The allogeneic effect: M-locus differences substitute for differences in the H-2 major histocompatibility complex.

The primary immune response against sheep red blood cells in T cell-deficient spleen cell cultures from nude mice was tested in the absence and presence of allogeneic spleen cells. The allogeneic spleen cells differed either in regard to the major histocompatibility complex (H-2) or only with respect to the M-locus. Surprisingly the M-locus different spleen cells were almost as efficient in enhancing the anti-sheep red blood cell response in nude cultures as were the cells differing on the complete H-2 complex. Evidence is presented that AKR anti-theta serum sensitive T cells are responsible for the M-locus-dependent effect edscribed. This effect is shown to be mediated by a factor released from actived T cells stimulated in M-locus different mixed lymphocyte cultures. Since almost identical parameters have been observed in both the M-locus-dependent situation as in the "classical" allogeneic situation we concluded that an allogeneic effect can be induced by T cells responding to a complete set of the major histocompatibility complex (H-2) as well as to lymphocyte-activating determinants (M-locus) alone.     

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.     

Further evaluation of the pregnancy-linked down-regulation of the paternal antigen-specific splenic cytotoxic T lymphocyte activity in allogeneically pregnant mice.

Cytotoxic T lymphocyte (CTL) activity directed against paternal alloantigen was examined in allogeneically pregnant mice using various allogeneic combinations. The spleen cells from pregnant C57BL/6 (H-2b) mice mated with BALB/c (H-2d) male mice generated less anti-H-2d CTL after in vitro sensitization than those from unpregnant or syngeneically mated C57BL/6 mice. Different allogeneic combinations including the incompatibility at only D region of H-2 or minor histocompatibility loci were effective for downregulating the anti-paternal CTL activity in pregnancy. The downregulation of anti-paternal CTL activity induced by allogeneic pregnancy occurred at day 10 to day 18 of pregnancy, most extensively at day 14. The allogeneic pregnancy also downregulated the allogeneic CTL activities that had been amplified by injecting alloantigens before mating.     

Cell-mediated inhibition of proliferation and activation of alloreactive cytotoxic lymphocytes: maintenance of response potential of precursors and dissociation between proliferation and effector function of activated cytotoxic lymphocytes

Adherent layers of macrophages (M phi-c) generated in vitro from splenic precursors inhibit lymphoproliferative responses to mitogen and to alloantigen without inhibiting the production of interleukin-2 (IL-2). Analysis of spleen cells stimulated for 48 hr in the presence of M phi-c indicated that both blastogenesis (increased cell mass) and expression of IL-2 receptors (7D4 determinants) were reduced. Analysis of BrdU incorporation (frequency of S-phase cells) and total cellular DNA revealed that the M phi-c inhibited the progression from G1 to S phase of cell cycle. The M phi-c not only inhibited the proliferative response to alloantigen but also prevented the generation of alloreactive cytotoxic T cells. The M phi-c were shown not to inhibit CTL responses by eliminating the stimulators or by inactivating precursors or inducing suppressors. The M phi-c were affecting the induction of CTL activity since the M phi-c did not affect the expression of cytolytic activity by activated CTL. The M phi-c did inhibit the proliferation of the activated CTL, suggesting that although cytolytic activity can be expressed in G1 phase of cell cycle, the activation of cytolytic activity in CTL-P may require a G1 to S phase transition. The cells recovered from 5-day MLC incubated in the presence of M phi-c were fully capable of generating a subsequent CTL response. This is in contrast to cells recovered from unstimulated cultures (no M phi-c) which have lost the ability to generate CTL responses. The M phi-c therefore prevent the generation of CTL responses in a totally reversible fashion, so as to allow activation and proliferation of CTL-P which have been removed from the influence of the M phi-c. These observations are discussed in the context of the currently hypothesized role of tissue macrophages in microenvironmental regulation.     

