Anti-CD3 epsilon F(ab')2 fragments inhibit T cell expansion in vivo during graft-versus-host disease or the primary immune response to nominal antigen

This study was undertaken to distinguish between several mechanisms responsible for graft-vs-host disease (GVHD) protection in anti-CD3epsilonF(ab')2 fragment (Fr)-treated recipients: TCR down-modulation, deletion, failure of expansion, or anergy induction. To quantify alloreactive T cell expansion and function, thoracic duct lymphocytes (TDL) were analyzed. Sixfold fewer donor TDL T cells were recoverable from anti-CD3epsilonF(ab')2 Fr as compared with irrelevant F(ab')2 Fr-treated recipients at the time of peak T cell expansion in vivo. Kinetic analysis revealed that donor T cell expansion was inhibited and not simply delayed by anti-CD3epsilonF(ab')2 Fr. Similar proportions of TDL T cells in irrelevant and anti-CD3epsilonF(ab')2 Fr were undergoing apoptosis. Although TCR modulation was observed, donor TDL T cells had intact anti-host alloresponses as compared with irrelevant F(ab')2 Fr-treated recipients. Because donor CD4+ T cells are primarily responsible for GVHD in this model, an adoptive transfer system was used in which the function and kinetics of expansion of OVA-specific CD4+ TCR transgenic cells could be physically tracked. Relevant Fr severely blunted CD4+ TCR transgenic T cell clonal expansion after OVA administration. Nonviable transgenic and nontransgenic T cells were proportionally similar in OVA-pulsed recipients, regardless of whether relevant or irrelevant F(ab')2 Fr were given. After discontinuing Fr, transgenic T cells were found to have intact in vitro OVA-specific responses. Our current and previous results suggest that reduced donor T cell expansion and T cell depletion both contribute to GVHD protection by anti-CD3epsilonF(ab')2 Fr. These data have implications for designing therapeutic approaches directed toward TCR targeting in humans.     

Use of anti-CD3 epsilon F(ab')2 fragments in vivo to modulate graft-versus-host disease without loss of graft-versus-leukemia reactivity after MHC-matched bone marrow transplantation

The use of T cell-specific mAb in vivo for prevention and treatment of graft-vs-host disease (GVHD) and its impact on graft-vs-leukemia (GVL) reactivity was examined in a murine model of MHC-matched bone marrow transplantation (BMT). F(ab')2 fragments of a CD3 epsilon-specific mAb were administered to irradiated AKR (H-2k) hosts after transplantation of BM plus spleen cells from B10.BR donors (BMS chimeras). The effects on GVH and GVL reactivity were Ab dose- and schedule-dependent. A short course of mAb (qe2d, days 0 to 8) prevented clinical evidence of GVHD and mortality. Anti-CD3 F(ab')2 mAb reversed clinical symptoms of acute GVHD when delayed up to 18 days post-transplant. Anti-host (Mls-1a)-specific V beta 6+ cells were absent from the spleens of GVH-negative control mice, but persisted in Ab-treated BMS chimeras despite the absence of GVHD. Leukemic mice given 16.7 micrograms of Ab on days 0, 2, and 4 survived leukemia-free without developing severe GVHD. A longer course of Ab completely prevented GVHD, but led to leukemia relapse in tumor-bearing hosts, despite engraftment of donor T cells. The GVL effect was quantitatively stronger when Ab was used for GVH therapy as compared with GVH prevention. Some Ab-treated, GVH-free chimeras relapsed with lymphomas in unusual sites, suggesting that occult tumor cells may persist in nonlymphoid tissues. These experiments demonstrate that T cell-specific mAb can be used successfully in vivo to avoid severe GVHD, but that excessive or ill-timed administration of Ab may eliminate GVL reactivity.     

