Split tolerance induced by chick embryo thymic epithelium allografted to embryonic recipients

To test the capacity of the epithelial component of the chick embryo thymus to induce tolerance to major histocompatibility complex (MHC) antigens, pre-colonized thymic rudiments were grafted into chick embryonic recipients. Semi-allogeneic or allogeneic transplantations were done between two lines of chickens histocompatible at the MHC locus. Approximately 10% of these thymic chimeras hatched and were studied 3 mo after hatching. Thymic grafts were not rejected by the allogeneic host. The tolerance of chimeric chickens to thymus donor MHC antigens was tested by using a skin graft rejection test and a graft-vs-host (GvH) assay. Chimeric chickens that received an MHC-incompatible thymic graft during the embryonic life tolerated skin graft with the MHC haplotype of the thymus donor. Nevertheless, the lymphocytes within the thymic graft, the host thymus, and the blood were tolerant to the host MHC antigens but were alloreactive in GvH reaction for the MHC antigens of the thymic graft type. These results suggest that the epithelial component of the thymus when taken before the starting of the colonization by hemopoietic precursors and grafted into an early chick embryonic host can induce a tolerance for the MHC determinants involved in allograft rejection but not in the GvH reaction.     

Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.

Graft-versus-host disease (GvHD) is a chief complication of allogeneic bone marrow transplantation. In HLA-identical bone marrow transplantation, GvHD may be induced by disparities in minor histocompatibility antigens (mHags) between the donor and the recipient, with the antigen being present in the recipient and not in the donor. Cytotoxic T lymphocytes (CTLs) specific for mHags of the recipients can be isolated from the blood of recipients with severe GvHD (ref. 3). A retrospective study demonstrated an association between mismatch for mHags HA-1, -2, -4 and -5 and the occurrence of GvHD in adult recipients of bone marrow from HLA genotypically identical donors. Tetrameric HLA-peptide complexes have been used to visualize and quantitate antigen-specific CTLs in HIV-infected individuals and during Epstein-Barr virus and lymphocytic choriomeningitis virus infections. Here we show the direct ex vivo visualization of mHag-specific CTLs during GvHD using tetrameric HLA-class and I-mHag HA-1 and HY peptide complexes. In the peripheral blood of 17 HA-1 or HY mismatched marrow recipients, HA-1- and HY-specific CTLs were detected as early as 14 days after bone marrow transplantation. The tetrameric complexes demonstrated a significant increase in HA-1- and HY-specific CTLs during acute and chronic GvHD, which decreased after successful GvHD treatment. HLA class I-mHag peptide tetramers may serve as clinical tools for the diagnosis and monitoring of GvHD patients.     

Natural regulation of immunity to minor histocompatibility antigens

MHC-matched hemopoietic stem cell transplantation is commonly used for the treatment of some forms of leukemia. Conditioning regimens before transplant act to reduce the burden of leukemic cells and the graft-vs-leukemia (GvL) effect can eliminate residual disease. The GvL effect results largely from the recognition of minor histocompatibility Ags by donor T cells on recipient tissues. These Ags are generally widely expressed and also provoke graft-vs-host (GvH) disease. Manipulation of immunity to promote GvL while curtailing GvH would greatly improve clinical outcome. To develop strategies that may achieve this, the parameters which control immunity to minor histocompatibility Ags need to be defined. In this study, we have analyzed responses to the mouse HY minor histocompatibility Ag using hemopoietic cell and skin grafts as surrogate GvL and GvH targets, respectively. We show that natural regulation of CD8 T cell responses to HY operates at multiple levels. First, CD4 T cell help is required for primary CD8 responses directed at hemopoietic cells. However, although CD4 T cells of H2(k) mouse strains recognize HY, they provide ineffective help associated with a proportion of recipients developing tolerance. This was further investigated using TCR-transgenic mice which revealed H2(k)-restricted HY-specific CD4 T cells are highly susceptible to regulation by CD25(+) regulatory T cells which expand in tolerant recipients. A second level of regulation, operating in the context of skin grafts, involves direct inhibition of CD8 T cell responses by CD94/NKG2 engagement of the nonclassical MHC class I molecule Qa1.     

