Lymphoid precursors in intestinal cryptopatches express CCR6 and undergo dysregulated development in the absence of CCR6

Small intestinal cryptopatches (CP) are the major anatomic site for extrathymic differentiation by precursors destined to become intestinal intraepithelial T lymphocytes (IEL). We found that mice deficient in CCR6 exhibited a 2.7-fold increase in the number of alphabeta TCR IEL, but little or no expansion of gammadelta TCR IEL. Among the alphabeta TCR IEL subsets, the CD4- CD8alphaalpha+ and CD4+ CD8alphaalpha+ subsets were preferentially expanded in CCR6 null mice. Because some CD8alphaalpha+ IEL can arise through extrathymic differentiation in CP, we investigated CCR6 expression by T lymphocyte precursors undergoing extrathymic differentiation in intestinal CP. In sections of CP, 50-60% of c-kit+ precursors were CCR6+. CD11c(+) cells concentrated at the periphery of CP did not express CCR6. A subset of c-kit+, Lin- cells in lamina propria suspensions was CCR6+, but CCR6 was absent from c-kit+ precursors in bone marrow. CCR6 was absent from the vast majority of mature IEL. CCR6 is present on lymphocyte precursors in cryptopatches, expressed transiently during extrathymic IEL development, and is required for homeostatic regulation of intestinal IEL.     

Regulatory function for murine intraepithelial lymphocytes. Two subsets of CD3+, T cell receptor-1+ intraepithelial lymphocyte T cells abrogate oral tolerance

The murine intraepithelial lymphocyte (IEL) population is enriched in T cells that express the gamma delta-TCR, however, the biologic function served by these T cells remains obscure. IEL are considered to be major effector cells in mucosal immunity, and we have investigated whether IEL subsets could reverse orally induced systemic unresponsiveness (oral tolerance; OT) and support secondary type responses when adoptively transferred to mice orally tolerized with SRBC. When purified CD3+ IEL from mice orally primed with SRBC were transferred to adoptive hosts and challenged with SRBC, splenic IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses were observed. However, CD3+ IEL from HRBC orally primed mice did not abrogate SRBC induced OT. Further, HRBC-primed CD3+, IEL converted HRBC-specific OT but not SRBC-specific OT. CD3+ IEL could be separated into four subsets based on expression of CD4 and CD8. CD3+, CD4-, 8+ T cells were the major subset (74.5%), with smaller numbers of CD4- and CD8- (double negatives, DN) (7.8%), CD4+, 8- (7.6%) and CD4+, CD8+ (double positives) (10.1%) T cells. Interestingly, both the CD3+, CD8+, and the CD3+, DN IEL subsets abrogated OT, resulting in significant IgM, IgG1, IgG2b, and IgA anti-SRBC plaque-forming cell responses when adoptively transferred to mice with OT. However, neither CD3+, CD4+, CD8-, nor double positive T cells affected OT when studied in this system. The CD3+, CD8+ IEL subset could be further separated into Thy-1+ (16.6%) and Thy-1- (83.4%) cells; adoptive transfer of Thy-1- cells abrogated oral tolerance whereas the Thy-1+ subset was without effect. When the expression of TCR on IEL with this biologic function was determined by use of monoclonal anti-alpha beta TCR (H57.597), TCR2-, CD3+ IEL possessed immunoregulatory function whereas the alpha beta-TCR+ (TCR2+) fraction did not abrogate OT. Immunoprecipitation of membrane fractions obtained from purified CD3+, CD4-, CD8+, Thy-1- IEL with polyclonal anti-delta peptide (Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) antibody revealed bands of 45 and 35 kDa, corresponding to the delta- and gamma-chains, respectively. These results suggest that gamma delta-TCR+ IEL possess a regulatory function, namely the restoration of immune responses in a state of oral tolerance. Further, both CD3+, CD4-, CD8+, Thy-1-, and CD3+, DN IEL T cells exhibit this effector contrasuppressor function.     

Novel function for intestinal intraepithelial lymphocytes. Murine CD3+, gamma/delta TCR+ T cells produce IFN-gamma and IL-5.