The requirements for antigen multivalency in class I antigen recognition and triggering of primed precursor cytolytic T lymphocytes

In vitro generation of a secondary cytolytic T lymphocyte (CTL) response to Class I alloantigen requires two signals: recognition of the Class I antigen by precursor CTL (Signal 1), and subsequent interaction with lymphokine(s) (Signal 2). Previous work using subcellular antigen stimulation has demonstrated that the required lymphokine(s) is produced as a result of adherent cell uptake, processing, and Ia-restricted presentation of alloantigen to helper T cells. This pathway could be bypassed by addition to the cultures of supernatant from Con A-stimulated rat spleen cells. When an optimal level of lymphokine(s) is provided by addition of Con A supernatant, the magnitude of the CTL response obtained is dependent on the effectiveness of alloantigen recognition and triggering of the primed precursor CTL (pCTL). By using this approach, we examined the cellular and molecular requirements for generation of Signal 1. Previous results had indicated that pCTL were able to directly recognize subcellular antigen, and that cellular presentation of the antigen to pCTL was not required. Further evidence for this was provided by the finding that pulsing of the responder population for short times with liposomes containing purified H-2Kk resulted in effective stimulation of the response. Exposure of cells to antigen for 1 to 2 hr at 4 degrees C generated responses of comparable magnitude to those obtained when antigen was continuously present in the cultures. Experiments were also done to directly examine the ability of alloantigen-pulsed splenic adherent cells (SAC) to deliver Signal 1. Although the antigen-pulsed SAC were very effective in presenting to helper T cells to result in factor production, they were found to be very ineffective in providing Signal 1 to the pCTL. Having obtained strong evidence for triggering of pCTL occurring via direct recognition of the subcellular alloantigen, we then examined the role of antigen multivalency in recognition and triggering. Purified H-2Kk was prepared in a variety of forms of differing multivalency, ranging from monovalent papain cleavage product to large, highly multivalent liposomes and plasma membranes. The magnitude of the CTL responses obtained was found to be critically dependent on the multivalency of the antigen preparation. Examination of the antigen dose-response curves and maximal responses obtained suggests that valency of the antigen may be important both in determining the avidity of interaction between the pCTL and the antigen-bearing structure, and in determining the extent to which localized receptor cross-linking occurs on the cell surface to result in triggering.     

Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

Thy.1lowCD3− Cells Sorted from Nylon Wool-Passed Bone Marrow Cells Can Augment the H-2 Identical but Not Non-Identical Cytotoxic T Lymphocyte Precursors in Mixed Lymphocyte Cultures

Thy. 1^lowCD3^– cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-α/β and -γ/δ on their cell surfaces. An extremely limited number of B10.BR (H-2^k) responder lymph node (LN) cells were stimulated with B10. D2 (H-2^d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1^lowCD3^– NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2^b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy. 1^lowCD3^– NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2⁺ cells or Thy.1⁺ L3T4^– (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1^low CD3^– NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1⁺ Lyt-2^– helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.     

Heterogeneity of immunologic memory T cells specific for H-2 antigens and detection of their receptors

The in-vivo-induced memory T cells (MC) of mice, specific to H-2 antigens, are assayed by the generation of the secondary cytotoxic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) activated by heat-killed stimulator cells. The MC are shown to adhere selectively to the corresponding target monolayer that gives rise to both the loss of MC activity in the population of non-adherent lymphocytes and gain in MC activity in the population adherent and eluted from the same monolayer. In addition to the revealing of MC H-2 antigen-binding receptors, the absorption-elution technique allows the separation of the MC into two categories: secondary CTL precursors bearing these receptors, and secondary amplifier cells non-adherent to the monolayer and assayed by promotion of the CTL generation from the primary precursors activated in MLC by heated stimulators. The difference in the receptor properties between the primary and secondary CTL precursors raises the possibility that the MC are generated not only in the amplifier cell population but also in the independent CTL precursor population.     

