Both gamma delta T cells and NK cells inhibit the engraftment of xenogeneic rat bone marrow cells and the induction of xenograft tolerance in mice

In murine allogeneic bone marrow transplantation recipients, treatment of the hosts with a nonmyeloablative regimen, including depleting anti-CD4 and anti-CD8 mAbs, allows establishment of long-term mixed chimerism and donor-specific tolerance. However, in the xenogeneic rat-to-mouse combination, additional anti-Thy1.2 and anti-NK1.1 mAbs are required. We have now attempted to identify the xenoresistant mouse cell populations that are targeted by anti-NK1.1 and anti-Thy1.2 mAbs. C57BL/6 (B6) wild-type, B6 TCRbeta(-/-), and B6 TCRdelta(-/-) mice received anti-CD4 and anti-CD8 mAbs, followed by 3 Gy of whole body irradiation, 7 Gy of thymic irradiation, and transplantation of T cell-depleted rat bone marrow cells. Anti-NK1.1 and anti-Thy1.2 mAbs were additionally administered to some groups. Increased rat chimerism was observed in TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-NK1.1 mAbs compared with similarly treated TCRbeta(-/-) mice. In TCRbeta(-/-) mice, but not in TCR delta(-/-) mice, donor chimerism was increased by treatment with anti-Thy1.2 mAb, indicating that CD4(-)CD8(-)TCRgammadelta(+)Thy1. 2(+)NK1.1(-) cells (gammadelta T cells) are involved in the rejection of rat marrow. In addition, chimerism was enhanced in both TCRbeta(-/-) and TCRdelta(-/-) mice treated with anti-CD4, anti-CD8, and anti-Thy1.2 mAbs by the addition of anti-NK1.1 mAb to the conditioning regimen. Donor-specific skin graft prolongation was enhanced by anti-Thy1.2 and anti-NK1.1 mAbs in TCRdelta(-/-) mice. Therefore, in addition to CD4 and CD8 T cells, gammadelta T cells and NK cells play a role in resisting engraftment of rat marrow and the induction of xenograft tolerance in mice.     

Breaking of CD8+ T cell tolerance through in vivo ligation of CD40 results in inhibition of chronic graft-versus-host disease and complete donor cell engraftment

In the DBA/2 --> unirradiated (C57BL/6 x DBA/2)F(1) model of chronic graft-vs-host disease (cGVHD), donor CD4(+) T cells play a critical role in breaking host B cell tolerance, while donor CD8(+) T cells are rapidly removed and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR (glucocorticoid-induced TNF receptor-related gene) can activate donor CD8(+) T cells, subsequently converting the disease pattern from cGVHD to an acute form. In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti-CD40 mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. The inhibition of cGVHD occurred because anti-CD40 mAb prevented donor CD8(+) T cell anergy such that subsequently activated donor CD8(+) T cells deleted host CD4(+) T cells and host B cells involved in autoantibody production. Additionally, functionally activated donor CD8(+) T cells induced full engraftment of donor hematopoietic cells and exhibited an increased graft-vs-leukemia effect. However, induction of acute GVHD by donor CD8(+) T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti-CD40 mAb, compared with mice that received anti-GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells, including leukemia vs induction of acute GVHD.     

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.     

A correlation between conditioning and engraftment in recipients of MHC-mismatched T cell-depleted murine bone marrow transplants