A critical role for CD48 antigen in regulating alloengraftment and lymphohematopoietic recovery after bone marrow transplantation

The binding of CD2, present on T cells, to its counterreceptor CD48 facilitates adhesion, signaling, alloantigen-induced cytokine production, and cytotoxic T-lymphocyte responses. Because these T-cell functions have been implicated in graft-versus-host disease (GVHD) pathogenesis, we have analyzed the effects of the CD2:CD48 pathway on GVHD mediated by CD4(+) and CD8(+) T cells infused into sublethally irradiated recipients. CD4(+) T-cell-mediated, and to a lesser extent, CD8(+) T-cell-mediated GVHD was inhibited by CD2 + 48 monoclonal antibody (MoAb) infusion. To assess the effects of combined MoAb infusion on alloengraftment, two different alloengraftment bone marrow transplantation (BMT) models were used. In both, MoAb infusion markedly inhibited alloengraftment and hematopoietic recovery post-BMT. To determine if the adverse effects on lymphohematopoiesis in the allogeneic BMT recipients were caused by an immune or nonimmune mechanism, studies were performed in congenic BMT recipients to preclude an immune mechanism as the cause for delayed recovery post-BMT. MoAb infusion resulted in impaired lymphohematopoietic recovery in congenic BMT recipients and markedly reduced day 12 colony-forming unit-spleen formation in syngeneic BMT recipients, consistent with a nonimmune mediated mechanism. Because the spleen is a site of early hematopoietic recovery post-BMT, studies were performed using adult splenectomized syngeneic BMT recipients. MoAb infusion delayed recovery in both nonsplenectomized and splenectomized recipients post-BMT, indicating that the delayed hematopoietic recovery was not the consequence of an abnormal homing pattern of hematopoietic progenitors to the spleen early post-BMT. CD48 MoAb was necessary and sufficient for the inhibition of GVHD lethality and delayed lymphohematopoietic effects of the combined MoAb regimen. CD48 MoAb was found to induce a profound modulation of CD48 antigen expression on BM cells, suggesting that the CD48 antigen may have an important function in hematopoiesis in the BM compartment. Taken together, these data provide evidence that the CD48 antigen plays a critical role in regulating hematopoiesis in post-BMT.     

3DinSight: an integrated relational database and search tool for the structure, function and properties of biomolecules

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, beta2-microglobulin-deficient mice (beta2m(-/-)) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. Beta2m(-/-) mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all beta2m(-/-) recipients were predominantly CD3+alphabetaTCR+CD4+ cells (15-20% of all splenocytes). In contrast, CD8+ cells engrafted in very small numbers (1-5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4+ T cells induced and maintained GVHD in mH-mismatched beta2m(-/-) mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic beta2m(-/-) cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Keratinocyte growth factor administered before conditioning ameliorates graft-versus-host disease after allogeneic bone marrow transplantation in mice

Keratinocyte growth factor (KGF) is important in tissue repair and wound healing and its administration can abrogate chemical- and radiation-induced tissue damage in rodents. We investigated KGF as a therapeutic agent for the prevention of graft-versus-host disease (GVHD)-induced tissue damage, morbidity, and mortality in an established murine allogeneic bone marrow transplantation (BMT) model. B10.BR (H2(k)) recipient mice were lethally irradiated and transplanted with C57BL/6 (H2(b)) bone marrow (BM) with spleen cells (BMS) as a source of GVHD-causing T cells. KGF-treated mice (5 mg/kg/d subcutaneously days -6, -5, and -4 pre-BMT) receiving BMS exhibited better survival than those not receiving KGF (P =.0027). Cyclophosphamide (Cy), a common component of total body irradiation (TBI)-containing regimens, was administered to other cohorts of mice at a dose of 120 mg/kg/d intraperitoneally on days -3 and -2 before BMT. KGF-treated mice again exhibited a better survival rate than those not receiving KGF (P =.00086). However, KGF-treated recipients receiving TBI or Cy/TBI BMS were not GVHD-free, as shown by lower body weights compared with BM groups. GVHD target tissues were assessed histologically during a 38-day post-BMT observation period. KGF ameliorated GVHD-induced tissue damage in the liver, skin, and lung (completely in some recipients) and moderately so in the spleen, colon, and ileum, even with Cy conditioning. These studies demonstrate that KGF administration, completed before conditioning, has potential as an anti-GVHD therapeutic agent.     