Natural killer cell alloreactivity and haplo-identical hematopoietic transplantation

In haplo-identical hematopoietic transplantation, donor vs. recipient natural killer (NK) cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory killer cell Ig-like receptors (KIR) for self-HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. When faced with mismatched allogeneic targets, these NK clones sense the missing expression of self-HLA class I alleles and mediate alloreactions. KIR ligand mismatches in the GvH direction trigger donor vs. recipient NK cell alloreactions, which improve engraftment, do not cause GvHD and control relapse in AML patients . The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated in mouse models. The infusion of alloreactive NK cells ablates (i) leukemic cells, (ii) recipient T cells that reject the graft and (iii) recipient DC that trigger GvHD, thus protecting from GvHD.     

Shift of graft-versus-host-disease target organ tropism by dietary vitamin A

Gut-homing of donor T cells is causative for the development of intestinal GvHD in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Expression of the gut-specific homing receptors integrin-α4β7 and chemokine receptor CCR9 on T cells is imprinted in gut-associated lymphoid tissues (GALT) under the influence of the vitamin A metabolite retinoic acid. Here we addressed the role of vitamin A deficiency in HSCT-recipients for donor T cell migration in the course of experimental GvHD. Vitamin A-deficient (VAD) mice were prepared by feeding them a vitamin A-depleted diet. Experiments were performed in a C57BL/6 into BALB/c model of acute GvHD. We found that expression of integrin-α4β7 and CCR9 in GALT was reduced in VAD recipients after HSCT. Competitive in vivo homing assays showed that allogeneic T cells primed in VAD mice did not home as efficiently to the intestine as T cells primed in mice fed with standard diet (STD). The course of GvHD was ameliorated in VAD HSCT-recipients and, consequently, their survival was prolonged compared to recipients receiving STD. However, VAD-recipients were not protected and died of clinical GvHD. We found reduced numbers of donor T cells in the intestine but increased cell counts and tissue damage in other organs of VAD-recipients. Furthermore, we observed high IFN-γ(+)CD4(+) and low FoxP3(+)CD4(+) frequencies of total donor CD4(+) T cells in VAD as compared to STD recipients. Taken together, these results indicate that dietary vitamin A deficiency in HSCT-recipients changed target organ tropism in GvHD but also resulted in fatal inflammation after HSCT.     

Recombinant MHC tetramers for isolation of virus-specific CD8<Superscript>+</Superscript> cells from healthy donors: Potential approach for cell therapy of posttransplant cytomegalovirus infection

Patients undergoing allogeneic hematopoietic stem cell transplantation have a high risk of cytomegalovirus reactivation, which in the absence of T-cell immunity can result in the development of an acute inflammatory reaction and damage of internal organs. Transfusion of the virus-specific donor T-lymphocytes represents an alternative to a highly toxic and often ineffective antiviral therapy. Potentially promising cell therapy approach comprises transfusion of cytotoxic T-lymphocytes, specific to the viral antigens, immediately after their isolation from the donor’s blood circulation without any in vitro expansion. Specific T-cells could be separated from potentially alloreactive lymphocytes using recombinant major histocompatibility complex (MHC) multimers, carrying synthetic viral peptides. Rapid transfusion of virus-specific T-cells to patients has several crucial advantages in comparison with methods based on the in vitro expansion of the cells. About 30% of hematopoietic stem cell donors and 46% of transplant recipients at the National Research Center for Hematology were carriers of the HLA-A*02 allele. Moreover, 94% of Russian donors have an immune response against the cytomegalovirus (CMV). Using recombinant HLA-A*02 multimers carrying an immunodominant cytomegalovirus peptide (NLV), we have shown that the majority of healthy donors have pronounced T-cell immunity against this antigen, whereas shortly after the transplantation the patients do not have specific T-lymphocytes. The donor cells have the immune phenotype of memory cells and can be activated and proliferate after stimulation with the specific antigen. Donor lymphocytes can be substantially enriched to significant purity by magnetic separation with recombinant MHC multimers and are not activated upon cocultivation with the antigen-presenting cells from HLA-incompatible donors without addition of the specific antigen. This study demonstrated that strong immune response to CMV of healthy donors and prevalence of HLA-A*02 allele in the Russian population make it possible to isolate a significant number of virus-specific cells using HLA-A*02–NLV multimers. After the transfusion, these cells should protect patients from CMV without development of allogeneic immune response.     

