I-region restriction of alloantigen recognition: alloantigen expressed on the surface of a hybridoma cell must be presented in the context of self class II major histocompatibility complex determinants for recognition by a dual-reactive T-cell clone

A helper-T-lymphocyte clone, designated A10, proliferated in response to both hen egg ovalbumin (OVA) presented in the context of self I-Ak and to the alloantigen I-As. The alloantigen source could be provided by irradiated H-2s spleen cells and also by paraformaldehyde-fixed H-2s spleen cells. However, for fixed allogeneic spleen cells to stimulate proliferation of the cloned cells, it was necessary to add irradiated syngeneic I-Ak-bearing spleen cells, as fixed H-2s spleen cells added, by themselves, to A10 cells were nonstimulatory. We have extended these findings by generating a monoclonal hybridoma cell which expressed the I-As allodeterminant. Similar to our results with fixed allogeneic spleen cells, this source of alloantigen could stimulate A10 cells to proliferate only if irradiated syngeneic spleen cells were added to the cultures. These proliferative responses were effectively inhibited by anti-I-Ak monoclonal antibody (mAb) and by anti-I-As mAb. Furthermore, the response of A10 cells to the alloantigen-bearing hybridoma cells were also inhibited by the anti-L3T4 mAb GK1.5. Collectively, these data indicate that, in some situations, alloreactivity may be mediated by self class II major histocompatibility complex restriction of alloantigen-driven proliferation.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Generation of cytolytic T lymphocytes in vitro. X. Induction of primary and secondary CTL responses by the mitogen sodium periodate.

Short-term (15 min) sodium periodate (NaIO4) treatment of mouse spleen cells previously primed in vivo or in vitro against alloantigens induced the formation of secondary (2 degree) cytolytic T lymphocytes (CTL) specific for the priming antigens. CTL formation was readily demonstrable within 24 hr after treatment.This early CTL response occurred equally well in the presence or absence of cytosine arabinoside (Ara C), indicating that NaIO4 could induce CTL independently of DNA synthesis. Forty-eight hours after periodate treatment, the lytic activity was similar to that observed in parallel cultures stimulated with irradiated allogeneic spleen cells, although the peak activity was reached earlier (day 4) and was somewhat lower than that induced by alloantigen. The addition of irradiated NaIO4-treated unprimed syngeneic spleen cells to cultures of untreated alloimmune spleen cells also led to CTL formation, which suggests an indirect mechanism of activation. In contrast to alloimmune spleen cells, normal spleen cells treated with NaIO4 developed only very low levels of cytotoxicity after 4 days of incubation. However, in the presence of PHA, such cells were capable of lysing syngeneic and allogeneic target cells.     

The induction of cytolytic T lymphocytes with syngeneic trinitrophenyl-coupled membranes.

Evidence is presented that trinitrophenyl-coupled tumor membranes are able to induce cytolytic T lymphocytes (CTL) when co-cultured with syngeneic spleen cells. These haptenated membranes stimulate spleen cells from naive and immune mice. The specificity of these CTL is determined by the H-2 antigens of the membranes used for stimulation.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.     

Isogeneic lymphocyte interactions (ILI) in CBA/N mice: Presence of ILI-Type 2 and absence of ILI-Type 1

The ability of spleen cells from immune defective CBA/N mice to stimulate two types of isogeneic lymphocyte interactions (ILI) was studied. In normal mice adult splenic cells induce proliferation of syngeneic neonatal thymocytes in Type 1 ILI and of syngeneic adult lymph node cells in Type 2 ILI. We have shown that CBA/N spleen cells are inoperative in ILI-Type 1 because the stimulating antigen, murine differentiation antigen 1 (MDA-1), is absent. Murine differentiation antigen 2 (MDA-2), however, is present and Type 2 ILI is evoked by CBA/N spleen cells. The results suggest that MDA-1 and MDA-2 are present on different subsets of B cells. In previous studies in other mice we have shown MDA-1 to be a strong contender for the role of receptor for T-cell signals. Our present findings lend further support to that hypothesis.     

Kinetics of CTL induction by concanavalin A.