Allospecific CD8+ Tc1 and Tc2 populations in graft-versus-leukemia effect and graft-versus-host disease

Allogeneic CD8+ T cells mediate both a graft-vs-leukemia (GVL) effect and graft-vs-host disease (GVHD). To evaluate whether CD8 cells of defined cytokine phenotype differentially mediate these processes, alloreactive donor CD8+ T cells preferentially secreting type I or type II cytokines were generated by alloantigenic priming in vitro in the presence of IL-12 or IL-4, respectively. Both cytokine-secreting subsets lysed allogeneic tumor targets in vitro ("Tc1" and "Tc2" subsets). A transplantation model was established (B6 into B6C3F1, 1050 cGy host irradiation) using the 32Dp210 myeloid line (bcr/abl transfected, H-2k; 1 x 10(4) tumor cells/recipient). Compared with leukemia controls (death at 12.9 days post-bone marrow transplantation), both Tc1 and Tc2 recipients were conferred a survival advantage. At cell doses of 2 to 2.5 x 10(7), the Tc1-mediated GVL effect (mean survival of 34.2 days) was more potent than the Tc2-mediated GVL effect (mean survival of 20.5 days; Tc1 > Tc2, p = 0.009). On day 15, histologic examination showed that Tc1 recipients had undetectable tumor burdens, whereas Tc2 recipients had extensive leukemic infiltrates. However, Tc2 recipients had essentially no histologic evidence of GVHD, whereas Tc1 recipients had mild to moderate GVHD (average GVHD scores of 1/40 and 9.3/40, respectively). In contrast, recipients of uncultured CD8+ donor T cells developed severe GVHD (average GVHD score of 26.7/40). Because in vitro-generated, alloreactive Tc1 and Tc2 populations mediated GVL with reduced GVHD, we conclude that both subsets may improve the therapeutic outcome of allogeneic T cell transfers in patients with leukemia.     

Prevention of marrow graft rejection without induction of graft-versus-host disease by a cytotoxic T-cell clone that recognizes recipient alloantigens

In allogeneic marrow transplantation, donor T cells that recognize recipient alloantigens prevent rejection but also cause graft-versus-host disease (GVHD). To evaluate whether the ability to prevent marrow graft rejection could be dissociated from the ability to cause GVHD, we generated a panel of four different CD8 cytotoxic T-lymphocyte clones specific for H2(d) alloantigens. Three of the clones caused no overt toxicity when as many as 20 x 10(6) cells were infused intravenously into irradiated H2(d)-positive recipients, and one clone caused acute lethal toxicity within 1 to 3 days after transferring 10 x 10(6) cells into H2(d)-positive recipients. One clone that did not cause toxicity was able to prevent rejection of (C57BL/6J x C3H/HeJ)F1 marrow in 800 cGy-irradiated (BALB/cJ x C57BL/6J)F1 recipients without causing GVHD. Large numbers of cells and exogenously administered interleukin-2 were required to prevent rejection. These results with different CD8 clones suggest that GVHD and prevention of rejection could be separable effects mediated by distinct populations of donor T cells that recognize recipient alloantigens.     

