Analysis of TCRAV and TCRBV Repertoires in Healthy Individuals by Microplate Hybridization Assay

Matsutani

Saitoh

et al. 2002

Summary:Acute graft-versus-host disease (GVHD) is a disorder involving the skin, gut and liver that is caused by mismatches of major and/or minor histocompatibility antigens between the HLA-identical donor and recipient. If T lymphocytes infiltrating GVHD lesions recognize antigens expressed in these organs, T cell clones should expand in inflammatory tissues. We previously reported that recipients of allogeneic bone marrow grafts have clonally expanded TCR␣␤ + T lymphocytes soon after transplantation, which leads to a skew of TCR repertoires. To establish whether or not the same antigens cause clonal expansion of T lymphocytes in both blood and GVHD tissues, we examined the usage of TCR ␣ and ␤ chain variable regions (TCRAV and TCRBV) and determined the complementarity-determining region 3 (CDR3) of T lymphocytes clonally expanded in circulating blood and GVHD lesions. We found that the repertoires and CDR3 diversity of TCRAV and TCRBV differed between the GVHD lesions and circulating blood, suggesting the selective recruitment of antigenspecific T cells into GVHD tissues. We also found that the usage of TCRAV and TCRBV by the clonally expanded T lymphocytes and their CDR3 sequences differed between the GVHD tissues and blood. These results suggest that the antigen specificity of TCR␣␤ + T lymphocytes clonally expanded in blood and GVHD lesions is different. Bone Marrow Transplantation (2002) 30, 915-923. doi:10.1038/sj.bmt.1703730 Keywords: human; graft versus-host disease; T lymphocytes; T cell receptors transplantation Acute graft-versus-host disease (GVHD) is a disorder involving the skin, gut and liver that is caused by mismatches of major and/or minor histocompatibility antigens between the donor and recipient. 1 Although several candidates for minor histocompatibility antigens have been reported, including the male-specific H-Y antigen, cellularly defined minor HA antigens and myxovirus-related protein, the mechanism of cellular injury in acute GVHD has not been fully elucidated. [2][3][4] We previously reported a skewing of repertoires of the T cell receptor-␤ chain variable region (TCRBV) and the TCR-␣ chain variable region (TCRAV) in recipients of allogeneic hematopoietic stem cell grafts from HLAmatched donors. 5 This results from the extensive clonal expansion of a limited number of T cells in the graft. 6 Skewed TCR repertoires accompany accumulation of the CD28 Ϫ fraction in both CD4 + and CD8 + T cells. 7,8 Since accumulating evidence indicates an association between the loss of CD28 expression and T lymphocyte aging, 9,10 posttransplant skewing of the TCR repertoires probably results from chronic antigen stimulation in vivo.The above results raise the issue of whether or not clonally expanded T cells in circulating blood recognize the discrepant histocompatibility antigens between donors and recipients, which may be responsible for the induction of acute GVHD. Moreover, crucial questions include whether or not T lymphocytes infiltrating acute GVHD lesions recognize antigens commonly expressed in...

Section: Repertoire Analysis Of Tcrav and Tcrbvmentioning

confidence: 99%

Distinct TCRAV and TCRBV repertoire and CDR3 sequence of T lymphocytes clonally expanded in blood and GVHD lesions after human allogeneic bone marrow transplantation

Matsutani

Saitoh

et al. 2002

“…15 Briefly, total RNA was extracted from PBMCs and converted to double-strand cDNA using the SuperScript cDNA synthesis kit (BRL, Bethesda, MD, USA). The P10EA/P20EA adaptors were ligated to the 5Ј end of cDNA prepared from PBMCs, and PCR was performed using either a biotinylated TCRCAspecific or TCRCB-specific primer, and a P20EA primer.…”

Section: Tcrav and Tcrbv Repertoire Analysismentioning

confidence: 99%

Identification of the T cell clones expanding within both CD8+CD28+ and CD8+CD28− T cell subsets in recipients of allogeneic hematopoietic cell grafts and its implication in post-transplant skewing of T cell receptor repertoire

