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Marsupials are a distinct lineage of mammals notable for giving birth to highly altricial (relatively less developed) young. The recent discovery of a unique TCR chain in marsupials, TCRµ, raises questions about its possible role in early development. Here we compare the timing of V(D)J recombination and appearance of TCRµ transcripts relative to the conventional TCRα, β, γ and δ mRNA during postnatal development in the opossum. There are two TCRµ transcript isoforms, TCRµ1.0 and TCRµ2.0. TCRµ1.0, which uses pre-joined V(D)J segments, is detectable as early as day 1, when the thymus is primarily undifferentiated epithelium. The other isoform, TCRµ2.0, which requires V(D)J recombination and contains an unusual double V configuration, is not detectable until day 13 when the thymus is histologically mature. Surprisingly we were able to detect TCRα, β and δ mRNA transcribed from loci that had completed V(D)J recombination as early as day 1 as well. At this early age there is apparent evidence for preference in the V segments used in the TCRα and β genes. In the case of Vα this preference appears to be associated with position in the TCRα/δ locus. In Vβ however preference may be due to the use of microhomology in the V, D, and J segments. Mature TCRγ transcripts were not detected until day 8 suggesting that, in contrast to eutherian mammals, in the opossum αβT cell development precedes γδT cell development. The results support that there may be differences in T cell subset development between marsupials and placental mammals.
The specific recognition of antigen by T cells is a critical step in the adaptive immune responses in all jawed vertebrates [1, 2]. This antigen recognition is mediated by the TCR complex, which is composed of the CD3 signal transduction molecules and either the TCRαβ or γδ heterodimers . Like Ig, the four TCR chains (α, β, γ and δ) are encoded by gene segments, called the V, D, and J for TCRβ and δ, and V and J for TCRα and γ, which undergo somatic DNA recombination to generate receptors with diverse binding specificities . Homologous TCRα, β, γ and δ genes have been described from all the major lineages of jawed-vertebrates, demonstrating their relatively high degree of conservation [2,4,5]. Recently a new, fifth TCR chain isotype was discovered during the analyses of the genomes of marsupial mammals [6,7]. This new TCR, designated TCRµ appears to be a bona fide TCR based on a number of characteristics including: i) Sequence similarity to other TCR chain C regions, ii) Predicted structural features shared with other TCRs including conserved residues involved in interactions with the CD3 complex, and iii) Being encoded by genes that undergo somatic V(D)J recombination in thymocytes . TCRµ is clearly distinct from TCRα, β, γ and δ however since it is encoded at a separate locus and has a number of features not found in the conventional TCR .
TCRµ is atypical for a TCR in that it is transcribed in two isoforms, TCRµ1.0 and TCRµ2.0, that both appear to be able to encode full-length, transmembrane proteins . Of the two isoforms, TCRµ1.0 most resembles a conventional TCR chain in structure and is predicted to encode a polypeptide containing a leader (L) sequence, single V and C domains, as well as TM, and cytoplasmic regions. TCRµ1.0 is unusual, however, in that the V domain is encoded by a joined V gene (Vµj)2, one in which the V, D, and J segments are already fused together in germline DNA rather than recombined somatically. The Vµj exon appears to have been generated by retrotransposition and is found in all marsupial species investigated so far and is the first known instance of a germline joined V in a TCR . The other isoform, TCRµ2.0, encodes a TM protein with an unusual double V organization. The N-terminal V domain is encoded by gene segments assembled by V(D)J recombination and the C-proximal V is encoded by Vµj. TCRµ2.0 would encode a receptor that is somatically diversified whereas TCRµ1.0 appears to encode an invariant receptor. Whether both isoforms are functional is not known. Transcripts for both are found in the thymus at similar levels, however TCRµ2.0 is the more abundant of the two in peripheral tissues .
What functions TCRµ+ T cells perform remain unknown. However, since the hallmark of marsupial biology is birth of extremely altricial young, we speculate that TCRµ might be a novel adaptation to their early development. At birth marsupial lymphoid tissue is undifferentiated, and in most species the thymus does not contain defined cortical and medullary regions until a week or more following birth [8,9]. The absence of a mature thymus at birth is consistent with functional studies reporting poor or absent conventional T cell mediated immune functions in neonatal marsupials, such as allograft rejection and T dependent antibody responses [9–11]. Therefore marsupials face peculiar immunological problems and may have evolved novel mechanisms for dealing with their unique reproductive strategy.