Differential display analysis of murine collagen-induced arthritis: cloning of the cDNA-encoding murine ATPase inhibitor

Perforin-deficient (-/-) mice were used as T-cell donors for infusion into irradiated major histocompatibility complex (MHC)-disparate recipients to investigate the requirement for perforin-mediated cytolysis during graft-versus-host disease (GVHD) generation. Administration of 5x10(6) C57BL/6 (H2b) perforin -/- splenocytes was significantly less effective in inducing GVHD lethality when given to MHC class I + II disparate B10.BR (H2k) recipients, as compared with wild-type (+/+) controls. Perforin expression by donor T cells was not required for GVHD induction because recipients given fivefold higher numbers of perforin -/- donor splenocytes uniformly succumbed to lethal GVHD. Because both CD4+ and CD8+ donor T cells are required for optimal GVHD lethality in this strain combination, to discern the relative contribution of perforin-mediated cytolysis by CD4+ and CD8+ T cells, additional studies were performed. For these latter studies, we used a sensitive assay involving the infusion of highly purified CD4+ or CD8+ T cells into sublethally irradiated MHC class II or I disparate recipients, respectively. As compared with recipients of perforin +/+ T cells, recipients of either CD4+ or CD8+ perforin -/- T-cell subsets had a significant reduction in GVHD-mediated lethality at T-cell doses that were uniformly lethal. T-cell dose titration studies established that GVHD lethality in recipients of perforin -/- CD4+ or CD8+ T cells was reduced by approximately threefold. These data are the first to indicate that approaches to limit perforin-mediated cytolysis should be similarly effective in situations in which CD4+ or CD8+ T cells dominate the GVHD response.     

Interleukin-12 prevents severe acute graft-versus-host disease (GVHD) and GVHD-associated immune dysfunction in a fully major histocompatibility complex haplotype-mismatched murine bone marrow transplantation model

BACKGROUND: We have recently reported that interleukin (IL)-12 prevents acute graft-versus-host disease (GVHD)-induced mortality in a full major histocompatibility complex- plus multiple minor antigen-mismatched A/J-->B10 bone marrow transplantation (BMT) model. Because most patients have access to a haploidentical, one haplotype-mismatched donor, we have now investigated the protective effect of IL-12 against GVHD and GVHD-associated immune dysfunction in a haploidentical CBD2F1 (H2kxd) --> B6D2F1 (H2bxd) strain combination. METHODS: GVHD was induced by injecting CBD2F1 marrow and spleen cells into lethally irradiated B6D2F1 mice. RESULTS: In untreated control mice, GVHD resulted in 87% mortality by day 8 after BMT, with no survivors beyond day 17. Treatment with a single injection of IL-12 on the day of BMT led to 87% long-term survival, with no significant weight loss, diarrhea or GVHD skin changes. The majority of T cells recovering in these mice showed the CD62L+, CD44low, CD45RBhigh naive phenotype. These T cells showed specific tolerance to both host and donor histocompatibility antigens, but normal anti-third party (H2s) alloresponses in vitro. B-cell proliferative responses to lipopolysaccharide were also normal in IL-12-protected mice. Moreover, normal negative selection of thymocytes bearing T cell receptors with Vbeta that recognize endogenous superantigens was observed among CD4+CD8- thymocytes, indicating a lack of GVHD-associated thymic selection abnormalities in IL-12-protected allogeneic BMT recipients. CONCLUSIONS: IL-12 provides permanent protection against an otherwise severe, rapidly lethal GVHD, with no clinical manifestations of chronic GVHD, immunosuppression or autoimmune features, in a full major histocompatibilty complex haplotype-mismatched murine BMT model.     

Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate engraftment of sorted allogeneic stem cells (SCs) in vivo. In the present study, treatment of BM with RAMBS+C' resulted in the depletion of approximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impaired stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCRalphabeta T cells in the BM, because BM cultures from TCR alpha-chain knockout mice supported normal numbers of exogenous CAs. Because CD8+/TCR- cells are enriched for FCs, we tested the effect of adding these cells back to the treated BM prior to culture. The sorted FCs alone did not produce CAs, but did improve the ability of the impaired stroma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that supports CA formation by SCs, and show that the number of CAs formed depends on the "quality" of the stroma as well as the number of SCs seeded. These findings further suggest that CD8+/TCR- BM cells may be important for the establishment of functional stroma.     

