TNF enhances CD4+ T cell alloproliferation,IFN-gamma responses,and intestinal graft-versus-host disease by IL-12-independent mechanisms

Inhibition of TNF/TNFR2 interactions ameliorates intestinal graft-vs-host disease (GVHD) and Th1 cytokine responses induced by transfer of B6 CD4(+) spleen cells into irradiated MHC class II disparate B6.C-H-2(bm12) (bm12) x B6 F(1) recipients. The present studies examined whether these effects of TNF are IL-12 dependent. T cell proliferative responses of B6.129S1-IL-12rb2(tm1Jm) (B6.IL-12R(-/-)) responder spleen cells were found to be comparable to those of control B6 spleen cells. TNF inhibition reduced T cell proliferation and IFN-gamma production in supernatants of MLC using either B6.IL-12R(-/-) or control B6 responder cells. GVHD induced wasting disease in recipients of B6.IL-12R(-/-) CD4(+) spleen cells that received a TNF inhibitor-encoding adenovirus (5.4 +/- 6.5% weight loss (n = 7)) was significantly reduced compared with levels of weight loss observed in recipients that had received a control adenovirus (25.7 +/- 12.2% weight loss (n = 11), p = 0.001). Furthermore, TNF inhibition was associated with a reduction in colonic GVHD scores (p = 0.039) and in the percentage of the splenic CD4(+) T cells that expressed IFN-gamma (16 vs 6%). These findings indicate that TNF promotes CD4(+) T cell alloproliferation, IFN-gamma responses, and intestinal GVHD by IL-12-independent mechanisms.     

IL-12-independent LIGHT signaling enhances MHC class II disparate CD4+ T cell alloproliferation, IFN-gamma responses, and intestinal graft-versus-host disease

Inhibition of LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor)/herpes simplex virus entry mediator (HVEM) and LIGHT/lymphotoxin beta receptor (LT beta R) interactions decreases mortality in MHC class I and II disparate graft-vs-host disease (GVHD). The present studies assessed the effects of these interactions on the generation of CD4+ T cell alloresponses in MHC class II-disparate MLC and GVHD. An inhibitor protein of LIGHT and LT alpha beta2 (LT beta R-Ig) and an inhibitor protein of LIGHT (HVEM-Ig) caused similar decreases in alloresponses of control B6 or B6.129S1-IL12rb2(tm1Jm) (B6.IL12R-/-) spleen cells (SpC) in a MHC class II-disparate MLC. GVHD-induced wasting disease in MHC class II-disparate recipients of B6 CD4+ SpC who received either the LT beta R-Ig-encoding adenovirus (LT beta R-Ig Adv; 13.1 +/- 10.9%; n = 10; p = 0.0004) or the HVEM-Ig-encoding adenovirus (HVEM-Ig Adv; 16.4 +/- 9.9%; n = 13; p = 0.0008) was significantly reduced compared with that in recipients of a control adenovirus (30.4 +/- 8.8%; n = 13). Furthermore, gut GVHD histologic scores of recipients of B6 CD4+ SpC who received the LT beta R-Ig Adv (0.8 +/- 0.8; n = 5; p = 0.0007) or the HVEM-Ig Adv (1.4 +/- 0.5; n = 5; p = 0.008) were reduced compared with scores of recipients of a control adenovirus (2.5 +/- 0.75; n = 11). In the intestine, both LT beta R-Ig Adv and HVEM-Ig Adv decreased CD4+ T cells (0.35 +/- 0.4 x 10(6) (n = 6) vs 0.36 +/- 0.02 x 10(6) (n = 9); p = 0.03 and p = 0.007) compared with control adenovirus (0.86 +/- 0.42 x 10(6); n = 9). LIGHT is critical for optimal CD4+ T cell alloresponses in MHC class II-disparate MLC and GVHD.     

CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6-->bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4(+) T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-gamma-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4(+) T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2(-/-) CD4(+) T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4(+) T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2(-/-) splenocyte-->bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.     