One indication that TCRµ may play a role in early development is from the presence of the joined V segments. One of the few examples of germline-joined V genes in other antigen receptors is in the Ig genes of cartilaginous fishes such as sharks [12,13]. Antibodies encoded by these germline joined V genes are expressed earliest in development, leading investigators to speculate that they may have a specific early ontogenic role [13–15]. Furthermore, we speculate that TCR chains containing pre-joined V might lack the need to undergo thymic selection and therefore could be utilized prior to maturation of the thymus, providing the newborn with a specific T cell repertoire, one that is hard-wired in the germline. One prediction of these hypotheses is that TCRµ expression might precede that of the conventional TCR in ontogeny. To test this prediction, here we compare the appearance of mature, conventional TCR and TCRµ transcripts during development in a model marsupial species, the gray short-tailed opossum, Monodelphis domestica. In addition we investigated V gene usage in the TCR repertoire during the period of time that the thymus in this species is undergoing its greatest maturational changes.
Estrus was induced by the presence of a male in the cage. The breeding pairs were allowed to mate and were kept in the same cage for two weeks. Males were then removed from the cage and females were monitored daily for the presence of newborns. Opossums typically give birth in the evening, after a 15 day gestation, to a litter of 8 to 9 pups . For the purposes of this study, day 1 was counted as the morning the pups were observed after having been born the evening before. Newborns were removed from the mother’s teat individually at different postnatal time points from day 1 through 16. Data on diversity of TCR transcripts and gene segment usage were pooled from two individual animals at days 1, 2, and 8. Data shown for week 2 is pooled from single individuals collected on postnatal days 12 through 16. Tissues were also collected from a 9 week old individual, an age which corresponds to one week after the young are weaned . Newborn opossums were also collected on days 3 through 7 and used to test for expression of specific TCR isotypes; for example 6 and 7 day old pups were tested for TCRγ expression to confirm that this does not occur prior to day 8. Due to the size of the neonates, collection of isolated lymphoid organs was not always possible. The upper thorax was utilized as the area that contained the thymus in newborns from postnatal days 1 through 16. For 9 week opossums isolated thymus tissue was easily collected. Fresh tissues were immediately processed to extract the RNA or stored in RNAlater® (Ambion, Austin, TX) at 4°C for 24 hours and −80°C thereafter. All animal procedures were performed under the approved Institutional Animal Care and Use Committee protocol No. 07UNM005 to R.D.M.
RNA extraction was performed using the Trizol RNA extraction protocol (Invitrogen, Carlsbad, CA) as previously described . Reverse transcription-polymerase chain reactions (RT-PCR) were performed using GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, CA). PCR reactions were performed using Advantage™- HF 2 PCR (BD Biosciences, CLONTECH Laboratories, Palo Alto, California) with previously described conditions .
Total RNA was used to perform 5’ RACE using the Gene Racer™ kit from Invitrogen (Carlsbad, CA). Reverse-transcription of the mRNA was done using SuperScript™ III RT and random primers. Amplification of 5’ cDNA ends was done using Advantage™- HF 2 PCR, as described previously . In these PCR reactions, a nested set of specific primers complementary to the C regions of each of the TCR were paired with GeneRacer™ 5’ PCR primers. The sequences of the C region specific primers were published previously .
