TNF-TNFR2 interactions are critical for the development of intestinal graft-versus-host disease in MHC class II-disparate (C57BL/6J-->C57BL/6J x bm12)F1 mice

TNF-TNFR2 interactions promote MHC class II-stimulated alloresponses while TNF-TNFR1 interactions promote MHC class I-stimulated alloresponses. The present studies were designed to evaluate whether TNF-TNFR2 interactions were involved in the in vivo generation of CD4(+) T cell-mediated intestinal graft-versus-host disease (GVHD) in the (C57BL/6J (hereafter called B6) --> B6 x B6.C-H-2(bm12) (bm12))F(1) GVHD model. Briefly, 5 x 10(6) splenic CD4(+) T lymphocytes from B6.TNFR2(-/-) or control B6 mice were transferred with 1--2 x 10(6) T cell-depleted B6 bone marrow cells (BMC) to irradiated MHC class II-disparate (bm12 x B6)F(1) mice. Weight loss, intestinal inflammation, and the surface expression of CD45RB (memory marker) on intestinal and splenic lymphocytes were assessed. IL-2 and IFN-alpha mRNA levels in intestinal lymphocytes were assessed by nuclease protection assays. A significant reduction in weight loss and intestinal inflammation was observed in recipients of the TNFR2(-/-)CD4(+) SpC. Similarly, a significant decrease was noted in T cell numbers and in CD45RB(low) (activated/memory) expression on intestinal but not CD4(+) T cells in recipients of TNFR2(-/-)CD4(+) spleen cells. IL-2 and IFN-alpha mRNA levels were reduced in the intestine in the recipients of TNFR2(-/-) splenic CD4(+) T cells. These results indicate that TNF-TNFR2 interactions are important for the development of intestinal inflammation and activation/differentiation of Th1 cytokine responses by intestinal lymphocytes in MHC class II-disparate GVHD while playing an insignificant role in donor T cell activation in the spleen.     

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.     

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.     

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.     

T cell TRAIL promotes murine lupus by sustaining effector CD4 Th cell numbers and by inhibiting CD8 CTL activity

T cells play an essential role in driving humoral autoimmunity in lupus. Molecules such as TRAIL exhibit strong T cell modulatory effects and are up-regulated in lupus, raising the possibility that they may influence disease severity. To address this possibility, we examined the role of TRAIL expression on pathogenic T cells in an induced model of murine lupus, the parent-into-F(1) (P-->F(1)) model of chronic graft-vs-host disease (GVHD), using wild-type or TRAIL-deficient donor T cells. Results were compared with mice undergoing suppressive acute GVHD. Although chronic GVHD mice exhibited less donor T cell TRAIL up-regulation and IFN-alpha-inducible gene expression than acute GVHD mice, donor CD4(+) T cell TRAIL expression in chronic GVHD was essential for sustaining effector CD4(+) Th cell numbers, for sustaining help to B cells, and for more severe lupus-like renal disease development. Conversely, TRAIL expression on donor CD8(+) T cells had a milder, but significant down-regulatory effect on CTL effector function, affecting the perforin/granzyme pathway and not the Fas ligand pathway. These results indicate that, in this model, T cell-expressed TRAIL exacerbates lupus by the following: 1) positively regulating CD4(+) Th cell numbers, thereby sustaining T cell help for B cells, and 2) to a lesser degree by negatively regulating perforin-mediated CD8(+) CTL killing that could potentially eliminate activated autoreactive B cells.     

CTL-promoting effects of CD40 stimulation outweigh B cell-stimulatory effects resulting in B cell elimination and disease improvement in a murine model of lupus

CD40/CD40L signaling promotes both B cell and CTL responses in vivo, the latter being beneficial in tumor models. Because CTL may also limit autoreactive B cell expansion in lupus, we asked whether an agonist CD40 mAb would exacerbate lupus due to B cell stimulation or would improve lupus due to CTL promotion. These studies used an induced model of lupus, the parent-into-F1 model in which transfer of DBA/2 splenocytes into B6D2F1 mice induces chronic lupus-like graft-vs-host disease (GVHD). Although agonist CD40 mAb treatment of DBA-->F1 mice initially exacerbated B cell expansion, it also strongly promoted donor CD8 T cell engraftment and cytolytic activity such that by 10 days host B cells were eliminated consistent with an accelerated acute GVHD. CD40 stimulation bypassed the requirement for CD4 T cell help for CD8 CTL possibly by licensing dendritic cells (DC) as shown by the following: 1) greater initial activation of donor CD8 T cells, but not CD4 T cells; 2) earlier activation of host DC; 3) host DC expansion that was CD8 dependent and CD4 independent; and 4) induction of acute GVHD using CD4-depleted purified DBA CD8+ T cells. A single dose of CD40 mAb improved lupus-like renal disease at 12 wk, but may not suffice for longer periods consistent with a need for continuing CD8 CTL surveillance. These results demonstrate that in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, CTL promotion is both feasible and beneficial and the CTL-promoting properties of CD40 stimulation outweigh the B cell-stimulatory properties.     