Continuous spinal anaesthesia

CD59 is a cell membrane-bound complement regulatory protein on glomerular cells that inhibits C5b-9 assembly and insertion. This report describes a recently developed model of immune thrombotic microangiopathy (TMA) induced by the renal artery perfusion of anti-glomerular endothelial cell (anti-GEN) antibody. To examine the role of CD59 in protecting the GEN from immune-mediated injury, rats underwent selective renal artery perfusion with F(ab')2 fragments of anti-CD59 monoclonal antibody to block CD59 activity or control mouse IgG followed by anti-GEN antibody or control goat IgG. Neutralization of CD59 in normal rats did not result in any significant functional or histologic changes. Perfusion with anti-CD59 did not change deposition of the pathogenic anti-GEN IgG used to induce the TMA model. However, neutralization of CD59 in the TMA model resulted in more C5b-9 formation in glomeruli, accompanied by increased platelet and fibrin deposition, more severe endothelial injury, and reduced renal function compared with the animals perfused with control F(ab')2 fragments. These results demonstrate directly that CD59 serves a protective role for GEN in this TMA model of rats, and confirm that C5b-9 formation has a critical pathogenic role in the mediation of the disease. CD59 may play an important role in protecting glomerular endothelium from other complement-mediated types of injury.     

IL-2 reduces graft-versus-host disease and preserves a graft-versus-leukemia effect by selectively inhibiting CD4+ T cell activity

We have recently demonstrated, in a fully MHC-mismatched murine bone marrow transplantation model, that administration of a short course of high dose IL-2 markedly diminishes graft-vs-host disease (GVHD) without compromising alloengraftment or the graft-vs-leukemia (GVL) effect of allogeneic T cells. We have now evaluated the mechanism of the dissociation of GVL and GVHD observed in this model. We demonstrate that CD4+ T cells were required to produce severe, acute GVHD in the fully MHC-mismatched plus minor histocompatibility Ag-mismatched A/J-->B10 strain combination. The GVHD-producing activity of A/J CD4+ T cells administered without CD8+ T cells was inhibited by IL-2 treatment. In contrast, CD8+ T cells alone mediated the GVL effect observed in the EL4 leukemia/lymphoma model, and CD4+ cells did not contribute to this effect. This CD8-mediated GVL activity was not inhibited by IL-2 treatment. Because naive A/J CD8+ T cells administered without CD4+ T cells did not produce acute GVHD, we were unable to evaluate the effect of IL-2 in this model. However, when A/J donors were presensitized with B10 skin grafts, CD4-depleted A/J spleen cells were capable of causing acute GVHD in B10 recipients. This CD8-mediated GVHD was not inhibited by treatment with IL-2. However, IL-2 did partially inhibit the GVHD produced by nondepleted presensitized A/J spleen cells, probably due to selective inhibition of the function of presensitized A/J CD4+ T cells. The dissociation of GVHD and GVL against the EL4 leukemia/lymphoma in IL-2-treated mice can therefore be explained by selective inhibition by IL-2 of CD4 activity.     

Lethal murine graft-versus-host disease induced by donor gamma/delta expressing T cells with specificity for host nonclassical major histocompatibility complex class Ib antigens