To amplify TCR transcripts containing specific V subgroups directly, primers specific for each V subgroup were paired with the reverse C specific primers. In the case of Cα and Cδ this would also include Vα/δ segments previously shown to be used with either C region . Since all products analyzed in this study were sequenced, the specificity of amplification in each case was confirmed. The primers used to amplify opossum TCRα, β, γ and δ V subgroups have also been published previously . PCR products were cloned using TOPO TA Cloning® Kit for Sequencing from Invitrogen (Carlsbad, CA) and plasmids were sequenced using BigDye Terminator Cycle Sequencing Kit v3 (Applied Biosystems, Foster City, CA) as described before . The total number of clones analyzed for each locus at each age is presented in Table I. Included is the number of unique sequences found in each pool of total clones (for example there were only nine unique sequences found amongst 36 separate clones for Cα at day 2). Also included is the number of different V segments and represented subgroups for each case. All 864 unique sequences identified can be found under the following GenBank accession numbers: TCRα: EU587537 - EU588001; TCRα/δ: EU588002 - EU588027; TCRβ: EU588028 -EU588347; TCRγ: EU587490 - EU587536; TCRδ: EU588348 - EU588383; where the specific V segment is annotated for each. Primers used to test for expression of TCRγ germline transcripts are: forward primer 5’-GCCCAAGGAAGAGTCTTGTAGTGG-3’ and reverse primer 5’-GGAAAAAGATGGGCTTTGGTGGC-3’. GenBank accession numbers for these TCR sterile transcripts are EU588384 - EU588390. RAG: forward primer: 5’-TCCCGAGGAACGCTACCATAGAA-3’; reverse primer: 5’-TGTGCTCACAGATCTGGCATGAG-3’. Tdt: forward primer: 5’-CCCAATATGCTTGCCAGAGACGG-3’; reverse primer: 5’-GCCCAAACTCCTTTCCCCTTCGG-3’. Reverse nested primers for C regions: Cγ: 5’-GGATGCCCAGTTTGGACCGATCAC-3’. Cβ: 5’-GGGGTCAGCACAGACCCTGAGCCC-3’. Cδ: 5’-GGAGTCGTTGCTACTGCAGATGGG-3’. Cα: 5’-CTGTGAGGAGGC AGACTGAAGTG-3’.
Opossum germline TCR V, D, and J gene segments were deposited at the Somatic Diversification Analyses (SoDA) website (http://dulci.org/soda/). Identification of V, D, and J gene segments used in each recombinant, presence of P and N nucleotides at the V(D)J junctions, and presence of microhomology between the gene segments was aided by using the SoDA algorithm followed by hand analyses .
The recent completion of the opossum genome project has facilitated determining both the complete content and organization of the TCR loci and analyses of the ontogeny of TCR expression for the first time in any marsupial [7,17,19]. To investigate the timing of the appearance of transcripts encoding the two TCRµ isoforms, RT-PCR was performed using RNA isolated from opossums at different ages (Figure 1 and Figure 2). To distinguish TCRµ1.0 transcripts from TCRµ2.0, forward primers were located in either the FR1 of Vµ to amplify TCRµ2.0 transcripts or in the leader sequence of the Vµj to amplify the TCRµ1.0 isoform (Figure 1A). We were able to detect TCRµ1.0 transcripts as early as postnatal days 1 and 2 (Figure 1B). Mature TCRµ2.0 transcripts, however, were not detected prior to day 13 (Figure 1B). TCRµ2.0 requires V(D)J recombination for expression and its later expression corresponds well with the appearance of a histologically mature thymus in the opossum .
To determine when mature conventional TCR transcripts appear in opossum ontogeny, we designed PCR primers to amplify the TCRα, β, γ and δ messages by RT-PCR. For clarity, the term “mature transcripts’ is used to denote those transcribed from loci that had undergone complete V(D)J recombination. All efforts were made to ensure that such transcripts would be detected independent of the V and/or C region genes were being utilized. Primers were first designed for the C regions of each of the conventional TCR genes . TCRα, γ, and δ have single C regions and a single C-specific oligonucleotide for each was able to prime all possible transcripts. TCRβ on the other hand has four C region genes, however they share > 97% nucleotide identity and one primer sequence was found to amplify TCRβ transcripts containing any one of the four C regions . Using these primers, 5’ RACE PCR was performed using RNA from opossums at postnatal day 2, 8 and at week 2. 5’ RACE was used in these experiments so that transcripts might be detected independent of which V(D)J combination were used. Using this approach we were able to detect mature TCRα, β, γ and δ transcripts on days 13 and 14, but not days 2 and 8 (not shown).
