Host-residual invariant NK T cells attenuate graft-versus-host immunity

Invariant NK T (iNKT) cells have an invariant TCR-alpha chain and are activated in a CD1d-restricted manner. They are thought to regulate immune responses and play important roles in autoimmunity, allergy, infection, and tumor immunity. They also appear to influence immunity after hemopoietic stem cell transplantation. In this study, we examined the role of iNKT cells in graft-vs-host disease (GVHD) and graft rejection in a mouse model of MHC-mismatched bone marrow transplantation, using materials including alpha-galactosylceramide, NKT cells expanded in vitro, and Jalpha18 knockout mice that lack iNKT cells. We found that host-residual iNKT cells constitute effector cells which play a crucial role in reducing the severity of GVHD, and that this reduction is associated with a delayed increase in serum Th2 cytokine levels. Interestingly, we also found that host-residual iNKT cause a delay in engraftment and, under certain conditions, graft rejection. These results indicate that host-residual iNKT cells attenuate graft-vs-host immunity rather than host-vs-graft immunity.     

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

Kinetics of the anti-recipient cytotoxic cell response of mice with minor histocompatibility antigen graft-vs-host disease

The kinetics of the anti-recipient cytotoxic cell response of spleen cells from mice undergoing graft-vs-host disease (GVHD) induced to minor histocompatibility antigens were studied. Two population of cytotoxic cells were identified. Cytotoxic T lymphocytes (CTL) were present in recipient spleens 2 and 3 wk after transplantation but disappeared from the spleens before the onset of clinical disease. Cytotoxic T lymphocyte precursors (CTLp) were first detected in recipient spleens 2 wk after transplantation and were present during clinical disease. CTL may function as effectors in GVHD induced to minor histocompatibility antigens.     

Mechanism of protection from graft-vs-host disease in murine mixed allogeneic chimeras. I. Development of a null cell population suppressive of cell-mediated lympholysis responses and derived from the syngeneic bone marrow component

Splenocyte populations from whole body-irradiated recipients of mixed T cell-depleted (TCD) syngeneic and allogeneic (complete H-2 disparity) bone marrow, or of TCD syngeneic marrow alone, contain cells with the ability to suppress the generation of cell-mediated lympholysis responses in vitro. This activity, which is present by 8 days after bone marrow transplantation and persists for several weeks, has been analyzed for possible veto-like or other specificity. Although reproducible patterns of suppression were observed, depending both on host strain and on the genetic combination of the response examined, the overall suppression in vitro most closely resembles that which has been ascribed to "natural suppressor" cells in other systems. The suppression appears to be mediated by a non-T cell, non-B cell, nonadherent, asialo GM1-negative population. Cold target inhibition and CTL activity of chimeric cells have been ruled out as factors contributing to the observed suppression. Significantly, in mixed chimeras, suppression was found to be mediated exclusively by cells which were syngeneic to the recipient in both recipient strains tested. The rapid development of this suppressive activity may explain the resistance to graft-vs-host disease conferred on whole body-irradiated mice by the addition of TCD syngeneic marrow to an allogeneic graft-vs-host disease-producing inoculum.     

Lymphocyte function in human bone marrow. I. Characterization of two T cell populations regulating immunoglobulin secretion

We analyzed the regulation of immunoglobulin (Ig) production in short-term cultures of human (rib) bone marrow cells. In contrast to blood or tonsil cell cultures, large quantities of IgG and IgA, but not IgM, were secreted by unstimulated marrow cells. The addition of pokeweed mitogen or phytohemagglutinin resulted in the suppression of this Ig secretion. Both mitogens induced the production of high levels of interleukin 2 (IL 2) in marrow cultures, and the addition of IL 2 alone mimicked the suppressive effect of mitogens. Incubation of marrow cells with Epstein Barr virus resulted in enhanced Ig secretion, primarily of the IgM isotype. The addition of mitogen or IL 2 suppressed Ig production in these cultures as well. The mitogen-induced suppression of Ig secretion in stimulated or unstimulated marrow cultures was inhibited by the monoclonal anti-TAC (IL 2 receptor) antibody. Cell separation experiments indicated that the induction of suppressor activity in marrow cultures involved two distinct populations of marrow-resident T lineage cells. The first population responds to activation by mitogens with the production of IL 2. This population has a surface phenotype appropriate for helper T cells. The second T cell population expresses T8 and TAC determinants. These cells acquire suppressor cell activity after exposure to IL 2. The expression of suppressor function does not require additional (e.g., mitogenic) activation signals. The IL 2-dependent marrow suppressor T cells represent a newly recognized T lymphocyte subset. The regulatory pathway delineated may be important for the regulation of antibody formation in bone marrow, the major site of Ig production in man.     

Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation

Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.     

Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients

4-1BB is expressed on activated CD4(+) and CD8(+) T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8(+) T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4(+) and CD8(+) T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4(+) T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8(+) and CD4(+) T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. alpha 4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4(+) and CD8(+) T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.     

Naive and memory T cells induce different types of graft-versus-host disease

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4(+) naive (CD62L(high)CD44(low)) T cells and CD4(+) effector memory (CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-gamma in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.     

Use of lymphokine-activated killer cells to prevent bone marrow graft rejection and lethal graft-vs-host disease

Prompted by our recent finding that lymphokine-activated killer (LAK) cells mediate both veto and natural suppression, we tested the ability of adoptively transferred LAK cells to block two in vivo alloreactions which complicate bone marrow transplantation: resistance to transplanted allogeneic bone marrow cells, and lethal graft-vs-host disease. Adoptive transfer of either donor type B6D2 or recipient-type B6 lymphokine-activated bone marrow cells, cells found to have strong LAK activity, abrogated or inhibited the resistance of irradiated B6 mice to both B6D2 marrow and third party-unrelated C3H marrow as measured by CFU in spleen on day 7. The ability of lymphokine-activated bone marrow cells to abrogate allogeneic resistance was eliminated by C lysis depletion of cells expressing asialo-GM1, NK1.1, and, to a variable degree, Thy-1, but not by depletion of cells expressing Lyt-2, indicating that the responsible cells had a LAK cell phenotype. Similar findings were obtained by using splenic LAK cells generated by 3 to 7 days of culture with rIL-2. Demonstration that allogeneic resistance could be blocked by a cloned LAK cell line provided direct evidence that LAK cells inhibit allogeneic resistance. In addition to inhibiting allogeneic resistance, adoptively transferred recipient-type LAK cells prevented lethal graft-vs-host disease, and permitted long term engraftment of allogeneic marrow. Irradiation prevented LAK cell inhibition of both allogeneic resistance and lethal graft-vs-host disease. These findings suggest that adoptive immunotherapy with LAK cells may prove useful in preventing graft rejection and graft-versus-host disease in human bone marrow transplant recipients.     

CD101 surface expression discriminates potency among murine FoxP3+ regulatory T cells

CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25-30% of CD4+CD25+ Treg and approximately 20% of conventional memory T cells. CD101(high) Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101(low) Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101(high) Treg, compared with CD101(low) Treg. Moreover, treatment with CD101(high) Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101(high) Treg all of the in vivo suppressor activity was contained within the CD62L(high) subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.     

Leucyl-leucine methyl ester treatment of donor cells permits establishment of immunocompetent parent----F1 chimeras that are selectively tolerant of host alloantigens

Treatment of murine lymphocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte precursors, and the capacity to cause lethal graft-vs-host disease, whereas bone marrow stem cell function and alloantigen-induced L3T4+ T helper function remains intact. The present studies assess the immunocompetence of allogeneic bone marrow chimeras established by reconstituting irradiated (C57BL/6 X DBA/2)F1 (B6D2F1) mice with Leu-Leu-OMe-treated C57BL/6 (B6) bone marrow and spleen cells. Spleen cells from such chimeras were found to have normal B and T cell mitogenic responses. Furthermore, levels of natural-killer cell function were comparable to those observed in B6----B6 syngeneic radiation chimeras established without Leu-Leu-OMe treatment of donor cells. Spleen cells from B6----B6D2F1 mice were identical with B6----B6 or B6 mice in allostimulatory capacity and thus contained no discernible cells of non-H-2b phenotype. Whereas B6----B6D2F1 spleen cells demonstrated alloproliferative and allocytotoxic responses toward H-2k bearing spleen cells, no H-2d specific proliferative or cytotoxic responses could be elicited. B6----B6D2F1 spleen cells did not suppress the generation of anti-H-2d or anti-H-2k proliferative or cytotoxic responses from control B6 spleen cells. Furthermore, addition of rat concanavalin A supernatants did not reconstitute anti-H-2d responses of B6----B6D2F1 chimeric spleen cells. Thus, Leu-Leu-OMe treatment of B6 donor cells not only prevents lethal graft-vs-host disease, but also permits establishment of long-lived parent----F1 chimeras that are selectively tolerant of host H-2 disparate alloantigens, but fully immunocompetent with respect to natural killer cell function, B and T cell mitogenesis, and anti-third party alloresponsiveness.     

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.     

Mechanism of protection from graft-versus-host disease mortality by IL-2. III. Early reductions in donor T cell subsets and expansion of a CD3+CD4-CD8- cell population.

