Genetics of graft-versus-host reactions

Intracardiac injection of lymph node cells into newborn allogeneic recipients incompatible with congenic donors at multiple loci within theH-2 complex (B10B10.A) led to GVH splenomegaly, retarded growth, and mortality. In contrast,H-2I region differences [B10.A(4R)B10.A(2R)] required fortyfold more cells to produce a comparable splenomegaly, and did not produce runting or GVH mortality despite a strong proliferative response in the spleen. However, when 4R donors were preimmunized with host lymphoid cells, 51 percent of 2R recipients died of allogeneic disease. GVHR was also observed in the reciprocal combination (2R4R), in contrast to previous reports. The phenotypic determinants governing GVHR in the 4R2R combination are probably coded for byI-C subregion genes. Disparities at theI-B orI-J subregions did not evoke detectable GVHR. The data indicate thatI-C subregion loci have the impact of so-called minor or moderate histocompatibility (MoH) loci. Possible implications for allogeneic human bone marrow transplantation are discussed.Portions of this work were presented at the 60th annual meeting of the Federation of American Societies for Experimental Biology on April 13, 1976 in Anaheim, California     

Sex-dependent effects of non-H-2 loci on lethal GVH reaction induced across an H-2 barrier

We have studied the influence of DBA/2 non-H-2 antigens on the lethal graft-versus-host reaction (GVHR) developed across an H-2 barrier. (DBA/2 x B10.D2)F₁ x B10.D2 (H-2 ^d) backcross (BC) mice were typed for their allelic constitution at nine genetically independent chromosome markers and used as individual cell donors simultaneously for two to three (DBA/2 X B10.D2)F₁ recipients incompatible for DBA/2 non-H-2 antigens alone and two to three (DBA/2 x B10.BR)F₁ recipients incompatible for DBA/2 non-H-2 antigens and H-2^k. The results showed that, when compared with that developed in a control group incompatible for H-2 ^kalone [B10.D2(B10.D2xB10.BR)F₁], the GVHR mortality seen in the presence of an additional incompatibility for DBA/2 non-H-2 antigens [(DBA/2 X B10.BR)F₁recipients] is significantly delayed but only in female mice. An analysis of individual BC donors indicated that this protective effect of DBA/2 non-H-2 antigens correlates with incompatibility for gene(s) linked to the Pgm-1 chromosome marker. In contrast, incompatibility for gene(s) linked to Mod-1 and Es-3 markers accelerates GVHR mortality, but only in male mice. Finally, the results obtained with (DBA/2 x B10.D2)F₁ and (DBA/2 x B10.BR)F₁ recipients were compared; they showed that the intensity of the GVHR developed by cells from individual BC donors against a given set of DBA/2 non-H-2 antigens correlates well with that developed by the same BC donor against the same set of non-H-2 antigens plus H-2^k. We conclude that certain non-H-2 genes (and antigens) can modulate the intensity of the GVHR developed across an H-2 barrier. The number of such genes is probably great; their effects are strong and complex, and can be sex-dependent.     

Role of L3T4+ and Lyt-2+ donor cells in graft-versus-host immune deficiency induced across a class I, class II, or whole H-2 difference

The i.v. injection of parental T cells into F1 hybrid mice can result in a graft-vs-host (GVH)-induced immune deficiency that is Ag nonspecific and of long duration. The effect of the GVH reaction (GVHR) on the host's immune system depends on the class of F1 MHC Ag recognized by the donor cells. To determine the role of different subsets of donor-derived T cells in the induction of GVHR, donor spleen cells were negatively selected by anti-T cell mAb and C, and the cells were injected into F1 mice that differed from the donor by both class I and II MHC Ag or by class I or class II MHC only. The induction of GVHR across class I + II differences was found to require both L3T4+ and Lyt-2+ parental cells. Induction of GVHR across a class II difference required only L3T4+ parental T cells in the combination tested [B6-into-(B6 x bm12)F1]. In contrast, B6 Lyt-2+ cells were sufficient to induce GVHR across a class I difference in (B6 x bm1)F1 recipients. In addition, a direct correlation was observed between the cell types required for GVH induction and the parental T cell phenotypes detected in the spleens of the GVH mice. The number of parental cells detected in the unirradiated F1 hosts was dependent upon the H-2 differences involved in the GVHR. Induction of a class I + class II GVHR resulted in abrogation of both TNP-self and allogeneic CTL responses. In contrast, induction of a class II GVHR resulted in only a selective loss of TNP-self but not of allogeneic CTL function. Unexpectedly, the induction of a class I GVHR also resulted in the selective loss of the TNP-self CTL response. Thus, these class I and class II examples of GVH both result in the selective abrogation of L3T4+ Th cell function. The data are discussed in terms of respective roles of killer cells and/or suppressor cells in the induction of host immune deficiency by a GVHR, and of the selective deficiency in host Th cell function induced by different classes of GVHR.     