Although T-cell receptor (TCR) alpha/beta expressing cells have a well-known role in graft-versus-host disease (GVHD) generation, the role of TCR gamma/delta expressing cells in this process has remained unclear. To elucidate the potential function of TCR gamma/delta cells in GVHD, we have used transgenic (Tg) H-2d mice (termed G8) that express gamma/delta heterodimers on a high proportion of peripheral T cells. In vitro, G8 Tg gamma/delta T cells proliferate to and kill C57BL/6 (B6) (H-2b) which express gene products (T10b and T22b) from the nonclassical major histocompatibility complex (MHC) class Ib H-2T region. The infusion of G8 Tg (H-2Td) TCR gamma/delta cells into lethally irradiated [900 cGy total body irradiation (TBI)] B6 (H-2b) mice resulted in the generation of lethal GVHD characterized histologically by destruction of the spleen, liver, lung, and colon. Lethal GVHD was prevented by the injection of anti-TCR gamma/delta monoclonal antibodies. Immunohistochemical analysis of B6 recipients post-bone marrow transplantation (BMT) confirmed that G8 Tg TCR gamma/delta cells infiltrated GVHD target tissues (skin, liver, colon, and lung) and were absent in recipients treated with anti-TCR gamma/delta monoclonal antibodies (MoAbs) but not anti-CD4 plus anti-CD8 MoAbs. In contrast, injection of TCR gamma/delta+ cells into irradiated (900 cGy TBI) B6.A-TIaa BoyEg mice that do not express either T10b or T22b did not induce lethal GVHD. Similarly, in a different GVHD system in which sublethal irradiation without bone marrow (BM) rescue was used, B6 but not B6.A-TIaa/BoyEg mice were found to be susceptible to TCR gamma delta+ cell mediated GVHD-induced lethality characterized by an aplasia syndrome. These results demonstrate that TCR gamma/delta cells have the capacity to cause acute lethal GVHD in mice and suggest that nonclassical MHC class Ib gene products expressed on GVHD target organs are responsible for G8 Tg TCR gamma/delta+ cell mediated lethality.     

Bispecific monoclonal antibodies for intravenous treatment of carcinoma patients: immunobiologic aspects

Immunobiologic parameters measured during a phase I trial of intravenously (i.v.) administered bispecific monoclonal antibodies (BsmAb) in renal cell carcinoma (RCC) patients are described. The BsmAb used, BIS-1, is reactive with a pancarcinoma-associated 38 kDa transmembrane glycoprotein, EGP-2, as well with the CD3 complex. Patients received during a 2 h i.v. infusion F(ab')2 fragments of BIS-1 at doses of 1, 3, or 5 micrograms/kg body weight during concomitantly applied subcutaneous (s.c.) IL-2 treatment. Acute but transient BIS-1 F(ab')2-related toxicity was observed at the 3 and 5 micrograms/kg dose level, and the maximum tolerated dose (MTD) was set at 5 micrograms/kg. A dose-dependent binding of BIS-1 F(ab')2 to circulating T lymphocytes was found. The in vivo occupancy of CD3 molecules on T lymphocytes was highest at teh end of the infusion period and then rapidly decreased, as shown by flow cytometry. A much slower decrease of BIS-1 F(ab')2 binding was observed in vitro, suggesting migration of BIS-1 F(ab')2-loaded T lymphocytes from the circulation. A strong but transitory leukopenia was observed, in which LFA-1 alpha bright, CD3/CD8 double positive T cells left the circulation preferentially. This phenomenon was most likely induced by elevated TNF-alpha and IFN-gamma plasma levels, which were at a maximum shortly after the end of the infusion. Isolated peripheral blood mononuclear cells obtained from patients directly after treatment with BIS-1 F(ab')2 at the 3 and 5 micrograms/kg dose level showed increased EGP-2-directed antitumor activity.     

Tumor-specific activation of mitogen-activated protein kinase in human colorectal and gastric carcinoma tissues

In the present study we investigated the effect of anti-CD3 stimulation on IL-3-induced histamine, IL-6, and IL-4 synthesis by murine hematopoietic precursor cells. These activities were strikingly decreased in splenocytes from mice that had received a single intravenous injection of 10 microg of anti-CD3 monoclonal antibody (mAb) 24 hours previously. A similar inhibition occurred after 24-hour in vitro stimulation of normal spleen cells with 1 microg/mL of anti-CD3 mAb. In both situations the inhibitory effect depended on T cell activation in that treatment with F(ab')2 fragments of anti-CD3 did not diminish secretion of histamine and cytokines. Cross-linking of Fas antigen on spleen cells mimicked the action of anti-CD3, provided that interferon (IFN)-gamma was present during the incubation period. Substantial amounts of this cytokine were detected in spleen cell supernatants, which were able to replace recombinant IFN-gamma during Fas receptor cross-linking. This effect was entirely mediated by IFN-gamma, as assessed by its neutralization in the presence of anti-IFN-gamma mAbs. In contrast to splenocytes, bone marrow cells responded normally to IL-3 after in vivo or in vitro stimulation with anti-CD3. They were also not affected by combined treatment with anti-Fas mAb and IFN-gamma. Together, our data support the notion that the decrease in IL-3-induced histamine and IL-6 production by splenocytes pretreated with anti-CD3 is mediated, at least in part, by Fas/FasL interactions, suggesting that the activity of extramedullary myeloid precursor cells can be modulated by molecules involved in apoptosis.     