These results are consistent with the analysis of the TCRµ2.0 transcripts in that it appears that those TCR loci requiring V(D)J recombination are not detectable until a time-point when the opossum thymus is more developed. However, to ensure that we were not missing rare or lowcopy conventional TCR transcripts by using the 5’ RACE approach, we also paired the C region primers with primers specific for each of the V subgroups determined previously . In our experience, the approach of targeting specific V regions is a more sensitive way of detecting rare, mature TCR transcripts than is 5’ RACE, however this sensitivity is at the sacrifice of any quantitative analyses of the transcripts since we are targeting specific V subgroups. In other words all we are able to conclude is if a particular transcript is present or not. Using this approach, surprisingly, we were able to detect mature TCRα, β, and δ transcripts containing particular V subgroups as early as postnatal days 1 and 2 (Figure 2 and Figure 3 and data not shown). Sequencing of the clones amplified at these early time points confirmed that the transcripts contained sequences that matched TCR gene segments that contain canonical recombination signal sequences and are un-rearranged in the germline DNA and, therefore, had undergone V(D)J recombination (not shown) . Furthermore, there is no evidence of germline joined V segments in any of the conventional TCR loci that could have encoded these transcripts in the absence of somatic V(D)J recombination . In other words, as early as the first postnatal day of life in the opossum evidence of developing T cells that have completed V(D)J recombination at the TCRα, β, and δ loci was detectable. Consistent with this conclusion, we were able to detect expression of RAG1 using RT-PCR as early as day 1 and all subsequent days demonstrating that V(D)J recombination is possible at these early time points (data not shown).
In contrast to TCRα, β and δ, mature TCRγ transcripts were not detected prior to postnatal day 8 (Figure 2 and data not shown). To determine if there was evidence of recombination at the TCRγ locus at earlier time points, we investigated the presence of the sterile transcripts that are thought to contribute to the chromatin remodeling that is required for RAG mediated recombination, and which are indicative of initiation of V(D)J recombination . These transcripts typically contain a non-coding exon that is located 5’ of the J segments in addition to the C exon. While performing 5’ RACE for all four of the conventional TCR loci at postnatal days 13 and 14 we were able to identify mRNA transcribed from loci likely in the germline state (not shown). Since mature TCRγ transcripts were undetectable until at least a week later than TCRα, β and δ, initiation of recombination at the TCRγ locus was examined at earlier time points by RT-PCR using primers specific for the non-coding exon. Such transcripts were detected as early as day 2 consistent with at least the initiation of recombination at the TCRγ locus at earlier time points (not shown).
Detecting TCR transcripts using V subgroup specific primers also allowed for analyses of V gene use in T cell development early in ontogeny. The TCRα locus contains 41 Vα subgroups which were numbered by the convention where Vα1 is the most 5’ or distal subgroup and Vα41 is the most 3’ or C proximal subgroup . This is presented in Figure 3 as a pie chart where the subgroups are generally arrayed 5’ to 3’ in a clockwise fashion. In some cases there is minor interspersion of gene segments from neighboring subgroups and in one case, Vα2, there are members located both at the extreme 5’ end and in the middle of the locus . This exception does not affect the conclusions drawn below. What emerged from this analysis is that only four Vα subgroups are used by TCRα transcripts on day 2 and these are four of the five most 3’ or C proximal subgroups. This bias was found in both productive and non-productive V(D)J recombinants suggesting the bias in favor of 3’ Vα subgroups is not likely due to thymic selection but rather a bias in the gene segments used for recombination (Figure 4 and data not shown). These subgroups have also been shown to be used later in development by both TCRα and TCRδ chains and are, by definition Vα/δ . By day 8 the apparent positional affect of V subgroup use is gone and by week 2 all subgroups are being used (Figure 3). Recombination between Vα and Jα gene segments appears to be random with five different Vα segments recombined with eight different Jα segments in the day 2 old opossum (Figure 4 and data not shown). Although there was a preference for location of Vα segments, there is no apparent positional affect for the Jα segments that are expressed at day 2, as the eight Jα used were scattered amongst the 53 segments available (Figure 4 and see map of the TCRα/δ locus in reference 17 for location). This may be due to a limited number of sequences analyzed in this study. Also, there was no apparent preference for specific Vα -Jα recombinations, since none of the unique sequences isolated at day 2 used the same V–J pair.
