Gamma delta T lymphocytes regulate the induction and maintenance of oral tolerance.

Oral administration of Ag, over a period of several days, induces a state of tolerance that is associated with activation of CD8+ T cells that can transfer unresponsiveness to naive syngeneic hosts. We previously demonstrated that these T cells are not CTL precursors and that they inhibit responses by CD8+ CTL, as well as Ab and CD4+ T cell responses. Activation of noncytolytic, CD8+ suppressor T cells by oral Ag is a process that is not understood. In these studies, we asked whether depletion of the gamma delta T cells altered induction of oral tolerance. Injection of the anti-delta-chain Ab (GL3) down-modulated the expression of gamma delta TCR and inhibited the induction of oral tolerance to OVA, as measured by Ab, CD4+, and CD8+ T cell responses. GL3 did not activate IL-2 secretion that could be detected in the serum, nor did it induce IL-2R expression by intraepithelial lymphocytes, suggesting that GL3 inhibited the function of gamma delta T cells rather than activating them. This interpretation is supported by our observation that oral administration of Ag did not induce tolerance in TCR-delta knockout mice. These data suggest that gamma delta T cells play a critical, active role in tolerance induced by orally administered Ag.

Tracking H-2 alleles in transgenic mice by RFLP and heteroduplex analysis.

Transgenic mice provide valuable tools for biological research including many areas of immunology. In studies involving the major histocompatibility complex (MHC), it is often necessary to place the desired transgene in a specific H-2 (the murine MHC) environment. In this regard, the strains commonly used for the production of transgenic mice also carry well characterized H-2 alleles and provide an appropriate genetic background for MHC related experiments. In this study, a highly polymorphic microsatellite of tetranucleotide repeats from the second intron of the class II Eb gene within the H-2 complex was used in order to identify the corresponding alleles. The relevant H-2 allele(s) along with the transgene were then tracked throughout the production of a chicken ovalbumin-specific transgenic strain. The technique involved PCR-amplification of a DNA sequence encompassing the H-2 specific microsatellite followed by RFLP and heteroduplex analyses. This approach is likely to find wide application in the background checking of transgenic mice, especially in immunological research requiring a defined H-2 background.

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

Extrathymic T cell maturation. Phenotypic analysis of T cell subsets in nude mice as a function of age.

T cell maturation in an extrathymic environment has been studied using as a model the congenitally athymic nude mouse. Phenotypic analyses as a function of age were conducted on lymphocytes obtained from the spleens and lymph nodes of nude mice through use of mAb recognizing T cell surface markers and multiparameter flow cytometry. The data show that nude mice accumulate increasing numbers of lymphocytes bearing Thy-1, CD3, CD4, and CD8 with age characterized by a progression from heterogeneous dim to more homogeneous bright expression. In contrast, the expression of heat-stable Ag (HSA), a marker of immature thymocytes, decreases with age. By analogy to intrathymic maturation, spleens and lymph nodes in nude mice contain T cells defined as immature, transitional, and mature based on the expression of these markers. Although the proportion of CD4+ and CD8+ T cells associated with bright CD3 expression increases with age, at no age are significant numbers of CD4+8+ cells observed, in contrast to intrathymic T cell maturation. In addition to the frequently observed inversion in the ratio of CD4 to CD8, the CD8 T cell subpopulation in older nude mice contains mainly mature cells (CD8+, CD3+, HSA-) whereas only 50% of CD4+ T cells express the mature (CD4+, CD3+, HSA-) phenotype. At any age, the spectrum of phenotypes observed indicates that lymph nodes contain more mature T cells than spleen, suggesting a role for environmental Ag in driving extrathymic maturation, a process occurring most efficiently among CD8+ T cells. Because extrathymic maturation mirrors some but not all aspects of the intrathymic pathway, we propose that the nude mouse may be a useful model for further dissecting those interactions crucial to establishing the T cell repertoire in euthymic individuals as well as elucidating the contribution of extrathymically derived T cells to the peripheral immune system.

CD8+ alpha/beta or gamma/delta T cell receptor-bearing T cells from athymic nude mice are cytolytically active in vivo.