We studied engraftment in a murine model of allogeneic bone marrow (BM) transplantation. Recipient C57BL/6 (H-2b) mice were conditioned with single-dose (9 or 7.5 Gy) total body irradiation (TBI), fractionated (4 X 3.3 Gy) TBI, hyperfractionated (8 X 1.65 Gy) TBI, 2 X 120 mg/kg cyclophosphamide (CY) followed by 7.5 Gy TBI, or 300 mg/kg CY followed by 9 Gy total lymphoid irradiation (TLI). Conditioned mice were transplanted with BALB/c (H-2d) BM supplemented with splenocytes (BMS) to facilitate graft-vs-host disease (GVHD). Ex vivo T cell depletion of the BMS with anti-Thy-1.2 antibody and complement protected recipients from lethal GVHD. Engraftment was measured in transplanted animals by serotyping peripheral blood mononuclear cells with anti-H-2-specific antibodies and complement. Mice that were given a T cell-depleted BMS transplant after conditioning with 9 Gy TBI, fractionated TBI, or CY plus TBI showed a 99 to 100% incidence of engraftment. However, if the T cell-depleted graft was given to mice conditioned with hyperfractionated TBI, 7.5 Gy TBI, or CY plus TLI, only 3 to 32% of the animals engrafted. BM which was not T cell-depleted engrafted in 63 to 100% of the mice regardless of the conditioning used. Nonengrafted mice tested with anti-host type antibody demonstrated autologous recovery. We conclude that engraftment or failure/rejection of BM in transplanted mice is determined in part by a dynamic equilibrium between T cells present in the donor graft and the surviving hemopoietic cells in the conditioned recipient. More intensive conditioning of the recipient allows engraftment of T cell-depleted, mismatched BMS. Such conditioning is not limited to a single modality, but can be achieved with single-dose TBI, fractionated TBI, or with TBI combined with CY. These findings have timely and important implications for the current understanding of engraftment in human allogeneic BM transplantation following T cell depletion.     

Mouse CD94 participates in Qa-1-mediated self recognition by NK cells and delivers inhibitory signals independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94(bright) and CD94(dull) cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94(bright) but not CD94(dull) cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and "missing self" target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.     

Ly-49s3 is a promiscuous activating rat NK cell receptor for nonclassical MHC class I-encoded target ligands

Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region. Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes. Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3(+) NK cells were reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses.     

Induction of immune tolerance during administration of monoclonal antibody to L3T4 does not depend on depletion of L3T4+ cells

Treatment of mice with mAb to L3T4 profoundly depletes T helper cells. This treatment inhibits humoral and cellular immunity, retards autoimmunity, and permits the induction of Ag-specific tolerance. Treatment of BALB/c mice with F(ab')2 anti-L3T4 inhibits humoral immunity without depleting L3T4+ cells, which is evidence that mAb to L3T4 may inhibit T helper cell function in vivo. In this report, we demonstrate that F(ab')2 anti-L3T4 also permits the induction of immune tolerance in a manner that is independent of T cell depletion. C57BL/6 mice were treated with 1 mg of F(ab')2 anti-L3T4 every other day for 18 days and from the onset were challenged weekly with the immunogen 2C7, a rat mAb to chicken ovalbumin. These mice failed to respond to 2C7 not only during the treatment period but also for at least 5 mo thereafter. This immune tolerance was Ag-specific; the mice rapidly produced antibodies to subsequent challenge with another Ag, human gamma-globulin. Unlike intact anti-L3T4, which immediately depletes L3T4+ cells by greater than 90%, F(ab')2 anti-L3T4 did not initially deplete cells and caused only a partial reduction by the end of the 18-day treatment. This partial reduction of L3T4+ cells did not contribute to the induction of tolerance because mice that were first challenged with 2C7 3 days after stopping the F(ab')2 anti-L3T4 treatment, when L3T4+ cells were lowest, had a normal Ir to 2C7. These findings demonstrate that mAb to L3T4 permits induction of Ag-specific immune tolerance by a mechanism independent of its ability to deplete L3T4+ cells. They also show that F(ab')2 anti-L3T4 treatment does not impair humoral immunity when immunization is initiated after treatment is stopped. Because L3T4 is homologous to CD4 in humans, our findings suggest that F(ab')2 anti-CD4 may offer significant advantages over the use of intact anti-CD4 as an immunosuppressive agent in humans.     

