Tolerance to Mls-disparate cells induces suppressor T cells that act at the helper level to prevent in vivo generation of cytolytic lymphocytes to hapten-altered self

We have been examining the mechanisms that control in vivo development and down regulation of cytolytic T lymphocytes (CTL) to trinitrophenyl (TNP)-altered self antigens. In vivo generation of hapten-specific CTL requires an auxiliary antigenic stimulus, which can be provided by H-2 compatible but Mls-disparate cells. These experiments were designed to study the effect of tolerization with such Mls-disparate cells on CTL development. C3H/HeN (H-2k, Mlsc ) mice sensitized in the footpads with C3H-TNP spleen cells plus CBA/J (H-2k, Mlsd ) spleen cells develop CTL in the draining lymph nodes that will lyse 51Cr-labeled TNP-modified C3H targets. However, we have found that if C3H/HeN mice are given tolerizing doses of CBA/J spleen cells 5 to 7 days before sensitization, a splenic suppressor T cell (Ts) appears. This Ts will suppress CTL development in its tolerant host, and can be transferred adoptively to function in naive mice. Ts and its precursor are cyclophosphamide insensitive and therefore different from the naturally existing suppressor cell present in mice. When triggered by cells with Mlsd , the Ts produces a factor (TsF) that hinders helper factors from functioning in an in vitro CTL assay. Furthermore, TsF acts to prevent utilization of IL 2 by an IL 2-dependent cell line. Thus, evidence has been provided that the in vivo generation of CTL toward hapten-altered self can be down regulated at the level of helper signals by a Ts. The latter is inducible by the Mls-disparate cells that are needed at a different site to trigger the helper factors in this CTL system.     

H2Kk can influence whether cytotoxic T lymphocytes recognize trinitrophenyl in association with H2Dk unique or H-2Kk and H-2Dk shared self determinants

The present study investigates the effect of trinitrophenyl- (TNP) modified H-2Kk (TNP-Kk) antigens on the generation of anti-TNP-Dk restricted cytotoxic T lymphocyte (CTL) responses. C3H.OH mice were primed to TNP-self by skin-painting with trinitrochlorobenzene, and spleen cells from these primed mice were subsequently stimulated in vitro with TNP-self. The effector cells generated exhibited appreciable lysis of TNP-modified C3H.OH blast target cells. Cold target inhibition studies demonstrated the generation of two effector cell populations: one that recognizes TNP in association with unique Dk self determinants, and one that recognizes TNP in association with self determinants shared between TNP-Kk and TNP-Dk. This was in contrast to primed C3H/He spleen cells, which did not generate CTL that recognized TNP in association with unique Dk self determinants. When spleen cells from (C3H/He x C3H.OH)F1 mice primed to TNP were stimulated in vitro with TNP-C3H.OH cells, unique Dk self determinants were recognized in association with TNP. However, in vitro stimulation of the same F1 responding cells with TNP-C3H/He or TNP-F1 cells failed to elicit CTL that utilized these Dk-unique self determinants. The findings of this study demonstrate that unique or shared H-2Dk determinants can be differentially utilized by CTL populations, depending on the H-2 alleles expressed by the stimulator cells.     

Pretreatment with minor histocompatibility antigens prevents the development of functional CTL helper cell activity

We have been studying the regulation of allogeneic cytotoxic cells (CTL) in vivo. CBA/J (H-2k, mls d) responder mice are unable to develop CTL after an allogeneic footpad immunization if they are pretreated i.p. with spleen cells from either C3H/HeN (H-2k, mls c) or B10.BR (H-2k, mls b) mice. These mouse strain combinations are H-2 compatible but differ at the Mls and other minor histocompatibility loci. We reported that this state of CTL unresponsiveness is specific and that the allogeneic cells used for footpad immunization and the pretreatment strain must share both minor antigens and part of the MHC. In this paper, we describe some of the characteristic features of this CTL unresponsiveness. The CBA host plays an active role and appears to down-regulate its subsequent response against minor antigens after the initial pretreatment. This statement is based on the following: 1) The inhibition of in vivo CTL generation can be achieved by injecting F1 or irradiated C3H cells, i.e., under conditions where GVHD was not a factor; and 2) the state of unresponsiveness is abolished by host treatment with cyclophosphamide. In addition, we demonstrate that the lack of CTL development in pretreated responder animals is the result of impaired helper cell activity. Draining LNC from unresponsive mice can become functionally cytolytic if cultured in a Con A-activated spleen cell supernatant. However, normal CTL responses were not restored after adult thymectomy or splenectomy. Thus, the state of CTL inhibition that is induced by the minor antigen pretreatment is the result of a host-mediated regulatory circuit.     