Role of CD28 in acute graft-versus-host disease

Because CD28-mediated T-cell costimulation has a pivotal role in the initiation and maintenance of T-cell responses, we tested the hypothesis that CD28 is critical for the development of graft-versus-host disease (GVHD). We compared the in vivo effects of CD28(-/-) T cells transplanted from B6 donor with the CD28 gene deleted by homologous recombination with those of CD28(+/+) T cells transplanted from wild-type C57BL/6 (B6) donor. Fifty million CD28(-/-) or CD28(+/+) splenocytes from B6 mice were transplanted into unirradiated (B6 x DBA/2)F1 (BDF1) recipients. Unlike CD28(+/+), CD28(-/-) T cells from B6 mice had lower levels of proliferation and interleukin-2 production, had a limited ability to generate cytotoxic T lymphocytes against the recipient, and did not induce immune deficiency, despite survival in the recipient for at least 28 days. The ability to prevent rejection was reduced by the absence of CD28, because as many as 1.0 x 10(7) CD28(-/-) CD8(+) cells were needed to prevent rejection of major histocompatibility complex (MHC) class-I incompatible marrow in sublethally irradiated (550 cGy) bm1 recipients, whereas 8.0 x 10(5) CD28(+/+) CD8(+) T cells were sufficient to produce a similar effect, indicating that CD28 on donor CD8(+) cells helps to eliminate host immunity. Two million CD4(+) CD28(-/-) or CD28(+/+) T cells were transplanted into sublethally irradiated (750 cGy), MHC class-II incompatible (B6 x bm12)F1 recipients. With CD28(-/-) cells, 44% of the recipients died at a median of 20 days compared with 94% at a median of 15 days with CD28(+/+) cells (P < .001). Two million CD8(+) CD28(-/-) or CD28(+/+) T cells were transplanted into sublethally irradiated (750 cGy), MHC class-I incompatible (B6 x bm1) F1 recipients. With CD28(-/-) cells, 25% of the recipients died at a median of 41 days compared with 100% at a median of 15 days with CD28(+/+) cells (P < . 001). (B6 x bm12)F1 and (B6 x bm1)F1 mice surviving after transplantation of CD28(-/-) cells recovered thymocytes, T cells, and B cells in numbers and function comparable with that of irradiation-control F1 mice. We conclude that CD28 contributes to the pathogenesis and the severity of GVHD. Our results suggest that the severity of GVHD could be decreased by the administration of agents that block CD28 function in T lymphocytes.     

Control of monocyte influx in glomerulonephritis in transplanted kidneys in the rat