Phenotypic analysis of lymphocytes that mature extrathymically in congenitally athymic nude mice has revealed a large population of CD3+ CD8+ T cells that express gamma/delta-TCR. In euthymic mice, significant numbers of cells with this phenotype are found only in the intestinal epithelium. Intestinal intraepithelial lymphocytes have been shown to be cytolytically active in vivo, as measured by the redirected lysis assay. In this communication, freshly harvested T cell subsets obtained from pooled nude mouse spleen and lymph nodes and separated by flow cytometric cell sorting were assayed for their ability to lyse FcR+ P815 targets in the presence of mAb to the epsilon-chain of the CD3 complex. CD8+, but not CD4+ or CD4- CD8-, T cells in nude mice were cytolytically active. CD8+ alpha/beta- and gamma/delta-TCR-bearing T cells from the spleen and lymph nodes of nude mice demonstrated similar cytolytic activity. No cytolytic activity of purified cell subsets was apparent in the absence of anti-CD3 mAb, even when NK-susceptible target cells were used. These data indicate that, in contrast to euthymic mice, a large proportion of CD8+ cells from the spleen and lymph nodes of nude mice are cytolytically active in vivo. In addition, these results suggest that the intestinal epithelium is not the only anatomical location where constitutively cytolytic CD8+ alpha/beta- or gamma/delta TCR-bearing T cells may be found.

T cell receptor expression by T cells that mature extrathymically in nude mice.

The expression of TCR by T cells that mature extrathymically in nude mice was determined by staining Ig- cells from B10 nude mice that were 5 months of age or older with mAbs specific for CD3, alpha/beta or gamma/delta TCR. Although the majority of Ig- cells in older nude mice express TCR, the distribution of alpha/beta and gamma/delta TCR in relation to CD4 and CD8 expression is markedly different compared to T cells from euthymic mice. Approximately half of the CD3+ T cells found in the spleen and lymph nodes of nude mice express gamma/delta TCR that is equally distributed between CD4-8- double-negative and CD8+ single-positive T cells. These data provide the first quantitative measure of the expression of TCR by T cells that mature in the absence of a thymus and suggest that the extrathymic environment, although not efficient, is permissive for the maturation of T cells that express alpha/beta and gamma/delta TCR.

Tolerance induced by physiological levels of secreted proteins in transgenic mice expressing human insulin.

We have used transgenic mice to study immune tolerance to autologous, non-MHC encoded proteins that are expressed at physiological levels in the circulation. The transgenic mice used in these studies express the human preproinsulin gene and synthesize human proinsulin. Human and mouse insulin are secreted from the pancreatic islets of transgenic mice in response to normal physiological stimuli, such as glucose. Our data demonstrate that the transgenic mice have acquired tolerance to human insulin. The repertoire of T cells specific for exogenous antigens is shaped by the acquired tolerance to autologous proteins since pork but not beef or sheep insulin is also nonimmunogenic in the transgenic mice. We also found that the transgenic mice were tolerant to human proinsulin, the intracellular precursor of insulin. Unresponsiveness to human proinsulin most likely results from tolerance of insulin-specific and proinsulin-specific T cells that recognize the secreted enzymatic cleavage products of proinsulin, insulin and C-peptide.

Characterization of L-glutamic acid60-L-alanine30-L-tyrosine10-specific suppressor T cells in responder mice restricted by Igh-C-linked genes.

A Ts cell subset has been identified in the spleens of responder mice 3 to 6 wk after immunization with an optimally immunogenic dose of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). These Ts were positively selected by panning procedures by using a mAb (1248 A4.10) produced by immunization of rats with semipurified mouse GAT-specific, single polypeptide chain suppressor factor. These Ts cells inhibited the activity of virgin Th cells but not memory Th cells and this activity was genetically restricted by genes which are linked to the Ig H chain (Igh) locus on chromosome 12. Use of the Igh recombination strain, BAB.14, which has a crossover near the VHCH region junction, demonstrated that the genes regulating the Igh restriction map telomeric to the VH genes. The Igh-linked restriction regulated the interaction of A4.10+ Ts cells with virgin T cells and not B cells. However, A4.10+ Ts did not act directly on Lyt-2-Th cells, but required the presence of Lyt-2+ cells for suppression. Suppression by GAT-primed A4.10+-Ts cells also required syngenicity at Igh-linked genes by both Lyt-2- and Lyt-2+ T cells. These results indicated that A4.10+-Ts cells were inducer Ts cells which activated Lyt-2+ effector Ts cells which prevented primary GAT specific Th cell activity. The interaction between A4.10+-Ts inducer and effector Ts cells and/or the interaction of the effector Ts and its target cell were restricted by genes linked to the Igh constant region.

Regulation of immune responses by T cell subsets. Role of helper and suppressor T cells in the development and expression of MHC-restricted antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) by (responder X responder)F1 spleen cells.

