H-2-restricted graft-versus-host reaction: Foreign determinants and restriction elements

Nylon-wool-purified T cells from radiation chimeras cause a lethal graft-versus-host reaction (GVHR) in irradiated, bone-marrow-protected recipients only if the recipient shares a restriction element with the T-cell donor and also expresses antigens foreign to the donor. Class I molecules (H-2K and H-2D) can act as restriction elements, but restriction to class II molecules could not be demonstrated. However, class II molecules as well as H-2K and some non-H-2 determinants could serve as foreign antigens.     

HLA-A2-restricted protection against lethal lymphocytic choriomeningitis

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.     

H-2-restricted helper activity mediated by immunoglobulin-bearing lymphocytes

The genetic requirements for helper activity mediated by a unique, Ig-bearing lymphocyte population were studied. This Lyt-1+, I-A+, Thy-1- population, called BH, preferentially helps expression of NPb idiotypic plaque-forming cells when added to T cell-depleted responder cultures. Furthermore, the BH population can directly bind NPb idiotypic determinants. Using H-2 congenic mice, we show that BH helper activity can be expressed only when BH cells share I-A subregion alleles with responder B cell populations. This H-2 restriction is not a result of thymic influences, because the activity of BH cells from athymic mice are also H-2 restricted. Macrophages present in the BH population do not contribute to the H-2 restriction. Results are presented that definitively rule out the possible role for T lymphocytes in BH activity and demonstrate that a single helper population expresses both Lyt-1 and I-A determinants. These results indicate that Ig-bearing cells serve a regulatory as well as an effector role in immune responses and that, like other regulatory lymphoid subsets, their activity is regulated in part by MHC-encoded determinants.     

Establishment of T-cell clones recognizing difference in H-2K antigen and inducing graft-versus-host disease

From the spleen cells of C57BL/6 (B6) mice, T-cell clones were established which responded to mutant H-2K antigen of B6. B-H-2bml (Hz1) mice. One clone and its subclones were maintained in culture medium containing lower concentration (2.5%) of IL-2 (culture supernatant of rat spleen cells stimulated with concanavalin A). They were shown to have a capability to induce graft-versus-host reaction in (B6 X Hz1)F1 recipients as well as to exert cytotoxicity against concanavalin-A-activated spleen cells of Hz1 mice and also their kidney fibroblasts. They lacked cytotoxicity against syngeneic and also third-party Con-A-stimulated spleen cells. These reactive clones were Thy-1.2 and Lyt-2 positive and Lyt-1 negative. On the other hand, clones which were maintained in culture medium containing higher concentration (20%) of IL-2 were devoid of activities in vivo as well as in vitro. These clones were Thy-1.2 positive but Lyt-1.2 and Lyt-2.2 negative.     

Preliminary characterization of the cells causing an H-2-restricted GVH reaction

An H-2-restricted graft-versus-host reaction can be demonstrated when irradiated bone marrow-protected recipients receive injections of cells from radiation chimeras. The cells responsible are Thy-1-positive, thymus-dependent, radiation-sensitive and pass through a nylon wool column, i. e., they are T cells. Treatment of the cells with anti-Lyt-1 or anti-Lyt-2 serum and complement reduces but does not eliminate the activity which can be eliminated by treatment with both antisera. Combining anti-Lyt-1-treated and anti-Lyt-2-treated cells does not restore the original activity.     

Acute lethal graft-versus-host disease stimulates cellular proliferation in the adult rat liver

