Search

We previously showed that skin-homing CD4 T cells in peripheral blood can be subdivided into three populations on the basis of the expression pattern of the cutaneous lymphocyte Ag (CLA) and fucosyltransferase VII (FucT-VII): FucT-VII(+)CLA(-), FucT-VII(+)CLA(+), and FucT-VII(-)CLA(+). In view of the known late appearance of CLA during T cell differentiation, T cells programmed to attain skin-homing properties may start to generate E-selectin-binding epitopes at early stages of differentiation before induction of CLA expression. To this end, the in vitro differentiation from naive to CLA(+) memory T cells was followed after activation with anti-CD3 mAb. Here we demonstrate that naive skin-homing CD4 T cell precursors undergo a linear differentiation process from the FucT-VII(+)CLA(-) phenotype to the FucT-VII(+)CLA(+) phenotype and eventually to the FucT-VII(-)CLA(+) phenotype. The appearance of the FucT-VII(+)CLA(-) subset coincided with or could be immediately followed by the generation of E-selectin binding epitopes, and even after E-selectin-binding epitopes were no longer detectable, CLA remained expressed for prolonged periods of time, suggesting that induction of functional E-selectin ligands depends primarily on the expression of FucT-VII, but not CLA. Immunofluorescence and confocal microscopy studies of these T cells confirm that most E-selectin ligands were found independently of CLA expression.

Although regional lymph nodes (LN) have been extensively studied as rich sources of effector T cells in contact hypersensitivity (CH), it remains unknown whether T cell responses in the LN reflect those in effector skin sites. We previously showed that repeated elicitation of CH results in a shift in the time course of Ag-specific CH from a delayed-type hypersensitivity response to an early-type response, a reflection of a shift in cutaneous cytokine expression from a type 1 to a type 2 profile. To investigate whether repeated elicitation of CH could also drive T cell development to the type 2 phenotype in the regional draining LN, sequential cytokine gene expression after hapten application was assessed during both the acute and the chronic phase of CH. In the draining LN the shift to type 2 cytokine production was also observed, but more mixed patterns of responses were induced than in the corresponding skin sites. The chronic LN cells (LNC), when stimulated in vitro, produced markedly lower levels of type 1 cytokines and higher levels of type 2 cytokines than the acute LNC. A successful passive transfer of an early-type response by the LNC was only induced in the recipient mice when the skin sites chronically treated with hapten were elicited. These results indicate that an early-type response by regional LNC would take place only in a milieu with sufficient levels of type 2 cytokines.

Recombinase-activating genes (RAG-1 and RAG-2) are expressed in immature T or B lymphocytes and possess activity to induce V(D)J rearrangement in TCR and Ig genes. We examined their expression in human decidual and non-pregnant endometrial samples using a highly sensitive RT-PCR technique. Expression of RAG-1 and 2 was noted in all (13/13) pregnant decidual tissues obtained at different gestational stages. After anti-human Ig treatment to rule out the possibility of RAG-1,2 expression from decidual B-cells, strong expression of RAGs was still noted in B cell depleted decidual cells in contrast to lymph nodes and PBMC that lost RAG mRNA expression after this treatment. After FACS mediated cell sorting, strong expression of RAG-1,2 was noted in CD16-CD56bright cells and weak expression in CD3+ cells. Although CD16-CD56bright cells lack surface CD3, they express CD3 epsilon mRNA only detectable by RT-PCR. Our results suggest the nature of decidual CD16-CD56bright cells as a progenitor of extrathymic T cells that possess RAG-1 and 2 mRNA as markers of their immaturity, and possibly differentiate into CD3+ extrathymic T-cells in the decidua under the influence of trophoblastic cells. We propose the human decidua as a new site of extrathymic T-cells differentiation and propose possible roles of trophoblastic cells to attract progenitor lymphocytes of bone marrow origin and trigger their TCR rearrangement as thymic epithelial cells.

