γδTCR⁺ cells of the pregnant ovine uterus express variable T cell receptors and contain granulysin

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Animals and tissue collection
  • 2.2. Purification of uterine intraepithelial γδ T cells
  • 2.3. Anchor PCR
  • 2.4. SDS-PAGE and amino acid sequencing
• 3. Results
  • 3.1. γδ T cells in sheep uterine epithelium express a diverse TCR repertoire
  • 3.2. Granulated intraepithelial uterine γδ T cells contain high levels of granulysin
• 4. Discussion
• Acknowledgment
• References
• Copyright

Abstract 

γδ T cells are a prominent granulated cell population in the ruminant pregnant uterus and both their number and granule size increase dramatically during pregnancy. Anchor-RT-PCR was used to assess TCRδ gene usage by γδ T cells from the uterine epithelium of pregnant sheep. The TCRδ genes obtained exhibited distinct combinatorial and junctional diversity and only two out of nine V–D–J rearrangements sequenced were identical. Furthermore, two of the Vδ elements used are also expressed in peripheral blood, indicating that γδTCR use in sheep epithelia is neither restricted nor site-specific, similar to humans but in contrast to findings in mice. Protein analysis of purified, granulated uterine γδ T cells revealed the presence of large amounts of the antimicrobial peptide, granulysin. The results of the present study indicate that ovine uterine γδTCR⁺ intraepithelial lymphocytes have the potential to recognise diverse antigens and may have a role in protecting the uterus from infection during pregnancy and parturition. A similar protective role for γδ T cells may exist in the human decidua parietalis.

Keywords: Intraepithelial lymphocytes, γδ T cells, Granulysin, Pregnancy, Uterus, Sheep

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1. Introduction 

Although γδ T cells represent a substantial or dominant proportion of intraepithelial lymphocytes (IEL) within various surface epithelia in humans, ruminants and mice (Kronenberg and Havran, 2007, Meeusen et al., 2001), their exact function and antigen recognition properties have not been fully determined. γδ T cells in murine skin and reproductive tract epithelium express homogeneous TCR and have therefore a limited capacity to recognise diverse antigens; it has been proposed that they respond to a conserved antigen expressed by epithelial cells after exposure to stressful stimuli (Asarnow et al., 1988). In support of this proposal, murine γδ T cells can respond to conserved heat shock proteins and to stressed keratinocytes in vitro (Chien and Konigshofer, 2007, Kronenberg and Havran, 2007). These results in mice contrast to humans, where extensive TCR diversity has been found in γδ T cell clones from female reproductive tissues (Christmas et al., 1993, Christmas et al., 1995). γδTCR diversity is generated by rearrangement of pools of V, J, (D) and C elements during T cell development. Diversity is increased by junctional modifications including addition of non-germline encoded (N) nucleotides and P nucleotides, exonucleolytic deletion of nucleotides from the 3′ ends of rearranging elements and simultaneous use of more than one D element in TCRδ genes (Chien and Konigshofer, 2007, Kronenberg and Havran, 2007).

γδ T cells represent up to 60% of lymphocytes in the peripheral blood of sheep and express extremely diverse TCR using a particularly large number of Vδ elements (Hein and Dudler, 1993). γδ T cells are also a prominent population in the uterine epithelium of sheep (Meeusen et al., 1993). In contrast to the majority of blood γδ T cells, ovine uterine γδTCR⁺ IEL express a unique, WC1⁻, CD45R⁺, CD8^+low surface phenotype and contain distinct cytoplasmic granules (Fox et al., 1998, Meeusen et al., 1993). From mid to late pregnancy, uterine γδTCR⁺ IEL disappear from the placentomes but their number increases dramatically in the non-invaded inter-placentomal areas (from ∼25% to 70% of IEL) (Lee et al., 1992, Majewski et al., 2001, Meeusen et al., 1993). In addition, both the number and size of their cytoplasmic granules increase significantly during pregnancy (Meeusen et al., 1993). At birth, there is a sudden and dramatic decline in the number of uterine granulated γδ T cells and they show clear signs of degranulation, suggesting a distinct role for granule proteins during parturition (Lee et al., 1992, Nasar et al., 2002). In the present study, the diversity of TCR usage by the uterine γδTCR⁺ IEL was assessed, by characterizing TCRδ gene usage, and the identity of a major granule constituent was revealed by N-terminal peptide sequencing.

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2. Materials and methods 

2.1. Animals and tissue collection 

Pregnant Merino cross ewes were purchased from a commercial farm and housed indoors at the School of Veterinary Science, The University of Melbourne, under close veterinary supervision. Ewes were killed at late pregnancy (110–140 days of gestation) by intravenous injection of an overdose of sodium pentobarbital. All experimental sheep carried a healthy foetus and showed no signs of infection or inflammation after gross and histological examination by a veterinary pathologist. Tissues were collected from the inter-placentomal (i.e. non-invasive) endometrial areas only for further studies.