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

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

Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease.

Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.     

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.     

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.     

G-CSF-treated donor CD4(+) T cells attenuate acute GVHD through a reduction in Th17 cell differentiation

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.     

Alloreactive memory T cells are responsible for the persistence of graft-versus-host disease

Graft-vs-host disease (GVHD) is caused by a donor T cell anti-host reaction that evolves over several weeks to months, suggesting a requirement for persistent alloreactive T cells. Using the C3H.SW anti-C57BL/6 (B6) mouse model of human GVHD directed against minor histocompatibility Ags, we found that donor CD8(+) T cells secreting high levels of IFN-gamma in GVHD B6 mice receiving C3H.SW naive CD8(+) T cells peaked by day 14, declined by day 28 after transplantation, and persisted thereafter, corresponding to the kinetics of a memory T cell response. Donor CD8(+) T cells recovered on day 42 after allogeneic bone marrow transplantation expressed the phenotype of CD44(high)CD122(high)CD25(low), were able to homeostatically survive in response to IL-2, IL-7, and IL-15 and rapidly proliferated upon restimulation with host dendritic cells. Both allogeneic effector memory (CD44(high)CD62L(low)) and central memory (CD44(high)CD62L(high)) CD8(+) T cells were identified in B6 mice with ongoing GVHD, with effector memory CD8(+) T cells as the dominant (>80%) population. Administration of these allogeneic memory CD8(+) T cells into secondary B6 recipients caused virulent GVHD. A similar allogeneic memory CD4(+) T cell population with the ability to mediate persistent GVHD was also identified in BALB/b mice receiving minor histocompatibility Ag-mismatched B6 T cell-replete bone marrow transplantation. These results indicate that allogeneic memory T cells are generated in vivo during GVH reactions and are able to cause GVHD, resulting in persistent host tissue injury. Thus, in vivo blockade of both alloreactive effector and memory T cell-mediated host tissue injury may prove to be valuable for GVHD prevention and treatment.     

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.     

The novel costimulatory programmed death ligand 1/B7.1 pathway is functional in inhibiting alloimmune responses in vivo

The programmed death ligand 1 (PDL1)/programmed death 1 (PD1) costimulatory pathway plays an important role in the inhibition of alloimmune responses as well as in the induction and maintenance of peripheral tolerance. It has been demonstrated recently that PDL1 also can bind B7.1 to inhibit T cell responses in vitro. Using the bm12 into B6 heart transplant model, we investigated the functional significance of this interaction in alloimmune responses in vivo. PD1 blockade unlike PDL1 blockade failed to accelerate bm12 allograft rejection, suggesting a role for an additional binding partner for PDL1 other than PD1 in transplant rejection. PDL1 blockade was able to accelerate allograft rejection in B7.2-deficient recipients but not B7.1-deficient recipients, indicating that PDL1 interaction with B7.1 was important in inhibiting rejection. Administration of the novel 2H11 anti-PDL1 mAb, which only blocks the PDL1-B7.1 interaction, aggravated chronic injury of bm12 allografts in B6 recipients. Aggravated chronic injury was associated with an increased frequency of alloreactive IFN-γ-, IL-4-, and IL-6-producing splenocytes and a decreased percentage of regulatory T cells in the recipients. Using an in vitro cell culture assay, blockade of the interaction of PDL1 on dendritic cells with B7.1 on T cells increased IFN-γ production from alloreactive CD4(+) T cells, whereas blockade of dendritic cell B7.1 interaction with T cell PDL1 did not. These data indicate that PDL1 interaction with B7.1 plays an important role in the inhibition of alloimmune responses in vivo and suggests a dominant direction for PDL1 and B7.1 interaction.     

Progression from acute to chronic disease in a murine parent-into-F1 model of graft-versus-host disease

The parent-into-immunocompetent-F(1) model of graft-vs-host disease (GVHD) induces immune dysregulation, resulting in acute or chronic GVHD. The disease outcome is thought to be determined by the number of parental anti-F(1) CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 x DBA/2)F(1) (B6D2F(1)) mice (acute model) leads to extensive parental cell engraftment and early death, whereas injection of DBA/2 cells (chronic model) results in little parental cell engraftment and a lupus-like disease. This study demonstrated that injection of BALB/c splenocytes into (BALB/c x B6)F(1) (CB6F(1)) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F(1) initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F(1). However, the acute disease resolved, and the CB6F(1) mice went on to develop chronic GVHD with detectable Abs to ssDNA, dsDNA, and extractable nuclear Ags. Limiting dilution CTL assays determined that B6 splenocytes have CTL precursor frequencies of 1/1000 against both CB6F(1) and B6D2F(1), whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F(1)s. The Th cell precursor frequency for B6 anti-DBA/2 was 3-fold higher than that for B6 anti-BALB/c determined by limiting dilution proliferation assays. These results indicate the importance of adequate allospecific helper as well as effector T cells for the induction and maintenance of acute GVHD in this model, and presents an unexpected model in which initial acute GVHD is replaced by the chronic form of disease.     