Tumour-induced altered gastrointestinal steady-states: absence of MHC restriction in the paraneoplastic gastrointestine

Abstract The growth of a number of experimental rodent tumours including the Lewis lung tumour (LLca) progressively compromises the integrity of the host's gastrointestine by inducing cytokinetic alterations in the small bowel resembling those generally defining the intestinal phase of a graft-versus-host reaction (GVHR). To determine whether the induction of this paraneoplastic gastrointestine (PGI) involves, similar to a GVHR, a disparity between the MHC of the donor (LLca tumour) and the recipient (host), PGI development was evaluated in various LLca tumour-bearing murine strains that were either ‘syngeneic’[C57BL/6 and BL/10 (H-2^b)], ‘semisyngeneic’[B6D2F₁ (H-2^bd) and B6C3F₁ (H-2^bk)] or ‘allogeneic’[C3H/HeJ (H-2^k) and DBA/2 (H-2^d)] to the H-2^b LLca tumour. The temporal appearance and magnitude of a PGI developing in either LLca-syngeneic or semi-syngeneic hosts, but not the allogeneic strains, suggested that the mechanism(s) involved in PGI development, like the GVHR, was restricted by the MHC. Subsequent studies using congenic strains [B10.A (H-2^k) and B10.D2/nSn (H-2^d)], however, demonstrated that the mechanism(s) responsible for the PGI was restricted by the non-MHC loci of the C57BL mouse. These observations were supported by the appearance of a LLca-induced PGI in various B10.A congenic strains carrying mutations at the I-A or I-E/I-J loci of the MHC. Not unlike the intestinal phase of a GVHR, development of the PGI required the participation of enhanced mucosal mast cells which were limited in the WCB6F₁ (S1/S1^d) but not the (+/+) murine strains. These observations are discussed in light of the postulated premature migration of immature thymocytes that accompany tumour growth and their ability to non-specifically enhance (or suppress) cell mediated immune reactions in the host.     

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. II. Development of natural suppressor cell activity

We explored the immunoincompetence of mice undergoing a chronic graft-vs-host reaction (GVHR) across minor histocompatibility barriers. BALB/c and B10.D2 mice are H-2d and mls b, and differ only with regard to minor histocompatibility antigens (MiHA). A large number of BALB/c mice were unirradiated or were irradiated with 300, 600, or 900 R. They then were injected with 5 X 10(7) spleen cells from either allogeneic B10.D2 or syngeneic BALB/c mice. The spleen cells from these recipient mice were assayed at various times post-irradiation/injection for their proliferative response to Con A and LPS, their ability to suppress the mitogen responses of normal spleen cells, and for the genetic specificity of this suppression. Spleen cells from BALB/c mice that had received 600 or 900 R (but not 0 or 300 R), and allogeneic B10.D2 lymphocytes, became very hyporesponsive to mitogens and became suppressive in vitro by days 7 to 10 post-irradiation/injection. These phenomena persisted for the entire 49 days of the experiment. After an initial period of splenomegaly, the spleens of these mice gradually became depleted of viable lymphocytes. Initial characterization of suppressor cells found in the spleens of GVH mice showed that they were not removed by treatment with anti-Thy-1.2 plus complement. GVH suppressors also were not adherent to plates coated with antiserum directed towards murine Ig. In addition, these cells did not adhere to plastic plates. Thus, we believe that the suppressor cells found in mice undergoing GVHD across MiHA are not mature T cells, B cells, or macrophages, but belong to a class of suppressor cells termed natural suppressor (NS). Genetic analysis of NS cell activity showed that as early as 10 days post-irradiation/injection, NS cells inhibited mitogen responses of all mouse strains tested, the exception being the relative difficulty in suppressing the LPS response of B10.D2 (syngeneic with donor cells). By day 42, this had developed into an almost complete inability to suppress a B10.D2 LPS response, although at this time NS cells were still capable of inhibiting all the other mitogen responses of all strains tested, including the Con A response of B10.D2 spleen cells. Moderate amounts of mitogen unresponsiveness and suppressor activity were seen in the syngeneic groups (BALB/c----BALB/c) but only if recipients received 600 or 900 R. This was a transient phenomenon that was maximal at day 14, and which we believe to be a similar but less severe degree of immunoincompetence when compared with that seen with allogeneic stimulation in the B10.D2----BALB/c GVH model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical characterization of murine Ia antigens with xenoantisera