The roles of helper and suppressor T cells in the development and expression of antibody responses to GAT were studied in (responder X responder)F1 mice immunized with parental GAT-M phi. Spleen cells from (B10 X B10.D2)F1 mice primed in vivo with B10 or B10.D2 GAT-M phi developed secondary in vitro plaque-forming cell (PFC) responses only when stimulated by GAT-M phi syngeneic with the GAT-M phi used for in vivo priming. By contrast, virgin F1 spleen cells developed comparable primary PFC responses to both parental GAT-M phi Co-culture of T cells from (B10 X B10.D2)F1 mice primed in vivo by B10 GAT-M phi with virgin (B10 X B10.D2)F1 spleen cells demonstrated the presence of suppressor cells that inhibited the primary response of virgin spleen cells stimulated by B10.D2 GAT-M phi. Spleen cells from (B10 X B10.D2)F1 mice primed in vivo with B10.D2 GAT-M phi had suppressor T cells that suppressed primary responses stimulated by B10 GAT-M phi. The suppressor T cell mechanism was composed of at least two regulatory T cell subsets. Suppressor-inducer T cells were Lyt-2-, I-J+ and must be derived from immune spleen cells. Suppressor-effector T cells can be derived from virgin or immune spleens and were Lyt-2+ cells. When the suppressor mechanism was disabled by treatment with 1000 rad gamma irradiation or removal of Lyt-2+ cells, Lyt-2-helper T cells from (B10 X B10.D2)F1 mice primed with B10 GAT-M phi provided radioresistant help to virgin F1 B cells stimulated by B10 but not B10.D2 GAT-M phi. Suppressor inducer Lyt-2-,I-J+ cells from B10 GAT-M phi-primed (B10 X B10.D2)F1 mice were separated from the primed Lyt-2-,I-J-helper T cells. In the presence of Lyt-2+ suppressor effector cells, the Lyt-2-,I-J+ suppressor-inducer suppressed the primary response of virgin spleen or virgin T plus B cells stimulated by both B10 and B10.D2 GAT-M phi. Therefore, suppressor T cells were able to suppress primary but not secondary GAT-specific PFC responses stimulated by either parental GAT-M phi. These results showed that immunization of (responder X responder)F1 mice with parental GAT-M phi results in the development of antigen-specific helper and suppressor T cells. The primed helper T cells were radioresistant and were genetically restricted to interact with GAT in association with the major histocompatibility complex antigens of the M phi used for in vivo priming.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic restrictions in the development of antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 by nude mice implanted with semiallogeneic thymus glands.

Athymic nude mice implanted with F1 thymus glands were used to investigate genetic restrictions regulating T cell-macrophage (M phi) interactions in the development of antibody responses to GAT. Spleen cells from conventional mice developed comparable primary plaque-forming cell (PFC) responses when stimulated by syngeneic and allogeneic GAT-M phi. However, spleen cells from strain A nude mice implanted with (A X B)F1 thymus glands were tolerant of strain B alloantigens and developed GAT-specific PFC responses to strain A GAT-M phi and allogeneic strain C GAT-M phi, but failed to respond to strain B GAT-M phi. The lack of primary GAT-specific PFC responses by spleen cells from (A X B)thy----A nude mice stimulated by strain B GAT-M phi was not due to detectable suppressor mechanisms. However, an allogeneic effect stimulated by H-2- or non-H-2-disparate GAT-pulsed or unpulsed M phi was able to overcome the inability of spleen cells from (A X B)F1 thy----A nude mice to respond to strain B GAT-M phi. Furthermore, the inability to respond to strain B GAT-M phi was overcome by the addition of supernatant fluids from independent cultures of H-2-disparate cells. These results 1) demonstrate that T cells from A nude mice implanted with (A X B)F1 thymus glands did not recognize nominal antigen in the context of B MHC antigens, and 2) suggested that the T cell repertoire was altered in strain A nude mice implanted with (A X B)F1 thymus glands, such that T cells that could recognize GAT in association with strain B MHC antigens were functionally deleted.

The antibody response to specific immune complexes is under genetic control and correlates with the expression of a recurrent idiotype.