Induction of anti-Thy-1 nephritis in different strains of inbred rats results in phenotypically different types of renal diseases. In Wistar and Lewis (LEW) rats, a transient influx of ED1+ macrophages occurs 24 hours after injection of anti-Thy-1 antibodies, whereas this does not occur in F344 rats. The present experiments were designed to investigate the role of the kidney in the regulation of the monocyte influx in this model. To dissociate the role of the immune system from local intrarenal factors in the control of monocyte influx, anti-Thy-1 nephritis was induced in LEW rats with an F344 kidney transplant and in F344 rats with a LEW kidney allograft. Acute rejection episodes were prevented by treatment with an anti-CD4 monoclonal antibody. Control rats received a syngeneic kidney graft. Monocyte influx after injection of anti-Thy-1 antibodies was found in the glomeruli of both LEW and F344 kidneys removed from LEW recipients, whereas there was no demonstrable monocyte influx after infusion of anti-Thy-1 antibodies in either LEW or F344 kidneys removed from F344 recipients. Monocyte infiltration correlated with the subsequent expansion of the mesangial extracellular matrix. The inability to attract monocytes was not due to the lack of glomerular expression of chemokines, because F344 and LEW glomeruli demonstrated a similar expression of monocyte chemoattractant protein-1 (MCP-1). Differences in the ability to activate the complement system were excluded. We conclude that the immune system controls the glomerular influx of monocytes and that the reaction of the mesangial cells is probably controlled by combinations of cytokines produced during the inflammatory process.     

Different effects of methylxanthines on central serotonergic postsynaptic neurons in a mouse behavioral model

We have used genetic engineering to obtain secretion of anti-human CD5 antibody fragments from Escherichia coli for conjugation to the 30-kDa form of ricin A chain (RTA30). This was accomplished by introducing stop codons at two positions in the hinge region of the human IgG1 gene so that coexpression of the truncated heavy-chain genes (Fd') with a light chain would result in Fab' and/or F(ab')2 proteins containing either one or two interheavy-chain cysteines. An Fd' gene encoding both interheavy-chain cysteines yielded a mixture of F(ab')2 and Fab', which could be separated by size-exclusion chromatography. An Fd' gene encoding only one interheavy-chain cysteine yielded primarily Fab'. Purified F(ab')2 protein was equivalent to unlabeled chimeric IgG in competing for binding of IgG with CD5 antigen, while the molar concentration of the monovalent Fab' required for 50% binding inhibition was 4- to 5-fold higher than IgG. An immunoconjugate was prepared with Fab' by direct coupling to the unique free cysteine on RTA30. The bivalent F(ab')2 was conjugated to RTA30 after derivatization with the crosslinking agent 5-methyl-2-iminothiolane. These immunoconjugates efficiently killed a CD5+ T-cell line and human peripheral blood T cells.     

Treatment of donor mice with an alpha beta T-cell receptor monoclonal antibody induces prolonged T-cell nonresponsiveness and effectively prevents lethal graft-versus-host disease in murine recipients of major histocompatibility complex (MHC)-matched and MHC-mismatched donor marrow grafts

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57-597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H-2d]) marrow grafts. Transplantation of H57-597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57-597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57-597. Donor T cells exposed to H57-597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.     

IL-2-activated murine newborn liver NK cells enhance engraftment of hematopoietic stem cells in MHC-mismatched recipients

To explore the modulatory effects of IL-2-activated NK cells on hematopoietic stem cell (HSC) engraftment further, we used fresh newborn liver cells (NLC) and IL-2-activated newborn liver cells (ANLC) as combined sources, respectively, of transplanted HSC and IL-2-activated NK cells free of contaminating CD3+ T cells. As previously found with adult IL-2-activated spleen cells, NLC cultured with IL-2 for 7 days exhibited lymphokine-activated killer (LAK) activity, veto activity, and natural suppressor activity, and enhanced both short-term and long-term stem cell engraftment by intact co-injected syngeneic and allogeneic NLC in totally MHC-mismatched lethally irradiated recipients. However, unlike adult IL-2-stimulated adult spleen cells, IL-2-activated NLC lacked CD3+ T cells and failed to induce lethal GVHD. FACS analysis and cell sorting experiments showed that the cells in ANLC which enhanced short-term HSC engraftment belonged to the relatively immature CD3-NK1.1-2B4+ NK cell subset. By contrast, cells belonging to the more mature CD3-NK1.1+2B4+ NK cell subset showed no HSC-enhancing effects. Identification and isolation in humans of similar NK cell enhancers of HSC could lead to a new approach to improving stem cell engraftment in MHC-mismatched recipients without increasing the risk of GVHD.