No abstract

Many studies address the influence of the gut microbiome on the immune system, but few dissect the effect of T cells on gut microbiota and mucosal responses. We have employed larval thymectomy in Xenopus to study the gut microbiota with and without the influence of T lymphocytes. Pyrosequencing of 16S rRNA genes was used to assess the relative abundance of bacterial groups present in the stomach, small and large intestine. Clostridiaceae was the most abundant family throughout the gut, while Bacteroidaceae, Enterobacteriaceae and Flavobacteriaceae also were well represented. Unifrac analysis revealed no differences in microbiota distribution between thymectomized and unoperated frogs. This is consistent with immunization data showing that levels of the mucosal immunoglobulin IgX are not altered significantly by thymectomy. This study in Xenopus represents the oldest organisms that exhibit class switch to a mucosal isotype and is relevant to mammalian immunology, as IgA appears to have evolved from IgX based upon phylogeny, genomic synteny, and function.

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates.

Rearranged immunoglobulin (Ig) variable (V) regions undergo somatic hypermutation (SHM) in activated B cells during a T-dependent humoral immune response, allowing affinity maturation. TCR loci were believed not to undergo SHM, until the demonstration of SHM at the TCRγ locus of sandbar sharks. Our study in the nurse shark corroborates this seminal finding, as we have found SHM of TCRγ loci in spleen; we further show that TCRγ V regions are highly mutated in clones isolated from the spiral valve (shark intestine), suggesting a role for T cell SHM in mucosal immunity. Surprisingly, we also found SHM to occur extensively at shark TCRα loci. Unlike B cell SHM that follows antigen exposure in sharks and most other vertebrates, SHM at TCRα loci occurs during thymic development. TCRα SHM is supported with isolation of clusters of mutated clones having the same CDR3 rearrangement.We suggest that such SHM, in addition to TCRα receptor editing, permits developing T cells with evolving receptors to scan cortical epithelial cells for positive selection on self-peptide/self MHC. Additionlly, we have found that shark TCRδ can employ variable gene segments of IgM and IgW in rearrangements, a situation that may be similar to the IgHV use by TCRδ noted in higher vertebrates. Future studies must further study the lineage commitment of shark B and T cells and the receptor plasticity of these lineages in all vertebrates.

Somatic hypermutation (SHM) is a mechanism B cells use while responding to antigen for affinity maturation, and is also employed in the diversification of the primary B cell repertoire in the gut associated lymphoid tissues of ruminants. Recently it was discovered that SHM operates at the T cell receptor γ locus of the sandbar shark. We have looked for evidence of this AID-mediated mechanism at other T cell receptor loci in the nurse shark, Ginglymostoma cirratum. Our initial hypothesis invoked SHM at the γ and δ loci, but not at the (presumably) MHC-restricted α and β loci. Surprisingly, we find evidence for mutation at the α locus. We see evidence for canonical point mutations as well as the tandem mutations that have been a hallmark of SHM at traditional immunoglobulin, IgNAR and now T cell receptor loci of cartilaginous fish. Ongoing studies are assaying SHM at α in the shark thymus to determine if this phenomenon is being employed for affinity maturation of αβ T cell responses (a process we deem unlikely with MHC restriction). Alternatively, SHM may be used along with receptor editing at the α locus in the thymus to generate primary repertoire paratopes that can mutate to positive selection. The thymus was suggested as a “mutant breeding” organ for self tolerance by Neils Jerne forty years ago - future comparative studies will determine if SHM is an original tool of vertebrate T cells for thymic diversification or an adaptation singular to sharks.