Rabbit anti-Ia sera was produced by immunization with detergentsolubilized extracts from splenic, lymph-node and thymus cells. The antisera contained activity against H-2 as well as Ia molecules. By a sequential immunoprecipitation assay it was shown that the rabbit anti-mouseH-2 ^s serum precipitated a second Ia molecule in theH-2 ^s haplotype. Previous studies with alloantisera have shown only one Ia molecule associated with this haplotype. Sequential precipitations with alloantiserum against the wholeI region were used to show that this second Ia molecule is coded by genes within theI region. Since only I-A- and I-E-region coded molecules are immunoprecipitable in most haplotypes, we presume that the rabbit antiserum could be identifying the I-E-subregion coded molecule in theH-2 ^s haplotype. The rabbit antiserum reacts with an isotypic specificity on the molecule. The studies suggest that theI-E subregion does exist in theH-2 ^s haplotype even though alloantiserum cannot be produced to identify allotypic variants associated with this subregion.     

Sequential changes of thymocyte surface antigens with presence or absence of graft-vs-host reaction following allogeneic bone marrow transplantation

Lethally irradiated AKR mice were reconstituted with C57BL/6 bone marrow cells. Though the allogeneic marrow transplantation protected AKR recipients from acute irradiation deaths, the mice given unmanipulated marrow developed severe GVHR disease, and 80% died within 50 days. The thymus and spleen from the recipient mice, following recovery of body weight between the 10th and 20th days, gradually involuted and became miniscule after Day 30. Thymocytes from recipients were found to be entirely of donor cell type by Day 15. Thereafter, however, as the graft versus host reaction (GVHR) developed, changes in sensitivity of the thymocytes to four different alloantisera directed toward donor histocompatibility antigens (H-2^b, Thy 1.2, Lyt 1.2, and Lyt 2.2) were observed and these changes were associated with changes in antigen expression or quantity of Thy 1 antigens on the thymocytes. A different pattern of changes was observed in antigen expression on thymocytes in mice given B6 marrow cells that had been pretreated with anti-Thy 1 serum which prevented initiation of graft-vs-host disease and in the mice which received marrow not so treated and which regularly led to graft-vs-host disease. By contrast, the amount of H-2 antigen on the thymocytes from chimeras with or without GVHR was elevated equally. The mechanisms of these changes are discussed.     

Bone-marrow-cell grafts involving theH-2D andH-2L mutant haplotypes

TwoH-2 ^d mutants,H-2 ^dm2 (H-2L loss mutation) andH-2dm1 (gainplus-loss mutation involving bothH-2L andH-2D) were evaluated for any change in the immunogenicity of marrow stem cells. Grafts of 2 or 4 × 10⁶ BALB/c(C) or BALB/c-H-2^dm2 (C-H-2 ^dm 2) marrow cells were accepted by lethally irradiated B10.D2(H-2 ^d ) recipients and were rejected by irradiated B10(H-2 ^b ) recipients. Moreover, both (B6 × C)F₁ and (B6 × C-H-2 ^dm 2)F₁ mice, as irradiated recipients, resisted the growth of parental-strain B6(H-2 ^b ) marrow cells but accepted grafts from C or C-H-2 ^dm 2 parental-strain donors. Thus, theH-2 mutation involvingH-2L but notH-2D did not affect the expression ofH-2 ^d -associated Hemopoietic or Hybrid(Hh) antigens of marrow stem cells. Grafts of 2 to 8 × 10⁶ B10.D2 or B10.D2-H-2 ^dm 1 marrow cells were rejected by B10.BR(H-2 ^k ) and B6 hosts and were accepted by B10.D2 hosts. However, B10.D2-H-2 ^dm 1 marrow cells grew to a much greater extent than B10.D2 cells in irradiated (B6 × B10.D2)F₁ or (B6 × B10.D2-H-2 ^dm 1)F₁ host mice. Therefore, theH-2 ^dm 1 mutation has altered the expression of Hh antigens at least quantitatively, resulting in a relative loss of hybrid resistance with the retention of Hh determinants recognized by allogeneic recipient mice which are notH-2 ^d . Since the Hh determinants of B10.D2 marrow cells have been mapped 16 cM to the right ofH-2, this mutation atH-2D/H-2L may have affected a regulatory gene.     

