Irradiated Splenocytes Research Articles

Efficacy of donor splenocytes mixed with bone marrow cells for induction of tolerance in sublethally irradiated mice

Background: High dose of bone marrow cells (BMCs) has been reported to be essential to establish donor-specific tolerance. In clinical settings, a large quantity of BMCs is very difficult to be obtained. Our previous report demonstrated that even a low dose of BMCs could establish donor-specific tolerance if mixed with splenocytes (SPLCs). In the present study, various components of SPLCs were purified or removed and were investigated their contribution for enhancement of bone marrow engraftment leading to donor-specific tolerance in sublethally irradiated mice. Methods: Sublethally irradiated C57BL/6 recipient mice were intravenously injected 3×10 6 BMCs mixed with various components and various numbers of SPLCs harvested from BALB/c donor mice. One week after injection, skin grafting was performed. The degree of chimerism in peripheral blood lymphocytes (PBLs) and in SPLCs was analyzed by FACS 3 months after transplantation. Results: Recipients receiving 3×10 6 BMCs mixed with 10×10 6 T cell-enriched SPLCs established chimerism. Recipients receiving BMCs mixed with macrophage-depleted SPLCs also showed chimeirism and donor-specific tolerance. B cell-enriched SPLCs did not help small dose of BMCs to establish chimerism. Irradiated SPLCs were not effective to induce tolerance even with additional infusion to recipients. Conclusions: Active effects of splenic T cells were more important to help engraftment of small dose of BMCs than B cells, but the interaction between T and B cells might play some roles to enhance BMC engraftment. Splenic macrophages or dendritic cells might have some adverse effects against tolerance induction. Fatal graft-versus-host disease (GVHD) might be avoided by depleting adherent cells from SPLCs, so macrophages or dendritic cells were also considered as key components to induce donor-specific tolerance and prevent GVHD in this model.

Induction by a lactic acid bacterium of a population of CD4(+) T cells with low proliferative capacity that produce transforming growth factor beta and interleukin-10.

We investigated whether certain strains of lactic acid bacteria (LAB) could antagonize specific T-helper functions in vitro and thus have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular, Lactobacillus paracasei (strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the potential to induce and suppress Th1 functions, respectively, we addressed the impact of this bacterium on the outcome of CD4(+) T-cell differentiation. For this purpose, bacteria were added to mixed lymphocyte cultures where CD4(+) T-cells from naive BALB/c mice were stimulated weekly in the presence of irradiated allogeneic splenocytes. In these cultures, L. paracasei NCC2461 strongly inhibited the proliferative activity of CD4(+) T cells in a dose-dependent fashion. This was accompanied by a marked decrease of both Th1 and Th2 effector cytokines, including gamma interferon, IL-4, and IL-5. In contrast, IL-10 was maintained and transforming growth factor beta (TGF-beta) was markedly induced in a dose-dependent manner. The bacteria were not cytotoxic, because cell viability was not affected after two rounds of stimulation. Thus, unidentified bacterial components from L. paracasei NCC2461 induced the development of a population of CD4(+) T cells with low proliferative capacity that produced TGF-beta and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis.

T cell recognition of rat myelin basic protein as a TCR antagonist inhibits reciprocal activation of antigen-presenting cells and engenders resistance to experimental autoimmune encephalomyelitis.