Effects of in vivo administration of anti-CD3 monoclonal antibody on T cell function in mice. II. In vivo activation of T cells

Anti-CD3 mAb are known to be both immunosuppressive and mitogenic to T cells in vitro. However, only immunosuppression has been observed after in vivo administration of these mAb. The present study demonstrates that T cell activation does occur after in vivo administration of anti-CD3 mAb to mice, evidenced by increased IL-2R expression on T cells, CSF secretion, and extra-medullary hematopoiesis in the spleen. These effects required multivalent cross-linking of the mAb, since F(ab')2 fragments failed to induce them. However, the F(ab')2 fragments did induce modulation of CD3/TCR from the surface of T cells, demonstrating that TCR modulation is not sufficient to induce activation. In addition, interaction of the TCR with either intact or F(ab')2 fragments of the mAb led to increased expression of CD8 in vivo, suggesting that the F(ab')2 fragments of anti-CD3 mAb might be capable of inducing a T cell to undergo some, but not all, of the changes involved in reaching a fully activated state. Further study of the activating effects of anti-CD3 mAb might increase the understanding of the mechanisms of in vivo T cell activation and might also be exploited clinically to stimulate T cell function in immunocompromised states and to enhance hematopoiesis in myelodysplastic disorders.     

Characterization of the T cell-mediated cellular cytotoxicity during acute infectious mononucleosis

Primary infection with EBV during acute infectious mononucleosis (IM) is associated with a cytotoxic response against allogeneic target cells. C depletion with anti-CD3 (OKT3) and anti-CD8 (OKT8) mAb decreased the allogeneic cytolysis of two EBV-infected lymphoblastoid cell lines (LCL) by 96% and 89%, respectively. Complement depletion with the NK cell-specific mAb Leu-11b and NKH-1a resulted in only a slight decrease (less than 35%) in the lysis of these LCL. mAb inhibition studies with OKT3 and OKT8 inhibited the allogeneic lysis of two LCL by 87% and 82%, respectively. The alloreactive cytotoxic response was strongly inhibited by mAb specific for MHC class I determinants (W6/32, 65% inhibition and BBM.1, 58% inhibition). Acute IM lymphocytes lysed the allogeneic EBV-negative cell lines HSB2 (45%) and HTLV-1 T cell lines (16%). NK cell-depleted lymphocytes from an acute IM patient demonstrated preferential lysis of K562 transfected with human HLA-A2 (73%) compared with the K562 transfected control (20%). Cold target competition studies with allogeneic and autologous target and competitor LCL demonstrated no significant competitive inhibition between allogeneic and autologous cells. We interpret these results as evidence that 1) the acute IM-alloreactive cytotoxic response is mediated primarily by CTL; 2) these alloreactive CTL lyse allogeneic target cells irrespective of EBV antigenic expression; 3) MHC class I expression is sufficient for allogeneic recognition and lysis of target cells; 4) distinct effector CTL populations mediate lysis of autologous and allogeneic target cells; and 5) during acute IM, EBV infection results in the induction of both virus-specific and alloreactive CTL populations.     

Anti-CD3 epsilon F(ab')2 prevents graft-versus-host disease by selectively depleting donor T cells activated by recipient alloantigens

Transplantation tolerance is facilitated by activation-induced apoptosis of peripheral T cells triggered by specific AG: Abs specific for the nonpolymorphic CD3 component of the TCR complex bind to APCs through Fc-FcR interactions, mimic MHC-peptide, and activate polyclonal T cells. In contrast, F(ab')(2) of anti-CD3epsilon Abs do not activate naive T cells but induce apoptosis of Ag-activated, cycling T cells. Here, we report that treatment with anti-CD3epsilon F(ab')(2) can selectively induce apoptosis of donor T cells that recognize a recipient alloantigen, thereby preventing graft-vs-host disease initiated by a TCR-transgenic T cell population. The selective elimination of Ag-activated T cells by non-FcR-binding anti-CD3epsilon Abs could serve as an ideal strategy to prevent graft-vs-host disease and allograft rejection or to treat autoimmune disorders.     

CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.     

Successful allogeneic neonatal bone marrow transplantation devoid of myeloablation requires costimulatory blockade

A significant number of nonmalignant, progressive childhood disorders respond to bone marrow transplantation (BMT). Toxic myeloablative pretreatment regimens, graft failure, and graft-vs-host disease complicate the utility of BMT for neonatal treatment. We recently demonstrated high-dose BMT in neonatal animals enables chimeric engraftment without toxic myeloablation. Reagents that block T cell costimulation (anti-CD40L mAb and/or CTLA-4Ig) establish tolerant allogeneic engraftment in adult recipients. Donor lymphocyte infusion (DLI) re-establishes failing grafts and treats malignant relapse via a graft-vs-leukemia response. In this study, we tested the hypothesis that combining these approaches would allow tolerant allogeneic engraftment devoid of myeloablation in neonatal normal and mutant mice with lysosomal storage disease. Tolerant chimeric allogeneic engraftment was achieved before DLI only in the presence of both anti-CD40L mAb and CTLA-4Ig. DLI amplified allografts to full donor engraftment long-term. DLI-treated mice either maintained long-term tolerance or developed late-onset chronic graft-vs-host disease. This combinatorial approach provides a nontoxic method to establish tolerant allogeneic engraftment for treatment of progressive childhood diseases.     