Horiuchi

Kawabata

et al. 2001

Summary:We have previously reported that skewed repertoires of T cell receptor-␤ chain variable region (TCRBV) and TCR-␣ chain variable region (TCRAV) are observed at an early period after allogeneic hematopoietic cell transplantation. Furthermore, we found that T lymphocytes using TCRBV24S1 were increased in 28% of the recipients of allogeneic grafts and an increase of TCRBV24S1 usage was shown to result from clonal expansions. Interestingly, the arginine residue was frequently present at the 3Ј terminal of BV24S1 segment and was followed by an acidic amino acid residue within the CDR3 region. These results suggest that these clonally expanded T cells are not randomly selected, but are expanded by stimulation with specific antigens. This study was undertaken to elucidate the mechanisms of the posttransplant skewing of TCR repertoires. Since the CD8 ؉ CD28 ؊ CD57 ؉ T cell subset has been reported to expand in the peripheral blood of patients receiving allogeneic hematopoietic cell grafts, we examined the TCRAV and TCRBV repertoires of the CD8 ؉ CD28 ؊ T cell and CD8 ؉ CD28 ؉ T cell subsets, and also determined the clonality of both T cell populations. In all three recipients examined, the CD8 ؉ CD28 ؊ T cell subset appeared to define the post-transplant TCR repertoire of circulating blood T cells. Moreover, the CDR3 length of TCRBV imposed constraints in both CD8 ؉ CD28 ؊ T cell and CD8 ؉ CD28 ؉ T cell subsets. The DNA sequences of the CDR3 region were determined, and the same clones were identified within both CD8 ؉ CD28 ؊ and CD8 ؉ CD28 ؉ T cell subsets in the same individuals. These results suggest that the clonally expanded CD8 ؉ CD28 ؊ T cells after allogeneic hematopoietic cell transplantation derive from the CD8 ؉ CD28 ؉ T cell subset, possibly by an antigen-driven mechanism, resulting The CD28 molecule is a disulfide-linked homodimer expressed on the surface of T cells and binds to the natural ligand B7 family members, CD80/CD86, expressed on antigen-presenting cells, resulting in costimulation of T cell activation. 1 In humans, all thymocytes and the vast majority of peripheral blood T cells at birth express CD28. The proportion of CD8 ϩ T cells lacking CD28 expression increases during human aging, so that approximately 30% of CD8 ϩ T cells are negative for CD28 in adults. 2 Most of the CD8 ϩ CD28 Ϫ T cells express CD57. 2 A number of reports have shown that the proportion of CD8 ϩ CD28 Ϫ T cells is increased in HIV-infected patients. 3,4 The CD8 ϩ CD28 Ϫ CD57 ϩ T cell subset is also expanded in the peripheral blood of patients receiving allogeneic bone marrow transplants. 5,6 However, the mechanisms and biological relevance of this expansion remain to be elucidated. Moreover, it is still uncertain whether the CD8 ϩ CD28 Ϫ T cell subset arises from the CD8 ϩ CD28 ϩ T lymphocytes or whether both subsets belong to distinct T cell lineages.We have previously reported that the skewed TCRAV and TCRBV repertoires are observed at an early period after allogeneic hematopoietic cell transplantation. 7 We also fo...

“…17 Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll/Conray gradient centrifugation from 10 ml of heparinized blood. Total RNA was extracted from PBMCs and converted to double-strand cDNA using a SuperScript cDNA synthesis kit (BRL, Bethesda, MD, USA).…”

Section: Tcrbv Repertoire Analysismentioning

confidence: 99%

“…Matsutani et al 17 have reported variable expression levels of several TCRBV segments among healthy individuals, whereas expression levels of TCRAV repertoires do not vary much. The TCRBV repertoires of T lymphocytes in bone marrow grafts from 10 healthy donors for allogeneic transplantation were essentially similar to those of circulating blood T cells from healthy individuals (Figure 1).…”

Section: Clonality Of T Cell Populations With Skewed Tcrbv Repertoiresmentioning

confidence: 99%

Extensive clonal expansion of T lymphocytes causes contracted diversity of complementarity-determining region 3 and skewed T cell receptor repertoires after allogeneic hematopoietic cell transplantation

Matsutani

Horiuchi

et al. 2001

Summary:We previously described skewed repertoires of the T cell receptor-␤ chain variable region (TCRBV) and the TCR-␣ chain variable region (TCRAV) soon after allogeneic hematopoietic cell transplantation. To determine the characteristics of skewed TCRBV after transplantation, we examined the clonality of T lymphocytes carrying skewed TCRBV subfamilies and determined the CDR3 sequences of expanded T cell clones. In all 11 recipients examined, TCR repertoires were skewed, with an increase of certain TCRBV subfamilies that differed among individuals. In nine of 11 patients, clonal/oligoclonal T cell expansion was observed, although the expanded T cells were not necessarily oligoclonal. The extent of expansion after transplantation appeared to predict clonality. The arginine (R)-X-X-glycine (G) sequence was identified in clonally expanded T cells from four of five recipients examined, and glutamic acid (E), aspartic acid (D) and alanine (A) were frequently inserted between R and G. These results suggest that T lymphocyte expansion may result from the response to antigens widely existing in humans, and that the extensive clonal expansion of a limited number of T cells leads to contracted CDR3 diversity and post-transplant skewed TCR repertoires. Bone Marrow Transplantation (2001) 27, 607-614. Keywords: T cell clonality; CDR3; allogeneic hematopoietic cell transplantationWe have found skewed TCRAV and TCRBV repertoires soon after allogeneic hematopoietic cell transplantation in virtually all tested recipients. 1 Furthermore, we demonstrated that reconstitution of the ␣␤ T cell, but not the ␥␦ T cell repertoire was incomplete for at least 2 months after transplantation, and that the delayed reconstitution of TCR repertoire diversity was more profound in immunocompro- mised hosts. 2,3 We also found that TCR usage appears to be limited, and that amino acid sequences frequently contain an arginine residue followed by acidic amino acid residues in the complementarity-determining region 3 (CDR3) of TCR-␤ chains containing BV24S1, suggesting that skewing of the TCRAV and TCRBV repertoires is antigen-driven. 1 These features of post-transplant T cell regeneration are similar to those during extrathymic differentiation of T cell progenitors and peripheral expansion of mature T cell populations, which have been demonstrated by murine bone marrow transplantation studies. 4 Extrathymic differentiation of T cell progeny in bone marrow generates TCR␥␦ ϩ and CD4 Ϫ CD8 Ϫ TCR␣␤ ϩ T cells. 5,6 Peripheral expansion of mature T lymphocytes is driven by the antigenic milieu of the host 7,8 and gives rise to substantial numbers of TCR␣␤ ϩ CD4 ϩ and TCR␣␤ ϩ CD8 ϩ T cells. 5,9 However, an infusion of a limited number of T cells into athymic mice results in skewed TCR repertoires. 5 Clonal expansion of T lymphocytes is the principal mechanism of antigen-specific immune responses. 10,11 The CDR3 regions of TCRBV and TCRAV make the principal contact with an antigenic peptide that is bound to the major histocompatibility complex (MHC) and the...