The aim of this study was to assess whether T cell recognition of myelin basic protein (MBP) as a partially antagonistic self antigen regulates the reciprocal activation of professional antigen-presenting cells (APC). This study focused on the rat 3H3 T cell clone that recognized guinea pig (GP) MBP as a full agonist and self rat (R) MBP as a partial agonist. In cultures of 3H3 T cells and splenic APC, the agonist GPMBP elicited several responses by splenic APC, including production of nitric oxide, down-regulation of I-A, induction of B7.1 and B7.2, and prolongation of APC survival. RMBP stimulated a partial increase in production of nitric oxide, partially promoted survival of splenic APC, but did not alter expression of I-A, B7.1, or B7.2 on splenic APC. In the presence ofGPMBP, RMBP antagonized agonist-stimulated induction of B7 molecules, reversed the loss of I-A, and promoted the generation of I-A(+), costimulus-deficient APC. Furthermore, 3H3 T cells cultured with RMBP and irradiated splenocytes reduced the severity of EAE upon adoptive transfer into naive rat recipients subsequently challenged with an encephalitogenic dose of GPMBP/CFA. Overall, this study indicates that T cell receptor antagonism blocks T cell activation, inhibits feedback activation of splenic APC, and promotes T cell-dependent regulatory activities in EAE.

GIF inhibits Th effector generation by acting on antigen-presenting B cells.

Glycosylation-inhibiting factor (GIF) is a 13-kDa cytokine secreted from T cells. Administration of bioactive recombinant GIF inhibits IgG1 and IgE Ab responses in vivo. Treatment of B cells with the cytokine reduces the secretion of IgG1 and IgE induced by LPS and IL-4. To examine the effect on cognate T-B interaction, GIF was added to low-density B cells from MD4 transgenic (Tg) mice, which express B cell receptor specific for hen egg lysozyme (HEL). The B cells were subsequently pulsed with HEL-OVA conjugate and cultured with OVA-specific naive CD4 T cells from DO11.10 Tg mice. Treatment of Ag-presenting B cells with GIF reduced expansion and IL-2 secretion of naive T cells and rendered them hyporesponsive to antigenic restimulation, resulting in 50--95% reduction of IL-4 and IFN-gamma secretion upon restimulation with Ag. GIF dramatically inhibited Th effector generation when it was added to B cells before pulsing with HEL-OVA, whereas it showed little to no effect when added after B cells were pulsed with Ag. GIF was more effective when B cells from MD4 Tg mice were pulsed with HEL-OVA than when they were pulsed with OVA. This cytokine did not affect Th effector generation when B cells or irradiated splenocytes pulsed with OVA(323--339) peptide stimulated naive DO11.10 T cells. Confocal microscopy revealed that GIF inhibited internalization of HEL by B cells from MD4 Tg mice. Therefore, the cytokine may regulate early steps of Ag presentation involving B cell receptors to diminish Th effector generation from naive CD4 T cells.

IL-10 Kinetics in Thyroiditis-Prone BB/Wor Rats

Cytokines modulate the course of autoimmunity, but their role in the evolution of spontaneous disease is unclear. This study compared the cytokine kinetics of T cell cultures from thyroiditis (LT)-prone NB line BB/Wor rats with those of Wistar (Wis) rat controls following activation with the thyroid-specific antigen thyroglobulin (Tg) or Concanavalin A (Con A). Design: T cell enhanced splenocytes from 60 day old Wis and NB rats were activated with 0.5 μg/ml rat thyroglobulin (Tg) or Con A in the presence of homologous irradiated splenocytes as antigen presenting cells (APC's). In addition, the effect of APC's was determined in a crisscross experiment which examined NB T cell responses to Con A in the presence of Wis APC's. ELISA and RT-PCR were used to examine IL-2, IL-4, IL-10, TNFα, IFNγ, IL-10 concentrations and mRNA expression in the supernatant and cells from parallel cultures harvested at specific intervals. Frozen thyroids from 60 day old NB, Wis and Fisher rats were examined for the presence of IL-10 by immunohistochemistry. T cell proliferation was measured by H thymidine uptake. Results: Following activation with either Tg or Con A, IL-10 concentrations exceeded IFNγ in NB rat cultures, but IFNγ exceeded IL-10 in Wis cultures. Wis splenocytes significantly enhanced NB T cell proliferation and cytokine responses to Con A. Thyroids from 60 day NB rats contained IL-10, but no IFNγ. There was no IL-10 in thyroids from Wistar or Fisher rats. Conclusion: Splenocyte responses in LT-prone BB/Wor rats favor IL-10 production. Future investigations will examine the source of intrathyroidal IL-10 and its role in LT