Maturation of some T cell populations has been suggested to occur in epithelial tissues. The CD8 molecule, which is expressed as heterodimers made of alpha/beta-chains on virtually all peripheral T cells, is preferentially expressed as homodimers made only of alpha-chains on a subset of CD8+ mouse gut intraepithelial lymphocytes, which arise from bone marrow (BM) precursors in a thymus-independent mechanism. This unique population of CD8 alpha+ beta- T cells, however, has not been identified in other lymphoid tissues in normal adult mice. Because our previous study demonstrated that reconstitution of thymectomized irradiated mice with T cell-depleted BM cells resulted in the appearance of CD8+, TCR-alpha beta+ dendritic epidermal cells (DEC) and suggested that a proportion of the BM-derived DEC may mature within the epidermis, we asked whether the unique phenotype of CD8 alpha+ beta- could be detected among the CD8+ BM-derived DEC. In this report for the first time we identified this unique population of CD8 alpha+ beta- cells among the DEC. Although this population comprised the predominant fraction of the BM-derived DEC in the early post-transfer period (2 to 3 mo), gradual shift from the CD8 alpha+ beta+ phenotypes occurred during the late post-transfer period (4 to 6 mo) independently of the presence of the thymus. Kinetic studies on the BM-derived DEC revealed that this phenotypic shift could be caused by the subsequent expansion of the CD8 alpha+ beta+ DEC. In contrast, the CD8 alpha+ beta- population was the major subset of the BM-derived intraepithelial lymphocytes throughout the entire observation period and the phenotypic shift with age was never observed. These results indicate that CD8 alpha+ beta- cells are preferentially detected on T cells that home to the epithelial tissues and that in the epidermis, but not in the gut epithelia, the subsequent expansion of the CD8 alpha+ beta+ DEC would dilute the high number of the CD8 alpha+ beta- cells.

Our previous studies demonstrated that CTL responses to the newly identified minor H-43a and H-43b alloantigens induced in H-43b and H-43a responder mice, respectively, utilized the same H-2Kb (Kb) MHC class I restriction element, whereas many allelic class I products were not utilized. Also, a single i.v. injection of H-43b recipient mice with spleen cells (SC) from H-43 congenic mice induced specific and lasting CTL tolerance to H-43a rather than priming. We have produced a H-43b C3H.AU mouse strain (H-2p), which we regard as H-43 congenic to the C3H.NB (H-2p, H-43a) strain. Using this strain we demonstrate that Kp and/or Dp alleles can serve as class 1 restricting elements in the CTL responses to H-43a and H-43b alloantigens. Injection of H-43a recipient mice with antigenic H-43b SC from the paired H-43 congenic abrogated the anti-H-43b CTL response, irrespective of MHC class I specificity, i.e., in the contexts of Kb and of Kp and/or Dp elements. A corresponding result was obtained upon injection of H-43b recipient mice with congenic H-43a SC. The tolerance-inducing capacity of H-43a congenic SC was so strong that injection of these cells into H-43b responders that had previously received immunogenic H-43a SC completely blocked the previously activated Kb-restricted memory CTL activity specific for the H-43a alloantigen.

The majority of murine Thy-1+ dendritic epidermal T cells (DETC) express virtually identical TCR encoded by V gamma 5 and V delta 1 genes and are derived from early fetal thymic precursors. However, not consistent with this notion is an early finding that DETC arise continuously from bone marrow (BM) precursors by a thymus-independent mechanism. To address this issue, donor-type DETC were characterized in lethally irradiated mice that were reconstituted by Thy-1-disparate BM cells with or without a thymus. The BM-derived DETC, unlike their normal TCR-gamma delta counterparts, were found to express the TCR-alpha beta/CD3 complex and CD8, and their migration to the epidermis dermis occurred independently of the thymus. The numbers of the BM-derived DETC increased with time and reached a plateau 6 mo after BM transfer, at which time the TCR-alpha beta/CD3 complex was expressed on a small fraction of the DETC in athymic BM chimeras. Although no further increase in the number was observed at later times, at 1 yr after transfer most of the BM-derived DETC came to express the TCR-alpha beta/CD3 complex in the absence of thymic influence. By contrast, most of BM-derived T cells in other lymphoid organs from athymic BM chimeras still failed to express the TCR-alpha beta/CD3 complex even at 1 yr after transfer. These results suggest that extrathymic differentiation of BM-derived DETC could occur with the epidermal microenvironment.