Production and characterization of T-cell clones specific for trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self)

Using serial antigenic challenge as the method of selection and stimulation, continuous lines of cytotoxic T-lymphocytes (CTL) directed against TNBS-modified syngeneic spleen cells (TNP-self) have been generated. Spleen cells from C3H/HeJ (H-2k) mice were primed in vitro with autologous spleen cells modified with TNBS, and subsequently cloned by limiting dilution and in soft agar in the presence of IL2. These CTL clones grew continuously in medium supplemented with IL2 and in the presence of antigen. They are antigen specific and H-2 restricted in their target cell recognition. They all express Thyl and Lyt2 surface markers. None of the clones exhibit natural killer (NK) cell activity. All CTL clones tested so far are restricted in their target cell recognition to H-2Kk-TNP and none were found to be restricted to H-2Dk-TNP. These findings demonstrate at the clonal level the H-2K/D restriction of TNP-self specific CTL. These clones provide tools that may facilitate an understanding of the development and regulation of antigen specific CTL. They may also serve as models useful towards an understanding of the mechanism of lysis by CTL.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response.

Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Regulatory T cell subpopulations in pregnancy. I. Evidence for suppressive activity of the early phase of MLR.

Previous in vivo experiments have provided evidence of suppressive activity induced by multiple allogeneic pregnancies. The reactivity of maternal spleen cells toward paternal strain alloantigens was investigated by use of MLR microculture technique. A study of the kinetics of the MLR showed an early peak of reactivity (48-hr culture) followed by a decline leading to a decreased reactivity by 96 hr when spleen cells from allogeneically pregnant mice were compared to those of virgin or even isogeneically pregnant mice, suggesting the possible action of MLR regulatory cells. A strong suppression of a H-2k (CBA) anti-H-2a (A/J) or anti-H-2d (C57BL/Ks) MLR was observed when mitomycin-treated spleen cells from CBA mice multiparous by A/J or C57BL/Ks (but not CBA) males were added to the culture. This suppression was abolished by treating the regulatory cell population with anti-theta serum plus complement or replacing the 1% normal mouse serum in the medium by a proper antiidiotypic mouse serum.     

The target minor H antigen for F1 cytotoxic T lymphocytes induced by Igh-congenic parental spleen cells is coded for by gene linked to H-2

Graft-vs-host reaction (GvHR) induced in (B10.BR X CWB)F1 (BWF1; H-2k/b, Ighb/b) by i.v. injection with CWB (H-2b, Ighb) spleen cells resulted in complete suppression of cytotoxic T lymphocyte (CTL) responsiveness of the F1 host spleen cells (GvHR-associated immunosuppression). In contrast, GvHR induced in BWF1 mice with CSW (H-2b, Ighj; Igh-congenic to CWB) spleen cells did not affect CTL responsiveness of the F1 host spleen cells at all. The BWF1 hosts undergoing the CSW-induced GvHR generated anti-CSW CTL in their spleens, and the subsequent culture of such BWF1 spleen cells with CSW stimulator cells, augmented the CTL activity. The BWF1 anti-CSW CTL lysed both Con A- and LPS-induced splenic blasts from mouse strains carrying the Ighj allele in the context of self H-2Kb. However, determination of the Igh haplotype in the serum IgG and of the susceptibility of the splenic lymphocytes to the BWF1 anti-CSW CTL on backcross mice, which carry either Ighb/j or Ighb/b in the context of H-2b/b or H-2b/k, showed clearly that Ighj and the gene coding for the target antigen for the BWF1 anti-CSW CTL segregated at ratios close to 1:1. The study in which linkage between the gene(s) coding for the target antigen for the BWF1 anti-CSW CTL and H-2 was examined on CWB X (C3H X CWB)F1 backcross mice and (B10.BR X CSW)F1 X B10 mice demonstrated that the gene, most likely a single gene, coding for the target antigen for the BWF1 anti-CSW CTL is located at 8.5 +/- 4.3 cross-over units to the right or left of the H-2 complex. We designated the minor H antigen to be recognized by the BWF1 anti-CSW CTL as H-X+, and we discuss the distinction between the H-X+ locus and the other minor H loci on chromosome 17.     

Interleukin 3 augments the murine primary cytolytic T lymphocyte response to allogeneic tumor cells

The primary anti-H-2k allospecific cytolytic T lymphocyte (CTL) response by BALB/c (H-2d) spleen cells in vitro to x-irradiated RDM4 (H-2k) tumor cells is weak. This response has been shown to be augmented by CTL helper factor (CHF), a factor present in supernatants of spleen cells cultured with Sendai virus (SC-CM). Conditioned medium from WEHI-3 cells (WEHI-CM) also contains activity that augments the BALB/c anti- RDM4 CTL response. Attempts to separate the CHF activity from interleukin 3 (IL 3), also present in WEHI-CM, were unsuccessful. Purified IL 3 was then tested, and was found to increase the BALB/c anti- RDM4 CTL response by five- to 10-fold. IL 3 is apparently the only material in WEHI-CM that is active in this assay. The response is apparently a classical CTL response because: 1) the effector cells are sensitive to monoclonal anti-Thy-1.2 antibody plus C; 2) the response is dependent on antigen stimulation, and it peaks on day 5 or 6 of culture; and 3) the effector cells are specific for H-2k targets. IL 3 must be added very early during the in vitro culture period for maximal augmentation of the response, consistent with possible action of IL 3 as a differentiation factor.     