Intestinal intraepithelial lymphocytes (IEL) from mice are greater than 80% CD3+ T cells and could be separated into four subsets according to expression of CD4 and CD8. In our studies designed to assess the functions of IEL, namely, cytokine production, it was important to initially characterize the various subsets of T cells that reside in IEL. The major subset was CD4-, CD8+ (75% of CD3+ T cells), which contained approximately 45 to 65% gamma/delta TCR+ and 35 to 45% alpha/beta TCR+ T cells. Approximately 7.5% of IEL T cells were CD4-, CD8- (double negative) and gamma/delta+ population. On the other hand, CD4+, CD8+ (double positive) and CD4+, CD8- fractions represented 10% and 7.5% of CD3+ T cells, respectively, which were all alpha/beta TCR+. Inasmuch as CD3+, CD4-, CD8+ T cells are a major subset of IEL which contain both gamma/delta TCR or alpha/beta TCR-bearing cells, the present study was focused on the capability of this subset of IEL T cells to produce the cytokines IFN-gamma and IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL spontaneously produced IFN-gamma and IL-5, although higher frequencies of cytokine spot-forming cells were associated with the alpha/beta TCR+ subset. Approximately 30% of CD8+, gamma/delta TCR+ cells produced both cytokines, whereas approximately 90% of alpha/beta TCR+ T cells produced either IFN-gamma or IL-5. Both gamma/delta TCR+ and alpha/beta TCR+ IEL possessed large quantities of cytokine-specific mRNA, clearly showing that these IEL were programmed for cytokine production. When IEL were activated with anti-gamma/delta or anti-CD8 antibodies, higher numbers of IFN-gamma and IL-5 spot-forming cells were noted. The present study has provided direct evidence that a major function of IEL involves cytokine production, and this is the first evidence that gamma/delta TCR+ cells in IEL possess the capability of producing both IL-5 and IFN-gamma.     

Detection and characterization of cytotoxic T lymphocyte precursors in the murine intestinal intraepithelial leukocyte population

Highly purified preparations of intraepithelial leukocytes (IEL) were obtained from the small intestinal mucosa. Leukocytes from the lamina propria (LPL) were isolated and phenotypically compared with IEL to verify that IEL were minimally contaminated by LPL. Because approximately 80% of IEL expressed the Lyt-2 antigen usually associated with cytotoxic/suppressor T lymphocytes, we wished to determine if precursors for cytotoxic T cells were present in this population. In order to generate cytotoxic cells, IEL and spleen cells from CBA/J mice (H-2k) were co-cultured with irradiated allogeneic spleen cells (H-2d or H-2b) in a one-way mixed leukocyte reaction (MLR). Four to six days later, the cultured cells were assayed against 51Cr-labeled H-2d or H-2b tumor or Con A-stimulated lymphoblast target cells, and the specificity of alloantigen-stimulated IEL and spleen cells was compared. The cytotoxic cells derived from both tissues displayed antigen-specific lysis of the allogeneic targets. Treatment of effector cells, generated from intraepithelial or splenic precursors, with monoclonal antibodies against Thy-1.2, Lyt-1.1, or Lyt-2.1 antigens plus complement, decreased cytotoxicity 85 to 100%, even though only 20 to 50% of the cells were lysed. The alloantigen specificity and surface antigen phenotype of the cultured IEL cells were identical to those of spleen cells and allowed us to conclude that IEL contained a cytotoxic T lymphocyte precursor (CTLp). Further characterization showed that, like spleen, the intraepithelial CTLp was Thy-1+ and Lyt-1+ and their sedimentation velocity was the same but differed from intraepithelial natural killer cells. Although 80% of IEL were Lyt-2+, the frequency of CTLp in the IEL population was estimated to be threefold to fivefold lower than in spleen, and the Lyt-2+ cells were shown not to be an enriched source of CTLp. Thus, the function of the majority of the IEL is still not known. However, there exists within this population CTLp, which may be capable of being stimulated with luminal antigens.     

Induction of high level IL-2 production in CD4+8- T helper lymphocytes requires post-thymic development.