Evidence of biased recombination between V, D, and J gene segments in other species has been attributed to DNA microhomology at the 5’ and 3’ ends of the segments . To investigate the molecular nature of the biased use of Vα segments at day 2 in the opossum we examined the TCRα recombinants for the presence of microhomology and N and P nucleotides. Microhomology is evident as nucleotides that could be encoded by either of the adjacent gene segments, i.e. V-D, D-J, or V-J, and the absence of N and P nucleotides at the junctions. Of the nine unique TCRα recombinants isolated from day 2: four contained both N and P nucleotides, two contained only N nucleotides, one contained only P nucleotides, and two contained neither (Figure 4 and data not shown). Of the two lacking any N or P nucleotides only one contained evidence of microhomology between the V and J segment (Figure 4). Therefore there is little evidence that microhomology contributes to the choice of Vα subgroups early in ontogeny in the opossum. The presence of clear N nucleotide additions to the junctions indicates the presence of TdT activity early in development. To confirm this result we examined TdT expression by RTPCR, however the earliest time point we could detect transcripts for this enzyme was day 10 (data not shown). Therefore, TdT expression, although evident from the presence of N nucleotides is below the level of direct detection at the earliest days.
Similar to TCRα, the early TCRβ repertoire is characterized by a limited number of V subgroups. However, rather than being clustered at one end of the locus, the Vβ subgroups used at postnatal day 2 are scattered throughout the locus apparently making chromosomal position not a determining factor (Figure 3). Like Vα, the number of Vβ subgroups used increased with age and by week 2 all subgroups are used. Of the eight unique TCRβ recombinants isolated at day 2 only one contained an N nucleotide and one contained a P nucleotide. The remaining six, lacking N and P nucleotides, all contained evidence of microhomology between the gene segments used in the recombinants, both productive and non-productive (Figure 4 and data not shown). Therefore, and in contrast to TCRα, microhomology appears to play a role in the selection of TCRβ gene segments being recombined early in ontogeny. Opossum TCRβ locus contains four D-J-C clusters  and recombination between D gene segments with J gene segments located in different clusters was observed at all time points (Figure 4). At day 2 there appeared to be a slight preference (four out of eight recombinants) towards using the Dβ1, the lone Dβ segment in the most 5’ cluster, with the Jβ4.3 segment from the most 3’ cluster . By day 8, all Dβ and Jβ segments from the four clusters were found to be used, however there remained a preference for using Dβ1, since 72% of the sequences isolated at day 8 (82 of 109 unique recombinants) and week 2 (143 of 201 unique recombinants) contain this gene segment. At these later time points, the use of Jβ segments seems more widespread, although a preference towards using the Jβ gene segments from cluster four is maintained. A significant fraction of the sequences isolated on day 8 (72 of 109) and week 2 (98 of 201) contained Jβ gene segments cluster 4. Apparent from the analyses of the TCRβ recombinants isolated at day 8 and week 2 was a trend towards increasing CDR3 length that was not evident in TCRα (Figure 5). The longer CDR3 however was not due to an increase in the addition of P and N nucleotides, but rather to less trimming of germline encoded nucleotides at the V-D-J junctions (Figure 4).
Mature TCRγ transcripts were not detected until postnatal day 8 (Figure 2). The TCRγ locus contains only 4 V subgroups and only primers specific for Vγ4 were able to amplify transcripts on day 8. However, all four Vγ subgroups were being utilized by week 2 (Figure 3). Vγ4 is a single member V subgroup that is the second most C proximal V segment . The few unique TCRγ recombinants obtained at day 8 lacked N and P nucleotides, and microhomology was evident (Figure 4). Jγ1 was found in 58% of the TCRγ recombinants on day 8 (2 out of 2) and week 2 (8 out of 15) making it the most abundant used.