Regulation of the production of immune interferon and cytotoxic T lymphocytes by interleukin 2

The activation of alloantigen-specific cytotoxic T lymphocyte precursors is dependent upon the presence of both macrophages and helper T cells or regulatory molecules derived from these facilitative cells. Three biochemically distinct helper factors have been identified: interleukin 1 (macrophage-derived), Interleukin 2 (T cell derived), and immune interferon. All 3 factors are found in supernatants of mixed lymphocyte cultures (MLC), however, the removal of macrophages from these cultures completely ablates the production of these factors as well as the induction of cytotoxic T lymphocytes (CTL). The addition of IL 2 to these macrophage-depleted MLC restores the ability of responder T cells to: 1) bypass the requirement for macrophage soluble function, 2) produce immune interferon, and 3) generate CTL. The kinetics and dose response of immune interferon production in response to IL 2 correlates with the generation of CTL. The production of immune interferon as well as the generation of CTL requires T cells, alloantigen, and IL2. Furthermore, the induction of CTL by IL2 was neutralized by the addition of anti-immune interferon. These data suggest that: 1) the regulation of immune interferon production is based on a T to T cell interaction mediated by IL 2, and 2) immune interferon production may be required for IL 2 induction of CTL. These findings are consistent with the hypothesis that the induction of CTL involves a linear cell-factor interaction in which IL 1 (macrophage-derived) stimulates T cells to produce IL 2, which in turn stimulates other T cells to produce immune interferon and become cytotoxic.     

In vitro induction of immunological tolerance

IL-2 was previously shown to induce cytotoxic effectors with a broad spectrum of target specificities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effectors. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific. IL-2-activated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable "chamber insert" culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.     

Loss of specificity in cytolytic T lymphocyte clones obtained by limit dilution culture of Ly-2+ T cells

Nonspecific cytotoxicity developed reproducibly and with high frequency in limit dilution cultures consisting of low numbers of murine cells stimulated with concanavalin A in the presence of growth factors and irradiated filler cells. The individual clones in cultures showing nonspecific killing were all derived from single, Thy-1+, Ly-2+ cells. At early times of culture (day 5 or 6), clones appeared to be specific in their lytic activity, as expected of cytolytic T lymphocytes (CTL). On continued culture (day 8 or 9), most of the originally specific CTL clones became nonspecific, killing a range of murine target cells, both syngeneic and allogeneic. The lack of specificity was observed at all effector cell doses. The effector cells responsible for the nonspecific cytolysis were Thy-1+ and Ly-2+, as were most cells in the cultures. The effector cells had the normal DNA content for a dividing T cell population, and most cells in the cultures had a normal chromosome complement. In mixed cultures in which the responder cells and the irradiated filler cells were from different mouse strains, the nonspecific killers displayed the Thy-1 and H-2 allotypes of the responder, and not of the filler cells. The development of a broad cytotoxic potential appears to be a normal and rapid event when Ly-2+ T cell-derived CTL-clones are grown under these conditions; this is a caveat for the use of limit dilution cultures to determine the T cell specificity repertoire. The relationship between these nonspecific CTL, activated lymphocyte killers, and natural killer cells is discussed.     

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.     

Down-regulation of cytotoxic T lymphocyte development by a minor stimulating locus-induced suppressor cascade that involves Lyt-1+ suppressor T cells, IA- macrophages, and their factors

Down-regulation of the development of CTL has been studied in mice both in vivo and in vitro. To generate CTL to hapten-altered self Ag in vivo, an immunization protocol has been used in which the host's Th cells are stimulated by a minor locus histocompatibility Ag (Mlsd) and its precursor CTL are activated by trinitrophenylated syngeneic spleen cells. Injecting the H-2 compatible Mls-disparate spleen cells along with the TNP-coupled self cells into the hind paws causes TNP-self specific CTL to appear in popliteal lymph nodes within 5 days. We have previously reported that inducing Ts cells by i.v. injecting Mlsd-bearing cells prevents in vivo generation of TNP-self specific CTL after immunization in this way. Here the induced Ts cell as well as the mechanism by which it functions have been further examined. The suppression was seen to extend to allogeneic as well as TNP-self Ag, provided the Mlsd-tolerized animal was reexposed to Mlsd-bearing cells at the time of immunization for CTL. By transferring the Mlsd-induced suppression adoptively we have learned that the splenic suppressor cell bears Thy-1.2 as well as Lyt-1.1 Ag and inhibits the generation of CTL at the afferent limb. In addition, Mlsd-induced PEC of Mlsd-tolerized mice, but not of normal mice, mediated suppression of development of CTL in vivo. The active cells within the tolerized PEC have been identified as T cells and macrophages (M phi). Furthermore, PEC from mice tolerized to Mlsd suppressed generation of CTL directed toward TNP-self targets in vitro. T cells and M phi separated from PEC of Mlsd-tolerized mice achieved suppression best in culture when present together. In addition, Lyt-1+ splenic cells from tolerized but not normal mice cooperated to down-regulate CTL generation in vitro with peritoneal M phi from either tolerized or normal mice. Supernatants of 24- to 72-h cultures of PEC from tolerized mice were suppressive of CTL generation when incorporated at 40 to 50% of culture volume. Supernatants of T cells from tolerized PEC or spleen were suppressive in culture only when M phi from normal mice were also present. To achieve suppression dialyzed supernatants of M phi from tolerized mice could replace the M phi.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo activation of autoreactive cytotoxic T lymphocytes after bone marrow transplantation: evidence for the prethymic specificity of T cells?