Irradiated Donor Leukocytes Promote Engraftment of Allogeneic Bone Marrow in Major Histocompatibility Complex Mismatched Recipients Without Causing Graft-Versus-Host Disease

Graft rejection in allogeneic bone marrow transplantation (BMT) can occur when donor and recipient are mismatched at one or more major histocompatibility complex (MHC) loci. Donor T cells can prevent graft rejection, but may cause fatal graft-versus-host disease (GVHD). We tested whether irradiation of allogeneic donor lymphocytes would preserve their graft-facilitating activity while inhibiting their potential for GVHD. Infusions of irradiated allogeneic T cells did not cause GVHD in MHC-mismatched SJL → (SJL × C57BL6) F1, C57BL6 → B10.RIII, and C57BL6 → B10.BR mouse donor → recipient BMT pairs. The 60-day survival among MHC-mismatched transplant recipients increased from 2% (BM alone) to up to 75% among recipients of BM plus irradiated allogeneic splenocytes. Optimal results were obtained using 50 × 106 to 75 × 106 irradiated donor splenocytes administered in multiple injections from day −1 to day +1. Recipients of an equal number of nonirradiated MHC-mismatched donor splenocytes uniformly died of acute GVHD. The graft facilitating activity of the irradiated allogeneic splenocytes was mediated by donor T cells. Irradiation to 7.5 Gy increased nuclear NFκB in T cells and their allospecific cytotoxicity. Irradiated T cells survived up to 3 days in the BM of MHC-mismatched recipients without proliferation. Recipients of irradiated allogeneic splenocytes and allogeneic BM had stable donor-derived hematopoiesis without a significant representation of donor splenocytes in the T-cell compartment. Irradiated allogeneic T cells thus represent a form of cellular immunotherapy with time-limited biologic activity in vivo that can facilitate allogeneic BMT without causing GVHD.

Irradiated Donor Leukocytes Promote Engraftment of Allogeneic Bone Marrow in Major Histocompatibility Complex Mismatched Recipients Without Causing Graft-Versus-Host Disease

AbstractGraft rejection in allogeneic bone marrow transplantation (BMT) can occur when donor and recipient are mismatched at one or more major histocompatibility complex (MHC) loci. Donor T cells can prevent graft rejection, but may cause fatal graft-versus-host disease (GVHD). We tested whether irradiation of allogeneic donor lymphocytes would preserve their graft-facilitating activity while inhibiting their potential for GVHD. Infusions of irradiated allogeneic T cells did not cause GVHD in MHC-mismatched SJL → (SJL × C57BL6) F1, C57BL6 → B10.RIII, and C57BL6 → B10.BR mouse donor → recipient BMT pairs. The 60-day survival among MHC-mismatched transplant recipients increased from 2% (BM alone) to up to 75% among recipients of BM plus irradiated allogeneic splenocytes. Optimal results were obtained using 50 × 106 to 75 × 106 irradiated donor splenocytes administered in multiple injections from day −1 to day +1. Recipients of an equal number of nonirradiated MHC-mismatched donor splenocytes uniformly died of acute GVHD. The graft facilitating activity of the irradiated allogeneic splenocytes was mediated by donor T cells. Irradiation to 7.5 Gy increased nuclear NFκB in T cells and their allospecific cytotoxicity. Irradiated T cells survived up to 3 days in the BM of MHC-mismatched recipients without proliferation. Recipients of irradiated allogeneic splenocytes and allogeneic BM had stable donor-derived hematopoiesis without a significant representation of donor splenocytes in the T-cell compartment. Irradiated allogeneic T cells thus represent a form of cellular immunotherapy with time-limited biologic activity in vivo that can facilitate allogeneic BMT without causing GVHD.