Induction of maximal CTL activity was achieved within 12 hr of exposure to Con A in vitro in various mouse lymphoid cell populations. These included spleen cells from normal unsensitized mice, spleen cells from mice previously immunized with alloantigen, and mouse spleen cells exposed to alloantigen in long-term mixed leukocyte culture (LTMLC). Although induction of maximal incorporation of tritiated thymidine was accomplished within this same period in the cells obtained from LTMLC, a much longer period of Con A exposure (greater than 24 hr) was required for freshly prepared spleen cells from normal or previously immunized mice. These findings indicate that the increased tritiated thymidine uptake induced in freshly prepared spleen cells on continued exposure to Con A beyond 12 hr is not associated with the development of cytolytic activity, and that it probably represents stimulation of subpopulations no longer present in the LTMLC population where positive selection for cells responsive to cellular alloantigens has taken place.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

The immune response to a chemically induced fibrosarcoma

Summary A clone of C3H10T 1/2 fibroblasts transformed in vitro with the carcinogen 3-methylcholanthrene readily produced tumors when as few as 10³ cells were injected into immunocompetent adult syngeneic mice. A non-transformed clone of the same parentage did not produce tumors. Because the cell-mediated immune response has an important role in inhibiting the growth of tumors, we have compared the ability of both these transformed and non-transformed fibroblasts to stimulate and to act as targets in cell-mediated cytotoxicity (CMC) assays. This model is unique in that studies of the immune response to tumors rarely have or utilize appropriate normal controls. When both types of irradiated fibroblasts were used as stimulators in vitro, neither syngeneic nor allogeneic effector spleen cells capable of efficiently lysing the tumor fibroblasts were generated. In contrast, the normal fibroblasts could both stimulate and be lysed by allogeneic cytolytic T cells (CTL). However, the tumor fibroblasts could be lysed by allogeneic effector spleen cells that had been sensitized to C3H/He spleen cells. These results suggest that the expression of alloantigenic determinants necessary for stimulating a CMC response may vary substantially among normal cell types. They further indicate that the tumor cells are not resistant to lysis by appropriately stimulated effector cells. Thus, they must express antigenic determinants necessary for immune lysis and they do not inhibit the functional expression of cytolytic cells once generated. Consequently, tumor growth in vivo may be dependent, in part, upon a failure of the syngeneic host's immunocompetent cells to respond appropriately to the tumor cells. Additional data are provided which suggest that this failure is attributable in large part to immunosuppressive properties of the tumor cells.     

Ia-specific mixed leukocyte reactive T cell hybridomas: analysis of their specificity by using purified class II MHC molecules in synthetic membrane system

This study was undertaken to determine the nature of the antigens recognized in allogeneic and syngeneic mixed leukocyte reactions (MLR). Specifically, we wished to determine whether Ia antigens alone were recognized by MLR-reactive T cells, or whether the specificity was determined by the corecognition of non-MHC antigens together with syngeneic or allogeneic Ia. To do this we used 11 T cell hybrids that were characterized as being specific for Iad and were tested their capacity to respond to isolated I-Ad or I-Ed that had been incorporated into liposomes and had bound to the surface of glass beads. Of nine alloreactive T cell hybrids (five I-Ad-and four I-Ed-specific), seven were shown to be responsive to the relevant isolated Ia antigen on glass beads. Also, two of two syngeneic I-Ad-specific T cell hybrids responded to I-Ad on the glass beads. One of the two alloreactive T cell hybrids that failed to respond to the relevant Ia antigen on glass beads was shown to be specific for an antigen in fetal calf serum (FCS) that was recognized in the context of the allo-Ia antigen (I-Ed), because when intact accessory cells were used, a response by this hybrid was only observed when FCS was present in the assay culture medium or when the accessory cells were pre-pulsed with FCS. The possible involvement of FCS antigens and non-Ia accessory cell antigens in the stimulation of the nine T cell hybrids that responded to isolated Ia on glass beads was evaluated. T cell hybrids that were grown and were tested in serum free medium were still capable of reacting to Ia on beads. The isolated Ia preparations used were greater than 90% pure, and their capacity to stimulate the T cell hybrids did not correlate with the degree of contamination with non-Ia proteins. We conclude from these studies that the majority of T cells that respond to allogeneic or syngeneic Ia bearing stimulator cells are specific for the Ia antigens themselves, and do not require the co-recognition of other non-Ia antigens; nor is there any requirement for Ia antigen processing for this recognition.     