Lethal graft-vs-host disease induced by a class II MHC antigen only disparity is not mediated by cytotoxic T cells

Treatment of C57BL/6J (B6) murine splenocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes NK cells, CTL precursors, and the capacity to cause lethal graft-vs-host disease (GVHD) in irradiated B6 X DBA/2 F1 mice. In contrast, alloantigen-induced L3T4(+) Th cell function has been shown to be relatively preserved after exposure to this agent. The present studies assessed the effects of Leu-Leu-OMe treatment of donor cells on induction of lethal GVHD in other murine strain combinations. When irradiated B6 X CBAF1 mice were infused with T and NK cell-depleted B6 bone marrow cells and 3 to 30 X 10(6) B6 spleen cells, uniformly lethal GVHD was observed. However, B6 X CBAF1 recipients of T and NK-depleted B6 bone marrow cells and similar numbers of Leu-Leu-OMe-treated B6 spleen cells demonstrated 90 to 100% long term survival. In contrast, Leu-Leu-OMe treatment of B6 donor cells had no beneficial effect on mortality rates in irradiated (B6 X B6-C-H-2bm12)F1 (B6 X bm12F1) recipients. When B6 spleen cells were stimulated in vivo or in vitro with either B6 X CBAF1 or B6 X bm12F1 stimulator cells, the capacity to generate alloantigen-specific CTL was abolished comparably by Leu-Leu-OMe treatment. Thus, the dramatic difference between the effects of Leu-Leu-OMe treatment of B6 spleen cells on the course of GVHD in B6 x CBAF1 and class II MHC only disparate B6 x bm12F1 recipients could not be explained by unique resistance of bm12-specific CTL precursors to Leu-Leu-OMe. These findings indicate that T cell effector mechanisms distinct from classic cell-mediated cytotoxicity are sufficient to generate lethal GVHD in class II MHC only disparate B6----B6 X bm12F1 mice.     

Intestinal lamina propria retaining CD4+CD25+ regulatory T cells is a suppressive site of intestinal inflammation

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (T(REG)) cells play an important role in the normal intestinal homeostasis, but also that its dysregulation of immune response leads to the development of inflammatory bowel disease. In this study, we demonstrate that murine CD4(+)CD25(+) T cells residing in the intestinal lamina propria (LP) constitutively express CTLA-4, glucocorticoid-induced TNFR, and Foxp3 and suppress proliferation of responder CD4(+) T cells in vitro. Furthermore, cotransfer of intestinal LP CD4(+)CD25(+) T cells prevents the development of chronic colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into SCID mice. When lymphotoxin (LT)alpha-deficient intercrossed Rag2 double knockout mice (LTalpha(-/-) x Rag2(-/-)), which lack mesenteric lymph nodes and Peyer's patches, are transferred with CD4(+)CD45RB(high) T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LTalpha(+/+) x Rag2(-/-) mice transferred with CD4(+)CD45RB(high) T cells. Of note, when a mixture of splenic CD4(+)CD25(+) T(REG) cells and CD4(+)CD45RB(high) T cells are transferred into LTalpha(-/-) x Rag2(-/-) recipients, CD4(+)CD25(+) T(REG) cells migrate into the colon and prevent the development of colitis in LTalpha(-/-) x Rag2(-/-) recipients as well as in the control LTalpha(+/+) x Rag2(-/-) recipients. These results suggest that the intestinal LP harboring CD4(+)CD25(+) T(REG) cells contributes to the intestinal immune suppression.     

IL-5 mediates eosinophilic rejection of MHC class II-disparate skin allografts in mice.

CD4 T cells play a crucial role in the acute rejection of MHC class II-disparate skin allografts, mainly by Fas/Fas ligand-mediated cytotoxicity. Because recent observations indicate that eosinophils may be found within allografts rejected by CD4 T cells, we evaluated the role played by IL-5, the main eosinophil growth factor, and by eosinophils in the rejection of MHC class II-disparate skin grafts. C57BL/6 mice rapidly rejected MHC class II-disparate bm12 skin grafts. Rejected skins contained a dense, aggressive eosinophil infiltrate. Lymphocytes isolated from lymph nodes draining rejected bm12 skin were primed for IL-5 secretion, and IL-5 mRNA was present within rejected grafts. The IL-5/eosinophil pathway played an effector role in allograft destruction, because the rejection of bm12 skin was significantly delayed in IL-5-deficient mice as compared with wild-type animals. The role of the IL-5/eosinophil pathway was further investigated in MHC class II-disparate donor-recipient strains unable to establish Fas/Fas ligand interactions. Fas ligand-deficient gld/gld mice rejected bm12 skins, and bm12 mice rejected Fas-deficient lpr/lpr C57BL/6 skins. Neutralization of IL-5 prevented acute rejection in both combinations. We conclude that MHC class II-disparate skin allografts trigger an IL-5-dependent infiltration of eosinophils that is sufficient to result in acute graft destruction.     

Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease

Alloantigen expression on host APCs is essential to initiate graft-vs-host disease (GVHD); however, critical APC subset remains to be elucidated. We compared the ability of dendritic cells (DCs) and B cells to initiate acute GVHD by an add-back study of MHC class II-expressing APCs (II(+/+)) into MHC class II-deficient (II(-/-)) mice that were resistant to CD4-dependent GVHD. Injection of host-derived, but not donor-derived, II(+/+) DCs or host-derived II(+/+) B cells, was sufficient to break GVHD resistance of II(-/-) mice and induced lethal acute GVHD. By contrast, host-derived II(+/+) B cells, both naive and LPS stimulated, failed to induce activation or tolerance of donor CD4(+) T cells. Similarly, in a model of CD8-dependent GVHD across MHC class I mismatch injection of allogeneic DCs, but not B cells, induced robust proliferation of donor CD8(+) T cells and broke GVHD resistance of chimeric recipients in which APCs were syngeneic to donors. These results demonstrate that host-derived DCs are critical in priming donor CD4(+) and CD8(+) T cells to cause GVHD, and selective targeting of host DCs may be a promising strategy to prevent GVHD.     

CD30/CD30 ligand (CD153) interaction regulates CD4+ T cell-mediated graft-versus-host disease

CD30, a TNFR family member, is expressed on activated CD4(+) and CD8(+) T cells and B cells and is a marker of Hodgkin's lymphoma; its ligand, CD30L (CD153) is expressed by activated CD4(+) and CD8(+) T cells, B cells, and macrophages. Signaling via CD30 can lead to proliferation or cell death. CD30-deficient (-/-) mice have impaired thymic negative selection and increased autoreactivity. Although human alloreactive T cells preferentially reside within the CD30(+) T cell subset, implicating CD30 as a regulator of T cell immune responses, the role of CD30/CD153 in regulating graft-vs-host disease (GVHD) has not been reported. We used a neutralizing anti-CD153 mAb, CD30(-/-) donor mice, and generated CD153(-/-) recipient mice to analyze the effect of CD30/CD153 interaction on GVHD induction. Our data indicate that the CD30/CD153 pathway is a potent regulator of CD4(+), but not CD8(+), T cell-mediated GVHD. Although blocking CD30/CD153 interactions in vivo did not affect alloreactive CD4(+) T cell proliferation or apoptosis, a substantial reduction in donor CD4(+) T cell migration into the gastrointestinal tract was readily observed with lesser effects in other GVHD target organs. Blockade of the CD30/CD153 pathway represents a new approach for preventing CD4(+) T cell-mediated GVHD.     

Defining the herpes simplex virus-specific CD8+ T cell repertoire in C57BL/6 mice

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (β), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ～84 HSV-1 proteins are recognized by CD8(+) T cells, and most (～80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 β and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.     

Characterization of reciprocal Lmb1-4 interval MRL-Faslpr and C57BL/6-Faslpr congenic mice reveals significant effects from Lmb3

