Knockout Donor Research Articles

Differential requirement for IFN-gamma in CTL maturation in acute murine graft-versus-host disease.

Although IFN-gamma is the archetypal Th1 cytokine, its role in CTL maturation is uncertain. We used an in vivo mouse model of CTL development, parent-into-F(1) acute graft-vs-host disease (AGVHD), to evaluate this issue. In AGVHD, transfer of naive parental T cells into F(1) hosts stimulates the development of allospecific CTL effectors that eliminate host lymphocytes, particularly B cells. Complete elimination of IFN-gamma, using IFN-gamma-deficient donors and administering anti-IFN-gamma mAb, suppressed B cell elimination, down-regulated TNF-alpha production, and enhanced Th2 cytokine production, but did not allow the B cell expansion characteristic of chronic GVHD (CGVHD). Because complete CTL inhibition results in full-blown CGVHD that is IFN-gamma independent, these observations indicate that IFN-gamma elimination only partially blocks CTL development. IFN-gamma elimination did not inhibit donor T cell engraftment or activation in the AGVHD model, but almost completely blocked Fas/Fas ligand (FasL) gene expression, protein up-regulation, and Fas/FasL-mediated CTL killing. In contrast, IFN-gamma elimination only partially inhibited perforin gene expression and perforin-mediated CTL activity. The contributions of IFN-gamma to CTL development were indirect, because IFN-gamma receptor-deficient donor cells differentiated normally into allospecific CTLs. Consistent with the view that the Fas/FasL and perforin pathways each mediate CTL killing in AGVHD, the absence of both perforin and IFN-gamma (perforin knockout donor cells and anti-IFN-gamma mAb) converted AGVHD to CGVHD. Thus, both IFN-gamma-dependent induction of Fas/FasL and IFN-gamma-independent induction of perforin contribute to CTL-mediated elimination of host B cells in AGVHD. Suppression of both pathways is required for typical CGVHD development.

An absence of CCR5 on donor cells results in acceleration of acute graft-vs-host disease

Objective Chemokines have been postulated to play a role in the pathogenesis of graft-vs-host disease (GVHD) after allogeneic hematopoietic transplantation. Recent reports have indicated that the absence of donor expression of CCR5 on T cells ameliorates GVHD in models using no conditioning of the recipient. We therefore assessed the role of CCR5 on donor cells in models where intensive conditioning of the recipient occurs, thus more appropriately mirroring the clinical experience. Methods Lethally irradiated mice received allogeneic bone marrow transplants. Recipients were given full MHC-mismatched donor bone marrow and splenocytes from CCR5 knockout (KO) mice vs wild-type (WT) control donors. Results Recipients of CCR5 KO donor cells succumbed to acute GVHD at an accelerated rate compared to mice receiving WT cells. Donor CD8 + T cells expanded to a significantly greater extent in recipients of CCR5 KO vs WT control cells. T cells recovered from recipients of CCR5 KO cells produced more IFN-γ and TNF-α and proliferated to a T-cell mitogen at a significantly greater level then T cells from recipients of WT cells, indicating that CCR5 plays a role in downregulating donor alloreactive CD8 + T-cell expansion. Histological assessment of the mice indicated pathological lesions in the kidneys and a greater degree of liver pathological changes in mice that received CCR5 KO donor grafts. Conclusions These results indicate that the role of CCR5 in allogeneic bone marrow transplants and GVHD is more complex than initially thought. In a murine transplant model with intensive conditioning, the overall effect of absent CCR5 expression on donor cells results in greater GVHD and donor T-cell expansion.

P-glycoprotein and Mrp1 collectively protect the bone marrow from vincristine-induced toxicity in vivo.

ABC transporter proteins may protect haematopoietic progenitor cells from chemotherapy-induced toxicity. By using an in vitro colony-forming assay, we found that bone marrow of Mdr1ab, Mrp1, Mdr1ab/Mrp1 knockout (KO) mice was two-, five- to 10- and 25-fold, respectively, more sensitive to vincristine than wild-type mice bone marrow. To study the impact of ABC transporters on in vivo bone marrow sensitivity without the added complication of altered pharmacokinetics, we created chimeras of wild-type mice transplanted with bone marrow from wild-type, Mrp1, Mdr1ab or Mdr1ab/Mrp1 KO donor mice. Following a single bolus injection of vincristine, the chimeras transplanted with wild-type or Mdr1ab KO marrow cells showed no reductions in WBC. A significant reduction was observed in Mrp1 KO chimeras, but the most pronounced effect was observed in mice receiving bone marrow from Mdr1ab/Mrp1 KO mice. A pharmacokinetic analysis in wild-type and KO mice showed that the absence of P-gp reduced the body clearance of vincristine, but that no further reduction occurred when Mrp1 was also absent. However, the tissue accumulation of vincristine in tissues of these Mdr1ab/Mrp1 KO mice was further increased. This study demonstrates that the presence of multiple drug transporters protects the bone marrow, and probably other tissues as well, against chemotherapeutic insults.