A direct method for the calculation of alloreactive CD4+ T cell precursor frequency.

Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible due to the lack of a specific means of determining the absolute number of daughter cells generated with each division in a repertoire of stimulated T cells. Responder lymphocytes were fluorescently labeled and adoptively transferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation period, responder cells were analyzed by flow cytometry. The precursor frequency of dividing CD4+ T cells was determined both in vivo and in vitro. The observed number of alloreactive daughter cells generated with each round of division was used to calculate the frequency of alloantigen-specific CD4+ T cells. A novel method for the direct calculation of the frequency of alloreactive CD4+ T cells is described. This technique allows the determination of changes in the frequency of alloreactive T cells that might underlie tolerance to alloantigens.

Vβ, Jβ USAGE AND COMPLEMENTARY DETERMINING REGION 3 (CDR3) ANALYSIS OF NAIVE BABOONS AND DURING BABOON T CELL RESPONSE AGAINST XENOANTIGENS

1004 The pig to baboon combination is the most relevant preclinical model of xenotransplantation. Since strategies aimed at inhibiting hyperacute rejection (transgeneic donor, CR1s, antibody removal) have sufficiently prolonged graft survival time, the role of T cells in delayed and late rejection can be approached. Owing to the fact that donor pigs will be likely to be cloned or inbred animals, thus the search for public T cell receptor (TCR) alterations and the understanding of the nature of the T cell response in this specific combination is important. We have analysed, for the first time, the T cell repertoire of the Vβ chain in naive or stimulated (with pig splenocytes enriched for dendritic cells) peripheral blood lymphocytes (PBL) from baboons Papio anubis. Total RNA was first reverse transcribed from naive PBL into cDNA and then amplified using a set of 24 Vβ, 13 Jβ and Cβ potentially cross-reactive primers specific for human Vβ, Jβ, Cβ families. The elongation products were also further analysed for length distribution of the CDR3 using Immunoscope® software. This set of primers allowed to identify most of the baboon T cell repertoire: twenty two Vβ, twelve Jβ families and at least one Cβ segment. Only Vβ21, Vβ24 and Jβ1.5 families were not detected. Each Vβ family profile obtained from naive PBL showed a typical Gaussian pattern of CDR3 distribution (as found in naive human PBL). Then, variations within the baboon T cell repertoire were studied in xenogeneic mixed cultures, where irradiated pig splenocytes enriched for dendritic cells were added to purified baboon T lymphocytes. In this model, baboon T lymphocytes against pig dendritic cells proliferated, indicating a functionally direct pathway and a cross-reactivity between baboon TCR and pig MHC/peptide complexes. However, interestingly, the Vβ repertoire of stimulated baboon T lymphocytes was poorly altered, almost mimicking that found in PBL from naïve baboons, despite a significant proliferation index. In contrast, baboon T lymphocytes stimulated by allogeneic PBL exhibited a much strongly altered repertoire, with dominant CDR3 of stereotyped nucleotide lengths. These results suggest that, in vitro, as recently shown in vivo in the hamster to rat combination (S. Brouard et al, J. Immunol., in press), a large proportion of baboon T cells may expand in each Vβ family in response to pig stimulator cells. These data suggest a strong direct T cell response in the pig to baboon model. Similar experiments are currently being performed using T cells extracted from transgeneic pig kidneys grafted into baboons and presenting delayed or late rejection.

CLASS I MHC PROTEIN TOLEROGENS FOR RAT CARDIAC ALLOGRAFTS ACROSS MAJOR HISTOCOMPATIBILITY BARRIERS.