CD4+ T cells generated de novo from donor hemopoietic stem cells mediate the evolution from acute to chronic graft-versus-host disease

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.     

Increased frequency of suppressive regulatory T cells and T cell-mediated antigen loss results in murine melanoma recurrence

Therapeutic treatment of large established tumors using immunotherapy has yielded few promising results. We investigated whether adoptive transfer of tumor-specific CD8(+) T cells, together with tumor-specific CD4(+) T cells, would mediate regression of large established B16BL6-D5 melanomas in lymphopenic Rag1(-/-) recipients devoid of regulatory T cells. The combined adoptive transfer of subtherapeutic doses of both TRP1-specific TCR transgenic Rag1(-/-) CD4(+) T cells and gp100-specific TCR transgenic Rag1(-/-) CD8(+) T cells into lymphopenic recipients, who received vaccination, led to regression of large (100-400 mm(2)) melanomas. The same treatment strategy was ineffective in lymphoreplete wild-type mice. Twenty-five percent of mice (15/59) had tumors recur (15-180 d postregression). Recurrent tumors were depigmented and had decreased expression of gp100, the epitope targeted by the CD8(+) T cells. Mice with recurrent melanoma had increased CD4(+)Foxp3(+) TRP1-specific T cells compared with mice that did not show evidence of disease. Importantly, splenocytes from mice with recurrent tumor were able to suppress the in vivo therapeutic efficacy of splenocytes from tumor-free mice. These data demonstrate that large established tumors can be treated by a combination of tumor-specific CD8(+) and CD4(+) T cells. Additionally, recurrent tumors exhibited decreased Ag expression, which was accompanied by conversion of the therapeutic tumor-specific CD4(+) T cell population to a Foxp3(+)CD4(+) regulatory T cell population.     

CD99-dependent expansion of myeloid-derived suppressor cells and attenuation of graft-versus-host disease

CD99 is involved in many cellular events, such as the generation of Hodgkin and Reed-Sternberg cells, T cell costimulation, and leukocyte transendothelial migration. However, these studies have been limited to in vitro or in vivo experiments using CD99-deficient cell lines or anti-CD99 antibodies. In the present study, using CD99-deficient mice established by the exchangeable gene trap method, we investigated the physiologic function of murine CD99. In a B6 splenocytes → bm12 graft-versus-host disease model, wild-type cells were minimally lethal, whereas all mice that received CD99-deficient donor cells developed an early and more severe pathology. Graftversus-host disease in these mice was associated with insufficient expansion of myeloid-derived suppressor cells. This was confirmed by experiments illustrating that the injection of wild-type donor cells depleted of Mac-1(+) cells led to an almost identical disease course as the CD99-deficient donor system. Therefore, these results suggest that CD99 plays a crucial role in the attenuation of graft-versus-host disease by regulating the expansion of myeloid-derived suppressor cells.     

CD4 and CD8 T cells in susceptibility/protection to collagen-induced arthritis in HLA-DQ8-transgenic mice: implications for rheumatoid arthritis

To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8.CD4(-/-) mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8.CD8(-/-) mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8.CD8(-/-) and DQ8 mice produced rheumatoid factor. In addition, DQ8.CD8(-/-) mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4(-/-) mice. An increased frequency of CD3(+) double-negative (DN) T cells was found in DQ8.CD8(-/-) compared with DQ8.CD4(-/-) and DQ8 mice. These CD3(+) DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3(+) and CD3(+) DN T cells in DQ8.CD8(-/-) mice compared with DQ8.CD4(-/-) and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8(+) T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.     

Accelerated onset and increased severity of acute graft-versus-host disease following adoptive transfer of DR6-deficient T cells

DR6 is a recently identified member of the TNFR family. In a previous study, we have shown that DR6 KO mice have enhanced CD4(+) T cell proliferation and Th2 cytokine production. Acute graft-vs-host disease (GVHD) results from the activation and expansion of alloreactive donor T cells following bone marrow transplantation. In this article, we demonstrate that the transfer of donor T cells from DR6 KO mice into allogeneic recipient mice in a parent into an F(1) model of acute GVHD results in a more rapid onset of GVHD with increased severity. Recipients of DR6 KO T cells exhibit earlier systemic symptoms of GVHD, more rapid weight loss, earlier histopathological organ damage in the thymus, spleen, and intestines, and earlier mortality. The rapid onset of GVHD in these mice may be attributable to the enhanced activation and expansion of DR6 KO CD4(+) and CD8(+) T cells. Our findings support the hypothesis that DR6 serves as an important regulatory molecule in T cell immune responses. The identification and use of DR6 ligands and/or agonistic Abs to DR6 may represent useful therapeutics in the treatment of T cell-mediated diseases such as GVHD.