Triggering of the CD3:TCR complex by optimal concentrations of anti-CD3, anti-TCR beta-chain, and allogeneic stimulator cells induced dramatically higher levels (fivefold for anti-CD3, greater than 10-fold for anti-TCR beta-chain, 84-fold for alloantigen) of IL-2 production in spleen CD4+8- T cells than their thymic counterparts, despite comparable levels of CD3 and TCR beta-chain expression. The nature of the reduced IL-2 production was examined by analysis of anti-CD3-induced IL-2 production at the single cell level. The frequency of IL-2-producing cells in spleen CD4+8- T cells (40.0%) was approximately threefold that of thymus CD4+8- T cells (14.5%). Furthermore, the average IL-2 levels among positive IL-2 producers was also approximately threefold higher in spleen CD4+8- T cells than their thymic counterparts. Adoptive transfer of purified Thy-1.2+ CD4+8- T cells into Thy-1.1-congenic hosts provided a physiologic and histocompatible system that enabled identification of transferred donor (Thy-1.2+) among a sea of host (Thy-1.2-) CD4+ T cells, whose immune function with respect to IL-2 inducibility was examined after isolation by electronic cell sorting. Donor CD4+ T cells thus isolated from host spleen shortly (1 day) after i.v. transfer of thymus CD4+8- T cells were similar to freshly isolated thymus CD4+8- T cells in that they both produced little IL-2 in response to anti-CD3. However, by day 3 post-transfer, IL-2 production by donor CD4+8- T cells had more than doubled and by day 8, they produced IL-2 levels comparable to those of host spleen CD4+8- T cells. A similar acquisition of high level IL-2 inducibility in thymus CD4+8- T cells upon i.v. transfer into Thy-1.1-congenic hosts was also observed using allogeneic cells as the stimulus of IL-2 production. When thymus CD4+8- T cells were intra-thymically transferred into Thy-1.1-congenic hosts, those donor cells that emigrated to the periphery became high IL-2 producers in a time-dependent manner, whereas those that remained inside the thymus showed no signs of up-regulation in IL-2 inducibility. Intrathymic transfer of CD4-8- thymocytes revealed that the most recent thymic emigrant CD4+8- T cells contained few IL-2-producing cells and were not functionally mature with respect to high level IL-2 inducibility.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization.

Phenotype and commitment of thymus-colonizing precursors are unknown. Here we report the identification of T lineage-committed precursors (designated prothymocytes) in murine fetal blood at day 15.5 of development. Fetal blood pro-thymocytes are Thy-1+c-kit(low)CD3- in contrast to fetal blood-derived pluripotent hematopoietic progenitors which are Thy-1-c-kit+. Upon transfer into the thymus, fetal blood pro-thymocytes generate a single wave of CD4+CD8+ thymocytes and subsequently mature TCR alpha beta+ peripheral T cells. However, fetal blood pro-thymocytes lack multipotent progenitor potential since they fail to reconstitute B lymphocytes and myeloid and erythroid lineages. In contrast, T and B lymphocytes as well as myeloid and erythroid lineages are reconstituted from fetal blood-derived pluripotent progenitors. Pro-thymocytes are equally present in peripheral blood of athymic fetal mice, suggesting that this novel precursor population is T lineage-committed prior to thymus colonization and represents the earliest T lineage precursor identified.     

T cell-induced allotypic suppression. Origin of the CD8+ T cells maintaining IgG2ab suppression.

One of the problems raised by the T cell-induced allotypic suppression is the origin (donor or host) of the T cells responsible for the chronicity of the suppression. To address this point, we used T cells from Igha/a Thy-1.2 mice whose natural T cell activity against IgG2ab was enhanced in vivo. These T cells were injected into newborn Ighb/b Thy-1.1 mice where they induced complete suppression of IgG2ab expression in around 70% of these recipients. During a study that lasted more than 1 yr, we found that about 3% of the recipient splenocytes were T cells of the donor type. By means of suppression-transfer experiments, using either Thy-1.2+ or Thy-1.1+ cell-depleted splenocytes from mice suppressed in this manner we were able to unambiguously show that Thy-1.2+ cell-depleted splenocytes were incapable of transferring the suppression, whereas Thy-1.1+ cell-depleted splenocytes could. We thus demonstrated that suppression was maintained throughout the recipient's life by donor Thy-1.2+ T cells.     