MIs locus recognition by a cloned line of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

This report demonstrates the expression of strong MIs locus MIsd) recognition by a cloned line of H-2-restricted influenza virus-specific CTL. This clone of F1 (H-2b/d; MIsb) origin was found to specifically proliferate in response to uninfected cells of CBA/J (H-2k, MIsd) origin but not to uninfected B10.BR or CBA/CaJ cells (H-2k, MIsb). In addition, proliferation by this cTL line was observed in response to histocompatible cells expressing cross-reactive MIsa determinants (DBA/2, NZB; H-2d, MIsa). This recognition was observed only at the level of CTL proliferation. The CTL line exhibited no cytotoxic activity for target cells of these MIs types. These observations are contrasted with the response of another cloned H-2-restricted influenza-specific CTL line that simultaneously exhibits alloreactivity for H-2k. The significance of these results for T lymphocyte recognition is discussed.     

The effect of pepsin digestion in relation to the pre-S region on hepatitis B surface antigen-induced hypersensitivity.

The role of the pre-S region of the hepatitis B surface Ag (HBsAg) particle in hypersensitivity to HBsAg was evaluated in mice. Plasma-derived or recombinant HBsAg was digested with pepsin to prepare different forms of HBsAg with or without pre-S region. Strains of mice including AKR/J (H-2k), A.SW (H-2s), C3H/He (H-2k), and CBA/J (H-2k) did not respond to the major S protein with regard to hypersensitivity. However, the pre-S-containing HBsAg overcame this nonresponsiveness. In BALB/c (H-2d) and A/J (H-2a) mice, the pre-S-containing HBsAg induced higher hypersensitivity than did the major S protein. The enhancement induced by the pre-S region was demonstrated to occur during the induction phase by crisscross assay using pre-S-containing HBsAg and major S protein as Ag. The patterns of hypersensitivity induced by the major S, middle S (composed of major S and pre-S2), and large S (composed of middle S and pre-S1 proteins) were also compared. The middle S protein induced responses of 1-h and 24-h hypersensitivities in major S non-responder (C3H/He and CBA/J) mice, whereas the large S protein circumvented only the 1-h one. The effectiveness to stimulate hypersensitivities in vivo by HBsAg is in the following order: middle S greater than large S greater than major S. These data suggest that the pre-S region of HBsAg particle can enhance both the 1-h and 24-h hypersensitivities in the afferent phase.     

Peripheral tolerance induced in lymph nodes by syngeneic spleen cells inhibits generation of CTLs to hapten-altered self antigens but not to alloantigens.

The immunological tolerance that is induced in lymph nodes that have been exposed to syngeneic spleen cells has been examined. Development of cytotoxic T lymphocytes was used to assess the immunological status of the lymph node cells. The tolerance was studied from the viewpoint of its induction, its activation, and its specificity. We had already reported that injecting either T or B cells of splenic origin into a regional lymph node environment a week prior to immunization for CTL to hapten-altered self antigens prevents development of the CTL. Here, we confirm that syngeneic splenic cells but not lymph node cells will induce the suppression provided that spleen cells are not coupled with hapten. We now report that splenic cells that cannot replicate or synthesize and secrete protein are capable of inducing the suppression. The data suggest a preformed surface marker peculiar to spleen cells and perhaps on cells that traverse the thymus induces local tolerance that is mediated by suppressor cells. Triggering the induced suppressor T cells (previously identified as CD8-) was achieved by syngeneic spleen cells as well as by H-2-compatible, Mls-disparate spleen cells but not by syngeneic lymph node cells or apparently by allogeneic spleen cells. Furthermore, triggering suppression was achieved by hapten-coupled syngeneic spleen cells whereas such cells would not induce the suppression. Thus, activating the suppressor cells requires reexposure to splenic cells of the proper MHC haplotype, unaltered or coupled with either TNP or FITC. Once triggered, the suppression was manifested toward CTL generation against hapten-coupled syngeneic antigens on either spleen or lymph node cells but not against allogeneic antigens. Thus, the specificity of the tolerance was directed to altered self antigens despite its induction by unaltered spleen antigen. Furthermore, for suppression to be seen the spleen antigen was not required to be on the hapten-coupled syngeneic cells used for the CTL immunization. The relationship of the splenic cell "antigen" to hapten-altered self antigens and to other surface markers and its site of acquisition within the body and its significance for cell homing have become intriguing questions of importance. This information has been discussed from the viewpoint of its applicability to autoimmune diseases as well as to cessation of inflammatory reactions that may be mediated by lymph node cells.     