Susceptibility to severe lupus in MRL-Fas(lpr) mice requires not only the lpr mutation but also other predisposing genes. Using (MRL-Fas(lpr) x B6-Fas(lpr))F2 (where B6 represents C57BL/6) intercrosses that utilize the highly susceptible MRL and poorly susceptible B6 backgrounds, we previously mapped CFA-enhanced systemic lupus-like autoimmunity to four loci, named Lmb1-4, on chromosomes 4, 5, 7, and 10. In the current study, we generated and analyzed reciprocal interval congenic mice for susceptibility to CFA-enhanced autoimmunity at all four Lmb loci. Although all loci had at least a slight effect on lymphoproliferation, only Lmb3 demonstrated a major effect on lymphoproliferation and anti-chromatin Ab levels. Further characterization of Lmb3, primarily by comparing MRL-Fas(lpr) with MRL.B6-Lmb3 Fas(lpr) congenic mice, revealed that it also played a significant role in spontaneous lupus, modifying lymphoproliferation, IgG and autoantibody levels, kidney disease, and survival. The less susceptible B6 Lmb3 locus was associated with a marked reduction in numbers of CD4(+) and double-negative (CD4(-)CD8(-)) T cells, particularly in lymph nodes, as well as reduced T cell proliferation and enhanced T cell apoptosis, both in vivo and in vitro. IFN-gamma-producing CD4(+) T cells were also reduced in MRL.B6-Lmb3 Fas(lpr) mice. Further mapping using subinterval congenic mice placed Lmb3 in the telomeric portion of chromosome 7. Thus, Lmb3, primarily through its effects on CD4(+) and double-negative T cells, appears to be a highly penetrant lupus-modifying locus. Identification of the underlying genetic alteration responsible for this quantitative trait locus should provide new insights into lupus-modifying genes.     

APCs in the liver and spleen recruit activated allogeneic CD8+ T cells to elicit hepatic graft-versus-host disease

Host APCs are required for initiating T cell-dependent acute graft-vs-host disease (GVHD), but the role of APCs in the effector phase of acute GVHD is not known. To measure the effect of tissue-resident APCs on the local development of acute GVHD, we selectively depleted host macrophages and DCs from the livers and spleens, but not from the skin, peripheral lymph nodes (PLN), or mesenteric lymph nodes (MLN), of C57BL/6 (B6) mice by i.v. administration of liposomal clodronate before allogeneic bone marrow transplantation. Depletion of host hepatic and splenic macrophages and DCs significantly inhibited the proliferation of donor C3H.SW CD8(+) T cells in the spleen, but not in the PLN or MLN, of B6 mice. Such organ-selective depletion of host tissue APCs also markedly reduced the trafficking of allogeneic CD8(+) T cells into the livers and spleens, but not PLN and MLN, of B6 recipients compared with that of the control mice. Acute hepatic, but not cutaneous, GVHD was inhibited as well, resulting in improved survival of liposomal clodronate-treated B6 recipients. When C3H.SW CD8(+) T cells were activated in normal B6 recipients, recovered, and adoptively transferred into secondary B6 recipients, activated donor CD8(+) T cells rapidly migrated into the livers and spleens of control B6 recipients but were markedly decreased in B6 mice that were depleted of hepatic and splenic macrophages and DCs. Thus, tissue-resident APCs control the local recruitment of allo-reactive donor T cells and the subsequent development of acute GVHD.     

Alloreactive T cell responses and acute rejection of single class II MHC-disparate heart allografts are under strict regulation by CD4+ CD25+ T cells

Skin but not vascularized cardiac allografts from B6.H-2bm12 mice are acutely rejected by C57BL/6 recipients in response to the single class II MHC disparity. The underlying mechanisms preventing acute rejection of B6.H-2bm12 heart allografts by C57BL/6 recipients were investigated. B6.H-2bm12 heart allografts induced low levels of alloreactive effector T cell priming in C57BL/6 recipients, and this priming was accompanied by low-level cellular infiltration into the allograft that quickly resolved. Recipients with long-term-surviving heart allografts were unable to reject B6.H-2bm12 skin allografts, suggesting potential down-regulatory mechanisms induced by the cardiac allografts. Depletion of CD25+ cells from C57BL/6 recipients resulted in 15-fold increases in alloreactive T cell priming and in acute rejection of B6.H-2bm12 heart grafts. Similarly, reconstitution of B6.Rag(-/-) recipients with wild-type C57BL/6 splenocytes resulted in acute rejection of B6.H-2bm12 heart grafts only if CD25+ cells were depleted. These results indicate that acute rejection of single class II MHC-disparate B6.H-2bm12 heart allografts by C57BL/6 recipients is inhibited by the emergence of CD25+ regulatory cells that restrict the clonal expansion of alloreactive T cells.     