241 Soluble rat heavy chain class I MHC proteins were produced with an E. coli-based protein expression system. Site-directed mutagenesis using PCR-based SOEing was utilized to produce allochimeric class I major histocompatibility complex (MHC) proteins. The allochimeric protein α1hn-RT1.Ac contains donor Brown Norway(BN) amino acid (a.a.) residues of the highly polymorphic donor alpha-1-helical region on the background of the recipient PVG (RT1.Ac) class I MHC a.a. sequence. In a tolerogenic protocol, 50 μg wild-type RT1.An injected by portal vein (PV) at the time of transplantation to PVG rats plus a 7-day course of oral cyclosporine (CsA, 4 mg/kg/day) prolonged the survival of BN cardiac allografts to a mean survival time (MST) of>200days (n=7, p=0.03). Administration of CsA alone produced a MST of 11.0±1.0 days, (n=25). Administration of 10 μg of α1hn-RT1.Ac via PV at the time of transplantation to PVG rats with CsA prolonged the survival of BN heart allograft to a MST>200days, (n=6, p=0.006). In an oral tolerogenic protocol, PVG rats were pre-treated with 100 μg wild-type RT1.An daily for 5 days(days −4 to 0) before transplant of a BN cardiac allograft plus peri-operative CsA yielded a MST>180 days (n=3, p=0.001). Using a 3H-thymidine uptake assay, analysis of the pan-T-cells from long-term allograft survivors demonstrated markedly decreased proliferation upon challenge with irradiated BN splenocytes in vitro as compared to T-cells from rats with rejected allografts and naïve untreated rats. The data demonstrate that the MHC class I tolerogens are effective for the induction of specific tolerance across MHC barriers.

Coinfusion of irradiated splenocytes with low titer tumor-infiltrating lymphocytes augments antitumor efficacy in adoptive immunotherapy.

We hypothesized that adoptively increasing the density of antigen-presenting cells (APCs) at a tumor site would improve tumor-infiltrating lymphocyte (TIL) in vivo antitumor efficacy. Irradiated splenocytes were used as crude APCs. Alone, they did not have in vitro antitumor activity nor did they augment TIL efficacy in vitro. Pulmonary metastases were established by intravenous (i.v.) injection of 5 x 10(5) MC-38 tumor into irradiated C57B1/6 mice (500 cGy). After 3 days, MC-38 TIL (0.1, 0.5, and 1 x 10(6) cells) +/- irradiated splenocytes (5,000 cGy) as APCs were administered intravenously (0.25, 0.5, and 1 x 10(6) cells) to each group (n = 5/group). Interleukin-2 (60,000 IU) was injected intraperitoneally three times daily for 3 days. Mice were sacrificed 9 days later and metastases elaborated in blinded fashion. A titer of 1 x 10(6) TIL, completely eradicated pulmonary metastases. In two consecutive experiments, when increasing titers of irradiated splenocytes were coinfused with a constant titer of TIL that did not completely eradicate pulmonary metastases, a moderate reduction in pulmonary metastases was observed. The contribution of splenocytes to an improvement in TIL antitumor efficacy was not altered when irradiated splenocytes derived from mice bearing 10-day subcutaneous MC-38 tumors were used. The coinfusion of nonirradiated splenocytes did not improve TIL antitumor in vivo activity. Activated B cells (expressing ICAM-1, B7.1, and B7.2) had no effect on in vitro tumor lysis and did not augment in vivo TIL efficacy. The results show a modest but statistically significant improvement in adoptive immunotherapy antitumor efficacy with fewer TIL by coinfusion of irradiated splenocytes. Further studies to characterize the active potential APC cell subpopulation and to clarify the mechanism(s) responsible for in vivo augmentation of TIL antitumor efficacy are in progress.