The decline in murine splenic PHA and LPS responsiveness with age is primarily due to an intrinsic mechanism

Changes in splenic B and T lymphocyte number and mitogenic activity with age were quantitated in (A X C57BL/6)F1 (AB6F1) hybrid mice. Although both the B and T lymphocyte proliferative reactivity to their respective mitogens, lipopolysaccharide (LPS) and phytohemagglutinin (PHA), declined significantly with age, an earlier and more marked reduction was recorded for the T cell response. The decline in B and T lymphocyte mitogenic activity with age could not be correlated with a corresponding reduction in the percentage of splenic B or T lymphocytes. The main focus of this study was to determine if the reduction in T and B lymphocyte mitogenic activity with age results primarily from a mechanism intrinsic to the lymphoid lineage itself or from adverse extracellular factors that increase with age. Bone marrow cells (BMC) derived from individual young and old donor AB6F1 mice were transplanted into the neutral environment of young, lethally irradiated syngeneic recipients. Number and mitogenic activity of splenic T and B lymphocytes were recorded for the original BMC donors as well as for the recipients of the young and old BMC lines 9 mo after the BMC transplants. A predominance of the donor (male) rather than recipient (female) karyotype within the mitogen-responding populations of recipient mice confirmed a donor BMC take. The PHA and LPS response levels exhibited by the old donors were 30% and 70% of those of the young donors, respectively. These differences in PHA and LPS reactivity recorded between young and old donors were maintained between recipients of young and old donor BMC lines. Thus, even under the influence of a young recipient environment, old BMC were incapable of giving rise to mitogen responding cells with a functional competence equivalent to that of their younger counterparts. This finding would lend further support to the theory that an intrinsic mechanism is responsible for the decline in murine mitogenic activity with age.     

Minor histocompatibility antigens on canine hemopoietic progenitor cells

Adoptive immunotherapy with CTL against minor histocompatibility Ags (mHA) provides a promising way to treat leukemia relapse in allogeneic chimeras. Here we describe the in vitro generation of CTL against mHA in the dog. We tested their inhibitory effect on the growth of hemopoietic progenitor cells stimulated by hemopoietic growth factors in a 4-day suspension culture. CTL were produced by coculture of donor PBMC with bone marrow-derived dendritic cells (DCs). These DCs were characterized by morphology, high expression of MHC class II and CD1a, and the absence of the monocyte-specific marker CD14. Characteristically these cells stimulated allogeneic lymphocytes (MLR) and, after pulsing with a foreign Ag (keyhole limpet hemocyanin), autologous T cells. CTL were generated either ex vivo by coculture with DCs of DLA-identical littermates or in vivo by immunization of the responder with DCs obtained from a DLA-identical littermate. In suspension culture assays the growth of hemopoietic progenitor cells was inhibited in 53% of DLA-identical littermate combinations. In canine families mHA segregated with DLA as restriction elements. One-way reactivity against mHA was found in five littermate combinations. In two cases mHA might be Y chromosome associated, in three cases autosomally inherited alleles were detected. We conclude that CTL can be produced in vitro and in vivo against mHA on canine hemopoietic progenitor cells using bone marrow-derived DCs.     

