Biological effects of activation of human peripheral blood mononuclear cells by mitogenic oxidizing agents. I Alloantigen-independent activation of alloimmune memory cells

Mechanisms of activation of alloimmune memory cells by immunologically nonspecific signals were investigated utilizing the mitogenic oxidizing agents, neuraminidase and galactose oxidase (NAGO) and sodium periodate (IO₄^?). Direct activation of primary long-term human mixed-lymphocyte culture (MLC) cells (memory cells) with either NAGO or IO₄^? resulted in increased specific secondary cytolytic activity. Kinetics of the proliferative and cytotoxic responses resulting from such treatment of memory cells paralleled those resulting from treatment of memory cells with irradiated cells that were the stimulators in the primary MLC. Indirect activation of memory cells by NAGO or IO₄^?-treated and irradiated syngeneic cells also resulted in increased specific secondary cytolytic activity. In contrast, peripheral blood mononuclear cells (PBM) activated by the mitogenic oxidizing agents did not develop cytolytic activity toward syngeneic or multiple allogeneic target cells, despite extensive proliferative responses. Cytotoxicity generated by either direct or indirect activation of memory cells by IO₄^? was prevented by treatment of the oxidized cells with the reducing agent, sodium borohydride. Incubation of memory cells in supernatants from 24-hr cultures of PBM activated with either NAGO or IO₄^? resulted in proliferation and in an increase in cytolytic activity in memory cells, but not in PBM. These findings indicate that alloimmune memory cells can be activated by immunologically nonspecific lymphocyte-derived signals that do not depend on alloantigen or lectin.     

Studies on the induction and expression of T cell-mediated immunity. XIV. Antigen-nonspecific oxidation-dependent cellular cytotoxicity (ODCC) mediated by sodium periodate oxidation of cytotoxic T lymphocytes

Alloimmune murine thymus-derived cytotoxic lymphocytes (CTL) generated in vivo or in vitro are shown to lyse antigen-nonspecific target cells (tumor cells, Con A, and LPS blasts) following treatment of CTL with an oxidizing agent, sodium periodate (NaIO4). It has been shown that NaIO4 oxidizes terminal sialic acid residues of cell surface macromolecules. The presence of reactive aldehyde groups, generated by NaIO4 modification, is required for the expression of antigen-nonspecific cytotoxicity because treatment of modified cells with a reducing agent such as potassium borohydride (KBH4) resulted in the abrogation of cytotoxicity. However, KBH4 treatment of unmodified or NaIO4-modified CTL has no effect on antigen-specific cytotoxicity. The modification of CTL by NaIO4 is sufficient to lead to the formation of lymphocyte-target cell conjugates and lysis of bound targets. Monoclonal antibodies directed against the Lyt-2 antigens of CTL, but not Lyt-1 antigens, in the absence of complement inhibited the nonspecific cytotoxicity resulting from NaIO4 modification of effector lymphocytes. These findings suggest that the mere interaction with or perturbation of appropriate cell surface molecule(s) of effector lymphocytes such as Lyt antigens by receptor-ligand interaction in SCMC or by NaIO4 modification in ODCC may lead to the expression of cytotoxicity. The present studies demonstrate a functional role of surface carbohydrates on CTL in cell-to-cell recognition and interactions. Furthermore, the results suggest that target cell modification is not a requisite for recognition and lysis in an antigen-nonspecific cytotoxic system such as ODCC. However, partial blocking of ODCC by alloantibodies directed against the H-2 of unmodified target cells suggests that NaIO4-modified CTL recognize unrelated target H-2 antigens. The implication of these findings on the molecular mechanism of cell-mediated cytotoxicity is discussed.     

Induction of lymphocyte cytotoxicity by modification of the effector or target cells with periodate or with neuraminidase and galactose oxidase.

Treatment of mouse spleen cells with periodate (NaIO4) or with neuraminidase and galactose oxidase (NAGO) induces blastogenesis and renders the cells cytotoxic to mastocytoma (P815) target cells. Treatment of target cells (P815 cells and turkey erythrocytes) with NaIO4 or with NAGO renders them susceptible to cytolysis by untreated mouse spleen cells. The cytotoxicity induced by NaIO4 is reduced upon reacting the NaIO4-treated, effector or target cells with borohydride or hydroxylamine. Thus the formation of free surface aldehydes on either the effector or target cell induced a cytotoxic effect. It is postulated that cross-linkage via a Schiff base between effector and target cell initiates the cytotoxic effect. Cytotoxicity induced by NaIO4 or NAGO is immunologically nonspecific and is independent of major antigenic differences between effector and target cells. Phagocytic cells are not involved in NaIO4-or NAGO-induced cytotoxicity toward P815 target cells.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

Syngeneic mixed lymphocyte reaction in mice: strain distribution, kinetics, participating cells, and absence in NZB mice.