Resistance of C57BL/6 mice to amoebiasis is mediated by nonhemopoietic cells but requires hemopoietic IL-10 production

Resistance to intestinal amoebiasis is mouse strain dependent. C57BL/6 (B6) mice clear Entamoeba histolytica within hours of challenge, whereas C3H and CBA strains are susceptible to infection and disease. In this study, we show using bone marrow (BM) chimeric mice that mouse strain-dependent resistance is mediated by nonhemopoietic cells; specifically, B6 BM --> CBA recipients remained susceptible as measured by amoeba score and culture, whereas CBA BM --> B6 recipients remained resistant. Interestingly, hemopoietic IL-10 was required for maintaining the resistance of B6 mice, in that B6 IL-10-deficient mice and IL-10(-/-) BM --> wild-type recipients, but not IL-10(+/+) BM --> IL-10(-/-) recipients, exhibited higher amoeba scores than their wild-type controls. Additionally, C57BL/10 IL-10(-/-)Rag2(-/-) mice exhibited diminished amoeba scores and culture rates vs IL-10(-/-) mice, indicating that lymphocytes potentiated the susceptibility of IL-10-deficient mice. We conclude that nonhemopoietic cells mediate the natural resistance to intestinal amoebiasis of B6 mice, yet this resistance depends on hemopoietic IL-10 activity.     

CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major

Standard murine models of cutaneous leishmaniasis, involving s.c. inoculation of large numbers of Leishmania major promastigotes, have not supported an essential role for CD8(+) T cells in the control of primary infection. Recently, a L. major model combining two main features of natural transmission, low parasite dose and inoculation into a dermal site, has been established in resistant C57BL/6 mice. In the present studies, C57BL/6 mice with CD8(+) T cell deficiencies, including CD8(-/-) and CD8-depleted mice, failed to control the growth of L. major following inoculation of 100 metacyclic promastigotes into the ear dermis. The resulting dermal pathology was minor and delayed. Lesion formation in wild-type mice was coincident with the killing of parasites in the inoculation site. Both events were associated with the accumulation of CD8(+) T lymphocytes in the skin and with the capacity of CD8(+) T cells recovered from draining lymph nodes or infected dermis to release IFN-gamma following coculture with infected dendritic cells. Reconstitution of resistance to L. major in RAG(-/-) mice using T cells from naive donors was optimal when both CD4(+) and CD8(+) T cells were transferred. Primed CD8(+) T lymphocytes obtained from C57BL/6 mice during the acute stage of infection were able to mediate both pathology and immunity when transferred alone. The low dose, intradermal challenge model reveals that CD8(+) T cells play an essential role in both pathogenesis of and immunity to primary infection with L. major in the skin.     

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4(+) T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-gamma production. Furthermore, a potent IFN-gamma-inducing, D(b)-restricted CD8(+) epitope was identified using MHC class I mutant B6.C-H-2(bm13) mice and intracellular IFN-gamma and whole blood CD8(+) T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this D(b)-restricted PstS-3 epitope. IFN-gamma ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4(+) and CD8(+) T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2(b, p, and f) haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2(d, k, r, s, and q) haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2(bm12) mice could be overcome by DNA vaccination. IFN-gamma-producing CD8(+) T cells could also be detected against the D(b)-restricted epitope in H-2(p) haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4(+) and particularly CD8(+) T cell responses against mycobacterial Ags.     

Inter-strain tissue-infiltrating T cell responses to minor histocompatibility antigens involved in graft-versus-host disease as determined by Vbeta spectratype analysis