A novel functional cell surface dimer (Kp43) expressed by natural killer cells and gamma/delta TCR+ T lymphocytes. II. Modulation of natural killer cytotoxicity by anti-Kp43 monoclonal antibody.

In the present study we provide the first evidence supporting the fact that the Kp43 NK-associated cell-surface dimer may be involved in regulating MHC-unrestricted cytotoxicity. Our results indicated that incubation of IL-2-activated NK cells in a 51Cr-release assay with either the Kp43-specific mAb or its F(ab')2 fragments induced a significant cytolytic activity directed against normal autologous and allogeneic T cell blasts, which are relatively resistant to NK cell-mediated lysis. The cytotoxic effect was not observed in fresh CD3- CD16+ CD56+ Kp43+ lymphocytes and was only substantiated in IL-2-preactivated NK cells. Although stimulation with the Kp43-specific mAb did not significantly change the intracellular Ca2+ concentration, both Ca2+ and Mg2+ were required for the induction of cytotoxicity. The anti-Kp43-mediated activation of cytolysis was inhibited by anti-CD18 and CD11a mAb, whereas it was not significantly altered by either CD11b, CD11c, CD2, or LFA-3-specific mAb, rendering unlikely the participation of the latter. In contrast to these results the Kp43-specific mAb did not enhance the high levels of spontaneous cytotoxicity mediated by IL-2-activated NK cells against a panel of different tumor cell lines. An inhibitory effect mediated by anti-Kp43 mAb on the IL-2-dependent proliferation of NK cells was previously reported and appears, at least partially, secondary to the induction of an autolytic mechanism that is synergistically enhanced by anti-CD16 mAb. Altogether our results point out that interaction of the Kp43 dimer with its specific mAb is capable of inducing cytolytic activity and suggest that the molecule may play an important functional role in lymphokine-activated NK cells.     

Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated,sensitized T cells and adult T cell-depleted bone marrow in fetal mice

Although engraftment following in utero stem cell transplantation can readily be achieved, a major limitation is the low level of donor chimerism. We hypothesized that a lack of space for donor cells in the recipient marrow was one of the primary reasons for failure to achieve significant engraftment, and that donor T cells could make space in an allogeneic mismatched setting. We found that 3 x 10(5) C57BL/6 (B6) naive CD3(+) cells coinjected with B6 T cell-depleted bone marrow (TCDBM) into 14- to 15-day-old BALB/c fetuses resulted in multilineage engraftment (median, 68.3%) associated with severe graft-vs-host disease (GvHD; 62 vs 0% with TCDBM alone). When 1.5 x 10(5) CD4(+) or CD8(+) cells were used, low levels of engraftment were seen vs recipients of 1.5 x 10(5) CD3(+) cells (2.4 +/- 1.1 and 6.6 +/- 3.9 vs 20.4 +/- 10.4%, respectively). To test the hypothesis that proliferation of T cells in response to alloantigen resulted in GvHD and increased engraftment, we pretreated naive T cells with photochemical therapy (PCT) using S-59 psoralen and UVA light to prevent proliferation. GvHD was reduced (60-0%), but was also associated with a significant reduction in engrafted donor cells (53.4 +/- 4.2 to 1.7 +/- 0.5%). However, when B6 T cells were sensitized to BALB/c splenocytes, treated with PCT, and coinjected with TCDBM, there was a partial restoration of engraftment (13.3 +/- 2.4% H2Kb(+) cells) with only one of nine animals developing mild to moderate GvHD. In this study we have shown that PCT-treated T cells that are cytotoxic but nonproliferative can provide an engraftment advantage to donor cells, presumably by destroying host hemopoietic cells without causing GvHD.     