Reducing the graft-vs-host disease (GVHD)-promoting capacity of allogeneic T cells while maintaining alloengraftment and graft-vs-leukemia effects remains an important but elusive goal in clinical bone marrow transplantation (BMT). We have recently demonstrated that a short course of high dose IL-2 administered at the time of BMT has a powerful protective effect against GVHD mortality in mice. This short course of IL-2 is able to protect mice from both acute and chronic GVHD without sacrificing alloengraftment or graft-vs-leukemia effects of allogeneic T cells. Because the early administration of IL-2 seems to be crucial for this effect, we have studied the early lymphoid repopulation events after lethal irradiation and allogeneic BMT. These studies show that there are consistent delays in splenic repopulation by allogeneic cells after BMT in IL-2-treated animals compared with their untreated cohorts. Even greater percent reductions were seen in donor splenic T cell populations in the first few days after BMT in IL-2-treated animals. Splenic cells with the CD3+CD4-CD8- phenotype were increased in IL-2 treated animals at days 3 and 4 after BMT. This phenotype resembles that of bone marrow-derived cells which have been previously shown to inhibit GVHD, suggesting a possible mechanism for the protective effect of IL-2.     

Ex vivo-expanded CD4+CD25+ immunoregulatory T cells prevent graft-versus-host-disease by inhibiting activation/differentiation of pathogenic T cells

CD4+CD25+ immunoregulatory T cells (Tregs) can be administered to inhibit graft-vs-host disease (GVHD) while preserving graft-vs-leukemia activity after allogeneic bone marrow transplantation in mice. Preclinical studies suggest that it is necessary to infuse as many Tregs as conventional donor T cells to achieve a clinical effect on GVHD. Thus, it would be necessary to expand Tregs ex vivo before transplantation. Two strategies have been proposed: expansion of Tregs stimulated by anti-CD3/CD28-coated microbeads for polyclonal activation or by host-type allogeneic APCs for selecting Tregs specific for host Ags. In this study, we describe the mechanisms by which ex vivo-expanded Tregs act on donor T cells to prevent GVHD in mice. We demonstrate that expanded Tregs strongly inhibited the division, expansion, and differentiation of donor T cells, with a more pronounced effect with Tregs specific for host Ags. These latter cells permit the efficient and durable control of GVHD and favor immune reconstitution.     

Accelerated onset and increased severity of acute graft-versus-host disease following adoptive transfer of DR6-deficient T cells

DR6 is a recently identified member of the TNFR family. In a previous study, we have shown that DR6 KO mice have enhanced CD4(+) T cell proliferation and Th2 cytokine production. Acute graft-vs-host disease (GVHD) results from the activation and expansion of alloreactive donor T cells following bone marrow transplantation. In this article, we demonstrate that the transfer of donor T cells from DR6 KO mice into allogeneic recipient mice in a parent into an F(1) model of acute GVHD results in a more rapid onset of GVHD with increased severity. Recipients of DR6 KO T cells exhibit earlier systemic symptoms of GVHD, more rapid weight loss, earlier histopathological organ damage in the thymus, spleen, and intestines, and earlier mortality. The rapid onset of GVHD in these mice may be attributable to the enhanced activation and expansion of DR6 KO CD4(+) and CD8(+) T cells. Our findings support the hypothesis that DR6 serves as an important regulatory molecule in T cell immune responses. The identification and use of DR6 ligands and/or agonistic Abs to DR6 may represent useful therapeutics in the treatment of T cell-mediated diseases such as GVHD.     

Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "natural suppressor" cells

We developed a nonmyeloablative host conditioning regimen in a mouse model of MHC-mismatched bone marrow transplantation that not only reduces radiation toxicity, but also protects against graft-vs-host disease. The regimen of fractionated irradiation directed to the lymphoid tissues and depletive anti-T cell Abs results in a marked change in the residual host T cells, such that NK1.1+ or DX5+asialo-GM1+ T cells become the predominant T cell subset in the lymphoid tissues of C57BL/6 and BALB/c mice, respectively. The latter "natural suppressor" T cells protect hosts from graft-vs-host disease after the infusion of allogeneic bone marrow and peripheral blood cells that ordinarily kill hosts conditioned with sublethal or lethal total body irradiation. Protected hosts become stable mixed chimeras, but fail to show the early expansion and infiltration of donor T cells in the gut, liver, and blood associated with host tissue injury. Cytokine secretion and adoptive transfer studies using wild-type and IL-4(-/-) mice showed that protection afforded by NK1.1+ and DX5+asialo-GM1+ T cells derived from either donors or hosts conditioned with lymphoid irradiation is dependent on their secretion of high levels of IL-4.     

