Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells. VII. Generation of thyroid-specific cytotoxic effector cells

During primary syngeneic sensitization of lymphocytes on monolayers of thyroid epithelial cells (TEC), lymphoblasts are generated as assessed by thymidine uptake. When these thyroid-sensitized lymphoblasts are deposited onto 51Cr labeled TEC used as target cells, syngeneic cytotoxicity (SC) occurs after 5 hr. The effector cell of this SC is a T lymphocyte, belonging to the Lyt-2 T-cell subset. Moreover, blocking experiments demonstrated that SC involves class I major histocompatibility complex and thyroglobulin (Tg) antigens. These data demonstrate that SC can be compared to the classical hapten or virus self-mediated cytotoxicity, but in this case Tg plays the role of these agents. The in vivo relevance of this SC is discussed.     

Soluble suppressor of T cell proliferation in primary in vitro response to murine minor histocompatibility antigens

Normal mouse lymphocytes are not capable of mounting a primary cytotoxic T cell response to Mls encoded, non H-2, allodeterminants, although a strong lymphoproliferative response is observed in primary MLR between Mls incompatible cells. In this study it is reported that in the supernatant of primary cultures between AKR macrophages and CBA/H lymphocytes (H-2 identical, incompatible for Mls and other minor antigens) a suppressor of T cell proliferation in MLR is detected. By contrast, a suppressor is not detected in supernatants from primary cultures between BALB/C macrophages and CBA/H lymphocytes (H-2 incompatible, Mls identical), B10.BR macrophages and CBA/H macrophages and CBA/H lymphocytes (syngeneic) suggesting that the production of the suppressor factor occurs only when an Mls incompatibility exists. The suppressive activity of the Mls incompatible culture supernatant upon MLR between incompatible macrophages and lymphocytes is neither antigen specific nor Mls or H-2 restricted, nor is it due to an irreversible toxic effect on T lymphocytes or macrophages. The inhibition of T cell proliferation could be explained by inhibition of IL 2 production, by blocking its union to T cells or by a combination of both effects. Our findings could help explain previous observations that lymphocytes from mice preimmunized with Mls incompatible cells have a depressed proliferative response as well as depressed cytotoxicity against alloantigens.     

Stimulation of splenic T cells on syngeneic monolayers of epidermal basal cells (EBC) in mice. Regulation by Lyt 1+ and Lyt 2+ cell subsets

Syngeneic proliferative response of splenic T cells against monolayers of epidermal basal cells (EBC) was obtained with C57BL/6 and DBA/2 mice. Optimal response, as assessed by [³H]thymidine uptake, occurred on the 6th day of coculture. The level of [^3H]thymidine uptake by unseparated spleen cells was lower than by fractionated T cells from C57BL/6 mice, and null for DBA/2 mice. It was not significantly different when lymphocytes were cocultured with syngeneic or allogeneic epidermal cells. Ia antigens did not appear to be involved in the syngeneic response, since it was not prevented by pretreating stimulator monolayers with monoclonal anti-Ia^k antibody or by adding this antibody directly to the cultures. When the proliferative responses of separated Lyt 1⁺ and Lyt 2⁺ cell subsets were compared, the prominent role of Lyt 1⁺ cells was demonstrated. Enhancement of the T-cell reactivity by eliminating Lyt 2⁺ cells and suppression of the response of a constant number of Lyt 1⁺ cells by adding Lyt 2⁺ cells suggested that Lyt 2⁺ cells could suppress and modulate the Lyt 1⁺ cell proliferation.     

Human macrophage-lymphocyte interaction in proliferation to soluble antigen: II. Characterization of lymphocyte binding to antigen-pulsed macrophage monolayers