Co-culture of mouse spleen nonadherent (T-enriched cells with mitomycin C-treated unfractionated syngeneic spleen cells resulted in increased DNA synthesis in the responding T cells. The kinetics of this syngeneic mixed lymphocyte reaction (SMLR) showed that peak DNA synthesis occurred on day 5 of culture compared to day 4 for conventional mixed lymphocyte reaction (MLR). Anti-T cell antiserum plus complement treatment of the responding cell population abolished the reaction, and similar treatment of the stimulator population enhanced SMLR. These studies indicate that SMLR represents the response of T cells to non-T cells. Studies on the generation of cytotoxic T lymphocytes (CTL) in parallel cultures of T cells activated by syngeneic or allogeneic spleen cells showed no cytotoxicity of SMLR-activated cells for either PHA- or LPS-induced blasts but did show a good CTL response of allo-activated cells to both targets. Studies on the strain distribution of SMLR revealed that NZB mice manifested poor or no stimulation in SMLR whereas all other strains tested exhibited strong SMLR. This defect in NZB mice may be pathogenetically related to the autoimmune disease that develops in these mice.     

Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis.

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.     

Suppression of alloimmune cytotoxic T lymphocyte (CTL) generation by depletion of NK cells and restoration by interferon and/or interleukin 2

By using rabbit antiserum to a glycolipid, ganglio-n-tetraosylceramide (ASGM1), the accessory effect of natural killer (NK) cells on the generation of alloimmune CTL in mice was investigated. When normal C3H/He mice were immunized with C57BL/6 or BALB/c spleen cells, they generated alloimmune CTL with a surface marker phenotype of Thy-1+ Lyt-1-2+ ASGM1-, preceded by early augmentation of cytotoxic activity of NK cells with a Thy-1-Lyt-1-2-ASGM1+ phenotype. Administration of anti-ASGM1 (10 microliters) in mice resulted in a complete depletion of NK activity and ASGM1+ cells in the spleen even 1 day after injection, but no changes in the proportions of T (Thy-1+) cells and their Lyt-1 and Lyt-2 subsets as revealed by an immunofluorescence analyzer (FACS) and phagocytic cells. When these anti-ASGM1-treated mice were immunized with allogeneic cells, they showed neither augmented NK activity nor generation of alloimmune CTL, and spleen cells isolated from these anti-ASGM1-treated mice produced no CTL response to alloimmunization in vitro. Normal spleen cells treated with the antiserum and complement in vitro also showed a complete NK depletion without any deterioration of T cells and their Lyt-1 and Lyt-2 subsets, and when stimulated with allogeneic cells they generated no CTL. Spleen NK (ASGM1+) cells were purified by Percoll-gradient centrifugations followed by complement-dependent killing of T cells with the use of anti-Thy-1 monoclonal antibody, and were further purified by panning methods with anti-ASGM1, giving a preparation consisting of greater than 90% ASGM1+, Ly-5+ cells, and less than 0.5% of Thy-1+, Lyt-1+, and Lyt-2+ cells. These purified ASGM1+ Thy-1- cells alone generated no alloimmune CTL in response to alloantigens, suggesting that ASGM1+ NK cells contained no precursors of alloimmune CTL. When added into NK-depleted spleen cells, they restored the normal alloimmune CTL response of the spleen cells, indicating that ASGM1+ fractions contained cells to provide an accessory function for CTL generation. Lyt-1+ cells purified by panning methods did not restore the CTL response of NK-depleted spleen cells. These results indicate that ASGM1+ NK cells, but not Lyt-1+ helper T cells contaminating ASGM1+ fractions at undetectable levels, are responsible for the accessory function. When these purified ASGM1+ Thy-1- cells were stimulated with allogeneic cells, they produced IL 2 and IFN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of natural killer activity in sponge matrix allografts

NK cell activity, defined by the ability of infiltrating host cells to lyse the YAC-1 tumor target, can be detected in sponge matrix allografts across all genetic barriers tested. Nonspecific tumor cell killing cannot be detected either within bulk populations of host-infiltrating cells or in populations enriched for non-adherent lymphocytes. NK activity is also detected in cells infiltrating a syngeneic sponge matrix graft although to a much lesser extent than an allogeneic graft. NK cell functional activity at the graft site precedes the appearance of alloimmune CTL by several days. The surface phenotype of the NK cell is Thy-1.2+ and L3T4- as determined by depleting the various subpopulations with antibody and C. Systemic treatment of sponge-bearing animals with repeated injections of anti-asialo GM1 (AGM1) results in inhibition of both NK activity and CTL activity recovered from the graft on days 5 to 9 after grafting, but on days 11 to 13 after grafting both NK activity and CTL activity are found within the sponge graft. Treatment of sponge-associated cells with anti-AGM1 in vitro or intrasponge injection of anti-AGM1 at various times after grafting eliminates NK activity more readily than alloimmune CTL activity. The intimate association observed between NK cells and alloimmune CTL at the graft site prompts further investigation into the role of NK cells in the allograft response.     