Modulation of lymphocyte subsets in Peyer's patches of mice treated with monoclonal antibody against helper T-cells

Treatment of mice with anti-L3T4, a monoclonal antibody directed against helper T-cells, impairs clearance of intestinal Giardia muris infection. The present study examined the effect of anti-L3T4 treatment on mouse Peyer's patch cytoarchitecture and on the distribution of T-cell subsets within microenvironments of the follicle. Female BALB/c mice, aged 8 weeks, were given 4-7 weekly injections of either anti-L3T4 (1 mg/wk) or PBS (control group), and Peyer's patches were examined by immunohistochemistry or flow cytometry. In anti-L3T4-treated mice, Peyer's patch follicles (B-cell areas) were about two thirds the size of follicles in controls, and virtually all the size difference occurred in germinal centers. Peyer's patches were depleted of L3T4+ cells, yet the proportion of Thy-1.2+ (all T) cells was not decreased correspondingly, and the distribution of Thy-1.2+ cells in the patches was similar to that in control mice. In anti-L3T4-treated mice, Thy-1.2+ cells consisted of (a) Ly-2+ (cytotoxic/suppressor T) cells, and (b) a population of Thy-1.2+ cells that were neither L3T4+ nor Ly-2+. After treatment, Ly-2+ cells accounted for most of the T-cells in interfollicular areas and were also scattered in follicles, in germinal centers, and below the dome epithelium--in areas where L3T4+ cells predominated in control mice. Thy-1.2+ cells that were L3T4- and Ly-2- were mainly localized below the dome epithelium. These shifts indicate complex interrelationships among different lymphocyte subsets in Peyer's patches.     

Analyses of acute graft-versus-host-like reaction in [MRL/lpr----MRL/+] chimeric mice using MRL/lpr-Thy-1. 1 congenic mice.

When MRL/Mp(-)+/+(MRL/+) mice are lethally irradiated and then reconstituted with MRL/Mp-lpr/lpr (MRL/lpr) bone marrow and/or spleen cells, these MRL/+ mice develop "lpr-GVHD" which is similar to acute graft-versus-host disease (GVHD). Using a Thy-1 congenic strain of MRL/lpr mice (MRL/lpr-Thy-1.1), we analyzed T cell subpopulations in the thymus and spleen of MRL/+ mice suffering from lpr-GVHD. lpr-GVHD was induced in MRL/+ mice by transplantation of bone marrow cells (BMC) from MRL/lpr-Thy-1.1 mice; severe lymphocyte depletion associated with fibrosis was observed in the spleens after 7 weeks of bone marrow transplantation (BMT). Thymocytes of the host MRL/+ thymus were replaced with donor-derived cells from the early stage of lpr-GVHD, whereas in the spleen, a small number of host T cells (Thy-1.2+) (4-5%) were retained until the late stage of lpr-GVHD. Donor-type (Thy-1.1+) T cell subsets were not different from those of nontreated MRL/+ mice in the thymus, whereas in the spleen. CD8+ T cells (Thy-1.1+) reached a peak at 5 weeks after BMT, and CD4+ T cells (Thy-1.1+), a peak at 6 weeks. The elimination of T cells from MRL/lpr BMC had no evident effect on the prevention of lpr-GVHD. T cell subpopulations showed a similar pattern to GVHD elicited by MHC differences. Analyses of autoreactive T cells expressing V beta 5 or V beta 11 revealed that autoreactive T cells were deleted from the peripheral lymph nodes. Interestingly, the levels of IgG anti-ssDNA antibodies markedly increased, and both IgM and IgG rheumatoid factors slightly increased 5 to 7 weeks after BMT. These findings are discussed in relation to not only GVHD elicited by MHC differences but also autoimmune diseases.     

Phenotypic characterization of thymic prelymphoma cells of B10 mice treated with split-dose irradiation

Using an intrathymic injection assay on B10 Thy-1 congenic mice, it was demonstrated that thymic prelymphoma cells first developed within the thymuses from 4 to 8 days after split-dose irradiation and were detected in more than 63% of the test donor thymuses when examined at 21 and 31 days after irradiation. Moreover, some mice (25%) at 2 mo after split-dose irradiation had already developed thymic lymphomas in their thymuses. To characterize these thymic prelymphoma cells, the thymocytes from B10 Thy-1.1 mice 1 mo after irradiation were stained with anti-CD4 and anti-CD8 mAb and were sorted into four subpopulations. These fractionated cells were injected into the recipient thymuses to examine which subpopulation contained thymic prelymphoma cells. The results indicated that thymic prelymphoma cells existed mainly in CD4- CD8- and CD4- CD8+ thymocyte subpopulations and also in CD4+ CD8+ subpopulation. T cell lymphomas derived from CD4- CD8- prelymphoma cells had mainly CD4- CD8- or CD4- CD8+ phenotypes. T cell lymphomas developed from CD4- CD8+ prelymphoma cells mainly expressed CD4- CD8+ or CD4+ CD8+ phenotype. T cell lymphomas originating from CD4+ CD8+ prelymphoma cells were mainly CD4+ CD8+ but some CD4- CD8+ or CD4+ CD8- cells were also present. These thymic prelymphoma cells were further characterized phenotypically in relation to their expression of the marker defined by the mAb against J11d marker and TL-2 (thymus-leukemia) Ag, which is not expressed on normal thymocytes of B10.Thy-1.2 or B10.Thy-1.1 strain, but appears on the thymocytes of lymphomagenic irradiated mice. The results indicated that the prelymphoma cells existed in J11d+, TL-2+ cells.     