Restricted gene segment usage early in development was also found for TCRδ, where only Vδ2 is used on day 2 (Figure 3). However, there are only four functional Vδ segments, each their own subgroup, all of which can be used as Vα/δ later in development . Although the Vδ2 subgroup that is used at day 2 is not the most 3’ of the Vδ, like most Vδ it is amongst the most 3’ V segments in the TCRα/δ locus . At day 2 the Vδ2 segment is used with a single D, D2, and with both Jδ5 and Jδ6 (Figure 4). Similar to TCRα, N and P nucleotides were observed in all sequences and there was no evidence of the use of microhomology (Figure 4). There are two Dδ gene segments in the opossum TCRα/δ locus . All TCRδ sequences isolated from day 2 use only the Dδ2 gene segment. However, 58% of the TCRδ recombinants isolated at days 8 (2 of 4) and week 2 (17 of 29) contained both Dδ gene segments, making the CDR3 of these chains longer (Figure 4).
Marsupials are born at a much earlier stage of development than are eutherian mammals making them important models for early immunological development . Opossums are members of the family Didelphidae and two particular genera, Monodelphis and Didelphis have been utilized for biomedical research. Histological studies of the thymus in newborn and developing Didelphis virginiana revealed this lymphoid organ to be primarily undifferentiated epithelium lacking defined cortical and medullary regions, but containing large lymphocyte like cells [22,23]. By day seven medium size lymphocytes can be found localized at the edge and small lymphocytes in the center of the thymus, and the medullary region is apparent by day 12 in this species [22,23]. Similar results were reported for Monodelphis domestica. At postnatal day one in this species the thymus lacks defined cortical and medullary regions and Hassall’s corpuscles . By day seven, a distinct cortical region and Hassall’s corpuscles are observed, but the medullary region remains poorly defined . By the end of the second postnatal week, the opossum thymus appears mature with a well defined medulla . Functional studies at early stages of development in marsupials correlates with the histological studies and appears to reflect a general lack of immune competence. For example, D. virginiana less than two weeks of age fail to reject allografts . Studies of neonatal humoral responses in this species revealed that in most cases animals in the first week of life had impaired or absent T dependent antibody responses [11,23,25]. Such responses were not generally observed until the second postnatal week. Although these studies remain limited, they are consistent with development of functionally mature T cells primarily occurring postnatally in marsupials.
Most marsupials therefore face a critical period immediately after birth when their endogenous adaptive immune system remains undeveloped yet no maternal Ig has been transferred. M. domestica, like most marsupials, does not obtain maternal Ig until nursing begins . The rare exception to the tranfer of maternal Ig in marsupials are tammar wallabies, which appear to acquire IgG across the yolk sac [8,27]. Maternal IgG and IgA transfer through the milk has been demonstrated in several different marsupials [11,26,28,29]. Compared to placental mammals, the transfer of Ig in marsupials occurs for a longer period of time. For example in the quokka (Setonix brachyurus) and in the brush-tail possum (Trichosurus vulpecula) IgG can be transferred through milk until postnatal days 170 and 85 respectively . The long period of maternal Ig transfer and the newborn’s ability to absorb it are likely one of the mechanisms used to compensate for the undeveloped immune system at birth.
The relative immuno-incompetence of newborn marsupials raises a number of questions regarding the dependency on maternal immunity and the development of self-tolerance in the face of exposure to pathogens and environmental antigens. These differences raise the possibility that novel strategies have evolved to protect the newborn marsupial. To better understand the nature of development of immuno-competence in neonatal opossums, we wish to investigate the timing of appearance of mature T cells. Unfortunately, marsupial specific reagents such as mAb capable of identifying T cell subsets are currently unavailable. Therefore, as a proxy for identifying early time-points for the initiation of T cell development we have used the appearance of mature TCR transcripts. Such transcripts would occur relatively early in T cell ontogeny and therefore do not directly indicate the presence of mature, functional T cells, but rather indicate the presence of cells committed to the T cell lineage and the completion of V(D)J recombination.
It may be that early developing T cells are playing an important role in driving thymus differentiation in the newborn opossum. Interactions between the thymocytes and the nonlymphoid thymic stromal cells play an important role in T cell development in the thymus . Thymic stromal cells have an effect on cell proliferation, differentiation and apoptosis of T cells by production of cytokines and through cell to cell contact. Furthermore, this interaction is bidirectional since the differentiation and maintenance of stromal cells are also dependent on the developing thymocytes . Therefore, early T cell differentiation may be driving maturation of the thymus in newborn opossums making it unsurprising that mature TCR transcripts are detectable much earlier than the appearance of a histologically mature thymus.