Interleukin 2 signaling is required for CD4(+) regulatory T cell function.

Mice deficient in interleukin (IL)-2 production or the IL-2 receptor α or β chains develop a lethal autoimmune syndrome. CD4+ regulatory T cells have been shown to prevent autoimmune diseases, allograft rejection, and to down-regulate antibody responses against foreign antigens. To assess the role of IL-2 in the generation and function of regulatory T cells, we transferred CD4+ T cells from mice genetically deficient in IL-2 or IL-2Rα (CD25) expression. A small number of splenic or thymic CD4+ T cells from IL-2 knockout mice can protect mice from spontaneous experimental autoimmune encephalomyelitis (EAE). In contrast, splenic or thymic CD4+ T cells from CD25 knockout donor mice conferred little or no protection. We conclude that T cells with regulatory potential can develop, undergo thymic selection, and migrate to the peripheral lymphoid organs in the absence of IL-2, and do not protect from disease by means of IL-2 secretion. However, IL-2 signaling in regulatory T cells is essential for their protective function. Altogether, our results favor a model whereby IL-2 induces regulatory T cell activity.

Murine graft-versus-host disease induced using interferon-gamma-deficient grafts features antibodies to double-stranded DNA, T helper 2-type cytokines and hypereosinophilia.

Acute, lethal graft-versus-host disease (GvHD) develops in B6D2F1 hybrid recipients of wild-type, C57BL/6, parental strain grafts; however, when interferon-gamma (IFN-gamma) gene knockout (gko) donors are used, the disease is prolonged and associated with a higher level of engraftment, particularly of T cells. Lesions containing large, mixed cellular infiltrates develop in the skin, liver, pancreas, salivary gland, lung and kidney. In our current study, we wished to determine whether GvHD features a preponderance of T helper 2 (Th2) cytokines in the absence of donor-derived IFN-gamma, and whether autoantibody production, commonly associated with chronic GvHD, also occurs. Because mitogen responsiveness is consistently suppressed in mice with acute GvHD, we wished to measure this response in recipients of IFN-gamma gko grafts. Our findings indicate that spleen cells from the latter produce interleukin (IL)-4, IL-5 and IL-13 in culture, but respond poorly to concanavalin A (Con A) and lipopolysaccharide (LPS). Their sera contain anti-nuclear antibodies (ANA), some of which are specific for double-stranded (ds)DNA and are predominantly immunoglobulin (Ig)M and IgG1. We also noted the presence of numerous eosinophils in the infiltrates developing within the target organs. In some respects, this syndrome bears resemblance to both systemic lupus erythematosus (SLE) and chronic GvHD. However, histological evidence of glomerulonephritis is lacking and proteinuria fails to develop in recipients of IFN-gamma gko grafts, suggesting that IFN-gamma may be necessary for the development of lupus nephritis. On a broader scope, our findings underscore the importance of IFN-gamma in the pathogenetic mechanism of GvHD, and demonstrate that the absence of this cytokine promotes the development of chronic GvHD and autoimmunity.

Eradication of Cryptosporidium parvum infection by mice with ovalbumin-specific T cells.

CD154 is necessary for mice to clear a Cryptosporidium parvum infection, but whether this ligand has to be expressed on T cells with specificity for C. parvum has not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG(-/-) background with C. parvum and found that the infection was cleared within 6 weeks, while RAG(-/-) controls were unable to clear C. parvum infection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44(high) phenotype that characterizes memory cells. To determine whether a C. parvum-infected environment sufficed to activate transgenic T cells, we reconstituted C. parvum-infected BALB/c SCID mice with DO11.10 RAG(-/-) splenocytes. Fecal excretion of C. parvum antigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa of C. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44(high) memory phenotype in C. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear a C. parvum infection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response to C. parvum antigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide and C. parvum DNA sequences. C. parvum-infected SCID mice given RAG(-/-) CD8(+) T cells with a Listeria-specific transgene did not recover from C. parvum infection. Our data suggest that antigen-nonspecific CD4(+) T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication of C. parvum infection.

The use of transplanted mammary gland to study cancer signalling pathways.