Human peripheral blood T lymphocytes from individuals immune to tetanus toxoid (TT) or mumps soluble antigen can be depleted of cells capable of proliferating in response to these antigens by specific adsorption to antigen-pulsed macrophage (Mφ) monolayers. Specific adsorption is manifest by depletion of proliferative activity (measured by tritiated thymidine incorporation) in the fraction of T lymphocytes nonadherent to Mφ monolayers pulsed with the appropriate antigen. The occurrence of T lymphocyte-Mφ binding is supported by experiments which failed to detect cell-free factors or cells capable of inhibiting lymphocyte proliferation in the nonadherent lymphoid population. A quantitative analysis of the immunoadsorption assay suggests that proliferative activity representing at least 90% of that displayed by reciprocal control lymphocytes can be depleted on Mφ monolayers. Experiments employing varying numbers of adsorbing Mφ relative to T lymphocytes have established practical limits which predict the efficiency of adsorption. Kinetic studies indicate that specific adsorption occurs within 1 hr, with maximal levels or binding evident by 4–6 hr; binding remains stable for at least 16 hr. Specific adsorption is temperature dependent, occurring maximally at 37 °C, but not at all at 4 °C. T lymphocyte-Mφ interaction is complex and includes more than a recognition of pulsed antigen alone. This conclusion is supported by the finding that antigen-pulsed Mφ fail to absorb MLC-nonidentical allogeneic lymphocytes and that a heterologous antiserum directed against human Ia-like molecules (p23,30) inhibits specific T cell-Mφ binding.     

The differentiation of T lymphocytes. Density characterisation of thymic and peripheral T cells responding in syngeneic and allogenic mixed lymphocyte reactions.

Equilibrium density separation on continuous albumin gradients was used to separate and characterise the T cells responding by proliferation to both syngeneic and allogeneic stimulating cells in the one-way mixed leucocyte reactions (MLR). In CBA mouse spleen both light and dense T cells were capable of responding in an allogeneic MLR. No T cells responding to stimulation be syngeneic B lymphocytes could be isolated from adult or 7-day CBA mouse spleen. In adult CBA mouse thymus, cells responding to allogeneic stimuli were enriched in the light density region, along with the low theta subpopulation. Self-reactive cells, responding with proliferation when cultured with syngeneic adult CBA splenic lymphocytes, and found in adult and 4-day CBA mouse thymus, were also enriched in the light density zones. However, in adult thymus syngeneic MLR reactivity was also found in the dense zones, and the density distribution profiles of total syngeneic MLR responding cells revealed a series of peaks extending over the whole density range. It was suggested that these syngeneic MLR responders undergo a complete maturation process, including progressive density increases, within the thymus gland. Such a sterile differentiation pathway could be a censorship process, leading to death of self-reactive cells within the thymus.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: IV. Correlation with H-2 haplotypes

In vitro primary syngeneic sensitization on monolayers of thyroid epithelial cells was performed with 21 inbred strains of mice representing 11 original H-2 haplotypes. Significant differences in the proliferative responses, assessed by thymidine uptake, were found to be related to the major histocompatibility complex haplotype. This result was further confirmed using congenic resistant strains of mice. In comparison with the experimental autoimmune thyroiditis induced by syngeneic thyroglobulin and adjuvant, primary syngeneic sensitization on monolayers of thyroid epithelial cells appeared to be under the same genetic control (H-2^k strains being good responders, while H-2^b mice are poor responders).     

Characteristics of cytotoxic thyroglobulin-specific T cell hybridomas

Clones of cytotoxic thyroid-specific T cell hybridomas were generated by fusion of thyroglobulin-primed, "in vitro"-boosted CBA lymph node cells with the AKR-derived lymphoma cell line BW 5147. One hundred and thirty one clones were obtained. Among them, 15 were able to induce the lysis of 51Cr-labeled syngeneic thyroid epithelial cells after 5 h of incubation at 37 degrees C. Two T cell clones, HTC1 and HTC2, were further studied. These clones, which exhibit cell surface characteristics of cytotoxic cells, were specific for only syngeneic thyroid cells (allogeneic thyroid cells or syngeneic epithelial cells were never lysed by these hybridomas). Moreover, by using Ag-pulsed syngeneic macrophages as targets, syngeneic cytotoxicity was shown to be specific for thyroglobulin and not for a nonrelated Ag. The lysis obtained with these autoreactive thyroid-specific T cell clones is restricted to class I major histocompatibility Ag. This property is assessed by both the blocking of syngeneic cytotoxicity toward thyroid epithelial cells or thyroglobulin-pulsed macrophages only by anti-class I mAb and by the detection of specific lysis of target cells exclusively when effector hybrid cells and target thyroid epithelial cells or thyroglobulin-pulsed macrophages shared at least class I major histocompatibility Ag.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Induction of tolerance against the Mls antigen in mice no need for Mls-bearing cells