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.     

Dysfunction of irradiated thymus for the development of helper T cells

The development of cytotoxic T cells and helper T cells in an intact or irradiated thymus was investigated. C57BL/6 (H-2b, Thy-1.2) mice were whole body-irradiated, or were irradiated with shielding over either the thymus or right leg and tail, and were transferred with 1.5 X 10(7) bone marrow cells from B10.Thy-1.1 mice (H-2b, Thy-1.1). At various days after reconstitution, thymus cells from the recipient mice were harvested and a peanut agglutinin low-binding population was isolated. This population was further treated with anti-Thy-1.2 plus complement to remove host-derived cells and was assayed for the frequency of cytotoxic T cell precursors (CTLp) and for the activity of helper T cells (Th). In the thymus of thymus-shielded and irradiated mice, Th activity reached normal control level by day 25, whereas CTLp frequency remained at a very low level during these days. In the thymus of whole body-irradiated mice, generation of CTLp was highly accelerated while that of Th was retarded, the period required for reconstitution being 25 days and more than 42 days for CTLp and Th, respectively. Preferential development of CTLp was also seen in right leg- and tail-shielded (L-T-shielded) and irradiated recipients. Histological observation indicated that Ia+ nonlymphoid cells were well preserved in the thymus of thymus-shielded and irradiated recipients, whereas in L-T-shielded and irradiated recipients, such cells in the medulla were markedly reduced in number. These results suggest strongly that the generation of Th but not CTLp is dependent on radiosensitive thymic component(s), and that such components may represent Ia+ cells themselves in the medulla or some microenvironment related to Ia+ cells.     

Demonstration of a CD3+ lymphocyte subset in the epidermis of athymic nude mice. Evidence for T cell receptor diversity.

The existence of CD3/TCR-bearing lymphocytes in athymic and thymectomized chimeric mice implies that T cell maturation can occur in the absence of a thymus. Considering the possibility that the epidermis may be one of the organs providing T cell educating stimuli, we attempted to characterize the Thy-1+ epidermal lymphocyte population of athymic mice. Immunohistologic studies of epidermal sheets revealed (1) that Thy-1+ epidermal cells of C57BL/6 nu/nu mice are CD5-, CD4-, and predominantly CD8-, and (2) that a minor subset of these cells displays anti-CD3 epsilon reactivity. Although these CD3+ epidermal cells could hardly be detected at 6 wk of age, they comprised approximately 2% of all Thy-1+ epidermal cells in 12-mo-old athymic mice. Most of these CD3+ cells expressed TCR-gamma/delta, but TCR-alpha/beta+ cells were also present. TCR-gamma/delta+ epidermal T cells of athymic mice preferentially expressed TCR V gamma 2, V gamma 4, and V gamma 5 specificities rather than TCR V gamma 3 as found on DETC of euthymic mice. Using mitogenic stimuli, we have succeeded in establishing cell lines and clones from BALB/c nu/nu and C57BL/6 nu/nu epidermis. Their marker profile corresponds to that seen on resident CD3+ epidermal cells, as well as on a very small subset of CD3+ splenic and lymph node lymphocytes of athymic mice. The ontogenetic relationship, if any, between the epidermal and lymphoid CD3+, CD5-, CD4-, CD8- cells, has yet to be clarified. Cell lines/clones representative of resident CD3+ epidermal cells of nu/nu mice should provide a useful tool in the elucidation of homing patterns and functional properties of extrathymically matured T cells.     