One of the unexpected results to emerge from our data was not simply the early timing of the detection of mature TCR transcripts but also the order in which they appeared. In placental mammals and birds, the development of γδT cells generally precedes that of αβT cells in ontogeny . In the opossum, however, mature TCRα and β transcripts were detectable much earlier than were TCRγ transcripts making it possible that αβT cells appear prior to γδT cells. Perhaps this is an evolutionary adaptation to compensate for the extreme altricial nature of the marsupial young at birth. It may be that by producing αβT cells early the opossum will be better prepared for generating systemic cell mediated immune responses and T dependent antibody responses. It is also possible that there is a need to develop αβT cells early as a solution to the problem that exogenous antigens may create while negative selection in the thymus is ongoing. By initiating αβT cell development perinatally it is possible that some cells will have the chance to mature in the absence of environmental antigens and pathogens that the newborn will be exposed to immediately following birth. Alternatively a specific repertoire of T cells may be needed in the newborn opossum, a hypothesis that is consistent with the limited V(D)J combinations found in the earliest TCRα and β transcripts.
The TCRα and β transcripts isolated from newborn opossums were derived from V(D)J recombinations that appear to utilize a limited and select number of V gene segments. It is possible that this oligoclonal repertoire is not necessarily a programmed bias but rather simply a limited number of total T cells available at this early stage. This seems unlikely however given the evidence of a bias for C proximal V segments in TCRα and the prevalence of using micro-homology in TCRβ. In other words, the TCR repertoire at this early age is oligoclonal but not apparently random. Whether this is due to selection on random V(D)J recombinations or preselection bias in the V(D)J recombination is not known, however the latter seems more likely. One reason is that the bias in TCRα and TCRβ V(D)J combinations suggest different mechanisms: location of the V segments versus micro-homology, respectively. Whereas the positional bias observed in TCRα could be evidence that the chromatin remodeling necessary for V(D)J recombination  results in favoring C proximal V segments, the lack of positional bias in TCRβ suggests that opening the chromatin at these loci early in ontogeny is not a problem. Furthermore, non-productive rearrangements contain the same V segment bias as the productive rearrangements at day 2, suggesting this bias occurs prior to thymic selection. Therefore it seems more likely that a developmental program is selecting the rearrangements of TCRα and β gene segments rather than positive and negative selection. This restricted and biased TCRαβ repertoire could be an evolutionary adaptation to respond to pathogens commonly encountered in the earliest days of life. This bias could be analogous to that of the invariant γδT cells and the anti-phosphorylcholine antibody idiotype, T15, in mice [33,34]. Such early bias in TCR and Ig repertoires appear important for establishing in a programmed way responsiveness to common or evolutionarily important antigens and pathogens.
Although restricted use of V gene segments was evident, N nucleotide addition in the opossum was observed early in development, which is indirect evidence of early expression of Tdt in this species. Not surprisingly there is an inverse relationship between those recombinants containing N nucleotide additions and those utilizing micro-homology. N nucleotide addition at early time points in development varies in different species [34,35]. In the opossum TCRα, β and δ recombinants isolated at postnatal day 2 contained N nucleotides, however they more frequent in TCRα and δ recombinants. Lower frequency of N region additions in TCRβ compared with TCRα recombinants is likely evidence of TCRβ rearranging first, consistent with T cell development in other species . Addition of N nucleotides early in development has also been found in IgH chains from newborn sharks . This is in contrast to what is found in human IgH, mouse TCRβ or chicken TCRβ chains, in which addition of N nucleotides is absent early in development [34,38,39]. The expression of Tdt in young opossums may be an adaptive mechanism to diversify the early TCR repertoire, especially since few V gene segments are being utilized. TCRα and β recombinants obtained from the opossum at week 2 have slightly longer CDR3 regions than the respective recombinants isolated at day 8 (Figure 5). TCRα had longer CDR3 mostly due to increase in the addition of N nucleotides (Figure 5). TCRβ CDR3 regions in the opossum were shorter at day eight than at week 2, mostly due to increased trimming of the D segments at the earlier time point (Figure 4 and Figure 5). Reduced trimming of D segments later in development have been reported for IgW and IgM in newborn sharks . Opossum TCRδ CDR3 was longer later in development mainly due to the usage of two D segments per recombinant which is facilitated by the asymmetrical RSS that flank these gene segments . The increase in the CDR3 length of the opossum TCR at later time points in development may be the result of different selection mechanisms for T cells throughout development.
