Nonspecific suppressor cells in patients with chronic graft-vs-host disease after marrow grafting.

Forty-four human long-term survivors after marrow transplantation for aplastic anemia or hematologic malignancy were studied for the presence of circulating nonspecific suppressor cells. Twenty-two of the patients were healthy and 22 had mild to moderately severe chronic graft-vs-host disease (GVHD). Patient mononuclear cells (of donor origin) were tested for their ability to suppress the responses of lymphocytes obtained from the respective marrow donors to alloantigens in mixed leukocyte culture (MLC) and/or to concanavalin A (Con A). Tests were carried out between 199 and 2393 (median 376) days after transplantation. Cells from only 1 of 22 patients without chronic GVHD showed suppression of donor cell blastogeneis responses. In contrast, cells from 11 of 22 patients with chronic GVHD showed more than 30% suppression of donor cell responses in MLC and/or to Con A. The finding of suppressor cells was not related to the time of testing after grafting nor to immmunosuppressive therapy. Nonspecific suppressor activity was abrogated by irradiation with 1600 rads in vitro in five of six cases tested. Nonspecific suppressor cells may be one explanation for the severe combined immunodeficiency and the recurrent infectious complications characteristic of patients with chronic GVHD.     

Studies on cellular inhibition and serum-blocking factors in 28 human patients given marrow grafts from HLA identical siblings.

Fifteen patients with aplastic anemia and 13 with acute leukemia were studied 36 to 1547 days after treatment with high-dose cyclophosphamide and/or total-body irradiation and marrow transplantation from HLA identical siblings. Peripheral blood lymphocytes from patients and normals (marrow donors and healthy unrelated individuals) were tested for cell inhibition (CI) of cultured skin fibroblasts from both patients and donors by using the microcytotoxicity assay. In addition, blocking of CI by factors in patient serum was studied. Three groups of patients were studied. Patients in group I were stable long-term survivors without evidence of graft-vs-host diseases (GVHD) between 250 to 1547 days postgrafting. Patients in group II were short-term survivors with or without acute GVHD between 36 and 144 days postgrafting. Patients in group III had chronic GVHD either at the time of testing or developed chronic GVHD subsequent to CI testing between days 61 and 960 postgrafting. Eleven of 14 patients in group I showed absence of both CI and serum blocking and three showed CI and blocking. Patients in group II without acute GVHD showed absence of CI and serum blocking on three occasions, presence of CI and blocking on four occasions, and CI without blocking on three occasions. Patients in group II with acute GVHD showed absence of CI on one occasion and presence of CI and blocking on three occasions. Patients in group III showed absence of CI and blocking on seven occasions and CI without blocking on two occasions. These results suggest that the maintenance of stable graft-host tolerance in long-term survivors after marrow grafting from HLA identical donors does not depend on the presence of serum-blocking factors. Short-term survivors with and without GVHD showed a spectrum of in vitro reactivity with 50% of the patients showing serum-blocking factors, and these results did not appear to be correlated with presence or absence of acute GVHD. Finally, results of the microcytotoxicity assays failed to provide insight into the mechanism of chronic GVHD.     

IgG anti-tetanus toxoid antibody production induced by Epstein-Barr virus from B cells of human marrow transplant recipients

This investigation shows that Epstein-Barr virus (EBV)-activated human B cells from marrow transplant recipients can produce in vitro IgG anti-tetanus toxoid antibody (anti-TT) without booster immunizations with tetanus toxoid (TT). Purified B cells (E-rosette negative) from 8 normal subjects, 6 healthy long-term marrow graft recipients, and 15 long-term marrow graft recipients with chronic graft-vs-host disease (GVHD), were stimulated for 12 days with EBV to induce anti-TT production in culture supernatants. The amount of anti-TT in culture supernatants was quantitated using a enzyme-linked immunosorbent assay. B cells from all 8 normal controls produced in vitro IgG anti-TT after EBV stimulation. Five of 6 healthy recipients had B cells that produced anti-TT after EBV stimulation. Four of 15 recipients with chronic GVHD had B cells capable of producing anti-TT after EBV stimulation. The number of cultures making anti-TT responses was less in those with chronic GVHD than in those without chronic GVHD or normal individuals (P less than 0.001). B cells from patients with chronic GVHD had fewer responses exceeding the overall median of 0.7 ng/ml when compared with the other two groups (P less than 0.03). These data show that B cells of donor origin can produce in vitro IgG anti-TT antibody to tetanus toxoid antigen in a T-independent fashion.     