Our previous study showed that in cutaneous graft-vs-host disease (GVHD) induced by intradermal injection of autoreactive T cells the epidermal structures spontaneously recovered from the destruction became resistant to the subsequent attempts to induce the cutaneous GVHD and that the resistance correlated well with a 30-fold increase in the number of Thy-1+ epidermal cells (Thy-1+EC). We show that the resistance to the cutaneous GVHD was never induced in athymic nude mice and adult thymectomy lethal radiation and bone marrow reconstitution (ATXBM) mice under the same conditions, indicating that the induction of the resistance is dependent on the presence of thymus. A great increase in the number of Thy-1+EC was similarly observed in the epidermis of the athymic nude and ATXBM mice that spontaneously recovered from the cutaneous GVHD and that remained susceptible to the induction of the cutaneous GVHD. However, the results with B10Thy-1.1----B6 radiation chimeras clearly demonstrate that the vast majority of the increased Thy-1+EC found in the "susceptible" epidermis of the ATXBM mice were of donor bone marrow origin and there was no increase in the number of host-derived Thy-1+EC, whereas in the "resistant" epidermis of the XBM mice both Thy-1+EC populations were equally increased. The overall results indicate that the expansion of Thy-1+EC that mature in the thymus is crucial to the induction of the resistance, although the migration of bone marrow-derived Thy-1+EC precursors to the epidermis occurs quite independently of the presence of thymus.

Backcross progeny, (NC X TF/GnLe)F1 X TF/GnLe, was tested for C3 allotype controlled by C3-1 and the expression of mutant gene tf, repeated loss and regrowth of hair. The recombination frequency between these two loci both located in chromosome 17 of the mouse was 24%. Taken together with our previous linkage data, C3-1 is now localized to a position 11 cm more distal than H-2 on chromosome 17.

We have previously demonstrated that anti-H-43a CTL response of H-43b responder mice was exclusively restricted by self H-2Kb (Kb) but not by the other nine self MHC class I alleles from independent origins, i.e., Kbml,d,k,s and Db,d,k,q,s. In the present study, we verified that Kf,q,r and Df,r alleles could also not serve as restricting class I elements in the CTL response to H-43a alloantigen. Another notable observation made in the earlier study was the fact that, in H-43 incompatibility of the alternative combination, H-43a mice were incapable of generating CTL activity against H-43b alloantigen. However, by means of employing new in vivo immunization procedures, we discovered that some but not all genetically identical H-43a responder mice could mount anti-H-43b CTL response restricted by self Kb. Again, no anti-H-43b CTL activity could be generated in the context of self Kk, Kj, Db or Dk molecules. Although the number of class I alleles we examined is still limited, these results indicate that antigenic fragments derived from the processed H-43a and H-43b alloantigens possess an indistinguishable epitope (agretope), and that such agretope either interacts only with the privileged Kb molecules or allows to bestow the immunogenic conformation of allodeterminants on the fragments solely in the context of the restricting Kb element.

Structural polymorphism of murine factor H protein was demonstrated by using three different methods. 1) By prolonged agarose electrophoresis and immunofixation, factor H protein was visualized in the beta region as a single, distinct protein band in freshly bled EDTA-plasmas from many laboratory and wild mice. Two variants were detected among a large number of tested strains; one, referred to as H.1, moved faster to the anodal region (type strain, BALB/c), and the other, referred to as H.2, moved more slowly to the anodal region (type strain, STR). The F1 hybrid between BALB/c and STR exhibited a combining type of factor H protein, which was observed in each parent. 2) Two-dimensional peptide mapping analysis was carried out with tryptic peptides of these two factor H allotypes. Almost all of the spots in the maps of tryptic peptides were common to both allotypes. However, three distinct spots among the 57 spots detected in the map of tryptic peptides of the H.1 allotypes were not detected in that of H.2 allotype, whereas two spots among the 56 spots in the map of H.2 allotype were unique for this allotype. The F1 hybrid between BALB/c and STR showed a combining type of the map of parent. 3) Alloantisera against each of H allotypes were successfully produced in BALB/c or BALB/c-H.2 (a congenic strain with H.2 allotype) by repeated injection of each purified factor H protein either from the BALB/c or the STR strain. These findings indicated that the observed variants of factor H represent antigenically and structurally distinguishable allotypes. The allotypes of murine factor H protein are controlled by a single codominant locus located between the Hc locus and the beta 2M locus on the second chromosome of the mouse. This was shown by phenotyping the Hc locus and H locus with backcross progenies between A/J (one of strain with H.1) and MoA (one of strain with H.2). The recombination frequency between these two loci was 0.17 +/- 0.046.