2.2. Purification of uterine intraepithelial γδ T cells 

Cells were prepared from the inter-placentomal uterine epithelium of a pregnant ewe at 110–140 days of gestation as described previously (Meeusen et al., 1993). For RNA preparation, γδ T cells were purified by indirect positive selection with sheep anti-mouse IgG(Fc) conjugated magnetic beads after reaction of the cells with anti-γδ TCR mAb, 86D, according to the manufacturer's protocol (Immunotech S.A. Marseille, France). For gel fractionation, cells were incubated with CD45R mAb and conjugated magnetic beads in PBS with 0.1% azide and 5mM EDTA before separating with a magnet. Bound and unbound fractions were collected, washed in PBS and stored at −70°C until use.

2.3. Anchor PCR 

RNA was extracted by the acid guanidinium thiocyanate–phenol–chloroform method and converted to cDNA with M-MLV reverse transcriptase (Unites States Biochemical, USA) and oligo dT. cDNA was filtered through a S300 Microspin™ column (Pharmacia) and RNA hydrolysed as described previously (Troutt et al., 1992). The anchor oligonucleotide sequence and preparation by 5′ phosphorylation and 3′ blocking was as described by Troutt et al. (1992). The anchor was ligated to the cDNA using T4RNA ligase (New England Biolabs) according to previously described methods (Tessier et al., 1986). Primers complimentary to the anchor (Troutt et al., 1992) and to a stretch of the TCRδ chain constant region (Hein and Dudler, 1993) were used for PCR. Amplifications were carried out using a hot start followed by 35 cycles of 94°C for 30s, 56°C for 30s and 72°C for 1min. PCR products were cloned into a T vector prepared from pBluescript^® SK(+/−) phagemid as described previously (Holton and Graham, 1991). Cloned products were fully sequenced in both directions and alignments carried out using FastA and Gap software (Wisconsin Sequence Analysis) with default settings.

2.4. SDS-PAGE and amino acid sequencing 

CD45R bound and unbound cells were solubilised in non-reducing SDS buffer and run on a 3 tier tricine SDS-PAGE gel (16% lower gel, 10% spacer gel and 4% stacking gel) and stained with Coomassie blue. Preparations containing large amounts of the selected band were blotted onto Problott membranes (Applied Biosystems, Inc), proteins visualized by staining with Coomassie blue and the selected band excised from the blot and subjected to Edman degradation sequencing as described previously (Tkalcevic et al., 1995).

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3. Results 

3.1. γδ T cells in sheep uterine epithelium express a diverse TCR repertoire 

TCRδ genes expressed by γδ T cells in sheep uterine epithelium were assessed by cloning and sequencing the products of anchor-PCR. Nine clones were fully sequenced in both directions and found to encode productive TCRδ gene rearrangements (EMBL Accession No's AJ005903–910). Since genomic sequences for sheep TCRδ elements are not available, the V–D and J–D boundaries were predicted by comparing sequences to each other and to genomic sequences for human, mouse and sheep TCRδ elements in GenBank. The predicted V elements were also translated into amino acid sequences for comparison. The nine clones derived from uterine IEL encoded seven clearly different V elements (Table 1). Clones 3 and 9 used the same V element and were the only two completely identical clones. Clones 4 and 8 also appeared to use the same V element with only a single base difference in the C terminal amino acid which probably reflects exonuclease activity and N nucleotide addition rather than a genomic difference. The seven V elements differed from each other by at least 9 amino acids. Differences were mainly clustered around position 30 and 60, equivalent to the complimentarity determining regions 1 and 2. All V elements were 342bp in length except for that of clone 2 which was 348bp long.

Table 1. TCRδ genes expressed by γδ T cells from uterine epithelium.

Clone	Vδ element comparison			Junctional region sequence (–N–D–N–)a	Jδ element nameb
	Best alignment	Nucleotide identity (%)	Amino acid identity (%)
1	3/9	94.7	91.7		3
2	8	89	80.7		1
3c	9c	100	100		3
4	8	99.7	99.1		1
5	2	64	53.5		3
6	7	94.7	92.1		1
7	6	94.7	92.1		3
8	4	99.7	99.1		1

Regions of homology within the junctional sequences are underlined. A single line represents homology to junctional regions of sheep PBL TCRδ genes (13), a dotted line homology to junctional regions of the other clones, and a double line homology to both junctional regions of the other clones and of PBL TCRδ genes.