B cell development and regulation after T cell-depleted marrow transplantation

Antibody secreting B lymphocytes from immunized donors can be adoptively transferred after T cell-depleted marrow transplantation to produce protective levels of antibody in the recipient. We have investigated whether these transferred lymphocytes remain subject to continued clonal selection and subsequently became memory B cells even in the initial absence of T cells. Twenty-eight donor/recipient pairs were randomized pretransplant to be immunized or not with tetanus toxoid (TT). The recipients were then vaccinated with TT at 3, 6, and 12 mo posttransplant, and the anti-TT antibody response (IgG and IgM) was measured. Only when both donor and recipient were immunized pretransplant could the recipient respond to antigen challenge within the first year posttransplant. Examination of the spectrotype pattern of the recipient anti-TT antibody shows that selection of B cell clones continues, so that T cell depletion does not prevent the appearance of oligoclonal antibody responses. However, because the spectrotype pattern of the recipient did not match the donors, B cell regulatory mechanisms in donor and recipient are nonidentical. These data contrast with observations made in recipients of non-T cell-depleted marrow and serve to illustrate the role of T lymphocytes in the induction and regulation of secondary antibody responses in man. The results also suggest that optimal humoral responses to any antigen after T cell depletion can only occur when both donor and recipient are immunized pretransplant, a prediction borne out by studies on the influence of donor cytomegalovirus status on the severity of cytomegalovirus infection in the recipient.     

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.     

Role for CXCR6 in recruitment of activated CD8+ lymphocytes to inflamed liver

Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6(-/-), H-2D(b) lymphocytes into BDF(1), H-2D(bxd) recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6(-/-) or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6(-/-) CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6(-/-) and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.     

Modulation of GvHD by suicide-gene transduced donor T lymphocytes: clinical applications in mismatched transplantation

In allogeneic hematopoietic cell transplantation (allo-HCT), donor lymphocytes play a central therapeutic role in both GvL and immune reconstitution. However, the full exploitation of these therapeutic properties is limited by the occurrence of GvHD. Different strategies have been investigated to obtain all the benefits derived from donor lymphocytes while avoiding the risk of GvHD. The genetic engineering of donor lymphocytes with the herpes simplex virus-thymidine kinase (HSV-TK) suicide gene confers the ability to modulate GvHD by invivo ganciclovir-induced elimination of the transduced cells. The suicide-gene strategy has applications in both donor lymphocyte infusion (DLI) for disease relapse and in add-back infusions after T-cell depleted allo-HCT. TK cell DLI resulted in anti-tumor activity in a relevant proportion of treated patients. Haplo-identical stem cell transplantation (haplo-HCT) is a promising therapeutic option for patients with high risk hematologic malignancies lacking an HLA-matched donor. However, the profound T-cell depletion required to overcome the risk of lethal GvHD has been associated with a marked delayed T-cell recovery with a prolonged risk of post-transplant viral, fungal and other opportunistic infections. TK cell add-backs efficiently promote early immune reconstitution after haplo-HCT and prevent disease relapse, providing a unique tool for the control of GvHD. The genetic manipulation of donor lymphocytes with a suicide gene is a promising strategy to increase feasibility and safety of allo-HCT.     

Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes.

While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-gamma only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-gamma-producing PBLs. Studies of donor-specific IFN-gamma-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.     

Characterization of effector cells of graft vs leukemia following allogeneic bone marrow transplantation in mice inoculated with murine B-cell leukemia

Summary It is now widely accepted that immunocompetent lymphocytes in allogeneic bone marrow grafts exert an antileukemic effect that contributes to the cure of leukemia. Graft vs leukemia (GVL) effects independent of graft vs host disease were investigated in allogeneic bone marrow chimeras tolerant of host and donor alloantigens. The role of Thy1.2, L3T4 and Lyt2 T lymphocytes as effector cells of GVL were investigated in (BALB/c × C57BL/6)F1 mice inoculated with murine B-cell leukemia and subsequently conditioned with total lymphoid irradiation and cyclophosphamide (200 mg/kg). Mice were reconstituted with C57BL/6 bone marrow cells depleted of well-defined T-cell subsets or enriched for stem cells by the soybean agglutination method. Detection of residual tumor cells, an indicator for efficacy of GVL, was carried out by adoptive transfer of peripheral blood or spleen cells obtained from treated chimeras into secondary naive BALB/c recipients at different time intervals following bone marrow transplantation. Treatment of the primary marrow inoculum with monoclonal anti-Thy 1.2 or anti-Lyt2 abolished the GVL effects and all secondary BALB/c recipients developed leukemia within 60 days. On the other hand, the treatment with monoclonal anti-L3T4 did not influence the effect of GVL and all treated recipients remained without leukemia. The data suggest that T cells may mediate GVL effects in the absence of graft vs host disease and in circumstances where tolerance to conventional alloantigens is elicited. Effector cells of GVL across the major histocompatibility complex (MHC) in the murine B-cell leukemia tumor model system appear to be Thy 1.2⁺ Lyt2⁺ L3T4—. Induction of GVL effects by allogeneic cells tolerant of host MHC suggests that these effects may be independent of graft vs host disease.     