Antigen feeding increases frequency and antigen-specific proliferation ability of intraepithelial CD4+ T cells in alphabeta T cell receptor transgenic mice

To study how intestinal intraepithelial lymphocytes (IEL) are affected by orally ingested antigen, the phenotypes and responses of the IEL in mice expressing a transgenic T cell receptor alphabeta (TCR alphabeta) specific for ovalbumin (OVA) were analyzed after feeding OVA. In the OVA-fed mice, the abundance of alphabeta-IEL as a proportion of the total IEL population increased and the frequency of CD4+ cells increased within the TCR alphabeta+ IEL population. CD4(+) IEL from OVA-fed transgenic mice proliferated in vitro more markedly in response to antigen stimulation than IEL from mice fed the control diet. These results indicate that antigen-specific proliferation of CD4+ IEL was amplified as a result of oral administration of antigen.     

T cells in cryptopatch aggregates share TCR gamma variable region junctional sequences with gamma delta T cells in the small intestinal epithelium of mice

The role of cryptopatch aggregates in the development of intestinal intraepithelial lymphocytes (IEL) is a matter of controversy. Therefore, an important question is whether T cells in cryptopatch aggregates are lineally related to IEL. We hypothesized that if gammadelta+ IEL derive from T cells in cryptopatch aggregates, then a clonal relationship would exist between the two populations. To test this hypothesis, we compared the sequence of rearranged TCR gamma variable region 5 genes in gammadelta+ IEL and cryptopatch cells. We purified IEL by FACS and cryptopatch cells were isolated from frozen sections of the intestine by laser-assisted microdissection. PCR showed that TCR gamma variable region 5 was rearranged in gammadelta+ IEL and in CD3+ cryptopatch cells, but not in CD3- cryptopatch cells. DNA sequence analysis showed that the frequency of in-frame junctions in cryptopatch aggregates was at a level consistent with positive selection in both wild-type and athymic nude mice. In addition, the predicted amino acid sequences of V-J junctions present in gammadelta+ IEL and cryptopatch cells were encoded by identical nucleotide sequences. By contrast, the frequency of in-frame joints was significantly reduced in cryptopatch cells isolated from TCR delta-deficient mice, indicating that the enrichment of in-frame joints in cryptopatch cells must normally depend on expression of surface gammadelta TCR. Our results are consistent with the hypothesis that a subset of gammadelta+ IEL are related to T cells in cryptopatch aggregates. The precise role of cryptopatch aggregates in intestinal gammadelta+ T cell homeostasis still needs to be determined.     

MHC class I allele dosage alters CD8 expression by intestinal intraepithelial lymphocytes

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.     

Sequential analysis of T cells in the liver during murine listerial infection.

Phenotypes and functions of T cells in the liver were studied after an i.p. inoculation with viable Listeria monocytogenes in mice. T cells in the liver of untreated C3H/HeN mice (C3H; H-2k, Mls-2a) contain Thy-1.2+TCR-alpha beta + cells as a majority and Thy-1.2+TCR-gamma delta + cells and Thy-1.2-TCR-gamma delta + cells as minorities. The liver of untreated C3H mice did not contain T cells expressing V beta 3 and V beta 11, which are potentially autoreactive against self-superantigens of Mls-2a and Dvbl, respectively. On days 3 to 6 after infection, Thy-1.2-CD4lowTCR-alpha beta + T cells or Thy-1.2-TCR-gamma delta + T cells increased significantly in number and proportion in the liver whereas T cells with these phenotypes were hardly detected in the spleen, lymph nodes, peripheral blood, and peritoneal cavity during the course of the infection. The Thy-1.2-CD4lowTCR-alpha beta T cells contained V beta 3 or V beta 11-bearing cells in high frequencies. The potentially autoreactive V beta 3- or V beta 11-bearing T cells disappeared from the liver on day 7 after infection. Furthermore, the V beta 3+ and V beta 11+ cells but not V beta 8+ cells disappeared after culture for 24 h at 37 degrees C. In vitro stimulation of liver T cells using anti-V beta 11 mAb showed no proliferative response. These results suggest that the potentially autoreactive clones with Thy-1.2-CD4low phenotypes, which increased in number after listerial infection, may be anergized after interaction with self-Ag and may be programmed to die. These potentially autoreactive clones induced in the liver of Listeria-infected mice may not be functionally relevant to the host defense against Listeria.