In addition to using multiple V elements, the nine sheep uterine IEL TCRδ genes sequenced used two different J elements identified previously in sheep peripheral blood as Jδ1 and Jδ3 (Hein and Dudler, 1993). The junctional regions between the V and J elements also varied greatly in sequence and length between the clones. The factors contributing to this junctional diversity cannot be determined since germline sequences for sheep Dδ elements are unknown, however, comparison of uterine IEL TCRδ and sheep PBL TCRδ sequences reported by Hein and Dudler (Hein and Dudler, 1993) revealed that several stretches of 6, 9 and 12 nucleotides were shared within the junctional regions (Table 1). The areas of nucleotide alignment spanned the junctional regions of several sequences and were separated from each other and the V and J elements by several nucleotides. This pattern may represent the use of multiple D elements separated by N nucleotides. In addition, junctional regions preceding Jδ3 elements ended in an AT or T, while those preceding a Jδ1 element ended in AG or G. Since these nucleotides precede and are apparently dependent on the J element, they resemble P nucleotides which are thought to result from cleavage of the 5′ dinucleotide of one strand of a germline element and ligation onto the 3′ end of the complimentary strand (Lafaille et al., 1989).

The V elements of uterine IEL TCRδ genes sequenced in the present study were compared to those used by sheep γδTCR⁺ PBL, reported in a study by Hein and Dudler (Hein and Dudler, 1993) (Table 2). Two of the V elements described in the present study were clearly the same as Vδ elements previously identified in sheep peripheral blood. The Vδ element used by clones 3 and 9 differed from PBL Vδ1.4 by only one nucleotide encoding an amino acid change at position 2. However, while clones 3 and 9 use Jδ3, Vδ1.4 is rearranged with Jδ1 and these TCRδ genes have different junctional sequences. Similarly, while the Vδ element of clone 7 is equivalent to PBL Vδ1.11, with only one amino acid difference between these sequences, they recombine with different junctional regions and J elements.

Table 2. Best alignments between Vδ elements expressed in sheep uterine epithelium and previously identified sheep PBL Vδ elements in GenBank.

The clones sequenced in the present study, with the exception of clone 5, contained Vδ elements with greater than 80% nucleotide identity and are therefore likely to be related members of a family. Furthermore, all of these Vδ elements had highest levels of identity to members of the Vδ1 family in peripheral blood. In contrast, the Vδ element of clone 5 was highly homologous to Vδ2 in peripheral blood (Table 2).

3.2. Granulated intraepithelial uterine γδ T cells contain high levels of granulysin 

Previous studies have shown that all uterine granulated γδTCR⁺ IELs strongly express surface CD45R (Fox et al., 1998, Meeusen et al., 1993). Positive selection of IEL from late pregnant uteri on CD45R-coated magnetic beads confirmed that the majority of cells bound by the beads contained large cytoplasmic granules (Fig. 1A). When bound and unbound cells were run on SDS-PAGE gels, a 9kDa protein was consistently observed enriched in the bound fractions and generally depleted in the unbound fractions (Fig. 1B). Sequencing was performed on the 9kDa band after blotting of three separate samples and resulted in a clear N-terminal sequence of GLLCGPCRKIIKSLEDMVG. This sequence was 100% identical with ovine granulysin (GenBank ABR67873.1) using the BLAST program, although the peptide sequence was not at the N-terminal end of the gene sequence.

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• Fig. 1. 

  Identification of granulysin in isolated uterine γδ T cells. (A) Cells isolated from the pregnant uterine epithelium using mAb-conjugated magnetic beads (orange) shown under phase contrast light microscopy. The nucleus (n) appears grey and granules (G) blue. (B) Tricine SDS-PAGE gel showing a series of samples of uterine lymphocytes fractionated into unbound fractions (U) and bound fractions (B) with CD45R labeled magnetic beads. The 9kDa protein is enriched in the bound fractions as indicated by the arrow and was N-terminally sequenced. Low molecular weight markers are indicated on the right. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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4. Discussion 

The present study could find no evidence of site-specific Vδ element usage in ovine uterine γδ T cells. Multiple Vδ elements were used by uterine IEL and there was remarkable combinatorial and junctional diversity. Furthermore, several of the Vδ elements used by uterine γδTCR⁺ IEL have previously been identified in sheep peripheral blood (Hein and Dudler, 1993). These findings are in contrast to those in mice where γδ T cells in both pregnant and non-pregnant reproductive tract epithelium express homogeneous TCR with site-specific V element usage (Itohara et al., 1990). TCRδ genes expressed in normal bovine skin are also extremely heterogeneous as a result of multiple Vδ element use, as well as combinatorial and junctional diversity (Hein and Dudler, 1997). Similarly, γδTCR⁺ IEL from human reproductive tract use up to six different Vγ elements and three different Vδ elements (Christmas et al., 1993) and exhibit junctional diversity (Christmas et al., 1995). It is possible that inbreeding and housing of mice in controlled environments may contribute to the lack of γδTCR diversity as opposed to outbred humans, cows and sheep.