Mammary epithelium can be genetically manipulated by reconstituting a mammary gland, in an animal, from epithelium and a mammary fat pad from which the endogenous epithelium has been removed at 3 weeks of age. Genes can be introduced into the epithelium before transplantation using retrovirus vectors. To remove genes from the epithelium at present requires epithelium to be transplanted from knockout donor mice, but this is a valuable extension of knockout technology, as (a) it creates knockout epithelium in a normal stromal and systemic environment, or vice versa, and (b) where the knockout mouse does not survive into adulthood, epithelium can be rescued from embryos after about 12 days of gestation, and grown to form mature mammary epithelium in a normal recipient mammary fat pad.

Role of Immunoregulatory Donor T Cells in Suppression of Graft-Versus-Host Disease Following Donor Leukocyte Infusion Therapy

In murine models of allogeneic bone marrow transplantation (BMT), MHC-mismatched recipients given a delayed infusion of donor leukocytes (DLI) at 21 days posttransplant develop significant GVHD whereas MHC-matched recipients do not. The current study was initially designed to test the hypothesis that small numbers of T cells in the MHC-mismatched donor bone marrow (BM) graft exacerbated graft-vs-host disease (GVHD) when DLI was administered at 21 days after BMT. Ex vivo depletion of Thy1+ cells from the donor BM had no impact on the severity of GVHD after DLI. However, depletion of donor T cells in vivo with a Thy1 allele-specific mAb given after BMT resulted in significantly more severe GVHD after DLI. Similar results were obtained in a MHC-matched model of allogeneic BMT, indicating that this was a general phenomenon and not model dependent. These results indicated that a population of donor-derived Thy1+ cells suppressed graft-vs-host reactivity after DLI. Results of experiments with thymectomized recipients demonstrated that an intact thymus was required for generation of the immunoregulatory donor cells. Experiments using TCR beta-chain knockout mice as BM donors indicated that the immunosuppressive Thy1+ cells coexpressed alphabetaTCR heterodimers. Similar experiments with CD4 and CD8 knockout donor BM suggested that the immunoregulatory Thy1+alphabetaTCR+ cells consisted of two subpopulations: a CD4+CD8- subpopulation and a CD4-CD8- subpopulation. Together, these results show that thymus-derived, Thy1+alphabetaTCR+ donor cells generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT.

Liver and bile duct pathology following Cryptosporidium parvum infection of immunodeficient mice.

Patients with acquired immune deficiency syndrome (AIDS) and boys with mutations of the CD154 gene (causing congenital X-linked immunodeficiency with hyper-IgM [XHIM]) are susceptible to chronic infections of the biliary tract with Cryptosporidium parvum (CP) that may lead to biliary sclerosis and ultimately to cholangiocarcinoma. To determine whether the CP infection and the consequent immune response contribute independently to this morbidity, we infected mice with severe combined immunodeficiency (SCID) or with disrupted genes for CD154, CD40, or interferon gamma (IFN-gamma) with CP. Even when CP infection persisted for 16 weeks, the SCID mice developed only mild triaditis, without apoptosis of biliary epithelial cells (BEC). Fifty percent of the CD154 knockout mice developed lobular hepatitis with acute and chronic triaditis. The CD40 knockout mice developed marked triaditis, and the IFN-gamma knockouts either succumbed to enteritis or survived to develop marked triaditis, portal fibrosis, biliary sclerosis, necrosis with dilation of duct-like structures within the porta hepatis, and dysplastic changes. CP-infected SCID mice reconstituted with T cells from IFN-gamma knockout donors either developed severe enteritis or survived to develop triaditis, cholangitis, lobular hepatitis with periductular sclerosis, and scarring. Mice with disruptions of both the CD40 and IFN-gamma genes remained infected by CP and developed bile duct and liver disease, but not enteritis. Our results suggest that T-cell cytokines are required for the inflammatory and sclerosing responses to CP infection in immunodeficient animals. The response of immunodeficient mice to CP infection may model at least the initial steps toward the development of sclerosing cholangitis or bile duct cancers in XHIM patients.

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.