Lymphocytes delay kinetics of HSV-1 reactivation from in vitro explants of latent infected trigeminal ganglia

A persistent immune response to herpes simplex virus type 1 (HSV-1) is evidenced by the expression of cytokine transcripts along with infiltrating mononuclear cells in the ganglia of latently infected mice. In trigeminal ganglion (TG) explant co-cultures, the presence of nonirradiated or irradiated primed splenocytes significantly reduced HSV-1 reactivation as defined by secreted infectious HSV-1 found in the supernatants of TG explant cultures. Primed splenocytes depleted of CD4 + or CD8 + cells did not antagonize HSV-1 reactivation. Cytokines including interleukin (IL)-2, IL-6, IL-10, and IL-12 were all detected in the TG explant cultures containing splenocytes. To further study the role of cytokines in HSV-1 reactivation, dissociated TG cell cultures were treated with exogenous recombinant cytokines including IFN-α or -γ, IL-4, 6, 10, 12 or tumor necrosis factor (TNF)-α at concentrations ranging from 2.0 pg to 2.0 ng/culture (or 0.3–300 units/culture for the IFNs). While no cytokines tested at any concentration significantly modified HSV-1 reactivation, neutralizing antibody to IL-6, but not to IFN-α/β, significantly antagonized HSV-1 reactivation. Collectively, the results suggest that IL-6 is directly or indirectly involved in HSV-1 reactivation in TG explant cultures.

An ex vivo study of T lymphocytes recovered from the lungs of I/St mice infected with and susceptible to Mycobacterium tuberculosis.

I/St mice, previously characterized as susceptible to Mycobacterium tuberculosis H37Rv, were given 10(3) or 10(5) CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44(-) CD45RB+ cells disappeared within 2 weeks postinfection, while the number of CD44(+) CD45RB-/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3(+) CD4(+) CD8(-) T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-gamma] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-gamma+ IL-4(-)) clone and one Th0-like (IFN-gamma+ IL-4(+) IL-10(+)) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-gamma responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-gamma production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

A novel monoclonal antibody against rat LFA-1: blockade of LFA-1 and CD4 augments class II MHC expression on T cells.

Previously, we have shown that myelin basic protein (MBP)-specific Lewis rat GP2.E5/R1 (R1) T cells cultured with antigen and irradiated syngeneic splenocytes (IrrSPL) in the presence of anti-CD4 and LRTC1 monoclonal antibodies (MAbs) become highly effective antigen presenting cells (APC). The purpose of these studies was to identify the ligand for the LRTC1 MAb and to determine whether this MAb affected MBP-stimulated IL-2 production and expression of MHC class II molecules by T cells. In the current studies, we show that the LRTC1 MAb specifically immunoprecipitated molecular species of approximately 95, 150, and 180 kD. Commercially available anti-CD18 (beta2 integrin, beta-chain of LFA-1, MAC-1, and p150, 95) and LRTC1 MAb immunoprecipitated proteins with identical mobilities on 1-D and 2-D SDS-PAGE gels. Moreover, anti-CD18 and LRTC1 immunoprecipitates also showed identical mobilities on 1-D gels after enzymatic cleavage of N-linked oligosaccharides and thereby had the same patterns of differential glycosylation. Anti-CD4 MAb W3/25 and LRTC1 MAb synergistically inhibited T-cell IL-2 mRNA and IL-2 bioactivity, but augmented antigen-stimulated surface I-A on R1 T cells. In conclusion, these studies describe the characteristics of a novel anti-LFA-1 MAb, LRTC1, which should prove useful in studying costimulatory and adhesion pathways among rat leukocytes.