The role of suppressor T cells in BCNU-mediated rejection of a syngeneic tumor

The present investigation was initiated to determine the mechanism by which 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) treatment of tumor-bearing mice results in a high percentage of surviving mice which are resistant to subsequent homologous tumor challenge. Spleen cells from C57BL/6 mice bearing the syngeneic LSA ascites tumor failed to demonstrate significant tumor-specific cytotoxic T lymphocyte (CTL) activity when stimulated in vitro with irradiated tumor cells. This lack of CTL activity correlated with the presence and high activity of two types of CTL-regulatory suppressor T cells (Ts), tumor-specific Thy-1+, Lyt-1-2+ and tumor-nonspecific Thy-1+, Lyt-1+2+ cells, as demonstrated by a double-positive selection technique. In contrast, spleen cells from BCNU-treated tumor-bearing mice generated high tumor-specific CTL activity when stimulated in vitro with irradiated tumor cells. This CTL activity correlated with the lack of demonstrable tumor-specific Ts and greatly diminished tumor-nonspecific Ts activity. The tumor-specific helper activity of Thy-1+, Lyt-1+,2- cells was found to be similar in both BCNU-treated and untreated tumor-bearing mice. BCNU-treated mice that survived a primary LSA tumor challenge (referred to as BCNU-cured mice) resisted subsequent challenge with the homologous (LSA) but not with a heterologous syngeneic tumor (EL-4). However, rejection of a secondary challenge with LSA tumor by BCNU-cured mice was inhibited by adoptive transfer of spleen cells from either normal mice or mice bearing LSA tumors. Furthermore, LSA tumor cells that failed to evoke tumor-specific CTL activity in normal mice could induce high CTL activity in BCNU-cured mice. The present study suggests that, in addition to its direct tumoricidal activity, BCNU inhibits the induction of tumor-specific Ts, thereby explaining why a high percentage of mice survive a primary syngeneic tumor challenge after treatment with BCNU, and also resist subsequent rechallenge with the homologous tumor.     

Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. II. Development of syngeneic cytotoxicity in the absence of specific antigenic stimulation

Two out of four long-term murine allospecific cytotoxic T lymphocyte (CTL) clones tested could develop high levels of cytotoxicity against syngeneic target cells when cultured under appropriate conditions. All CTL clones maintained strict allospecificity so long as they were cultured with both appropriate allogeneic stimulator cells and growth factor (supernatant from secondary mixed lymphocyte cultures). In two of the clones, syngeneic reactivity rapidly developed when the allogeneic stimulator cells were replaced with syngeneic or third party stimulator cells, and when the supernatant from EL4 thymoma cells stimulated with phorbol ester was used as growth factor. In addition to killing the appropriate allogeneic target, clones with syngeneic reactivity could kill both syngeneic C57BL/6 targets and H-2-congenic BALB.B targets but not third party unrelated targets, suggesting that the self structure recognized was coded for within the major histocompatibility complex. Such clones did not kill the natural killer (NK) target YAC. The results obtained from cold target inhibition and from subcloning at limiting dilution of clones with syngeneic reactivity suggested that both allogeneic and syngeneic reactivity could be expressed by the same individual cell in the CTL clone. The specificity for syngeneic H-2 as opposed to third party H-2 and NK-sensitive target cells, and the observation that both allospecific and syngeneic killing could be partially blocked by anti-Lyt-2 antibody treatment of the CTL, strongly suggested that different recognition structures are involved in CTL-mediated syngeneic cytotoxicity and NK cytotoxicity.     