Both ongoing suppression and clonal elimination contribute to graft-host tolerance after transplantation of HLA mismatched T cell-depleted marrow for severe combined immunodeficiency

Lymphocytes from children with severe combined immunodeficiency who had been immunologically reconstituted with haploidentical T cell-depleted bone marrow were analyzed with regard to their immunologic recognition of donor, host, or third party alloantigens. When compared with freshly isolated donor lymphocytes, the engrafted donor cells exhibited markedly reduced to absent responses toward host Ag in primary or secondary MLC and cell-mediated lympholysis assays. However, under limiting dilution conditions, cytotoxic responses to host Ag could be demonstrated, indicating that small numbers of host reactive cells were present, although down-regulated at high responder cell doses. These results are consistent with prior observations in limiting dilution cultures that indicate that cells with the potential to lyse autologous target cells exist in the peripheral blood of all normal individuals. The number of host reactive cells present in these patients is significantly less than that present in cells isolated directly from the marrow donors, and is also less than the number of autocytotoxic cells normally seen in peripheral blood. Together, these observations indicate that two mechanisms contribute to donor host tolerance in these patients. The majority of host reactive cells appear to have undergone clonal deletion or inactivation, whereas the small residual host-reactive population appears to be under ongoing immunoregulatory control.     

Induction of transplantation tolerance in guinea pigs by spleen allografts. II. Responses in the mixed leukocyte reaction correlate with the tolerant state

We have developed a model for the induction of transplantation tolerance in the guinea pig by vascularized spleen allografts. Spleen allografts from strain 13 to strain 2 hosts frequently survived in healthy recipients without clinical GVHD or induced clinical GVHD. (2 x 13)F1 to strain 2 spleen allografts survived indefinitely without inducing GVHD. In contrast, strain 2 spleen allografts were rejected by strain 13 hosts. An excellent correlation was observed between the clinical course and the degree of reactivity to donor strain stimulator cells in the MLR. Animals that had rejected their grafts had normal or enhanced proliferative responses in the MLR. Strain 2 hosts with long-term surviving strain 13 or (2 x 13)F1 grafts had markedly suppressed anti-13 responses. Animals with GVHD had a suppressed MLR toward donor strain stimulator cells with simultaneous reactivity to host strain stimulator cells. Cells capable of suppressing the response of normal host strain cells to donor strain stimulators were present in some long-term surviving animals and may be responsible in part for the maintenance of the tolerant state.     

Cellular interactions in marrow-grafted patients. I. Impairment of cell-mediated lympholysis associated with graft-vs-host disease and the effect of interleukin 2

Forty patients with hematologic malignancy or aplastic anemia were given allogeneic marrow after conditioning with high-dose cyclophosphamide alone or in combination with total body irradiation. Between 28 and 3857 days after transplantation, their peripheral blood mononuclear leukocytes were tested for reactivity in indirect cell-mediated lympholysis against normal leukocytes from unrelated individuals, and the results were compared to those with cells from their healthy marrow donors. An impairment of cell-mediated lympholysis was found with cells from most patients with acute and chronic graft-vs-host disease (GVHD) whereas cells from most short-term and long-term patients without GVHD had cell-mediated lympholysis reactivity comparable to that of cells from the marrow donors. When interleukin 2 was added to the mixed leukocyte cultures during the sensitization phase, the impaired cell-mediated immunity of cells from most short-term patients with acute GVHD, but not that of cells from most patients with chronic GHVD, could be restored to normal levels. These results suggest the impairment of cell-mediated immunity seen in cells of short-term patients with acute GVHD is attributable to helper cell defects or to ineffective communication between antigen-presenting cells and helper T cells. The impairment in cell-mediated immunity seen in patients with chronic GVHD, however, may reside on the effector cells (or their precursors) or may be due to the presence of suppressor cell activity.     

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.     

Sex chromatin analysis of lymphocytes invading host organs in transfusion-associated graft-versus-host disease

We report a method of sex chromatin analysis of lymphocytes separated from host organs in transfusion-associated graft-versus-host disease (GVHD), which enabled the demonstration of invasion by donor lymphocytes. The lymphocytes examined were separated from deparaffinized tissue blocks of skin, spleen and bone marrow from two female patients with transfusion-associated GVHD by incubation in 0.5% pepsin. The tissues had been removed at autopsy and fixed in formalin. Sex chromatin analysis was performed by fluorescence microscopy on separated lymphocytes stained with 0.005% quinacrine dihydrochloride. By this means Y chromatin-positive (i.e. male) lymphocytes were demonstrated in the skin, spleen and bone marrow of both female patients.     