Abstract. The present investigation was designed to analyse the effects of acute lethal graft-versus-host disease (GVHD) in adult (DA x LEW)F₁ rats on cellular proliferation within the liver. The influence of the host thymus on GVHD-induced proliferation was also assessed. From 1–28 days after initiation of GVHD [³H]thymidine ([³H]-TdR) was injected i.v. and rats were killed one hour later. Percentage labelled cells (LI) of periportal infiltrating cells (PIC), hepatocytes (H), and sinusoidal lining cells (SC) were counted. Mean values for control rats were 0.3 ± 0.1% (H), 0.4 ± 0.1% (SC) and 0.2 ± 0.1% (PIC). GVHD rats demonstrated a significant increase in LI of PIC (days 1–21), SC (days 2–17) and H (days 2–17). Most labelled cells in PIC were large lymphocytes. Peak LI values were 7.0 ± 1.0% PIC (day 17), 6.8 ± 0.9% SC (day 17), and 5.2 ± 0.9% H (day 7), with all cellular compartments returning to near normal LI values by day 28. Stimulation of cellular proliferation occurred in all three liver cell compartments in neonatally thymectomized (TXM) rats. The intensity of GVHD-induced cell proliferation was significantly decreased at day 7 in all compartments and PIC was dramatically decreased at day 21 in TXM-GVHD rats as compared to non-TXM-GVHD rats. It is hypothesized that the general stimulation of hepatocyte cell proliferation in GVHD is related to the secretion of lymphokines by primarily donor and secondarily host T cells in the periportal infiltrate.     

Recipient nonhematopoietic antigen-presenting cells are sufficient to induce lethal acute graft-versus-host disease

The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100-1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.     

H-2-restricted cytolysis of L cells doubly transformed with a cloned H-2Kb gene and cloned retroviral DNA

We report the construction of target cells by the double transformation of mouse L cells with a cloned H-2Kb gene and molecular clones of Akv or Gross murine leukemia virus (MuLV) or the cloned gag gene of Akv MuLV. Cytolytic T lymphocytes (CTL) generated in BALB.B mice specific for Gross MuLV (closely related to Akv MuLV) kill these doubly transformed cells. This is a direct indication that a CTL subpopulation recognizes H-2Kb antigen in association with a viral antigen encoded by the gag gene of Gross MuLV.     

Target antigens for H-2-restricted vesicular stomatitis virus-specific cytotoxic T cells.

Cytotoxic thymus-derived lymphocytes from mice infected with vesicular stomatitis virus (VSV) are H-2 restricted and virus specific for the Indiana and New Jersey strains of VSV. VSV-Indiana-immune T cells can lyse target cells infected with the temperature sensitive (ts) mutant ts 045 about 30 times better when target cell infection occurs at the permissive rather than the non-permissive temperature. Since this mutant fails to express the glycoprotein at the cell surface when grown at the nonpermissive temperature, our results support the view that the viral glycoprotein is involved in defining the major target antigen for VSV-specific T cells. However, the tl 17 mutant that expresses a mutant glycoprotein at the cell surface was lysed, suggesting that the expressed mutated glycoprotein can cross-react with Indiana wild-type glycoprotein. Targets infected at the nonpermissive temperature with VSV ts G31 (mutant in the matrix protein) are still susceptible to T cell-mediated lysis but at a lower level of sensitivity. These results are compatible with the interpretation that for VSV-specific T cell lysis of infected target cells, the viral glycoprotein is a major target antigen and must be expressed, and that the matrix protein plays a lesser role, probably by indirectly influencing glycoprotein configuration at the cell surface.     

Identification of the immunodominant H-2K(k)-restricted cytotoxic T-cell epitope in the Borna disease virus nucleoprotein

Borna disease virus (BDV)-induced immunopathology in mice is most prominent in strains carrying the major histocompatibility complex H-2k allele and is mediated by CD8(+) T cells that are directed against the viral nucleoprotein p40. We now identified the highly conserved octamer peptide TELEISSI, located between amino acid residues 129 and 136 of BDV p40, as a potent H-2K(k)-restricted cytotoxic T-cell (CTL) epitope. When added to the culture medium of L929 target cells, TELEISSI conferred sensitivity to lysis by CTLs isolated from brains of BDV-infected MRL mice with acute neurological disease. Vaccinia virus-mediated expression of a p40 variant with mutations in the two K(k)-specific anchor residues of the TELEISSI peptide (p40(E130K,I136T)) did not sensitize L929 target cells for lysis by BDV-specific CTLs, whereas expression of wild-type p40 did. Furthermore, unlike vaccination with wild-type p40, vaccination of persistently infected symptomless B10.BR mice with p40(E130K,I136T) did not result in central nervous system inflammation and neurological disease. These results demonstrate that TELEISSI is the immunodominant CTL epitope of BDV p40 in H-2k mice.     