COSTIMULATORY PATHWAYS ARE ACTIVE IN XENOGENEIC IMMUNE RESPONSES

36 Costimulatory blockade has been shown in this and other laboratories to prevent allograft rejection. Recent advances in the control of the innate humoral response to discordant xenografts have focused attention on potential means of preventing subsequent cellular and acquired humoral reactivity. A variety of costimulatory pathway blockers have been tested in mixed lymphocyte reactions (MLR) for their ability to prevent human peripheral blood mononuclear cell (PBMC) activation when stimulated by irradiated porcine splenocytes. The results that human CTLA4-Ig is capable of attenuating the xenoreactive PBMC proliferation by 60%, anti-human CD40L(5C8) by 52%, anti-human CD80 (1F1) by 13%, and anti-human CD86 (3D1) by 28%. Furthermore, combination of these reagents proves to have synergistic effects. The combination of hCTLA4-Ig/5C8 attenuates the response by 76% and the 1F1/3D1/5C8 combination by 72%. Consistent with this observation, LPS stimulated porcine splenocytes have been shown by flow cytometry to specifically bind hCTLA4-Ig, 1F1, and 3D1. A panel of 12 anti-human CD80 and CD86 antibodies have been screened with only 1F1 and 3D1 exhibiting cross reactivity to pig. In addition, using irradiated human PBMCs as stimulators and porcine PBMC's as the responding cells revealed that the combination of 5C8 and hCTLA4-Ig is also capable of blocking pig cell proliferation. It would thus appear that costimulatory ligands are sufficiently homologous, such that they support cross species ligand/receptor interaction which is important in the xenoreactive response between human and porcine cells. Preliminary evidence also suggests that blockade of CD86 on the porcine microvascular endothelial cell (PMVEC) inhibits their killing by human NK cells. This is consistent with the constitutive expression of CD86 on PMVEC's that we have observed. Furthermore, we have demonstrated that CD86 is upregulated by exposure to human T and NK cell and human T cell conditioned media. We conclude that costimulatory molecules play an important role in cellular xenoreactivity and that the same reagents used to prevent allograft rejection may function in the prevention of xenograft rejection. These results and the known effects of 5C8 on isotype switching and maturation of acquired humoral responses efficacy of these reagents in modulating both the humoral and cellular immune response. We are now in the process of initiating these studies in a pig to primate model.

COSTIMULATORY PATHWAYS ARE ACTIVE IN XENOGENEIC IMMUNE RESPONSES.

36 Costimulatory blockade has been shown in this and other laboratories to prevent allograft rejection. Recent advances in the control of the innate humoral response to discordant xenografts have focused attention on potential means of preventing subsequent cellular and acquired humoral reactivity. A variety of costimulatory pathway blockers have been tested in mixed lymphocyte reactions (MLR) for their ability to prevent human peripheral blood mononuclear cell (PBMC) activation when stimulated by irradiated porcine splenocytes. The results indicate that human CTLA4-Ig is capable of attenuating the xenoreactive PBMC proliferation by 60%, anti-human CD40L (5C8) by 52%, anti-human CD80 (1F1) by 13%, and anti-human CD86 (3D1) by 28%. Furthermore, combinations of these reagents proves to have synergistic effects. The combination of hCTLA4-Ig/5C8 attenuates the response by 76% and the 1F1/3D1/5C8 combination 72%. Consistent with this observation, LPS stimulated porcine splenocytes have been shown by flow cytometry to specifically bind hCTLA4-Ig, 1F1, and 3D1. A panel of 12 anti-human CD80 and CD86 antibodies have been screened with only 1F1 and 3D1 exhibiting cross reactivity to pig. In addition, using irradiated human PBMCs as stimulators and porcine PBMC's as the responding cells revealed that the combination of 5C8 and hCTLA4-Ig is also capable of blocking pig cell proliferation. It would thus appear that costimulatory ligands are sufficiently homologous, such that they support cross species ligand/receptor interaction which is important in the xenoreactive response between human and porcine cells. Preliminary evidence also suggests that blockade of CD86 on the porcine microvascular endothelial cell (PMVEC) inhibits their killing by human NK cells. This is consistent with the constitutive expression of CD86 on PMVEC's that we have observed. Furthermore, we have demonstrated that CD86 is upregulated by exposure to human T and NK cell and human T cell conditioned media. We conclude that costimulatory molecules play an important role in cellular xenoreactivity and that the same reagents used to prevent allograft rejection may function in the prevention of xenograft rejection. These results and the known effects of 5C8 on isotype switching and maturation of acquired humoral responses suggests efficacy of these reagents in modulating both the humoral and cellular immune response. We are now in the process of initiating these studies in a pig to primate model.