Post-natal ontogenesis of graft-versus-host reactivity of peanut agglutinin lectin-negative thymocytes in the chicken

Chicken thymocyte fractionation by peanut agglutinin lectin yields two cell subpopulations which differ in their GvH competence when injected into major histocompatibility complex (MHC)-(B locus) incompatible embryos. The nonagglutinated PNA^? fraction displayed a degree of alloreactivity similar to that of peripheral blood lymphocytes while the PNA⁺ cells were nonreactive. Analysis of chickens at the age of 4 to 24 weeks showed that the development of GvH-reactive PBL preceded by 12 weeks the maturation of GvH-reactive PNA^? thymocytes. Possible interpretations for this difference between the time of appearance of GvH reactivity of PNA^? thymocytes and PBL are discussed.     

In Vivo imaging of differences in early donor cell proliferation in graft-versus-host disease hosts with different pre-conditioning doses

Graft-versus-host disease (GVHD) results from immunemediated attacks on recipient tissues by donor-originated cells through the recognition of incompatible antigens expressed on host cells. The pre-conditioning irradiation dose is a risk factor influencing GVHD severity. In this study, using newly generated luciferase transgenic mice on a B6 background (B6.Luc^Tg) as bone marrow and splenocyte donors, we explored the effects of irradiation doses on donor cell dynamics in major histocompatibility complex (MHC)-matched allogeneic GVHD hosts via bioluminescence imaging (BLI). Results from BLI of GVHD hosts showed higher emission intensities of luminescence signals from hosts irradiated with 900 cGy as compared with those irradiated with 400 cGy. In particular, BLI signals from target organs, such as the spleen, liver, and lung, and several different lymph nodes fluctuated with similar time kinetics soon after transplantation, reflecting the synchronous proliferation of donor cells in the different organs in hosts irradiated with 900 cGy. The kinetic curves of the BLI signals were not synchronized between the target organs and the secondary organs in hosts irradiated with 400 cGy. These results demonstrate that pre-conditioning doses influence the kinetics and degree of proliferation in the target organs soon after transplantation. The results from this study are the first describing donor cell dynamics in MHC-matched allogeneic GVHD hosts and the influence of irradiation doses on proliferation dynamics, and will provide spatiotemporal information to help understand GVHD pathophysiology.     

Regulation of the intensity of delayed-type hypersensitivity to sheep erythrocytes by the K-region of the major histocompatibility complex in mice

The injection of 6 x 10(9) sheep red blood cells (SRBC) to mice suppressed the delayed type hypersensitivity (DTH) in situ and activated spleen T cells which prevent sensitization of syngeneic recipients. Similar effect was obtained when suppressor cells induced in F1 hybrids were transferred to parental mice. Suppression was also reached in allogeneic strain combination if suppressor cells of donors and recipients shared the major histocompatibility complex (MHC). Studied performed with recombinant and mutant strains revealed that the prerequisite for interaction of DTH suppressors and effectors was the identity of K-region of MHC. Passive transfer of DTH to SRBC was also possible if donors and recipients were identical in K-region of MHC. It is believed that interaction between DTH suppressors and effectors is restricted by a region of MHC whose product takes part in antigen representation.     