The light density fraction (A + B, i.e., remaining above the 26% concentration in the discontinuous BSA gradient) of BCF1 (H-2b X H-2k) mouse bone marrow contains cells that after injection into irradiated syngeneic recipients give rise to autoreactive Lyt-2+, Thy-1+ CTL. After injection of unfractionated bone marrow cells, the levels of these CTL were low or undetectable, suggesting that either the precursors were highly enriched in the A + B fraction or that bone marrow cells with higher density have a suppressive function. The specificity of the killing was not directed toward all the available class I MHC antigens: only targets carrying H-2Kb-coded determinants were killed. There was no overlapping between the autoreactive and alloreactive precursors: cells from the A + B fraction could not respond to an alloantigen in vitro, not even in the presence of an interleukin 2-containing supernatant, and the autoreactive CTL activated in vivo could not kill allogeneic targets. The induction of the autoreactive CTL did not require the presence of the appropriate MHC antigen in the maturation environment, thus differing from the activation of mature T cells. The observed CTL specificity, together with the previous findings showing that prethymic T cells are locating in the same BSA fraction as the precursors for these autoreactive cells, support the idea that the prethymic T cell repertoire is, at least partially, directed to recognize self-MHC antigens.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Induction of linked suppression in addition to the donor H-2 class I-specific unresponsiveness in recipient T cells by transfusing class I plus class II-disparate, but not class I alone-disparate, bone marrow cells

This study was undertaken to determine whether bone marrow (BM) cells contain a cell population with the capacity to induce an unresponsiveness of T cells specific to the BM self-H-2 class I antigens in vivo, i.e., veto cell population. Recombinant or congenic mice were infused intravenously with H-2-incompatible BM cells. One to several weeks later, donor H-2-and irrelevant H-2-specific responses in mixed lymphocyte reaction cultures of recipient T cells were assessed. Transfusion of H-2-incompatible BM of C57BL/10 (B10) recombinant strains caused a long-lasting cytotoxic T lymphocyte (CTL) unresponsiveness to the donor class I antigens in recipient lymph node cells. When class I plus class II-disparate BM cells were transfused, an anti-donor class I CTL response and a response against a third-party class I antigen, which was presented on the stimulator cells coexpressing the donor class I and class II, were significantly suppressed. This linked suppression lasted for less than 2 weeks after transfusion. Transfusion of class I-alone-disparate BM induced the donor class I-specific CTL unresponsiveness, but not the linked suppression. The induction of linked suppression was prevented considerably by transfusing nylon wool-nonadherent BM or by treating recipients with cyclophosphamide 2 days before transfusion. An anti-third-party class I CTL response, stimulated in vitro with fully allogeneic spleen cells, was not hampered by the BM transfusion. Coculturing the lymph node (LN) cells obtained from the class I plus class II-disparate BM recipient with normal LN cells interfered with the generation of both anti-donor class I and anti-linked third-party class I CTL, whereas, coculturing LN cells from the class I alone-disparate BM recipient inhibited neither specificity of CTL generation. Transfusion of class I plus class II-disparate BM resulted in a significant suppression of the donor class II-specific proliferative response. In contrast, transfusion of class I alone-disparate BM did not suppress any proliferative responses, including even a "linked" third-party class II-specific response. Transfusion of bm 1, (B6 X bm 1)F1, or (bm 1 X bm 12)F1 BM to B6 did not induce unresponsiveness in bm 1-specific CTL responses. However, the transfusion resulted in a significant suppression of bm 1-reactive proliferative response of recipient LN cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular requirements for the generation of primary cell-mediated lympholysis responses to Qa-1 antigens