An H-2-restricted CML target antigen controlled by a gene linked to theH-2 complex

(B10 × BALB/c)F₁ anti B10.D2/n effector cells obtained after in vitro restimulation of spleen cells from in vivo primed mice react in the CML assay with B10.D2/n target cells and target cells from certain otherH-2D _d carrying strains. The gene controlling the antigen involved maps proximal toH-2K.     

Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences

Information was sought on the phenotype of lymphoid cells causing lethal graft-vs-host disease (GVHD) in irradiated mice expressing whole or partial H-2 differences. In all strain combinations tested, pretreating donor lymph node (LN) cells with anti-Thy-1 monoclonal antibodies (MAb) plus complement (C) abolished mortality. With GVHD directed to class I H-2 differences, pretreating LN cells with anti-Lyt-2 MAb prevented mortality, whereas MAb specific for Ly-1 or L3T4 cell surface determinants caused severe mortality. These data imply that lethal GVHD directed to class I H-2 differences is mediated by L3T4-, Lyt-2+ cells; this subset of T cells was shown previously to control GVHD directed to multiple minor histocompatibility antigens, i.e., antigens seen in the context of self-class I molecules. With whole H-2 differences, GVHD appeared to be controlled largely but not exclusively by L3T4+, Lyt-2-T cells. This T cell subset was also the predominant cause of GVHD directed to class II differences. With class II incompatibilities, depleting donor cells of L3T4+ T cells, either by pretreatment with anti-L3T4 MAb + C or by fluorescence activated cell sorter selection, greatly reduced but did not completely abolish GVHD. These data might imply that L3T4-, Lyt-2+ cells have some capacity to elicit anti-class II GVHD. A more likely possibility, however, is that the residual GVHD to class II differences observed with Lyt-2+-enriched cells reflected minor contamination with L3T4+ cells.     

Binding of antigen-specific, H-2-restricted T cell hybridomas to antigen-pulsed adherent cell monolayers

Two methods were investigated for studying the binding of radiolabeled hybridoma T cells to antigen (Ag) and H-2 products for which they bore receptors. In both cases hybridoma T cells were labeled with tritiated thymidine. In one method labeled cells were added to adherent splenic cells prepulsed with antigen, and the mixture was incubated overnight at 37 degrees C before nonadherent cells were gently washed away. The percent of adherent hybridoma T cells was then estimated by harvesting the adherent monolayers and measuring tritium counts bound. In a second method radiolabeled hybridoma T cells were added to adherent antigen-pulsed B cell lymphomas or hybridomas for between 15 min and 1 hr at 37 degrees C before removal of nonadherent cells and harvesting of the adherent monolayers. In both cases binding was both antigen- and I-region specific. In the second case binding was also rapid; significant binding could be measured after 15 min incubation. These techniques were used to study subclones of one of our T cell hybridomas that were thought by a functional assay (interleukin 2 release) to have lost receptors for Ag/H-2. It was found that subclones of the hybridoma that no longer secreted interleukin 2 in response to Ag/H-2, even though they continued to secrete interleukin 2 in response to concanavalin A, also no longer bound specifically to Ag-pulsed monolayers of the appropriate H-2 type. This confirmed the idea that these subclones had lost the ability to synthesize receptors for Ag/H-2. It is hoped that assays of this type will be useful in the future for the study of Ag/H-2 receptors on T cells.     

H-2-restricted GVH reaction caused by T cells from normal donors of certain strains

A comparison has been made of lethal graft-versus-host (GVH) reactions caused by T cells from radiation chimeras and from normal mice. In the recipient strains tested, T cells from both chimeric and normal A, A.SW, and CBA mice showed H-2-restricted killing, while T cells from chimeric and normal C57BL/6 failed to show any evidence for H-2-restricted GVH reactions. With other strains tested as donors, T cells from chimeras showed H-2-restricted GVH reactions, while the corresponding normal cells showed some similarities to the chimeras but not complete H-2-restriction.[/p]     

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

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

Elimination of leukemia in the absence of lethal graft-versus-host disease after allogenic bone marrow transplantation