Investigations into the nature of Igh-V region-restricted T cell interactions by using antibodies to antigens on methylcholanthrene-induced sarcomas. I. Analysis of an Igh-V-restricted suppressor-inducer factor

We have previously demonstrated the relationship between antigens on BALB/c methylcholanthrene (MC)-induced fibrosarcomas and T cell regulatory molecules by using a variety of antisera raised to these sarcomas in BALB/c and BALB/c X C57BL/6 (CB6F1) mice. One such pool of antiserum, a CB6F1 anti-CMS 4 (Pool XIV) serum, was used to investigate the nature of the T cell regulatory structures recognized by these antibodies. Pool XIV antiserum was capable of blocking the induction of feedback suppression by Ly-1 TsiF, an SRBC-specific suppressor T cell factor secreted by Ly-1+, 2- I-J+ T cells. Ly-1 TsiF induces suppression by interacting with an Ly-1+,2+ I-J+ T cell target. Successful interaction of Ly-1 TsiF with its target cell requires genetic homology between inducer and target cells at the variable region of the immunoglobulin heavy chain gene complex (Igh-V). The addition of Pool XIV antiserum to primary in vitro anti-SRBC cultures resulted in blocking the ability of Ly-1 TsiF from Igha (BALB/c) and Ighj (CBA/J) mice to induce suppression on syngeneic cells, whereas suppression induced by Ly-1 TsiF in Ighb (B6), Ighc (DBA/2), Ighd (A/J), and Ighe (AKR) mice are unaffected by addition of the Pool XIV antiserum. The ability of Pool XIV antiserum to block Ly-1 TsiF activity is linked to the Igh region, because Pool XIV antiserum can block Ly-1 TsiF from BALB/c (H-2d, Igha) and the Igh congenic B.C9 (H-2b, Igha) while not affecting Ly-1 TsiF activity on B6 (H-2b, Ighb) or its Igh congenic C.B20 (H-2d, Ighb). In CB6F1 animals, Pool XIV antiserum could block the ability of CB6F1 Ly-1 TsiF to suppress BALB/c spleen cells but not B6 spleen cells. Conversely, Pool XIV antiserum could block the ability of BALB/c Ly-1 TsiF to suppress CB6F1 spleen cells, whereas B6 Ly-1 TsiF showed normal suppressive activity in the presence of Pool XIV antiserum. In contrast, Pool XIV was capable of blocking the ability of Ly-1 TsiF from BALB/c into CB6F1 bone marrow chimeras (BMC) to suppress both BALB/c and B6 mice, whereas the activity of Ly-1 TsiF from B6 into CB6F1 BMC on BALB/c or B6 spleen cells was unaffected by the addition of Pool XIV antiserum. We then investigated the molecular nature of the molecule recognized by Pool XIV antiserum on the Ly-1 TsiF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chimeric donor cells play an active role in both induction and maintenance phases of transplantation tolerance induced by mixed chimerism

Donor hemopoietic cell engraftment is considered to be an indicator of allograft tolerance. We depleted chimerism with cells specifically presensitized to the bone marrow donor to investigate its role in mixed chimera-induced tolerance. Three experimental models were used: model A, B10.A cells presensitized to B6 (a anti-b cells) were injected into (B6 x D2)F(1) --> B10.A mixed chimeras grafted with DBA/2 skin; model B, anti-B6 presensitized cells prepared in DBA/2 --> B10.A mixed chimeras, thus unresponsive to DBA/2 (a anti-b/tol-d cells), were injected into (B6 x D2)F(1) --> B10.A mixed chimeras grafted with DBA/2 skin; and model C, (BALB/c x B6)F(1) cells presensitized to CBA (d/b anti-k cells) were injected into (B6 x CBA)F(1) --> BALB/c mixed chimeras grafted with B6 skin. Skin was grafted on day 30. Injection of each cell type before skin grafting abolished hemopoietic cell engraftment and prevented allograft acceptance. Injection of presensitized cells after skin grafting resulted in different outcomes depending on the models. In model A, injection of a anti-b cells completely depleted chimerism and caused allograft rejection. In model B, injection of a anti-b/tol-d cells markedly reduced, but did not deplete, peripheral chimerism and maintained skin allograft survival. In model C, d/b anti-k cells reduced chimerism to the background levels but failed to cause graft rejection, probably due to persistence of injected cells which share MHC with skin grafts. Together, the results show that presence of chimeric donor cells is essential in both the induction and maintenance phases of tolerance induced by mixed chimerism.     

NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (beta(2)-microglobulin(-/-) (beta(2)m(-/-))) failed to engraft. Recipient mice lacking CD8(+), CD4(+), or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with beta(2)m(-/-) HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these beta(2)m(-/-)-->B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, beta(2)m(-/-) chimeras accepted beta(2)m(-/-) HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to beta(2)m(-/-). Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.     

Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation

Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were implicated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting properties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic alloresponses refractory to current immunosuppression.     

Alloimmune response results in expansion of autoreactive donor CD4+ T cells in transplants that can mediate chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is considered an autoimmune-like disease mediated by donor CD4(+) T cells, but the origin of the autoreactive T cells is still controversial. In this article, we report that the transplantation of DBA/2 donor spleen cells into thymectomized MHC-matched allogeneic BALB/c recipients induced autoimmune-like cGVHD, although not in control syngeneic DBA/2 recipients. The donor-type CD4(+) T cells from the former but not the latter recipients induced autoimmune-like manifestations in secondary allogeneic BALB/c as well as syngeneic DBA/2 recipients. Transfer of donor-type CD4(+) T cells from secondary DBA/2 recipients with disease into syngeneic donor-type or allogeneic host-type tertiary recipients propagated autoimmune-like manifestations in both. Furthermore, TCR spectratyping revealed that the clonal expansion of the autoreactive CD4(+) T cells in cGVHD recipients was initiated by an alloimmune response. Finally, hybridoma CD4(+) T clones derived from DBA/2 recipients with disease proliferated similarly in response to stimulation by syngeneic donor-type or allogeneic host-type dendritic cells. These results demonstrate that the autoimmune-like manifestations in cGVHD can be mediated by a population of donor CD4(+) T cells in transplants that simultaneously recognize Ags presented by both donor and host APCs.     

Murine cytomegalovirus infection can enhance hybrid resistance through modulation of host natural killer activity

Studies were undertaken to assess the effect of murine cytomegalovirus (MCMV) in two different models involving injection of parental cells into F1 hosts. In both of these systems, MCMV-induced enhancement of hybrid resistance was found. In the first model, parent-into-F1 graft-vs-host reaction, MCMV infection of (C57BL/6 x C3H)F1 (B6C3F1) hosts was found to prevent the GVHR normally induced by injection of B6 parental splenocytes into the F1 hosts. The second model involved injection of parental bone marrow into lethally irradiated B6C3F1 and (C57BL/6 x DBA/2)F1 (B6D2F1) hosts. These irradiated hosts are known to exhibit resistance to engraftment by parental C57BL/6 (B6) bone marrow. This resistance was found to be markedly enhanced by injection of the hosts with MCMV 3 days before irradiation and bone marrow injection. In contrast, engraftment into B6C3F1 hosts of syngeneic marrow, or bone marrow from the C3H parent, was not affected by MCMV infection. Engraftment of DBA/2 marrow into B6D2F1 hosts was reduced at lower doses of injected marrow, suggesting enhanced resistance against the minor Hh Ag Hh-DBA. To test whether the MCMV-induced enhancement of resistance was mediated by NK cells, splenic NK activity (YAC-1 killing) and frequency (NK1.1 staining) were assessed. Both parameters were found to be elevated at 3 days after MCMV infection but to return to normal levels by 9 days. B6 bone marrow engraftment was in fact found to be normal when the marrow was administered to F1 mice 9 days after MCMV infection. Furthermore, anti-asialoGM1 administration prevented MCMV-induced enhancement of resistance to marrow engraftment. Thus, the NK enhancement resulting from MCMV infection appears to play a major role in the enhanced HR observed in the marrow engraftment model. This effect may be of importance in clinical bone marrow transplantation, a situation in which patients are susceptible to viral infection.