Mixed leukocyte cultures (MLC) between NZB responder spleen cells and Qa-1-disparate stimulator spleen cells were employed to determine the cellular requirements for the generation of primary anti-Qa-1 cell-mediated lympholysis (CML) responses. Although primary anti-Qa-1 cytotoxic lymphocytes (CTL) were generated during H-2-homologous stimulation, anti-Qa-1 CTL were not detectable from MLC in which the stimulators were H-2 allogeneic. Anti-Qa-1 CTL also were not generated from MLC in which the stimulators were semiallogeneic. Thus, H-2 identity between responder and stimulator cells was not sufficient to permit the generation of primary anti-Qa-1 CTL when H-2 disparity was also present. The capacity for H-2 disparity to prevent anti-Qa-1 CML responses was further demonstrated in MLC containing both H-2-allogeneic and H-2-homologous stimulator cells. Therefore, in subsequent studies we employed NZB responders and H-2-homologous, Qa-1-disparate stimulators. When various subpopulations of stimulator cells were studied for their ability to induce anti-Qa-1 CTL, nylon wool-adherent cells were found to be potent stimulators, but nylon wool-nonadherent cells were not. Furthermore, depletion of macrophages from the stimulator population abrogated the generation of anti-Qa-1 CML responses, despite the presence of responder macrophages in the culture. In contrast, all fractionated subpopulations stimulated anti-H-2 CML responses. When macrophage-enriched cells were used as stimulators, anti-Qa-1 CTL could be generated with approximately 80-fold fewer stimulator cells than when unfractionated splenocytes were used as stimulators. These findings indicated that stimulator macrophages were essential for the generation of primary anti-Qa-1 CTL. Direct evidence for macrophage expression of Qa-1-antigens was obtained by using a Qa-1b-specific CTL clone. These studies provide i) the first evidence for Qa-1 expression on macrophages, ii) a basis for comparison of the cellular interactions necessary to generate CTL against H-2K/D-encoded vs Qa-1-encoded class 1 antigens, and iii) a model for investigating the mechanisms responsible for the immunodominance of H-2K/D alloantigens.     

AKR.H-2b lymphocytes inhibit the secondary in vitro cytotoxic T-lymphocyte response of primed responder cells to AKR/Gross murine leukemia virus-induced tumor cell stimulation.

We have previously shown that AKR.H-2b congenic mice, though carrying the responder H-2b major histocompatibility complex haplotype, are unable to generate secondary cytolytic T-lymphocyte (CTL) responses specific for AKR/Gross murine leukemia virus (MuLV). Our published work has shown that this nonresponsive state is specific and not due to clonal deletion or irreversible functional inactivation of antiviral CTL precursors. In the present study, an alternative mechanism based on the presence of inhibitory AKR.H-2b cells was examined. Irradiated or mitomycin C-treated AKR.H-2b spleen cells function as in vitro stimulator cells in the generation of C57BL/6 (B6) anti-AKR/Gross virus CTL, consistent with their expression of viral antigens. In contrast, untreated viable AKR.H-2b spleen cells functioned very poorly as stimulators in vitro. Viable AKR.H-2b spleen cells were also able to cause dramatic (up to > or = 25-fold) inhibition of antiviral CTL responses stimulated in vitro by standard AKR/Gross MuLV-induced tumor cells. This inhibition was specific: AKR.H-2b modulator spleen cells did not inhibit allogeneic major histocompatibility complex-specific CTL production, even when a concurrent antiviral CTL response in the same culture well was inhibited by the modulator cells. These results and those of experiments in which either semipermeable membranes were used to separate AKR.H-2b modulator spleen cells from AKR/Gross MuLV-primed responder cells or the direct transfer of supernatants from wells where inhibition was demonstrated to wells where there was antiviral CTL responsiveness argued against a role for soluble factors as the cause of the inhibition. Rather, the inhibition was dependent on direct contact of AKR.H-2b cells in a dose-dependent manner with the responder cell population. Inhibition was shown not to be due to the ability of AKR.H-2b cells to function as unlabeled competitive target cells. Exogenous interleukin-2 added at the onset of the in vitro CTL-generating cultures partially restored the antiviral response that was decreased by AKR.H-2b spleen cells. Positive and negative cell selection studies and the development of inhibitory cell lines indicated that B lymphocytes and both CD4- CD8+ and CD4+ CD8- T lymphocytes from AKR.H-2b mice could inhibit the generation of AKR/Gross virus-specific CTL in vitro. AKR.H-2b macrophages were shown not to be required to demonstrate AKR/Gross MuLV-specific inhibition, however, confirming that the inhibition by T-cell (or B-cell)-depleted spleen populations was dependent on the enriched B-cell (T-cell) population per se.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mixed lymphocyte response of senescent mice: sensitivity to alloantigen and cell replication time.