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.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. VI. Migration and activity in vivo in acute and persistent infection

Cloned cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were adoptively transferred to syngeneic mice acutely or persistently (carrier mice) infected with LCMV. Although infectious virus was cleared from the spleens during acute LCMV infection begun 24 hr earlier and the spleens remained clear of virus for the 4 days of testing, there was no concomitant reduction of viral titers in lymph nodes. In contrast, adoptive transfer of cloned CTL into animals with persistent rather than acute LCMV infection resulted in deaths of syngeneic but not allogeneic recipients. LCMV-immune spleen cells taken 30 to 50 days after a primary immunization and activated by in vitro stimulation before transfer also caused death of syngeneic carrier mice. However, LCMV-immune spleen cell per se provoked no clinical manifestations when transferred but cleared infectious virus and viral nucleic acid sequences from syngeneic carrier mice. The migration of 51Cr-labeled, LCMV-specific, H-2-restricted cloned CTL was assessed in vivo. The circulation of these CTL clearly differed from that of spleen cells freshly isolated from uninfected mice and from non-LCMV-specific CTL clone. Further, the circulatory pattern of LCMV-specific, H-2-restricted, cloned CTL in carrier mice was markedly different than in uninfected animals; only 7% of the injected cells remained in the lungs of uninfected mice 8 hr after injection, whereas 30% had accumulated in the liver. However, 55% of the cells injected into carrier mice still remained in their lungs 8 to 16 hr later. Hence, LCMV-specific, H-2-restricted, cloned CTL have unique trafficking patterns in the presence of LCMV antigens and immune activities in vivo.     

Alloimmune response results in expansion of autoreactive donor CD4+ T cells in transplants that can mediate chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is considered an autoimmune-like disease mediated by donor CD4(+) T cells, but the origin of the autoreactive T cells is still controversial. In this article, we report that the transplantation of DBA/2 donor spleen cells into thymectomized MHC-matched allogeneic BALB/c recipients induced autoimmune-like cGVHD, although not in control syngeneic DBA/2 recipients. The donor-type CD4(+) T cells from the former but not the latter recipients induced autoimmune-like manifestations in secondary allogeneic BALB/c as well as syngeneic DBA/2 recipients. Transfer of donor-type CD4(+) T cells from secondary DBA/2 recipients with disease into syngeneic donor-type or allogeneic host-type tertiary recipients propagated autoimmune-like manifestations in both. Furthermore, TCR spectratyping revealed that the clonal expansion of the autoreactive CD4(+) T cells in cGVHD recipients was initiated by an alloimmune response. Finally, hybridoma CD4(+) T clones derived from DBA/2 recipients with disease proliferated similarly in response to stimulation by syngeneic donor-type or allogeneic host-type dendritic cells. These results demonstrate that the autoimmune-like manifestations in cGVHD can be mediated by a population of donor CD4(+) T cells in transplants that simultaneously recognize Ags presented by both donor and host APCs.     

Helper cells in the autologous mixed lymphocyte reaction. I. Generation of cytotoxic T cells recognizing modified self H-2

The autologous mixed lymphocyte reaction (AMLR) in mice measures the proliferative response of T cells to determinants on syngeneic non-T spleen cells. Normally, cytotoxic T lymphocytes (CTL) are not generated in this reaction. However, the addition of trinitrophenyl-modified mitomycin C-treated syngeneic T cells (TNP-Tm) to the AMLR results in the generation of TNP-specific CTL but does not alter the proliferative response. Significant cytotoxic activity is not detectable against TNP in association with Ia unless TNP is present on cells bearing those determinants. Thus, if unselected spleen cells are TNP-modified and used as stimulators in the AMLR, the proliferative response is enhanced and CTL are generated that recognize TNP in association with K, D, and I region-encoded determinants. The CTL generated in the AMLR are H-2 restricted and dependent on the presence of adherent cells in the sensitization cultures. The experiments presented here suggest that the AMLR can provide the help necessary for generating cytotoxic T cells in vitro.     

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.     

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.     

Induction of cytotoxic T lymphocytes against herpes simplex virus type i: role of accessory cells and amplifying factor

Spleen cells from mice primed with herpes simplex virus type 1 (HSV-1) could be induced to differentiate into cytotoxic T lymphocytes (CTL) by in vitro culture with infectious HSV-1 but not by heat-inactivated virus. Induction of CTL failed to occur if the spleen cells were depleted of adherent cells by passage over columns of nylon wool before culture with virus. The CTL response could be restored by adding normal syngeneic peritoneal cells (PC) or L cell fibroblasts but not by allogeneic PC or BALB/c 3T3 fibroblasts. Thus, the induction of HSV-1-specific CTL was H-2 restricted. The response of HSV-1-stimulated nylon wool-depleted spleen cells could also be restored by adding amplifying factor (AF) produced in 24 hr mixed lymphocyte cultures. The addition of AF to nondepleted spleen cells also permitted the generation of CTL with heat-inactivated HSV-1 as a viral stimulant. Our results indicated that induction of a HSV-1 CTL response requires two signals, one provided by virus and a second, presumably nonspecific, by helper T cells. It was suggested that only the helper cells require H-2 restriction and need to be presented virus in the context of a macrophage.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.