Irradiated Donor Lymphocyte Infusion, A Novel Approach To Prevent Graft Failure During Allogeneic Bone Marrow Transplant • 797

Efforts to limit graft-vs-host disease (GvHD) by T-cell depletion (TCD) have been complicated by graft failure. Donor T-cells favor engraftment, but can lead to GvHD. We have explored the effect of low dose radiation on the ability of donor lymphocytes to facilitate engraftment without causing GvHD in a mouse model of allogeneic bone marrow transplantation (BMT). We have observed that proliferation of murine T-cells both in vitro and in vivo is inhibited by exposure to > 5 Gy of ionizing radiation. We tested the ability of irradiated allogeneic spleen cells to facilitate engraftment by allogeneic bone marrow (BM) cells in the C57BL6 H2b B10BR H2k combination of MHC mis-matched donor-recipient pairs. B10BR (H2k) mice that received multiple injections of irradiated H2b allogeneic splenocytes had 65% survival at day +60 post-transplant compared to <10% survival among recipients of T-cell depleted bone marrow alone, or T-cell depleted bone marrow plus non-irradiated allogeneic splenocytes (p<0.001 compared to recipients of non-irradiated allogeneic splenocytes or TCD BM alone). TCD of the allogeneic spleen cells eliminated the graft facilitating effect in this model system, indicating that irradiated T-cells retained short-term ability to enhance engraftment activity without the potential for in vivo proliferation or GvHD. Irradiated mouse T-cells had enhanced cytolytic activity against allogeneic targets compared to non-irradiated cells. Molecular analysis of the activation state of irradiated mouse splenocytes indicated up-regulation of nuclear factor κB(NFκB) in T-cells at the radiation doses used to treat the allogeneic donor splenocytes. These data suggest that irradiated donor lymphocytes represent a novel therapeutic modality which allows transplant across major histocompatibility barriers by preventing the graft failure usually associated with T-cell depleted grafts.

Antigen presentation by T cells: T cell receptor ligation promotes antigen acquisition from professional antigen‐presenting cells

The purpose of this study was to determine whether the clonotypic specificity of the T cell receptor influences the specificity of T cell-mediated antigen presentation. We have previously shown that myelin basic protein (MBP)-specific Lewis rat GP2.E5/R1 (R1) T cells cultured with antigen, irradiated syngeneic splenocytes (IrrSPL) and tolerogenic monoclonal antibody become highly effective antigen-presenting cells (APC). In the current studies, we investigated the transfer of specific (MBP) and unrelated (conalbumin) antigens from antigen-pulsed SPL to R1 T cells. R1 T cells cultured with IrrSPL that were pulsed simultaneously with both MBP and conalbumin acquired and presented both antigens to the appropriate T cell responders in a secondary assay. These results suggested a physical transfer of major histocompatibility complex (MHC)/peptide complexes from professional APC to R1 T cells. Transfer of conalbumin from professional APC to R1 T cells required specific recognition of MBP and was optimal when both conalbumin and MBP were presented on the same group of professional APC. Antigens transfer did not occur when allogeneic SPL were used as APC. The anti-I-A mAb OX6 inhibited antigen transfer but only when added during the initiation of culture. OX6 also inhibited antigen acquisition by R1-trans, a variant of the R1 T cell line which constitutively synthesizes high levels of I-A, from MBP-pulsed IrrSPL but blockade of I-A did not inhibit antigen acquisition when soluble MBP was added directly to the culture. Despite constitutive synthesis of I-A, R1-trans T cells did not acquire guinea pig MBP from pulsed allogeneic APC. These studies demonstrate that although T cells of a particular specificity can present unrelated antigens, the cognate interaction of the T cell antigen receptor with the appropriate antigen/self-MHC complex strongly promotes acquisition of these complexes from professional APC.

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts.

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells. (Hepatology 1997 Nov;26(5):1287-95)