Effect of selective T cell depletion of host and/or donor bone marrow on lymphopoietic repopulation, tolerance, and graft-vs-host disease in mixed allogeneic chimeras (B10 + B10.D2----B10)

Reconstitution of lethally irradiated mice with a mixture of T cell-depleted syngeneic plus T cell-depleted allogeneic bone marrow (B10 + B10.D2----B10) leads to the induction of mixed lymphopoietic chimerism, excellent survivals, specific in vivo transplantation tolerance to subsequent donor strain skin grafts, and specific in vitro unresponsiveness to allogeneic donor lymphoid elements as assessed by mixed lymphocyte reaction (MLR) proliferative and cell-mediated lympholysis (CML) cytotoxicity assays. When B10 recipient mice received mixed marrow inocula in which the syngeneic component had not been T cell depleted, whether or not the allogeneic donor marrow was treated, they repopulated exclusively with host-type cells, promptly rejected donor-type skin allografts, and were reactive in vitro to the allogeneic donor by CML and MLR assays. In contrast, T cell depletion of the syngeneic component of the mixed marrow inocula resulted in specific acceptance of allogeneic donor strain skin grafts, whether or not the allogeneic bone marrow was T cell depleted. Such animals were specifically unreactive to allogeneic donor lymphoid elements in vitro by CML and MLR, but were reactive to third party. When both the syngeneic and allogeneic marrow were T cell depleted, variable percentages of host- and donor-type lymphoid elements were detected in the mixed reconstituted host. When only the syngeneic bone marrow was T cell depleted, animals repopulated exclusively with donor-type cells. Although these animals had detectable in vitro anti-host (B10) reactivity by CML and MLR and reconstituted as fully allogeneic chimeras, they exhibited excellent survival and had no in vivo evidence for graft-vs-host disease. In addition, experiments in which untreated donor spleen cells were added to the inocula in this last group suggest that the presence of T cell-depleted syngeneic bone marrow cells diminishes graft-vs-host disease and the mortality from it. This system may be helpful as a model for the study of alloresistance and for the identification of syngeneic cell phenotypes, which when present prevent engraftment of allogeneic marrow.     

Destruction of autologous human lymphocytes by interleukin 2-activated cytotoxic cells

Human and murine lymphocyte populations differentiate into lymphokine activated killer (LAK) cells after in vitro or in vivo exposure to interleukin 2 (IL 2). LAK cells mediate destruction of neoplastic tissue in vitro and have been reported to spare normal tissue. However, systemic toxicity is observed in mice and patients receiving IL 2 infusions. Some aspects of this toxicity are similar to that seen in graft-vs-host disease, suggesting that IL 2 may cause an immune-mediated destruction of normal tissues. We have evaluated this issue by examining the destructive potential of fresh human lymphocytes cultured in media containing highly purified recombinant human IL 2. In the absence of any exogenous antigen or allogeneic stimulating cells, strong proliferative responses were induced after 6 days of exposure to IL 2. Lymphocytes harvested from these 6-day cultures were highly cytotoxic to K562 and Daudi target cells. These IL 2-activated cells were also cytotoxic against autologous and allogeneic normal lymphocyte target cells. This autologous lymphocyte destruction was detected in media containing autologous serum and was directly dependent on the concentration of IL 2 added to the cultures. These studies demonstrate that populations of IL 2-activated lymphocytes, containing LAK activity, can mediate low-level but significant destruction of normal lymphocytes in vitro.     

Defective thymic education of L3T4+ T helper cell function in graft-vs-host mice

Our study investigates the effect of a prior graft-vs-host (GVH) reaction on the subsequent ability of irradiated, bone marrow-re-populated mice to develop T cell function. The results indicate that such GVH-bone marrow transplanted (BMT) mice do not generate CTL responses to trinitrophenyl-modified syngeneic cells (TNP-self), but do generate strong CTL activity to H-2 alloantigens. This selective deficiency in TNP-self CTL response potential appeared as early as 10 days after GVH, and required both L3T4+ and Lyt-2+ donor T cells. The in vitro addition of either soluble Th factors or L3T4-enriched spleen cells from normal mice circumvented the defect in the TNP-self response in GVH-BMT mice. These results indicate that T effector function was not defective, and instead suggest a Th defect. Cell depletion and antibody-blocking, as well as IL-2 production experiments, indicate that the Th defect was selective for L3T4+ Th population and not for Lyt-2+ Th population. This defect in L3T4 Th function is not accounted for by the approximate twofold reduction in L3T4 cell numbers in GVH-BMT mice, because IL-2 production and CTL generation to L3T4-dependent Ag were at least eightfold below control levels. Rather, defective L3T4 Th function appears to be the consequence of a GVH-induced defect in thymic maturation because the defect was corrected in vivo by a neonatal parental thymus graft before irradiation and bone marrow transplantation. This system may be useful for elucidating the role of the thymus in the maturation of Th cells. Our findings raise the possibility that impaired development of T cell function occurring in marrow grafted patients who have undergone a GVH reaction could be partly due to a GVH-induced thymic defect.