Combined prophylactic suppression of graft-versus-host and host-versus-graft reactions following treatment of prospective bone marrow recipients with rat IgG 2b anti-mouse T cell antibodies

A new approach to avoid typical complications from bone marrow transplantation into MHC different mice was studied. Rat monoclonal anti-Thy-1 antibodies of the IgG 2b isotype were identified, which inhibit T lymphocytes in vivo so that transplanted donor T cells as well as residual T cells of the conditioned marrow recipient were suppressed. A single injection of these antibodies after irradiation and before marrow transplantation did not only prevent graft-versus-host mortality but suppressed also host-versus-graft reactivity so that the radiation dose necessary for engraftment of donor cells differing in H-2, IA (both haplotypes) major histocompatibility antigens could be reduced to 6.0 Gy. In addition an anti-T leukemic cell effect from the injected monoclonal T cell antibodies was observed.     

Histocompatibility requirements for cellular cooperation in the chicken: generation of germinal centers.

Cyclophosphamide-treated newly hatched chicks were transplanted with histocompatible, semiallogeneic and allogeneic combinations of B (bursa) and T (thymus) cells from newly hatched donors. At the age of 5 weeks the birds were studied for an anti-SRBC response and for the generation of germinal centers in the spleen. The results of these experiments are summarized as follows. i) Allogeneic bursal stem cells have the capacity to restore the bursal structures of CY-treated recipients, but not the germinal center or anti-SRBC formation. ii) When allogeneic B cells are combined with T cells histocompatible or semiallogeneic with them, a restoration of the germinal center formation is achieved, but not to the same level as observed in normal birds or in CY-treated birds transplanted with histocompatible or semiallogeneic B cells. iii) Allogeneic B cells, even when complemented with T cells histocompatible with them, fail to restore the antibody production against SRBC; this is achieved only after transplantation of B cells histocompatible or semiallogeneic with the recipient. These findings indicate that germinal center formation is dependent on cooperation of histocompatible or semiallogeneic B and T cells, and furthermore, that an additional factor provided by the host is involved. Studies with transplantation of histocompatible and histoincompatible 'empty' splenic stromata revealed that the additional factor is not related to the splenic stroma.     

Relationship between the expression of class I antigen and reactivity of chicken thymocytes

The expression of major histocompatibility complex (MHC) class I antigens in ontogenesis and the distribution of B-F⁺ cells, defined by means of a monoclonal antibody, were studied by indirect membrane immunofluorescence tests on suspensions of thymus, bursa, spleen, peripheral blood lymphocytes (PBL) and red blood cells (RBC) from 18-day-old chicken embryos and chickens from 1–90 days after hatching. At 18 days of incubation and at the first day after hatching, RBC, PBL, and the cells from bursa and thymus are negative. The percentage of positive PBL and bursal cells increases up to 9 days after hatching. By 2 weeks after hatching almost 100 % of the RBC, PBL, bursa, and spleen cells were positive whereas the thymus showed only 20% positive cells. Analysis on 4-m-thick, frozen acetone-fixed tissue sections of thymus showed that medullary cells are positive, while the cortical area is negative. The graft-versus-host (GvH) competence of these thymus subpopulations was compared after sorting by the fluorescence-activated cell sorter and injection into MHC incompatible embryos. GvH reactivity was associated primarily with the B-F⁺ population. Double staining studies with peanut agglutinin (PNA)-fluorescein isothiocyanate and a rabbit-anti-Ig tetramethyl isothiocyanate-conjugate proved that the PNA^– thymocytes are identical with B-F⁺ thymocytes.Abbreviations used in this paper: FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC-Ig fluorescein isothiocyanate-conjugated immunoglobulin - GvH graft-versus-host - HAT hypoxanthineaminopterin-thymidine - HBSS Hanks' balanced salt solution - IIF indirect immunofluorescence - MCA monoclonal antibody - MHC major histocompatibility complex - NWL normal white Leghorn - OS Obese strain - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline - PNA peanut agglutinin - RBC red blood cells - TRITC-Ig tetramethyl isothiocyanate-conjugated immunoglobulin