Lethal graft-vs-host disease (GVHD) can be induced between MHC-matched murine strains expressing multiple minor histocompatibility Ag differences. In the B6->BALB.B model, both CD4(+) and CD8(+) donor T cells can mediate lethal GVHD, whereas in the B6->CXB-2 model, only CD8(+) T cells are lethal. TCR Vbeta CDR3-size spectratyping was previously used to analyze CD8(+) and CD4(+) T cell responses in lethally irradiated BALB.B and CXB-2 recipients, which showed significant overlap in the reacting repertoires. However, CD4(+) T cells exhibited unique skewing of the Vbeta2 and 11 families in only BALB.B recipients. These Vbeta family reactivities were confirmed by immunohistochemical staining of lingual epithelial infiltrates, and by positive and negative selection Vbeta family transfer experiments for GVHD induction in BALB.B recipients. We have now extended these studies to examine the T cell repertoire responses involved in target tissue damage. Infiltrating B6 host-presensitized CD8(+) and CD4(+) T cells were isolated 8-10 days post-transplant from the spleens, intestines and livers of CXB-2 and BALB.B transplant recipients. For both T cell subsets, the results indicated overlapping tissue skewings between the recipients, also between the tissues sampled within the respective recipients as well as tissue specific responses unique to both the BALB.B and CXB-2 infiltrates. Most notably, the CD4(+) Vbeta 11(+) family was skewed in the intestines of BALB.B but not CXB-2 recipients. Taken together, these data suggest that there are likely to be target tissue-related anti-multiple minor histocompatibility Ag-specific responses in each of the strain recipients, which may also differ from those found in peripheral lymphoid organs.     

Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35-55 from myelin oligodendrocyte glycoprotein (pMOG(35-55)) consistently activate a high proportion of CD8+ alphabetaTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG(35-55). CNS lesions in pMOG(35-55) CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1(-/-) mice, but not in isogeneic beta2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1(-/-) mice in which disease was induced adoptively with <1 x 10(6) T cells sensitized to pMOG(35-55). It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.     

Host conditioning is a primary determinant in modulating the effect of IL-7 on murine graft-versus-host disease

Interleukin-7 has been shown to enhance T cell reconstitution after allogeneic bone marrow transplantation, in part, by expansion of mature donor T cells, but whether IL-7 also exacerbates graft-vs-host disease (GVHD) remains unresolved. To address this issue, we examined the effect of IL-7 on GVHD induction using a well-defined murine GVHD model (B6-->B6AF1/J). Administration of IL-7 to nonirradiated B6AF1/J recipients of B6 T cells resulted in expansion of splenic donor CD4(+) and CD8(+) T cells and increased GVHD mortality. In contrast, administration of IL-7 on the same schedule failed to increase GVHD mortality in either sublethally or lethally irradiated animals that received graded doses of T cells designed to induce varying degrees of GVHD severity. Moreover, IL-7 failed to increase the number of alloreactive T cells when examined in a murine model (B6-->BALB.B) that allowed for direct quantitation of graft-vs-host-reactive T cells. The combination of irradiation and transplantation of alloreactive donor T cells resulted in significantly increased levels of endogenous splenic IL-7 mRNA when compared with nonirradiated transplanted animals, providing a potential explanation for why exogenous IL-7 did not increase GVHD severity in these mice. We conclude that host conditioning modulates the ability of exogenous IL-7 to exacerbate GVHD and that this occurs through induction of endogenous IL-7 production.     

Effects of Aluminum on Immune Functions of Cultured Splenic T and B Lymphocytes in Rats

The effects of Aluminum (Al) exposure on immune functions of cultured splenic T and B lymphocytes of rats were studied. The lymphocytes were isolated from spleen of healthy male Wistar rats weighing 110-120 g. The cultured cells in RPMI-1640 medium were exposed to 0 (control group), 0.035 (low-dose group), 0.07 (medial-dose group), and 0.14 (high-dose group) mg/mL Al(3+) as aluminum trichloride (AlCl(3)) in an incubator under 5% CO(2) at 37°C for 24 h. The T and B lymphocyte proliferation was measured with a tetrazolium dye colorimetric assay. The levels of interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were determined by iodine [(125)I] IL-2, IL-6, and TNF-α radioimmunoassay kits, respectively. The proportions of CD3(+), CD4(+), and CD8(+) T lymphocytes were measured with a flow cytometer. The results showed that the T and B lymphocyte proliferation, the levels of IL-2, IL-6, TNF-α, the proportions of CD3(+) and CD4(+) T lymphocytes, and the ratio of CD4(+)/CD8(+) T lymphocytes were lowered by Al treatments, while the proportion of CD8(+) T lymphocytes was increased. These findings indicate that Al exposure can inhibit the immune functions of splenic T and B lymphocytes of rats in vitro.