Early Histocompatibility: Color the Mechanism Green and Redby Gary W. Litman, Larry J. Dishaw

Current Biology



tF bu id h u ra in genetics, how these molecules actually

Hydractinia are mediated, it’s worth homophilic trans protein–protein engineering [6] of the Alr genes and specific alleles in in vivo allorecognition departure from homophilic binding, it could be predicted if the interactionsexploring the underlying biology, which is fascinating. Asexually-expanding polyps are joined by colonial tissue through vascular-type canals. When the leading made a major phylogenetic leap to incorporate a mammalian read-out system, in which epitope tags — short peptides that can be recognized with between Alr proteins are along limited contact interfaces; a weaker binding may be biologically amplified but below the detection threshold of the colorwhereby allorecognition outcomes in undertook some straightforward geneticeffect allorecognition has not been clear.

New work from the Nicotra laboratory published in this issue of Current Biology [6] reveals that the basis for fusion or rejection of adjacent colonies involves homophilic trans protein–protein interactions.

Before getting to the mechanism interactions. But how do you test this in a model system in which the effector cells are not known and the animal is not amenable to many of the experimental manipulations that we take for granted in other experimental transplantation systems?

To solve the problem, the authors studies (Figure 1). Aggregation was detected in one case in which cells were expressing alleles that differ at four amino acids of which three are in the predicted protein spacer region and the other is in an extracellular domain.

Whereas this single disparate result could be inferred to represent aEarly Histocompa and Red

Gary W. Litman* and Larry J. Dishaw

Department of Pediatrics, University of South *Correspondence: glitman1@jhmi.edu http://dx.doi.org/10.1016/j.cub.2015.09.053

Allorecognition in Hydractinia, a cn transmembrane proteins. Using a fl specificity involves homophilic inte

Self–non-self recognition in multicellular organisms takes on a variety of forms. The adaptive immune system of vertebrates likely is the most complex, highly integrated and diverse form of self–non-self recognition, in which some of the molecules involved in the response to pathogens are themselves determinants of graft rejection [1,2]. Other life forms that extend deep into metazoan phylogeny utilize different approaches for allorecognition [3]. Transplantation experiments in invertebrates have revealed the genetic basis for histo(in)compatibility in two main models: a cnidarian, Hydractinia; and a colonial tunicate, Botryllus [3,4]. In

Hydractinia, allorecognition is controlled by two genes, Alr1 and Alr2, that encode single-pass transmembrane proteins that are distantly related to the immunoglobulin gene superfamily [3,5].

Although much is known about theiredge of the colonial tissue comes into contact with opposing tissue, projections known as stolons interact. The resulting vascular connectivity that forms then allows stem cells of one genetic type to migrate into the vasculature of the fusion

R1042 Current Biology 25, R1032–R1050, Noibility: Color the M lorida, 140 Seventh Avenue South, St. Peters arian, is governed by two different, orescent cell read-out system, a new ctions between extracellular doma partner, with potentially devastating loss of population fitness through germline parasitism. Years of observations have confirmed that three distinct experimental outcomes occur in

Hydractinia transplantation assays: fusion, transitory fusion and rejection [3,7,8]. If colonies share at least one allele at both genes, they fuse. If an allele is shared at only one of the two genes, colonial fusion is transitory. If they do not share any allele at either gene, the colonies reject.

Based on comparisons of outcomes of fusion assays and patterns of variation in amino acid sequence, allorecognition is likely mediated through extracellular regions of the Alr1 and Alr2 molecules.

Given that the overall domain architecture of the proteins resembles certain other receptor and cell adhesion molecules, it was hypothesized that selfdistinction could be mediated throughhighly specific, commercially available monoclonal antibodies — were fused to

Alr1f, Alr1r, Alr2f or Alr2r (where the superscript f and r denote gene products of different Alr alleles). The epitopetagged fusion proteins then were vember 2, 2015 ª2015 Elsevier Ltd All rights reechanism Green rg, FL 33701, USA ighly polymorphic genes encoding study now shows that the basis for s. transiently transfected into a mammalian cell line and their expression on the surfaces of the mammalian cells could be detected. Next, Alr-expressing cells were co-transfected with either a GFP (green) or RFP (red) stable reporter construct; the GFP or RFP signals mark those cells that express a specific allele of an Alr1 or Alr2 gene. When GFP+ and

RFP + cells expressing compatible alleles were combined, bicolor aggregates could be identified microscopically.

When cells expressing different alleles of either Alr1 or Alr2 were combined, only single color aggregates are seen (Figure 1). The lack of co-localization of the fluorescent markers in these latter experiments was taken as further evidence for isoform-specific, homophilic trans-interactions between different Alr isoforms. The isoform-specific patterns of aggregation in vitro are entirely consistent with the behavior of

Current Biology


As one of the two prominent models of non-vertebrate allorecognition, an obvious question in studies in

Hydractinia is whether or not the findings are relevant to those in Botryllus. served

Fusion rr/rr x rr/rr

Alr1r+ GFP

Alr1r+ RFP

Current Biology

DispatchesAnswers are not quite so simple because of controversy regarding which gene(s) in this system is most predictive of allograft outcomes.

Specifically, FuHc and fester represent two cell-surface molecules reported to govern allorecognition in

Botryllus [9,10]. Recently BHF, a polymorphic member of the fuhc genetic complex, has been implicated as a predictor of alloreactivity [11]; however, it is not a cell-surface protein. Are allorecognition mechanisms fundamentally different considering that the process may begin earlier in Botryllus since larvae can recognize conspecifics in this system but not in Hydractinia?