Infusion of CBA mice with spleen cells from the H-2-compatible, but Mis antigen-incompatible, strain C3H leads to a specific reduction of the MLC reactivity of the host's lymphocytes. One explanation of the reduced reactivity could be that the specifically Mls antigen-responsive CBA T cells become exhausted by intense antigen stimulation or that the infused cells actively neutralize specifically responsive cells. In this investigation, we have shown that depletion of membrane Ig-positive cells from C3H × CBA spleen cell preparations strongly reduces their capacity to stimulate CBA lymphocytes in the MLC, indicating that the Mls antigen is expressed on B but not on T cells. However, such B-cell depleted cell preparations were fully capable of reducing the MLC response of CBA hosts. Cell preparations of spleen and lymph nodes exhibited high stimulatory and inhibitory effects. Thymic cells lacked both these characteristics, whereas bone marrow cells were weak stimulators but relatively strong inhibitors. The results support the proposal that the observed reduction of MLC reactivity is due to an active process of the injected cells. The cell type which is working as an inhibitor has not been clearly defined yet.     

Herpes-simplex-virus-specific,H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2 ^k ) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected target cells expressing the D^d alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2D^k determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for D^d alloantigen could be positively selected on H-2^d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing D^d alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Characterization of the in vitro murine T-cell proliferative responses to murine and human thyroglobulins in thyroiditis-susceptible and -resistant mice

The in vitro proliferative response to autoantigenic mouse thyroglobulin (MTg) of lymph node cells (LNC) from thyroiditis-susceptible (high-responder) CBA/J (H-2k) mice was further characterized. The relatively weak response was enhanced by adding irradiated spleen cells from normal syngeneic mice to cultures of responding LNC. Furthermore, the adjuvant used for immunization was found to influence the magnitude of the response. Results of experiments varying both the adjuvant and the route of immunization (footpad versus subcutaneous) demonstrated that marked proliferative response to MTg in vitro was not necessarily a predictor of the severity of disease. However, the capacity to proliferate in response to MTg correlated with disease susceptibility, as reported previously. The response to MTg was dependent on Thy-1+, Lyt-1+2- cells and was inhibited by monoclonal I-A antibodies. Thus, proliferation is mediated by T cells of the helper/amplifier phenotype recognizing the autoantigen in association with Ia molecules. The determinants on human thyroglobulin (HTg) and MTg stimulating the proliferative responses of LNC from thyroiditis-susceptible and thyroiditis-resistant (low-responder) BALB/c (H-2d) mice were found to differ. Cells from resistant mice proliferated only in response to foreign determinants on HTg and not to shared or mouse-specific epitopes of MTg, whereas susceptible mice had T cells reactive to shared determinants expressed on MTg and HTg as well as to foreign determinants on HTg.     

Thymus-dependence of genetic resistance to (CBA x M523)F1 lymphocytes

The nature of cells responsible for the genetic resistance of lethally irradiated CBA mice to lymphocytes of (CBA x M523)F1 hybrids was studied. Preirradiation of the hosts was shown to abolish the resistance. The latter was generally recovered by syngeneic thymocytes or splenocytes while embryonic liver and bone marrow cells or splenocytes treated with anti-Thy-I serum plus complement before injection into host were ineffective. It is postulated that some cells with T cell characteristics are responsible for the phenomenon of parental resistance. These cells differ in several respects from T cells that mediate the transplantation immunity and from M cells that control other forms of the genetic resistance.     

Anti-immunoglobulin in combination with cytochalasin stimulates proliferation of murine B lymphocytes

The ability of cytochalasin to influence the stimulation of murine B lymphocytes through surface immunoglobulin was assessed during short term cultures. Modest doses of anti-immunoglobulin alone did not stimulate proliferation of mouse spleen cells at 2 days. Cytochalasin B alone also had no effect. However, anti-immunoglobulin in combination with cytochalasin B stimulated substantial proliferation as judged by [3H]thymidine incorporation. Cytochalasins A, E, and D, and dihydrocytochalasin B were all effective in promoting B cell proliferation. Spleen cells from xid-defective (CBA/N X DBA/2)F1 male mice failed to proliferate in response to anti-immunoglobulin plus cytochalasin, suggesting that this treatment affects the same subset of B cells as anti-immunoglobulin plus B cell growth factor. Moreover, proliferation that was stimulated by anti-immunoglobulin plus cytochalasin B was not affected by T cell depletion. Cytochalasin may circumvent the need for, or replace, a second signal for proliferation.     

