded from germinal centers (both in naïve and infected monkeys). Investigators conclude that the buildup of TFH population during pathogenic SIV infection may play the important role in shifting B cell activation status. Apart from that they suggest that germinal centers could form anatomical sites of potential viral escape due to the lack of CD8 T cells presence (and alleged insufficient control of infected CD4 T cells there).

The experimental setting of this publication is restricted to recording several immune parameters at time points that represent either acute or chronic SIV infection. However, it is interesting because it attempts the systematic documentation of changes in the immune response that may underlie the pathologic immune activation distinctive for some primate lentiviruses. The obvious question for the future is whether there may be any significant changes in germinal center reaction between species that develop pathogenic or non-pathogenic SIV infection.

Hong, J., Amancha, P., Rogers, K., Ansari, A., & Villinger, F. (2012). Spatial Alterations between CD4+ T Follicular Helper, B, and CD8+ T Cells during Simian Immunodeficiency Virus Infection: T/B Cell Homeostasis, Activation, and Potential Mechanism for Viral Escape The Journal of Immunology, 188 (7), 3247-3256 DOI: 10.4049/jimmunol.1103138

IgE class switching occurs in germinal centers

IgE antibody response is known to accompany infections with helminths. Organisms like Schistosoma mansoni, Nippostrongylus brasiliensis, Heligmosomoides polygyrus or Brugia malayi are able to induce the substantial class switching to IgE and increased levels of IgE serum antibody. IgE antibodies are also associated with allergies, asthma and generally Th2 effector responses. The other murine antibody class connected to Th2 is IgG1. Despite the importance of IgE response not all facts linked to basic IgE biology are entirely clear.  For example, it is not sure whether class-switching to IgE takes place in or outside germinal centers. Another elusive issue is the hypothetical presence of separate IgE memory population. There were some suggestions that IgE class switching needs to go through IgG1 intermediates and that IgE memory is maintained in IgG1 compartment. The recent publication uses IgE-reporter mouse strain to challenge the notion that IgE response occurs exclusively in tight connection with IgG1.

The link:
http://www.nature.com/ni/journal/v13/n4/abs/ni.2256.html

Authors use the fact that the secreted and membrane forms of antibody are translated from different mRNA splice variants. The IgE reporter strain they employ has the GFP sequence inserted downstream of exon that encodes the cytoplasmic domain of membrane IgE thus connecting the fluorescent signal with the membrane antibody version. Additionally, the IgE locus of reporter mice contains 52-amino acid region (called M1) derived from human antibody. Such approach allows detecting GFP signal as approximation of IgE membrane variant synthesis and subsequently verifying the presence of IgE with antibody against M1.

To confirm the validity of this strain for in vivo studies authors employ in vitro cultures with IgE response promoting factors (anti-CD40 and IL-4). It is established that GFP-positive population can be divided into GFPint and GFPhi subsets. The presence of soluble IgE correlates with appearance of GFPhi cells and only GFPhi fraction stains positively with anti-M1 antibody. Additionally, GFPhi cells express much more of IgE mature transcript than GFPint population which contains numerous cells positive for IgG1 presence. It is therefore concluded that only GFPhi fraction encloses IgE-switched cells.

To monitor how IgE-switched population comes into existence authors use the infection with Nippostrongylus brasiliensis. They aim to detect the germinal center (B220+IgDGL7+CD95+) and memory (B220+IgD+GL7CD38hi) compartments inside GFPhi (and thus IgE-switched) subset by flow cytometry. At the initial phase of infection all GFPhi cells bear features characteristic for germinal centers. At later stages some of them transform into memory cells. The appearance of IgE germinal center and memory populations occurs at the same time as analogous IgG1 subsets. Authors interpret these data as ruling against the possibility of IgG1 transitional stage for IgE-switched cells. Investigators also confirm the functionality of IgE memory population by adoptive transfer strategies. Additionally, the publication contains data about in vivo generation of IgE-secreting plasma cells and visualizations of germinal centers with IgE-switched cells (plus IgE memory and plasma cells) in lymph nodes of infected mice.

My first reaction after reading this paper was that of astonishment. Did they know so little about IgE response not to be sure whether there are IgE-switched cells inside germinal centers? The included data do not show anything very new or unexpected. They just extend basic facts from B cell biology for the IgE population.  Investigators show in the convincing way that IgE memory and plasma compartments derive from germinal center reaction and that memory IgE cells contribute to faster antibody response on re-infection. On the whole this is very interesting stuff which illustrates how many gaps are still in our knowledge.

Talay, O., Yan, D., Brightbill, H., Straney, E., Zhou, M., Ladi, E., Lee, W., Egen, J., Austin, C., Xu, M., & Wu, L. (2012). IgE+ memory B cells and plasma cells generated through a germinal-center pathway Nature Immunology, 13 (4), 396-404 DOI: 10.1038/ni.2256