Plasticity is the differentiated state of CD4 T cells

Cellular & Molecular Immunology (2013) 10, 375–378 ß 2013 CSI and USTC. All rights reserved 1672-7681/13 $32.00 www.nature.com/cmi RESEARCH HIGHLIGHT Plasticity is the differentiated state of CD4 T cells Alan G Baxter and Margaret A Jordan Cellular & Molecular Immunology (2013) 10, 375–378; doi:10.1038/cmi.2013.29; published online 5 August 2013 T he recent application of targeted mutation technologies to lymphocyte fate mapping1 has facilitated the in vivo analysis of CD4 T cells subsequent to their differentiation. The fate mapping of IL17-producing cells has shown them to be responsive to environmental cues in fungal infection and central nervous system autoimmunity.2 Now Stockinger’s group reports that many IL17-producing CD41 T cells home to intestinal Peyer’s patches (PP) and convert to a follicular helper T cell (TFH) phenotype, providing critical support for T-dependent IgA responses.3 The integrity of the intestinal mucosal barrier is dependent on suppression of inflammatory responses and control of intestinal flora. Secreted IgA, and the cytokines IL17 and TGF-b, play critical roles in intestinal integrity. Selective deficiency of IgA is the most common form of primary immunodeficiency in the West and can result in recurrent pulmonary and gastrointestinal infections.4 IgA is produced in the spleen and gut-associated lymphoid tissues (including the PP) by activated B cells and plasmablasts from two sources: (i) the T cell-independent effects of dendritic cell-derived APRIL and BAFF on ‘innate like’ B1 cells;5 and (ii) the influence of TGFb and cognate T cell help on (conventional) B2 cells.6–8 Comparative Genomics Centre, James Cook University, Townsville, Qld, Australia Correspondence: Dr AG Baxter, Comparative Genomics Centre, Molecular Sciences Building 21, James Cook University, Townsville 4811, Qld, Australia. E-mail: Received date: 27 May 2013; Revised date: No; Accepted date: 30 May 2013 Even in the complete absence of ab-T cells,9 isotype switching and protective antibody production occurs,7,8 reflecting the relative robustness of T-independent antibody production. Nevertheless, antibody responses to T-dependent antigens (such as cholera toxin or keyhole limpet hemocyanin) are reduced in the absence of CD41 T cells, particularly following oral immunisation,7,8 consistent with a dependence on TFH cells (reviewed in King et al.10). Studies of the differentiation pathways of TFH cells are complicated by the presence of excessive IL4 and IgE production in their absence.11,12 Currently, the best surface marker available for TFH cells is CXCR5, a chemokine receptor that mediates their recruitment to the follicular areas of lymphoid tissues via binding CXCL13, which is predominantly produced by follicular stromal cells.13 Tsuji et al.14 investigated the relationship between regulatory T (Treg) and TFH cells by the adoptive transfer of Treg cells into CD3e2/2 mice. In these experiments, Treg cells were tagged by the introduction of an internal ribosome entry site-enhanced green fluorescent protein (IRES-EGFP) cassette downstream of the Foxp3 stop codon (i.e., the locus is bicystronic). After adoptive transfer, Treg, but not conventional CD4 T cells, induced the formation of GC clusters in PP. Of the EGFP-tagged cells that homed to PP, 80% lost their GFP expression and sequential adoptive transfers showed that these cells were the most effective at supporting GC formation. Over half had acquired expression of CXCR5 and were located in the light zone of GC, and many expressed CD40L, ICOS, PD-1 and CD28, consistent with them having acquired a TFH phenotype.14 It should be emphasized that these cells were distinct from T follicular regulatory cells, which continue to express FOXP3 and suppress GC reactions.15,16 Plasticity in the phenotype of Treg cells, tagged with either bicistronic fluorochrome expression or an EGFP– FOXP3 hybrid molecule, was extensively reported, with up to 50% of tagged cells losing expression of the fluorescent marker and many producing IL2, IL17 and IFN-c 4 weeks after transfer.17–19 The possibility that this phenotypic plasticity was in part an artefact of the tagging technology was raised when these results were compared with experiments in which Treg cells were tagged with the cytoplasmic dye carboxyfluorescein succinimidyl ester20 or a transgene,21 in which only 5%–10% of Treg lost FOXP3 expression. In the case of mice bearing the EGFP–FOXP3 hybrid molecule, peripheral Treg induction and their regulatory function in inflammatory environments were inhibited by reduced interactions between FOXP3 and other transcription factors.22 While this mechanism is unlikely to affect Treg cells tagged with the IRESEGFP cassette, it remains possible that in the latter mice Treg commitment is inhibited by another mechanism, such as altered methylation of the Treg cell-specific demethylation region of the Foxp3 locus.20 Hirota et al.3 therefore re-examined the ability of Treg cells to differentiate into TFH cells14 using two different labelling technologies: congenic labelling with CD45.1, and a bicystronic red fluorescent protein expression cassette (IRESmRFP) inserted into the Foxp3 locus.23 Research Highlight 376 After adoptive transfer, Treg cells originally obtained from lymph nodes retained FOXP3 expression, were mainly restricted to lymph nodes, and did not acquire a TFH profile. Although Treg cells obtained from lamina propria and PP homed efficiently to PP, they also failed to acquire a TFH phenotype, induce GC B cells or support IgA production.3 In 2009, the relationship between IL4producing cells and TFH cells was examined in experiments that simultaneously used two reporters, one targeted to each Il4 allele.24–26 The 4get allele is bicistronic and contains an IRES-EGFP construct after the stop codon of the Il4 gene. It is expressed whenever the Il4 locus is transcribed. The second allele, the KN2 reporter, replaced the first two exons of the Il4 gene with a coding sequence for human CD2, which includes a transmembrane region that results in human CD2 being expressed on the cell surface of cells attempting to translate IL4.27 The resulting mice have a single functional copy of Il4 (i.e., the allele is hemizygous)—that associated with the bicystronic reporter.27 In infections with three different parasites—a nematode, a trematode and a trypanosome—virtually all IL4-secreting CD4 T cells in reactive lymph nodes (either peripheral or mesenteric nodes, depending on route of exposure) had become TFH cells, as defined by the expression of CXCR5, ICOS, PD-1, IL21 and BCL6.24–26 Similar studies have been performed to characterise the plasticity of Th17 cells.2,3 The tagging technology used in these cases replaced the first exon of the Il17a gene with sequence encoding Cre, intronic sequences and an SV40 polyA tail. To ensure that only Cre was transcribed from the locus, a cryptic start site in exon 2 of the Il17a locus was mutated. All m (...truncated)