In situ analysis of T cell subset composition in experimental autoimmune thyroiditis after adoptive transfer of activated spleen cells

T cells from genetically susceptible mice developing experimental autoimmune thyroiditis (EAT) proliferate in response to restimulation with mouse thyroglobulin (MTg) in vitro. The in vitro-activated cells adoptively transfer EAT as well as differentiate into cells cytotoxic for syngeneic thyroid monolayers. To examine the kinetics of T cell subset infiltration and distribution in situ after adoptive transfer, we applied the avidin-biotin-peroxidase labeling technique to thyroid sections, utilizing rat monoclonal antibodies followed by a biotinylated rabbit anti-rat antibody. Female CBA donor mice were immunized with MTg and lipopolysaccharide. Their spleen cells were obtained 7 days later, cultured with MTg, and transferred into recipient mice. The thyroids were removed on Days 7, 10, and 14 after transfer and serially sectioned. The early phase of transferred EAT showed a higher percentage of L3T4+ cells compared to Lyt-2+ cells, yielding a ratio of 2.3 and total T cells of about 35%. By Day 10, both T cell subsets had increased to a total of about 56%. However, the relative increase was greater in the Lyt-2+ subset; the nearly doubled percentage was statistically significant, resulting in a downward shift in the subset ratio to 1.7. Little change in the in situ distribution was seen on Day 14. The percentages of F4/80+ (macrophage) population in lesions examined on Days 10 and 14 were fairly constant and B cell involvement was minimal. These findings illustrate the pathogenic role of both T cell subsets in adoptively transferred EAT and the time-dependent changes in their relative proportions leading to thyroid gland destruction.     

Herpes-Simplex-virus-specific, H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2k) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected taraget cells expressing the Dd alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2Dk determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for Dd alloantigen could be positively selected on H-2d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing Dd alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Morphological Differences between Thymus- and Bone Marrow-Derived Lymphocytes

Simple morphological criteria are described, enabling one to distinguish two types of lymphocytes in normal unstimulated mice: 'Th', predominant in the thymus (90-92%) and 'Bm', predominant in the bone-marrow (85-93%). Their distribution in the peripheral lymphoid organs agreed significantly with the observed average distribution of T and B lymphocytes. Thy-mectomized and irradiated bone-marrow-restored CBA mice, as well as nude mice, showed a striking lack of Th cells. In vitro depletion of θ-bearing or Ig-bearing lymphocytes lead to a correlated depletion of Th or Bm lymphocytes, respectively: blood lymphocytes treated with anti-θ serum + complement showed a decrease in Th lymphocytes corresponding to the percentage of cells killed by the anti-θ serum. The selective retention of Ig-bearing spleen cells on Wigzell's anti-lg columns caused a depletion of Bm lymphocytes. It is concluded that the Th type is the morphological expression of the T lymphocytes, and the Bm type that of the B lymphocytes. The implications of this way of distinguishing T and B lymphocytes by simple morphology, and of the actual relationships between T and B cell differentiation, are discussed.     

Activation of CD4+ lymphocytes by syngeneic brain microvascular smooth muscle cells

Splenocyte proliferation as measured by [3H]thymidine incorporation was detected when brain microvessel smooth muscle cells (SM) were cocultured with syngeneic spleen cells. This report focuses on the role of different lymphocyte populations in this activation. The central role of CD4+ T cells in the proliferation response has been established by different sets of experiments. The phenotypic characterization of splenic lymphocytes before and after the co-culture showed that the only cell type present in higher number after the co-culture than before is the CD4+ T cell. When CD4+ cells were purified by flow microfluorimetry and co-cultured with SM a strong proliferative response was detected. In contrast, purified CD8+ cells in co-culture with SM cells did not proliferate. The activation of CD4+ cells by SM required direct cell-to-cell contact and could be detected on the fourth day, reaching maximal levels at the 6th and 7th days of the co-culture. The activation is more pronounced in the syngeneic system than under allogeneic conditions and is inhibited by anti-MHC II mAb, but not by anti-MHC II mAb. The finding that vascular smooth muscle cells can activate syngeneic T cells may have important implications concerning the mechanism of induction of vasculitis.     