Cell-mediated T lymphocyte responses against syngeneic cells modified with amino-reactive hapten (AED-NH2): H-2Dk serves as an element for cell-mediated lympholysis to amino-reactive hapten (AED-NH2)-modified self

Spleen cells from C3H/He mice immunized to the newly synthesized amino-reactive hapten, 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide (AED-NH2), were stimulated in vitro with AED-NH2 modified syngeneic cells. After 5 days of culture, effector cells were assayed for their cytotoxic activity against AED-NH2-modified target blast cells. In contrast to other amino-reactive haptens reported so far, a strong cytotoxic activity against AED-NH2-modified syngeneic cells was found in H-2b mice as well as in H-2k mice. Furthermore, Dk-restricted anti-AED-NH2 CTL recognition was observed in H-2k mice as shown by cold target inhibition. Previous studies have demonstrated the predominant influence of K over D region self determinants, and of the chemical reactivity of the haptenic reagent in Ir gene control of CTL response to hapten-self. The present report illustrates the importance of the hapten itself in genetic regulation of these CTL responses.     

CD3- bone marrow cells augment the generation of cytotoxic T lymphocytes showing a preference for the X-chromosome linked gene product of stimulator cells

Responder cells, composed of both a limited number of nylon wool-passed lymph node (NW-LN) cells and an excess number of CD3+ cell-depleted bone marrow (CD3- BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3- BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW-LN cells. C3H/He (H-2k, C3H background) responder cells were stimulated with either B10.D2 (H-2d, B10 background) or BALB/c (H-2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 x BALB/c) F1 male target cells than (BALB/c x B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c) x B10.D2) F1 male than (B10.D2 x BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H-2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He x B10.BR) F1 male target cells than B10.BR or (B10.BR x C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X-chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

Immunogenetic studies of spontaneous abortion in mice. II. Antiabortive effects are independent of systemic regulatory mechanisms

CBA/J females (H-2k) mated with DBA2/J males (H-2d) exhibit a high rate of fetal resorption. Fetal survival has been improved by pretreatment of CBA/J females with spleen cells from BALB/c J (H-2d) (but not from CBA/J or DBA/2/J) males. Similarly, three out of nine recombinant inbred strains (recombining BALB/c and DBA2 genomes at the homozygous state) possess antiabortive effects like the BALB/c parent. Previous studies showed that BALB/c pretreatment triggers the appearance of suppressor cells in the spleen and of IgG1 anti-H-2d antibodies in the serum of CBA/J females. Studies of these two immunological parameters after CBA/J preimmunization by the different recombinant strains suggest that local intrauterine immunoregulation is the determinant of success or failure of allopregnancy.     

The cellular basis for the Ia restriction in murine experimental autoimmune thyroiditis

Susceptibility to experimental autoimmune thyroiditis (EAT) in the mouse is linked to the I-A subregion of the major histocompatibility complex. EAT can be induced in susceptible strains of mice by immunization with mouse thyroglobulin (MTg) and adjuvant. We have described a cell transfer system wherein spleen cells from EAT-susceptible CBA/J mice primed in vivo with MTg and lipopolysaccharide (LPS) can be activated in vitro with MTg to transfer EAT to naive syngeneic recipients. This cell transfer system was used to elucidate the cellular basis for the I-A restriction in EAT. While the cell active in transferring EAT was Thy 1+ I-A-, depletion of I-A+ cells from the in vitro culture prevented the activation of EAT effector T cells. MTg-pulsed mitomycin C-treated naive syngeneic spleen cells as antigen-presenting cells (APCs) could replace the I-A+ cells in vitro. Allogeneic (Balb/c) APCs were ineffective. Using APCs from several recombinant inbred strains of mice, it was shown that C3H/HEN and B10.A(4R) APCs were effective in activating MTg/LPS-primed CBA/J spleen cells to transfer EAT while B10.A(5R) APCs were ineffective. This maps the H-2 restriction to the K or I-A subregions. Addition of polyclonal anti-Iak or monoclonal anti-I-Ak or anti-L3T4 during in vitro activation inhibited both the generation of EAT effector cells and the proliferative response to MTg. Irrelevant anti-Ia reagents, monoclonal anti-I-Ek, and monoclonal anti-I-Jk were ineffective. Thus the I-A restriction in murine EAT appears to result from an I-A restricted interaction between Ia+ APCs and Ia- EAT effector T cells.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.