Donor T cells are able to effect a graft-vs-leukemia (GVL) response but also induce graft-vs-host disease (GVHD) after allogeneic bone marrow transplantation. We used an AKR leukemia murine transplant model, analogous to human acute lymphoblastic leukemia, in which donor T cells expressed a thymidine kinase suicide gene, to test whether separation of GVL and graft-vs-host (GVH) responses was feasible by selectively eliminating alloactivated donor T cells at defined time points posttransplant. Under experimental conditions where untreated mice could not be cured of disease without dying from GVHD, mice transplanted with thymidine kinase-positive T cells and subsequently administered ganciclovir (GCV) could eliminate leukemia without lethal GVHD. Timing of GCV administration, donor T cell dose, and preexisting leukemia burden were observed to be critical variables. Eradication of leukemia without lethal GVHD in GCV-treated mice implied that the kinetics of GVL and GVH responses were asynchronous and could therefore be temporally dissociated by timely GCV administration. That this strategy was feasible in a murine leukemia model in which GVHD and GVL reactivity are tightly linked suggests that this approach may be relevant to the treatment of selected human leukemias where similar constraints exist. This strategy represents an alternative approach to separating GVL and GVH reactivity and challenges the current paradigm that separation of these responses is dependent upon the administration of donor T cells with restricted specificity for leukemia as opposed to host Ags.     

Allogeneic T cells treated with amotosalen prevent lethal cytomegalovirus disease without producing graft-versus-host disease following bone marrow transplantation

Infusion of donor antiviral T cells can provide protective immunity for recipients of hemopoietic progenitor cell transplants, but may cause graft-vs-host disease (GVHD). Current methods of separating antiviral T cells from the alloreactive T cells that produce GVHD are neither routine nor rapid. In a model of lethal murine CMV (MCMV) infection following MHC-mismatched bone marrow transplantation, infusion of MCMV-immune donor lymphocytes pretreated with the DNA cross-linking compound amotosalen prevented MCMV lethality without producing GVHD. Although 95% of mice receiving 30 x 10(6) pretreated donor lymphocytes survived beyond day +100 without MCMV disease or GVHD, all mice receiving equivalent numbers of untreated lymphocytes rapidly died of GVHD. In vitro, amotosalen blocked T cell proliferation without suppressing MCMV peptide-induced IFN-gamma production by MCMV-primed CD8(+) T cells. In vivo, pretreated lymphocytes reduced hepatic MCMV load by 4-log(10) and promoted full hemopoietic chimerism. Amotosalen-treated, MCMV tetramer-positive memory (CD44(high)) CD8(+) T cells persisted to day +100 following infusion, and expressed IFN-gamma when presented with viral peptide. Pretreated T cells were effective at preventing MCMV lethality over a wide range of concentrations. Thus, amotosalen treatment rapidly eliminates the GVHD activity of polyclonal T cells, while preserving long-term antiviral and graft facilitation effects, and may be clinically useful for routine adoptive immunotherapy.     

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

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

Two epitopes and one agretope map to a single HLA-A2 peptide recognized by H-2-restricted T cells

Mice immunized with syngeneic cells transfected with cloned genes coding for HLA class I molecules could recognize the human MHC Ag in the context of their own H-2 molecules. We obtained CTL clones from DBA/2 mice (H-2d) which had been immunized with P815 cells (a mastocytoma of DBA/2 origin) expressing either HLA-A2 or HLA-A3 or two different molecules containing recombined sequences of HLA-A2 and HLA-A3. Fourteen of these clones recognized a synthetic peptide corresponding to the region 170-185 of HLA-A2 in the context of H-2Kd. Moreover, from their activity on P815 cells expressing HLA-Cw3, two subpatterns could be distinguished: subpattern Cw3+, defined by those clones which lysed P815-Cw3, and subpattern Cw3- defined by those clones which did not lyse P815-Cw3. By testing the activity of clones of each subpattern on a series of modified synthetic peptides, we were able to define two epitopes on the same 170-185 peptide of HLA-A2. One of them was dependent on amino acids at positions 173 and 177, whereas the other was dependent on amino acid 177 alone. By using competition experiments, we were also able to define an agretopic region strongly dependent on the amino acid at position 178. Furthermore, experiments with L cells expressing molecules containing recombined sequences between H-2Kd and H-2Dd demonstrated the determinant role of residues 152, 155, and 156 from H-2Kd in the presentation to murine T cells of the 170-185 peptide of HLA-A2.