Modulation of natural cytotoxicity by alloantibodies. V. The mechanism of a selective augmenting effect of anti-H-2 antisera on the natural killer activity of mouse spleen cells

Treatment of mouse spleen cells with specific anti-H-2 antisera augments their natural killer (NK) activity against K562 cells but not against YAC target tumor cells. The same population of natural killer cells was found to lyse K562 as well as YAC target cells, since (a) depletion of YAC reactive NK cells by absorption on YAC monolayers resulted in a concomitant depletion of anti-K562 NK activity of mouse spleen cells, and (b) both K562 and YAC cells could inhibit their own as well as each others lysis in a cross-competition assay. Anti-H-2 antiserum could not induce anti-K562 NK activity in spleen cells previously depleted of NK cells by absorption on YAC monolayers, indicating that alloantiserum does not act by recruiting otherwise nonreactive cells to become cytotoxic toward K562 target cells. In a target-binding assay, K562 binding of NK cells (T-cell-, B-cell-, and macrophage-depleted spleen cells) increased five- to eightfold in the presence of anti-H-2 antiserum whereas YAC cells binding of NK cells was not increased. H-2 antigens per se did not appear to be involved in the alloantisera effect since anti-NK antiserum directed against a non-H-2 antigen selectively expressed on NK cells, showed a similar selective NK enhancing effect. Protein A, a reagent which binds to the Fc region of immunoglobulin molecules, completely blocked the alloantiserum induced augmentation of anti-K562 NK activity, but did not alter basal NK activity. Moreover, the F(ab)₂ fraction of alloantibodies failed to enhance anti-K562 cytotoxic activity of mouse spleen cells, indicating a crucial role for the Fc portion of the alloantibodies attached to the NK cells, in NK augmentation. Utilization of several target cell lines with or without membrane Fc receptors (FcR) revealed that alloantiserum enhanced the lysis of only FcR⁺ target cells. It is proposed that alloantibody-coated NK cells, as a result of a secondary interaction between attached alloantibody and Fc receptors on target cells, interact more readily with the target cells and thereby cause a higher level of lytic activity.     

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]     

Use ofH-2 mutations to quantitate alloreactivity: Alloreactivity is strongest against H-2 antigens which are closest to self

Lymph-node cells fromH-2 allogeneic, intra-H-2 recombinant andH-2 mutant congenic strains were sensitized in limiting dilution cultures to quantitate the cytotoxic T-lymphocyte precursor frequencies (CTL.Pf) against antigens encoded by different regions of theH-2 complex. When fourH-2K ^b mutants of C57BL/6 (B6) were tested, we observed anti-B6 CTL.Pf that were as high or higher than those of recombinant strains which differ from B6 at theK end of theH-2 complex. Relative to strains completelyH–2 allogeneic to B6, the CTL.Pf inH-2 ^bm1,H-2 ^bm3 andH-2 ^bm5 averaged 40–50 percent, andH-2 ^bm8 averaged 140 percent. Recombinant strains B10.A (4R) and B10.D2 (R103), which differ from B6 at theK end of theH-2 complex, averaged 60 percent of the completelyH-2 allogeneic value. Since the mutant and wild-type gene products have no serological and minimal structural differences relative to other alleles atH-2K, these results indicate that the CTL.Pf does not increase with increasing H-2 antigenic disparity between any two strains. Rather, the data suggests that the T-cell receptor repertoire recognizes those H-2 molecules or determinants closest to self.     