The age-dependent alteration in the proliferative response of C57B1/6J lymph node cells to stimulation by H-2- and M-locus alloantigens was examined in one-way mixed lymphocyte cultures (MLC). Balb/c (H-2^d, Mls^b) and DBA (H-2^d, Mls^a) spleen cells served as stimulating cells differing from C57B1/6J (H-2^b, Mls^b) at the H-2 and H-2 plus Mls loci, respectively. The day of peak response and the ratio of responder to stimulator cells required for optimal stimulation were the same for all the age groups (3 to 29 months) tested, irrespective of the stimulator strain used. Results obtained in MLC under optimal conditions showed a maximal response to both Balb/c and DBA/2 stimulation at the age of 6 months, followed by a gradual decline in the response with age. In order to determine whether the decline with age in mixed lymphocyte reactivity can be attributed to a reduction in the proliferative capacity of the responding lymphocytes of aged mice, cell cycle analyses were performed. Auto-radiographic studies of MLC containing lymphocytes from CS7B1/6J mice aged 6 and 24 months showed no difference in generation time, S, G₂, G₁, and M phases of the cell cycle. In addition, lymphocytes of both age groups underwent two identical mitotic waves within the period of examination. Our results determine that the functional decline with age in proliferative activity in mixed lymphocyte cultures is attributable to a neither decrease in sensitivity to alloantigen nor to a decrease in generation time or the ability to undergo several mitotic divisions, and suggest that such a decline is caused by fewer cells capable of response in old mice.     

Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses

Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study investigates the role of haptenic structure and hapten-cell surface reaction patterns in influencing the generation of the T cell specificity as well as the H-2-linked genetic control. CTL and helper T cell responses were generated against two structurally related haptens, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene-diamine (SH-reactive AEDANS; AED-SH) and 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (NH2-reactive form of AEDANS; AED-NH2) by immunizing C57BL/6N (H-2b) mice with these hapten-modified syngeneic spleen cells. Spleen cells from primed C57BL/6N mice generated strong CTL and helper T cell activities upon in vitro restimulation with the respective hapten-modified self. The generation of potent anti-AED-NH2 CTL and helper T cell responses in C57BL/6N mice sharply contrasted with the failure of NH2-reactive haptens studied thus far to generate strong anti-hapten cytotoxic responses in H-2b mice. Antibodies induced against the above two haptens exhibited extensive cross-reactivity detected by hemagglutination, whereas CTL and helper T cells clearly discriminated the structural difference between AED-NH2 and AED-SH haptens. The hapten specificity in T cell recognition was also observed between AED-NH2 and trinitrophenyl (TNP) haptens, which were demonstrated to functionally modify similar cell surface sites. These results indicate that hapten epitope structure and hapten-cell membrane conjugation patterns influence the generation of H-2-linked genetic control and T cell specificity in anti-hapten self cytotoxic as well as helper T cell responses.