Twenty-five sheep Vδ elements have previously been described (Hein and Dudler, 1993) and an additional five different sheep Vδ elements were identified in the present study. Similarly, 26 different bovine Vδ1 elements have been described (Hein and Dudler, 1997). In contrast, humans and mice have only single member Vδ element families and Vα, β and γ families contain far fewer than 30 elements (Chien and Konigshofer, 2007, Kronenberg and Havran, 2007). Members of the sheep Vδ1 family were up to 96.5% identical at the amino acid level compared to only 88% amino acid identify between related murine Vγ elements and 76–91% amino acid identity between related human Vγ1 elements (Lefranc et al., 1986). These findings indicate that ruminants have remarkably large Vδ element families in comparison to humans and mice. However, only rearranged ruminant TCRδ genes, and not germline sequences, have been examined in the studies to date. Therefore, it cannot be confirmed that the sheep Vδ1 elements represent different germline elements rather than the same germline elements which have undergone mutation. The high level of similarity between different members of the sheep Vδ1 family might support the latter.

The use of heterogeneous TCRδ genes by γδ T cells in sheep uterine epithelium suggests that they have the potential to recognise diverse antigens and are therefore unlikely to be reacting against conserved, endogenous stress proteins. Intraepithelial γδ T cells in the small intestine are thought to be fully activated and ready to act as a first line of defence against infection (Chien and Konigshofer, 2007). Similarly, γδ T cells in the uterine epithelium of both pregnant and non-pregnant sheep show a fully differentiated, effector phenotype (Fox et al., 1998) and could therefore rapidly respond to any invading pathogen. The hypothesis that γδ T cells in the ovine uterine epithelium have a role in defence against microbial infection of the reproductive tract is significantly strengthened by the finding of granulysin in purified γδ T cells. Granulysin and its homologue, NK-lysin, are antimicrobial peptides shown to be present in the granules of NK and T cells of humans (Pena et al., 1997), pigs (Anderson et al., 2003), horses (Davis et al., 2005) and bovine (Endsley et al., 2004), but no homologue has been detected in rodents. The ovine granulysin gene codes for a 13kDa peptide, but as in human granulysin and pig NK-lysin, the native ovine granulysin peptide was shown here to have a molecular weight of 9kDa, through posttranslational cleavage of the N-terminal. A common feature of all antimicrobial peptides is their cationic nature, small size and ability to permeabilise bacterial membranes, causing death within minutes. The 9kDa form of human granulysin has been shown to be highly cytolytic and can be constitutively secreted from CTL in vitro (Pena et al., 1997). Granulysin colocalises with perforin in cytotoxic granules, and both molecules cooperate in the penetration and lysis of intracellular pathogens (Stenger et al., 1998). Perforin expression by ovine uterine γδ T cells has previously been demonstrated (Fox and Meeusen, 1999), and, together with the present finding of abundant granulysin protein and diverse TCR recognition, is a strong indication that the role of the granulated γδ T cell population acts to protect the ovine uterus from various invading pathogens.

Uterine infections occur most commonly through the genital tract, and the uterus is particularly susceptible to infection during and soon after parturition when it is exposed to a host of environmental contaminants. Both the number and size of the granules in the uterine γδ T cells increase dramatically towards the end of pregnancy (Lee et al., 1992, Meeusen et al., 1993) and, at the same time, there is a gradual loss of LFA1 and MHC class I expression (Fox et al., 1998), suggesting a change in signal requirements. It has been shown that this dynamic change in lymphocyte populations is the result of general hormonal signals (Majewski et al., 2001). At parturition, there is a dramatic drop in the number of granulated IEL (Lee et al., 1992) and there are clear signs of migration and degranulation into the uterine lumen (Nasar et al., 2002). It is likely that parturition triggers a non-specific (possibly hormonal) release of granule content, including the potent antimicrobial proteins perforin and granulysin, into the uterine lumen to protect the female during this highly susceptible post-partum period.

Increased γδ T cell activity has been found in the decidua parietalis of normal term pregnancies in humans (Sindram-Trujillo et al., 2003). The decidua parietalis delineates the region of the pregnant human uterus where there is no trophoblast invasion and most closely resembles the ruminant inter-placentomal areas where granulated γδTCR⁺ IEL predominate. In contrast to the decidua basalis which forms part of the placenta, the decidua parietalis has not been studied in great detail in humans and it is possible that the γδ T cell population in the decidua parietalis provides a similar protection against infection of the human reproductive tract during pregnancy and birth.

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Acknowledgment 

AF was supported by a Dora Lush, National Health and Medical Research Council postgraduate fellowship.

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