Characterization of Effector Cells against B16 Melanoma in Mice Inoculated with Allogeneic Spleen Cells

Inbred C57BL/6 (B6) mice which had received an inoculation of allogeneic spleen cells showed remarkable antitumor activity against syngeneic tumor challenge with B16 melanoma cells 3 days after the allogeneic cell inoculation. This antitumor activity was not specific to the inoculated alloantigen, since the challenging B16 cells are syngeneic to B6 mice and since it was induced by BALB/c spleen cells as well as C3H/He spleen cells. The antitumor activity was sensitive to an in vivo treatment with anti-asialo GM1 (AGM1) antiserum or anti-Thy.1 monoclonal antibody (mAb) just before the tumor challenge and was resistant to an in vivo treatment with anti-CD8 (Ly. 2) mAb. These results suggest that AGM1+Thy.1+CD8– activated natural killer (NK) cells were generated by alloantigen inoculation and took an important part in the antitumor effect of the alloantigen inoculation.     

Lung tumor-reactive cytotoxic lymphocytes generated in mixed lymphocyte reaction between C3HfeB/HeN (H-2kb) and C3H/HeN (H-2k) strain mice.

The tumor-associated transplantation antigen expressed by several transplacentally induced lung tumors of C3HfeB/HeN mice (H-2kb haplotype) has previously been shown to exist as a normal tissue alloantigen in mice of known H-2k and H-2a haplotypes. This antigen is not expressed in normal tissues of C3HfeB/HeN mice but is expressed in C3H/HeN mice, the strain from which the C3HfeB/HeN mice were originally derived. The present study indicates that spleen cells from C3HfeB/HeN and C3H/HeN mice respond reciprocally in the mixed lymphocyte reaction. Cytotoxic T lymphocytes specific for the tumor-associated alloantigen can be readily generated in mixed lymphocyte reactions in which spleen cells from C3HfeB/HeN mice are reacted with x-irradiated spleen cells from C3H/HeN or A strain mice. These cells are effective in suppressing the growth of the C3HfeB/HeN-derived lung tumor 85 in x-irradiated syngeneic recipients.     

Dissociated and reconstituted subcellular alloantigen capable of stimulating mouse cytotoxic T lymphocytes in vitro.

Reconstituted subcellular membrane fragments were prepared from sonicated murine lymphoid tissue membrane fragments by detergent solubilization and dialysis. The reconstituted subcellular antigen was compared with sonicated cell membranes and x-irradiated cells for the ability to stimulate the formation of allogeneic cytotoxic thymus-dependent lymphocytes in vitro. The results showed that the reconstituted subcellular antigen had properties similar to those reported for the sonicated antigen. Both types of antigen could stimulate immune spleen cells but not normal spleen cells. Furthermore, soluble H-2 antigens, even of membrane origin, were incapable of the stimulation unless reconstituted into membrane fragments large enough to be centrifuged at 48,000 X G for 30 min.     

Induction of antitumor activities in spleen cells from mice and rats activated with lipopolysaccharide immobilized on beads

Summary Lipopolysaccharides (LPS) were coupled to polystyrene beads in order to apply the LPS without toxicity. The antitumor activity of the LPS-immobilizing beads was studied in experiments in vitro and in vivo. In vitro studies showed that spleen cells from C3H/HeN mice stimulated by beads immobilizing LPS fromEscherichia coli produced cytolytic activity as strong as that of lymphokine-activated killer (LAK) cells. Spleen cells from Sprague-Dawley rats stimulated by beads immobilizing LPS fromSalmonella minnesota produced cytolytic activity stronger than that of LAK cells. However, spleen cells stimulated by beads immobilizing each component of the LPS separately could not induce cytolysis. Contact stimulation, even for a brief period, sufficed for cytolytic activity, and was enhanced by culture for 48–72 h. Through in vivo studies, the suppression of tumor growth and a prolongation of the survival time were observed in tumorbearing mice injected with spleen cells activated by beads immobilizing LPS fromE.coli, and in mice injected with LAK cells. The effect of the activated spleen cells was stronger than that of the LAK cells. In rats bearing metastatic tumors, spleen cells activated by beads immobilizing LPS fromS.minnesota suppressed lung metastases more strongly than did LAK cells. These findings indicate that LPS immobilized by beads induced killer cells more strongly than interleukin-2. Ex vivo immunomodulation with LPS-immobilizing beads can be applied usefully as an anticancer treatment.     