Despite detailed genomic and phylogenetic analyses the origins of TCRµ remain mysterious [7,17]. What role the TCRµ1.0 isoform might be performing, given its expression early in development, remains unanswered. It is possible that TCRµ1.0 may not be translated but rather its transcription is part of the mechanisms that facilitate the chromatin remodeling that precedes V(D)J recombination. TCRµ1.0 could be the equivalent to sterile germline transcripts observed in conventional TCR . Sterile germline transcripts have been detected for the opossum conventional TCR as early as day 2 for all loci (data not shown). It is possible that for the TCRµ locus, the TCRµ1.0 isoform performs this role, being transcribed early to facilitate the V(D)J recombination that results later in the generation of the TCRµ2.0 isoform. This hypothesis is consistent with prior results revealing that TCRµ1.0 is abundant in the thymus but nearly undetectable by Northern blot and only rarely found by more sensitive RT-PCR analysis in the peripheral lymphoid tissues . Alternatively, TCRµ1.0 might be translated and provide an invariant chain receptor early in development. This possibility is supported by the observation that the sequence encoding the leader peptide upstream of the Vµj, which does not require mRNA splicing, is conserved across marsupial species . However, as previously shown the leader sequence is not conserved across all TCRµ clusters within the opossum, therefore not all clusters can encode the TCRµ1.0 isoform . If translated into protein, the invariant nature of TCRµ1.0 due to the use of the pre-joined V segments exclusively could provide an evolutionarily conserved response to a common epitope or epitopes that newborn animals are likely to encounter.
It is predicted that TCRµ forms heterodimers with another TCR chain, a prediction based on conserved residues involved in chain pairing . Which chain this might be is unknown, however. An intensive scan of the M. domestica whole genome sequence has not revealed any additional TCR loci other than TCRα/δ, TCRβ, TCRγ, and TCRµ, that might be the partner for TCRµ . Several observations support TCRγ being the most likely partner for TCRµ. First, as with Ig, TCR chains pair in a combination of H and L chains with the H encoded by genes that underwent recombination of V, D, and J segments, the L only V and J . If this rule were applied to TCRµ, which contains V, D, and J segments, it should pair with either TCRα or TCRγ, which are the two loci using only V and J . Secondly, the transmembrane regions of TCR chains contain conserved basic amino acids, lysine and arginine, that are important for chain pairing. Generally a TCR chain containing a single lysine pairs with a chain containing a lysine and arginine . TCRµ contains both the lysine and arginine, consistent with it likely pairing with TCRγ, which contains only a single lysine . At the present time this conclusion is merely an argument made by process of elimination and the true partner for TCRµ remains to be determined empirically. Furthermore, the inability to detect TCRγ expression coincident with the earliest transcription of TCRµ1.0 raises further questions as to what the latter’s partner might be if it were expressed as a heterodimer . It is possible that TCRγ is expressed earlier at levels below the sensitivity of detection of the RT-PCR performed here, however.
In the introduction to this paper we speculated that TCRµ might be present in marsupials and absent in placentals as a novel adaptation in the former to compensate for being extremely altricial at birth. We hypothesized that one exception of this speculation would be expression of TCRµ earlier than the conventional TCR in newborn marsupials. This is apparently not the case however, at least not for TCRµ2.0. Rather marsupials may have also evolved several strategies that include the conventional αβT cells bearing a restricted TCR repertoire as well. This does not mean that TCRµ+ T cells are not important in early development in marsupials. But rather they are likely part of a variety of mechanisms marsupials may have evolved to protect the immunologically vulnerable newborn.
1This publication was made possible by support from the National Institutes of Health Initiative for Maximizing Student Diversity (ZEP, JT) and Institutional Development Award (IDeA) program of the National Center for Research Resources (MLB and RDM), and awards from the National Science Foundation (RDM) and the Robert C. McNair program (AML).
2Vµj: Pre-joined Vµ gene.