Sex chromatin analysis of lymphocytes invading host organs in transfusion-associated graft-versus-host disease

We report a method of sex chromatin analysis of lymphocytes separated from host organs in transfusion-associated graft-versus-host disease (GVHD), which enabled the demonstration of invasion by donor lymphocytes. The lymphocytes examined were separated from deparaffinized tissue blocks of skin, spleen and bone marrow from two female patients with transfusion-associated GVHD by incubation in 0.5% pepsin. The tissues had been removed at autopsy and fixed in formalin. Sex chromatin analysis was performed by fluorescence microscopy on separated lymphocytes stained with 0.005% quinacrine dihydrochloride. By this means Y chromatin-positive (i.e. male) lymphocytes were demonstrated in the skin, spleen and bone marrow of both female patients.     

Human immunodeficiency disease: impairment of cellular interactions leading to abnormal mediator production in mixed lymphocyte culture reaction.

Leukocyte migration inhibitory factor (LMIF) production in mixed lymphocyte culture (MLC) reactions is the result of cellular interactions based on two separate phenomena: the capacity of lymphocytes to stimulate in MLC, and the capacity of lymphocytes to respond in MLC. Puromycin-treated lymphocytes are capable of stimulating allogeneic cells for LMIF production, but are unable to respond with synthesis of LMIF (one-way MLC-LMIF test). We have studied the stimulating and responding capacity of lymphocytes from patients with different immunodeficiency syndromes in a one-way MLC-LMIF assay. Lymphocytes from patients known to have qualitative and quantitative defects of T cell or B cell functions (Hodgkin's disease, mycosis fungoides, thymoma, chronic lymphatic leukemia) were found to respond poorly as measured by mediator production although their stimulating fuction was frequently retained. Patients with advanced solid tumors often had both MLC-stimulating and responding functions depressed. There was no apparent correlation between mitogen response and MLC-induced LMIF responses or between MLC proliferative response (as measured by thymidine incorporation) and mediator production. Studying of stimulatory and responding capacity of lymphocytes in the MLC-LMIF assay provides a new tool for assessing immunocompetence and allows for in vitro evaluation of cellular interactions that may play an important role in vivo.     

Deposition of IgM and complement at the dermoepidermal junction in acute and chronic cutaneous graft-vs-host disease in man.

The presence of cutaneous immunoglobulin and complement was investigated in 88 patients with and without graft-vs-host disease (GVHD) after transplantation of bone marrow from HLA identical siblings for the treatment of acute leukemia or aplastic anemia. For comparison, skin biopsies from the patients obtained before transplantation, from 58 healthy individuals (mostly marrow donors) and from four syngeneic marrow recipients were studied. A direct immunfluorescent staining technique was used. Dermo-epidermal IgM deposits were found in 11% of healthy individuals and patients before grafting but were present in 86% of patients with chronic and 39% of patients with acute GVHD. Patients with allogeneic grafts who never had GVHD or who had recovered from it and patients with syngeneic grafts showed findings not different from those in healthy individuals. Findings similar to those with IgM, although less striking, were made for C3, i.e., patients who had chronic or acute GVHD had a high incidence and intensity of C3 deposits at the dermo-epidermal junction. This observation raises the possibility that humoral immunity is involved in the development of GVHD.     