BCG-induced suppressor cells. I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro.

Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.     

Studies on the immune response to fixed antigens. III. Induction of helper function for antibody-dependent cellular cytotoxicity responses

Heavy trinitrophenylated sheep red cells (TNP128SRC) and glutaraldehyde-treated SRC (G-SRC) could not induce cellular cytotoxicity against 51Cr-SRC. In contrast, the native antigen SRC could stimulate a cytolytic response against the radiolabeled homologous target cell. However, fixed SRC could stimulate a priming function that accelerated and augmented the secondary cytotoxic response to SRC. Such fixed antigens could stimulate a delayed-type hypersensitivity (DTHS) response also. Thus, the immunologic memory to the chemically modified antigen, as well as the DTHS response, are completely dissociated from the primary cytotoxic responses. The primary and the secondary cytotoxic responses that were developed in the spleens of the injected mice were mediated by antibody-dependent cellular cytotoxicity (ADCC), since the active supernatant that was released from the spleen cells could lyse the target cells in the presence of normal splenocytes. The active supernatant was identified as antibody. We suggest that B effector cells cytolyzed the antibody-coated target cells. Normal cells from nude mice could mediate the cytolytic process as efficiently as spleen cells from other strains of mouse. The results are discussed in terms of selective stimulation of T cell subpopulations.     

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.     

Histamine production by alloantigen-activated mouse bone marrow cells

Histamine's contribution to the manifestations associated with graft-versus-host disease (GVHD) and/or hybrid resistance is unknown. Thus, we initiated studies to see whether or not mouse bone marrow cells could produce histamine upon alloantigen stimulation. Irradiated allogeneic spleen cells were shown to stimulate bone marrow cells to produce and secrete high levels of histamine. During 7 days of culture there was only a marginal increase in cell-associated histamine while the amount of histamine in the supernatant increased 10- to 20-fold. Optimal histamine production was dependent upon Lyt 1+2+ T cells resident in the bone marrow. Further, bone marrow cells from Nude mice failed to produce high levels of histamine following alloantigen stimulation. Soluble factors produced by alloantigen-stimulated bone marrow cells or by Con A-stimulated rat spleen cells induced high levels of histamine production in bone marrow cells in the absence of alloantigen. We suggest that histamine production by alloantigen-activated bone marrow cells may modulate immune functions following bone marrow transplantation.     

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells. II. Effect of a soluble "costimulator" factor(s) in primary and secondary mixed leukocyte culture

The ability of a concanavalin A-stimulated spleen cell supernatant (“costimulator”) to overcome the effects of impaired CD and LD antigen presentation by metabolically inactivated stimulator spleen cells was examined in the primary and secondary cytolytic T lymphocyte (T_c) response. (i) Cells inactivated by ultraviolet irradiation or mild glutaraldehyde treatment, which were unable to stimulate primary cytolytic activity on their own, generated near maximal responses in the presence of costimulator. The 30-fold lower efficiency of splenic membrane fragments as antigen in primary MLC with the supernatant indicated that the damage to immunogenicity caused by membrane isolation was not equivalent to that caused by uv light and glutaraldehyde, as has previously been assumed. (ii) Comparison of the relative effects of antigen and costimulator demonstrated that costimulator played the dominant regulatory role in primary MLC, increasing sensitivity to suboptimal antigen doses 10- to 30-fold; neither antigen nor the supernatant appeared preferentially to control the strength of the secondary response. (iii) Metabolically inactivated adult and untreated neonatal spleen cells failed to release costimulator activity in response to concanavalin A. However, the ability of the neonatal cells to induce a primary cytolytic response suggested that costimulator production by the stimulator cells themselves is not essential for primary T_c activation, and supports the hypothesis that the lack of primary immunogenicity of inactivated spleen cells reflects their failure to induce costimulator production by the responder population.