The primary antigen-specific antibody response of various strains of mice to TEPC-15/PnC immune complexes has been examined. We found that both BALB/c and C3H mice were good responders to the PnC antigen; however, C3H mice were low responders, whereas BALB/c mice were high responders to the TEPC-15/PnC complexes. Using congenic strains on the C3H and BALB/c background, we have shown that the response to the complexes is not restricted by gene products of the H-2 complex or by the Igh (allotype) locus. However, responsiveness may be controlled by genes linked to the Igh locus, since we have shown that strains that are Ighj, Ighd, and Ighf are low responders, whereas strains that are Igha, Ighb, and Ighe are high responders to the immune complex. Moreover, responsiveness correlates with the expression of the T15 Id as measured using the anti-T15 monoclonal antibody, AB1-2. Thus, strains such as BALB/c, BALB.B, BALB.K, and CB-20, which express high levels of T15 (AB1-2) Id in their PFC response to PnC are relatively high responders to TEPC-15/PnC complexes, whereas C3H, C3H.SW, and C3H-OH, which express low levels of the T15 (AB1-2) Id, are low responders to the complexes. Finally, we found that BALB/c mice are high responders to complexes formed with T15+ antibodies, whereas they are low responders to complexes formed using T15- antibodies. The results suggest that the antigen-specific response to these immune complexes is Id-restricted.

Target cell heterogeneity in murine leukemia virus infection. III. Identification of susceptible Lyt 1+ and resistant Lyt 2+ T-cell subsets following in vitro infection with Friend murine leukemia virus.

The susceptibility of splenic T-cell subpopulations to productive infection with Friend murine leukemia virus was determined after in vitro infection and stimulation with Con A. Con A enhanced the number of productively infected cells in unseparated spleen cells as well as in T-cell-enriched spleen cell fractions. Splenic T cells were fractionated into Lyt 1+ and Lyt 2+ subpopulations using both positive and negative selection techniques; susceptible splenic T cells were recovered in the Lyt 1+ fraction and specific cytotoxic treatment with anti-Lyt 1 antibody and complement reduced the number of infectious center-producing cells by greater than 87%. In marked contrast, Lyt 2+ splenic T cells were resistant to productive infection by Friend murine leukemia virus in vitro.

Loss of tolerance to thymus-donor strain antigens in nude mice bearing long-term allogeneic thymus grafts.

In contrast to lymph node cells from nonthymus gland-implanted nude mice, which failed to respond to foreign alloantigens in mixed lymphocyte reaction tests, lymph node cells from nude mice implanted with either syngeneic or allogeneic thymuses responded well following stimulation with third-party allogeneic cells from strains unrelated to the nude host or thymus-donor strain. Responses to thymus-donor strain alloantigens by lymph node cells from nude mice implanted with allogeneic thymuses were in part dependent upon the duration of the thymic implants; no responses were observed using cells from mice implanted 3 mth previously whereas the majority of experiments yield positive responses using cells from mice implanted 12 mth previously, suggesting a functional breakdown of the tolerant state with time post allogeneic thymus implantation. In no case were positive responses specific for the nude host strain observed.

Sendai virus-specific, H-2-restricted cytotoxic T lymphocyte responses of nude mice grafted with allogeneic or semi-allogeneic thymus glands.

The in vitro secondary cytotoxic T lymphocyte (CTL) response to Sendai virus-treated stimulator cells by primed spleen cells from thymus gland-grafted nude mice was examined. BALB/c (H-2d) nude mice grafted with allogeneic C57BL/10 (H-2b) thymus glands developed CTL responses directed exclusively to Sendai virus-infected H-2d target cells. (C57BL/6 X BALB/c)F1 nude mice grafted with thymus glands of either parent developed CTL responses preferentially against infected target cells expressing the MHC antigens present in the parental thymus graft, but also had detectable activity for infected target cells of the parental haplotype not expressed in the thymus. These results provide evidence against the concept that self recognition by MHC-restricted CTL is directed exclusively by the MCH type of the thymus.

Development of a localized hemolysis-in-gel assay for Vi antigen: characterization of the Vi-specific PFC response of nude and normal mice.

An assay to detect specific plaque-forming cells (PFC) to Vi antigen (Vi) was developed and the optimal conditions for sensitization of sheep erythrocytes (SE) and plaque development were determined. Using PFC and passive hemagglutination (PHA) assays, Vi-specific immune responses of athymic (nude) and normal mice were characterized. Vi was found to elicit only IgM PFC. No discernable secondary response was detected following a second injection of antigen. Nude and normal mice responded in a quantitatively similar manner to all doses of Vi tested and responded similarly on varying days following immunization. Also, both nude and normal mice produced the greatest number of Vi-specific PFC 4 days following immunization with an optimally immunogenic dose of Vi (1.0 microng/mouse). These results indicate that functional thymus-derived cells are not necessary to elicit an immune response against Vi antigen.