Genetic resistance of CBA and a mice to transplanted lymphoid and hemopoietic cells of CBA.M523 mutants and their F1 hybrids

(CBA × M523)F₁, (A × M523)F₁ and M523 lymphocytes grafted into lethally irradiated CBA or A mice temporarily lose their capacity to respond to test antigens (SRBC, Vi-antigenS. typhi). Immunoresponsiveness of F₁ cells is affected to a lesser degree in lethally irradiated M523 mice. Depression of response is absent in the CBA F₁ combination, in the syngeneic combination and in CBA mice which have received transplanted cells from F₁ hybrids which do not share theM523 mutation. The number of hemopoietic (CBA × M523)F₁ colonies was also reduced in CBA mice. Resistance of CBA mice to lymphoid (CBA × M523)F₁ cells develops 18 days after birth. It can be reduced by additional recipient preirradiation or preinoculation with (CBA × M523)F₁ spleen cells. The abrogated resistance can be partially restored by CBA spleen cells. The activity of (CBA × M523)F₁ lymphocytes passaged through CBA spleen is restored in syngeneic F₁ secondary recipients but inhibited again in the CBA secondary recipients. These results are consistent with the suggestion that resistance of lethally irradiated CBA mice to hemopoietic and lymphoid (CBA × M523)F₁ cells is mediated by immunologically competent, radioresistant recipient cells rapidly reacting to transplantation antigens coded by the mutantH-2K ^ka allele. These cells temporarily suppress the functional activity of transplanted cells but do not eliminate them.     

Transfer of experimental autoimmune thyroiditis with T cell clones

We have investigated three T lymphocyte clones isolated from CBA/CaJ mice primed with mouse thyroid extract (MTE) in adjuvant. All three clones are L3T4+, Ig-, and Lyt2- and proliferate to MTE, mouse thyroglobulin (MTG) and rat thyroid extract. Clones A7 and B7 transfer thyroiditis to irradiated (475 rad) syngeneic mice, but not to normal recipients. The thyroid lesion induced by the B7 clone is characterized by the infiltration of both mononuclear and polymorphonuclear cells. The thyroiditis is transient in that lesions are apparent 7 and 14 days after transfer, but thyroids return to normal by day 21. Clone B7 showed helper activity for trinitrophenyl-keyhole limpet hemocyanin-primed B cells in vitro when stimulated with trinitrophenyl-MTG and also stimulated the production of anti-MTG antibody in recipient mice. Clone A7 induced thyroid lesions characterized by infiltration of the thyroid with mononuclear cells, with virtually no polymorphonuclear cell infiltration. This clone has shown no helper activity following stimulation with trinitrophenyl-MTG. The third clone (D2) proliferates to and shows helper activity to MTG, but fails to transfer thyroiditis to syngeneic, irradiated mice. On continuous culture, clone B7 lost its surface Thy. The loss of Thy appears unrelated to the ability to transfer thyroiditis since subclones of B7 with markedly different percentages of Thy+ cells transferred disease equally well.     

Generation of cytotoxic lymphocytes to syngeneic tumor by using co-stimulator (Interleukin 2)

Cytotoxic lymphocytes (CL) highly active against the syngeneic mastocytoma, P815, were generated from spleen cells of DBA mice cultured with co-stimulator (Interleukin 2) and P815. More CL activity was generated from spleen cells of P815 tumor-bearing mice than from spleen cells of normal mice. Thymus cells from tumor-bearing mice, however, did not produce increased CL activity. Most of the CL were Thy 1 and Ly 1 positive. The optimal culture conditions and kinetics were similar to those for the generation of allogeneic cytotoxic T lymphocytes. The cytotoxic activity against syngeneic P815 was similar in magnitude to the response of DBA spleen cells to allogeneic tumor lines and to the response of allogeneic CBA spleen cells to P815. Although CL generated from tumor-bearing mice did not lyse normal DBA cells, they did lyse, to a much lesser degree, a number of tumor cell lines other than the sensitizing P815. This nonspecific lysis was not H2 restricted nor was it restricted to tumors of lymphoid origin. Generation of nonspecific cytolytic activity was antigen independent, occurring in the presence of co-stimulator alone.