Natural resistance of irradiated 129-strain mice to bone marrow allografts: Genetic control by theH-2K region

A major genetic determinant of natural resistance to bone marrow allografts, designated asHh-3, was mapped to theH-2K region. This gene may code for or regulate the expression of cell surface structures selectively expressed on donor hemopoietic cells and recognized by naturally occurring cytotoxic effectors. Resistance was observed as failure of donor cell growth in the spleen of irradiated 129-strain (H-2 ^bc ) recipients of H-2^k bone marrow cells. The mapping was accomplished by substituting donor cells bearingk alleles throughout theH-2 complex with cells of recombinant mouse lines bearingk alleles at definedH-2 regions. The host antigraft reaction underlying resistance was abrogated by pretreating 129-strain mice with either rabbit antimouse lymphocyte serum or the antimacrophage agent silica. Grafting of H-2K^k cells into mice ancestrally unrelated to 129 but sharing theH-2 ^bc or the similarH-2 ^b haplotype, and intoH-2 ^b/k ,H-2 ^k/bc , andH-2 ^k/d F₁ hybrids revealed that resistance was unique to 129 mice, since mice of the other strains, including F₁ hybrids, were susceptible to the grafts. Thus,Hh-3 incompatibility was a necessary but insufficient condition for the manifestation of allogeneic resistance; other genetic factors not associated withH-2 conferred responder status to 129-strain mice and nonresponder status to D1.LP, B10.129(6M), B10, B6, and possibly to F₁ hybrid mice. The possible relationships between allogeneic resistance to H-2^k marrow grafts, hybrid resistance to H-2^k lymphomas, and F₁ hybrid antiparental H-2^k cytotoxicity induced in vitro are discussed.     

Nonspecific suppressor elements in murine allogeneic radiation chimeras.

Spleen cells from long-term mouse allogeneic radiation chimeras were tested for their ability to modulate the graft-versus-host (GVH) or plaque-forming cell (PFC) response of normal lymphocytes transplanted in lethally X-irradiated recipients. In vivo GVH proliferation of normal lymphocytes (syngeneic to donor cells of the chimera) against antigens of host-type in which the chimeric state had been established was reduced by chimera cells. Inhibition varied, some chimeras suppressing GVH more than others and a few not suppressing at all. The suppressive effect was abrogated if the chimera cells were treated with anti-θ; treatment with anti-IgM did not eliminate this activity. When mixtures of normal donor lymphocytes and chimera cells were given to irradiated recipients genetically different from host or donor, reduction of donor cell GVH also occurred. Further, chimera cells reduced the GVH activity of normal host cells in irradiated recipients differing from the host at one H-2 locus and from the donor at minor histocompatibility loci. The modulating effect of spleen cells from chimeras on the PFC response by normal lymphocytes also varied. Six chimeras induced a 25 to 90% suppression, two enhanced the response, and one showed no effect. Where suppression occurred, treatment of chimera cells with anti-θ most often, but not always, restored PFC production. Our results show that the suppressive action of splenic lymphoid cells by chimeras is highly nonspecific and variable in expression. We suggest that tolerance in chimeras may be mediated by nonspecific suppressor elements leading to unresponsiveness to a variety of antigens including SRBC.     

Allosuppressor- and allohelper-T cells in acute and chronic graft-vs-host disease. IV. Activation of donor allosuppressor cells is confined to acute GVHD

Groups of nonirradiated BDF1 mice were injected with unseparated spleen cells from B10, B10.D2, or DBA/2 donors. The diverse clinical and pathologic symptoms that developed during the course of the ensuing graft-vs-host reaction (GVHR) were related to the functional subsets of donor-T cells activated in the host. The activation of F1-specific donor T suppressor (TS) cells was confined to those GVH F1 mice that developed acute GVH disease (GVHD) (donor B10 or B10.D2). Moreover, activation in these GVH F1 mice of the Lyt-1-2+ donor TS cells sharply preceded the onset of and coincided with (week 2 to 6) the suppressive pathologic symptoms characteristic of acute GVHD, such as pancytopenia and suppression of splenic IgG production. The activation of these alloreactive TS effector cells was briefly preceded by the activation of F1-specific Lyt-1+-2- donor T helper (TH) cells and stimulation of the host's lymphoid tissue. Thus, in acute GVHD, a sequential alloactivation first of donor TH and then of TS cells was found. Those F1 mice that recovered from acute GVHD and developed stimulatory pathologic symptoms showed a concomitant loss of donor TS cell activity. An initial activation of F1-specific Lyt-1 +2- donor TH cells was also found in that parent----F1 combination (donor DBA/2), which failed to develop acute GVHD. Significantly in that combination, the alloactivation of donor TH cells was not followed by activation of significant numbers of donor TS cells. Instead, the DBA/2-injected BDF1 mice directly developed a persistent increase in splenic Ig formation and lupus-like GVHD.     

Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient

Allo-chimerism and clonal elimination of self antigen (Ag) (Ia + Mls-1^a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti-Thy-1 monoclonal antibody (mAb) plus complement (C) (T^– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti-Thy-1 mAb alone (T⁺ chimeras). When lethally irradiated AKR (Mls-1^a) mice were reconstituted with BM cells from B10 or B10 H-2 congenic mice, both T⁺ and T^– chimeras were entirely free of signs of graft-versus-host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T⁺ chimeras but not in T^– chimeras. On the other hand, clonal elimination of Vβ6⁺ T cells reactive to the recipient Ag (Mls-1^a) was abolished in T⁺ chimeras but successfully induced in T^– chimeras. The Vβ6⁺ T cells not eliminated in T⁺ chimeras showed depressed responses against Mls-1^a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti-Thy-1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.     

Mlsa generated suppressor cells. I. Suppression is mediated by double-negative (CD3+CD5+CD4-CD8-) alpha/beta T cell receptor-bearing cells.

Grafting of cells from B10.D2 (H-2d) donors into H-2 compatible lethally irradiated (DBA/2 x B10.D2)F1 hosts results in a severe graft-vs-host reaction (GVHR), developed against DBA/2 non-H-2 Ag, with only 0 to 10% of animals surviving. This GVHR mortality rate is dramatically reduced (90 to 100% of animals survive) by donor preimmunization against Mlsa determinants. The protection against GVHR correlates with a decreased B10.D2 anti-DBA/2 proliferative response in vitro. Both in vivo and in vitro phenomena are associated with activation of CD5+ suppressor T cells in the spleens of immunized mice. The present work was designed to study the origin of these suppressor cells and to further characterize their phenotype. The results show that significant suppression is not inducible in "B" mice. In contrast, in mice that were only thymectomized or else pretreated in vivo with anti-CD4 or anti-CD8 mAb, the suppressor cells are activated as efficiently as in normal mice. The suppression of GVHR mortality and proliferative responses in vitro is lost after depletion from preimmunized splenocytes of CD5+ T cells and remains unaltered after depletion of CD4+ or CD8+ T cells or both. Depletion of asialo GM1+ cells removes all NK activity, whereas the suppression is decreased only slightly. FACS analysis showed that double-negative (DN) cells from normal and immunized mice contain both CD3+ and CD3- cells; the vast majority of the CD3+ DN T cells express the alpha/beta T cell receptor. Suppression of GVHR and of proliferative responses in vitro are abrogated after elimination of CD3+ cells. These results suggest that Mlsa generated suppressor cells: 1) are derived from post-thymic long-lived T cell precursors; 2) are low asialo GM-1+ but do not exhibit NK activity; 3) belong to a subset of peripheral CD5+ DN T cells bearing a CD3-associated alpha/beta-heterodimer.     

Adoptive transfer of H-2-incompatible lymphokine-activated killer (LAK) cells: an approach for successful cancer immunotherapy free from graft-versus-host disease (GVHD) using murine models

We investigated whether the adoptive transfer of H-2-incompatible lymphokine-activated killer (LAK) cells would efficiently demonstrate antitumor activity without damaging the normal host cells. Allogeneic LAK cells (5 X 10(7] did not cause graft-versus-host disease (GVHD) in irradiated recipients, whereas more than half of the mice transferred with the same dose of fresh allogeneic spleen cells developed GVHD. Repeated transfer (three times at 4-day intervals, 1.2 X 10(8) cells/mouse) did not result in GVHD. Graft-versus-host reaction (GVHR), which is detectable by spleen enlargement of recipients transferred with allogeneic lymphoid cells was also absent in LAK cell-transferred mice of all strain combinations tested. Host immune responses were not affected in these mice. Therefore, it is feasible to transfer allogeneic LAK cells. With the antitumor efficacy of allogeneic LAK cells, they preferentially lysed allogeneic tumor targets. Adoptive transfer of the allogeneic LAK cells led to a significant decrease in the lung-colonizing foci of intravenously inoculated B16 melanoma cells. Allogeneic LAK cells and syngeneic ones were equally active, in vivo. The use of allogeneic LAK cells may prove to be a valuable method for effective clinical antitumor immunotherapy.     