Immune reactivity in SL2 lymphoma-bearing mice compared with SL2-immunized mice

Summary We have studied the rather paradoxical phenomenon of the growth of an antigenic tumor in an immunocomponent host. This phenomenon was studied by comparing (a) the lymphocyte reactivity and (b) the macrophage cytotoxicity, during SL2 growth in DBA/2 mice (SL2-bearing mice) and in DBA/2 mice immunized against SL2 tumor cells (SL2-immune mice). Immune mice rejected a challenge of tumor cells. The immune T-lymphocytes rendered macrophages cytotoxic (arming) and were able to transfer tumor resistance to naive animals. Nonimmunized mice did not reject a challenge of SL2 cells. In these tumor-bearing mice various forms of immune reactivity were tested. Lymphocytes with the capacity to arm macrophages could not be found in the lymphoid organs. However, lymphocytes isolated from the tissue directly surrounding the subcutaneous SL2 tumor could arm macrophages in vitro.Shortly after subcutaneous tumor grafting cytotoxic macrophages were found in the peritoneal cavity. In the serum macrophage arming factors were detected that rendered macrophages cytotoxic in vitro. This cytotoxicity of the peritoneal macrophages and the presence of macrophage arming factors in the serum showed a similar biphasic pattern. The first phase of cytotoxicity between day 3 and 8 after tumor grafting was tumor (SL2) specific. The second phase from day 12 and onwards was not tumor specific. During the first 4 days after SL2 grafting the DBA/2 mice expressed a specific concomitant immunity to a second tumor graft. Then 7 or more days after grafting the first SL2 tumor, the concomitant immunity was nonspecific as the growth of a second SL2 tumor graft and a L5178Y (DBA/2) tumor graft were inhibited. In addition, the immune suppressive activity of serum and lymphocytes was tested. Neither serum nor lymphocytes from SL2-bearing mice suppressed the macrophage arming capacity of SL2 immune lymphocytes. Lymphocytes from tumor-bearing mice did not inhibit the capacity of SL2-immune lymphocytes to transfer resistance to naive animals. On the contrary, lymphocytes obtained from SL2-bearing mice 14 days after SL2 grafting transfered tumor resistance in a Winn-type assay. These data suggest that the growth of an antigenic tumor is due to the inability of the immune system to mount an effective antitumor effector cell population during tumor growth, rather than an immune suppression of the antitumor reactivity, as a limited immune reactivity could be detected in tumor-bearing mice, whereas immune suppression could not be detected.     

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

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

Donor APCs are required for maximal GVHD but not for GVL

Graft-versus-host disease (GVHD) is a major source of morbidity in allogenic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD in a mouse model across only minor histocompatibility antigens (minor H antigens). However, these studies did not address the function of donor-derived APCs after GVHD is initiated. Here we show that GVHD develops in recipients of donor major histocompatibility complex class I-deficient (MHC I(-)) bone marrow. Thus, after initial priming, CD8 cells caused GVHD without a further requirement for hematopoietic APCs, indicating that host APCs are necessary and sufficient for GHVD. Nonetheless, GVHD was less severe in recipients of MHC I(-) bone marrow. Therefore, once initiated, GVHD is intensified by donor-derived cells, most probably donor APCs cross-priming alloreactive CD8 cells. Nevertheless, donor APCs were not required for CD8-mediated graft-versus-leukemia (GVL) against a mouse model of chronic-phase chronic myelogenous leukemia. These studies identify donor APCs as a new target for treating GVHD, which may preserve GVL.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

Keratinocytes synthesize Ia antigen in acute cutaneous graft-vs-host disease

Keratinocytes express la antigen (Ia) during cutaneous graft-vs-host disease (GVHD); it is, however, unclear whether this Ia is adsorbed from alloactivated donor lymphocytes or from Ia-bearing host Langerhans cells (LC), or whether it is actively synthesized by host keratinocytes. We therefore sought to determine the origin of keratinocyte Ia in a murine model of GVHD. Lethally irradiated C3H/He (H-2k) mice developed characteristic histopathologic changes of acute cutaneous GVHD 7 days after injection of BALB/c (H-2d) bone marrow and spleen cells, and expressed keratinocyte Ia of host (Iak) but not donor (Iad) origin in immunofluorescence studies. To determine whether the Ia was synthesized by keratinocytes or adsorbed from host LC, we investigated GVHD that was induced in chimeric mice. Parental strain A mice were made chimeric by lethal irradiation and reconstitution with (A X B)F1 bone marrow cells as follows: (BALB/c X C3H/He)F1 (H-2d,k) leads to C3H/He (H-2k), B6C3F1 (H-2b,k) leads to C57BL/6 (H-2b), and B6C3F1 (H-2b,k) leads to C3H/He (H-2k). After 3 mo, the LC in the skin of these chimeric mice were mainly of F1 haplotype. The chimeric mice were again lethally irradiated and injected with marrow and spleen cells from a third strain of mouse (C57BL/6, H-2b or BALB/c, H-2d) histoincompatible with both F1 parental strains. In the ensuing GVHD, the chimeric recipients only expressed keratinocyte Ia syngeneic to the original haplotype of the animal (strain A), despite the fact that the majority of their LC were derived from F1 marrow and expressed Ia of both F1 parental strain haplotypes (strains A and B). Together, these findings indicate that keratinocyte Ia in GVHD is synthesized by keratinocytes and is not derived from donor lymphocytes or adsorbed from host LC.     

Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD

The opposing problems of graft-vs-host disease (GVHD) and failure of alloengraftment present major obstacles to the application of bone marrow transplantation (BMT) across complete MHC barriers. The addition of syngeneic T-cell-depleted (TCD) bone marrow (BM) to untreated fully allogeneic marrow inocula in lethally irradiated mice has been previously shown to provide protection from GVHD. We have used this model to study the effects of allogeneic T cells on levels of chimerism in recipients of mixed marrow inocula. The results indicate that T cells in allogeneic BM inocula eliminate both coadministered recipient-strain and radioresistant host hematopoietic elements to produce complete allogeneic chimerism without clinical GVHD. To determine the role of GVH reactivity in this phenomenon, we performed similar studies in an F1 into parent combination, in which the genetic potential for GVHD is lacking. The presence of T cells in F1 marrow inocula led to predominant repopulation with F1 lymphocytes in such chimeras, even when coadministered with TCD-recipient-strain BM. These results imply that the ability of allogeneic BM cells removed by T cell depletion to increase levels of allochimerism may be mediated by a population which is distinct from that which produces GVHD. These results may have implications for clinical BM transplantation.     

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.     

Donor CD8+ T cells mediate graft-versus-leukemia activity without clinical signs of graft-versus-host disease in recipients conditioned with anti-CD3 monoclonal antibody

Donor CD8(+) T cells play a critical role in mediating graft-vs-leukemia (GVL) activity, but also induce graft-vs-host disease (GVHD) in recipients conditioned with total body irradiation (TBI). In this study, we report that injections of donor C57BL/6 (H-2(b)) or FVB/N (H-2(q)) CD8(+) T with bone marrow cells induced chimerism and eliminated BCL1 leukemia/lymphoma cells without clinical signs of GVHD in anti-CD3-conditioned BALB/c (H-2(d)) recipients, but induced lethal GVHD in TBI-conditioned recipients. Using in vivo and ex vivo bioluminescent imaging, we observed that donor CD8(+) T cells expanded rapidly and infiltrated GVHD target tissues in TBI-conditioned recipients, but donor CD8(+) T cell expansion in anti-CD3-conditioned recipients was confined to lymphohematological tissues. This confinement was associated with lack of up-regulated expression of alpha(4)beta(7) integrin and chemokine receptors (i.e., CXCR3) on donor CD8(+) T cells. In addition, donor CD8(+) T cells in anti-CD3-conditioned recipients were rendered unresponsive, anergic, Foxp3(+), or type II cytotoxic T phenotype. Those donor CD8(+) T cells showed strong suppressive activity in vitro and mediated GVL activity without clinical signs of GVHD in TBI-conditioned secondary recipients. These results indicate that anti-CD3 conditioning separates GVL activity from GVHD via confining donor CD8(+) T cell expansion to host lymphohemological tissues as well as tolerizing them in the host.     

Recognition of human minor alloantigen(s) by cytotoxic lymphocytes in vitro

Lymphocytes from an extensively transfused patient with aplastic anemia were induced to cytotoxicity against target cells from several HLA-matched siblings by in vitro stimulation with allogeneic cells. Effective stimulating cells shared HLA-B7 with the patient, but not all B7 individuals were effective. An additional factor, which was found to segregate in both the patient's and an unrelated sibship, was also necessary. Segregation of this minor alloantigen, W, was also revealed among the patient's HLA-matched sibs by differential susceptibility to lysis by effectors from the patient. The ratio of six positive to four negative siblings suggests that the antigen difference might be coded by a single locus. Lymphocytes from a normal sib, who like the patient is lacking the minor antigen, could not be induced to cytotoxicity against positive targets. Thus in vivo sensitization of the donor of the responding cells appears to be necessary for the demonstration of the cytotoxic response to the minor antigen in vitro. No correlation was observed between the segregation pattern of W and of known blood group antigens, and no cytotoxic antibody to W was detected in the patient's serum in several trials.Abbreviations used in this paper MHC major histocompatibility complex - Tc thymus dependent cells capable of mediating cytotoxicity in the absence of Immoral antibody - GVHD graft versus host disease - CML cell mediated lympholysis - MLC unidirectional mixed lymphocyte culture - ADCC antibody-dependent cell mediated cytotoxicity