In vitro analysis of allogeneic lymphocyte interaction. III. Generation of a helper allogeneic effect factor (AEF) across an I-J subregion disparity.

Allogeneic effect factors (AEF) were produced across an I-J subregion incompatibility. The helper activity of these AEFs is H-2 restricted since they help B cells only of the stimulator haplotype and of other haplotypes that carry the same I-J subregion gene(s) as the stimulator haplotype. Immunoadsorption studies demonstrate that they consist of I-J determinants derived initially from the GVHR host and MLR stimulator cells and not the GVHR donor and MLR responder cells used to generate AEF. It is postulated that the genetic restriction of AEF helper activity is mediated in part by the ability of the GVHR activated donor T cells to acquire, in vivo, recipient T cell and/or macrophage derived I-J determinants. Cellular adsorption studies indicate that AEF helper activity may be adsorbed by B cells, but neither T cells nor macrophages, of the stimulator haplotype. The results suggest that an I-J-positive AEF interacts with an I-J subregion controlled complementary recognition structure on a target B cell and, after antigenic stimulation, activates that B cell to IgG antibody synthesis.     

Study of H-2 mutations in mice. IV. A comparison of the mutants M505 and Hz1 by skin grafting and serological techniques

The line B6.M505 is congenic with C57BL/6JY and carries a mutant form of theH-2 ^b haplotype designatedH-2 ^bd . The mutant site 505 was located by the F₁ tests in theK end of theH-2 gene complex. The M505 mice are histoincompatible with the B6.C(Hz1) line (haplotypeH-2 ^ba ) carrying another mutation in theK end ofH-2 ^b . Inability of M505 to complement Hz1 in tests with B6 skin grafting is considered as an evidence that the same gene was altered by both mutations. The gained H antigens of two mutants can cross-react in vivo as revealed by accelerated rejection of Hz1 skin grafts by B6 recipients presensitized with M505 spleen cells. The lost antigenic determinants are not identical as shown by accelerated rejection of B6 skin grafts by Hz1 hosts preimmunized with M505 spleen cells. Absorptions of the antiserum ASY-015, (d×a) anti-i, anti-H-2.33 with M505 spleen cells did not clear forH-2 ⁱ ,H-2 ^b andH-2 ^ba , and absorptions with Hz1 did not clear forH-2 ⁱ ,H-2 ^b , andH-2 ^bd . These results show that changes of histocompatibility determinants may be accompanied by loss of some haptenic determinants in the Hz1 and M505 mutations.     

Biochemical evidence for a separate,MHC-linked locus encoding H-2.28 antigens

In comparing the tryptic peptide maps of the H-2L and H-2D glycoprotein antigens isolated from NP-40 lysates of RADA1 (H-2 ^a ) leukemic cells, no more than 37% of the observed arginine-containing tryptic peptides are found to be homologous. Thus, the primary amino-acid sequences of these two antigens are probably less than 90% homologous. This constitutes the strongest evidence to date that the MHC-linkedH-2L region encodes H-2L antigens separately from theH-2D region, even though H-2L antigens bear D-end-associated antigenic determinants of the H-2.28 family. The anti-H-2.28 alloantiserum (k×r anti h2) used to precipitate H-2L antigens in this investigation was the NIH contract antiserum D28b. As the tryptic peptide maps also surprisingly revealed, D28b precipitates H-2D antigens as well and, thus, anti-H-2.4 immunoadsorbants were employed to isolate H-2L free of H-2D antigens. In light of the dual specificity of D28b, its reactivity with BALB/c-H-2 ^dm2 mutant cells was re-examined. Even though mutant lymphocytes, which lack H-2L but not H-2D antigens, are not cytotoxically lysed by D28b (as are parental H-2^d cells), D28b appears to precipitate H-2D antigens from NP-40 extracts of mutant splenocytes.