Acute, Lethal Graft‐Versus‐Host Disease in an F1‐Hybrid Model using Grafts from Parental‐Strain, T‐Cell Receptor‐δ Gene Knockout Donors

Gammadelta T cells have been implicated in the pathogenesis of acute graft-versus-host disease (GVHD). We therefore performed experiments to determine whether mortality from GVHD is reduced in C57BL/6 x DBA/2 F1-hybrid (BDF1-hybrid) mice when parental strain, T-cell receptor-delta (TCRdelta) knockout (KO) donors are used. We compared mortality, weight loss, interferon-gamma (IFN-gamma) production and cytotoxic activity in recipients of either wild-type or TCRdelta KO grafts. In both groups there was significant weight loss and an identical level of mortality. Elevated IFN-gamma levels were present in both groups, but recipients of TCRdelta KO grafts produced twice as much as recipients of wild-type grafts. Elevated natural killer (NK) and NK-like activity was also seen in both. These results demonstrate that TCRdelta KO grafts can induce GVHD as severe as that seen in recipients of wild-type grafts, a finding that is at odds with studies demonstrating reduced mortality when gammadelta T cells are purged from donor mice. We suggest that the inconsistency may lie in the higher levels of IFN-gamma seen with TCRdelta KO grafts and that the protection afforded by the absence of gammadelta T cells in the graft is overwhelmed by the higher levels of IFN-gamma.

T CELL COSTIMULATORY BLOCKADE SUPPRESSES INDIRECT BETTER THAN DIRECT ALLOIMMUNE RESPONSES IN VIVO

73 There are two pathways for stimulating CD4+ T cells during graft rejection, called the direct and indirect pathways of allorecognition. The relative contributions of these pathways to acute rejection and their susceptibility to immunosuppression have been hard to assess. We investigated these issues using skin grafts from donors and to recipients that have been genetically modified to eliminate one or other of the pathways for CD4+ stimulation. To examine the indirect pathway we placed skin grafts from B6 class II knockout donors (B6 II-) onto normal BALB/c recipients. To examine the direct pathway we placed normal BALB/c skin grafts onto B6 class II deficient recipients that have a reconstituted CD4+ population (B6 II-4+). B6 II-4+ mice were generated by crossing B6 II- mice with expressing a class II transgene only on thymic epithelium, and selecting offspring that express class II antigens only on thymic epithelium, thus achieving positive selection of CD4+ T cells without class II antigen expression in the periphery. We compared the effectiveness of combined CsA and anti-CD8 treatment or of combined CTLA4Ig and anti-CD40L treatment on skin allograft survival for the two types of transplants. CsA was given at 50 mg/kg/day sc from day 1, anti-CD8 was administered ip x3 prior to and weekly after transplantation. Both CTLA4Ig and anti-CD40L were given at 500µg/day ip on days 0,2,4,6 relative to the transplant. TableTableIn the absence of immunosuppression rejection depending on the indirect pathway was at least as fast as rejection depending on direct recognition (gp 4 vs 3). Both strategies for immunosuppression were more effective for BALB/c recipients than for B6 recipients (gps 6 vs 5 and 10 vs 9). Compared to their controls, neither the direct nor indirect pathway was especially sensitive to CsA and anti-CD8 immunosuppression (gps 7 vs 5 and 8 vs 6). Costimulatory blockade was more effective in the absence of a direct response (gp 12 vs 10) than in the absence of an indirect response; and only gp 12 had longterm surviving grafts, although they eventually rejected at >100 days. These results emphasize the importance of strain combinations when testing immunosuppressive strategies. They indicate that T cell costimulatory blockade suppresses the indirect pathway better then the direct pathway. They also suggest that T cell costimulatory blockade will be most effective when combined with strategies to prevent direct allo-responses.

T CELL COSTIMULATORY BLOCKADE SUPPRESSES INDIRECT BETTER THAN DIRECT ALLOIMMUNE RESPONSES IN VIVO

73 There are two pathways for stimulating CD4+ T cells during graft rejection, called the direct and indirect pathways of allorecognition. The relative contributions of these pathways to acute rejection and their susceptibility to immunosuppression have been hard to assess. We investigated these issues using skin grafts from donors and to recipients that have been genetically modified to eliminate one or other of the pathways for CD4+ stimulation. To examine the indirect pathway we placed skin grafts from B6 class II knockout donors (B6 II-) onto normal BALB/c recipients. To examine the direct pathway we placed normal BALB/c skin grafts onto B6 class II deficient recipients that have a reconstituted CD4+ population (B6 II-4+). B6 II-4+ mice were generated by crossing B6 II- mice with mice expressing a class II transgene only on thymic epithelium, and selecting offspring that express class II antigens only on thymic epithelium, thus achieving positive selection of CD4+ T cells without class II antigen expression in the periphery. We compared the effectiveness of combined CsA and anti-CD8 treatment or of combined CTLA4Ig and anti-CD40L treatment on skin allograft survival for the two types of transplants. CsA was given at 50 mg/kg/day sc from day 1, anti-CD8 was administered ip x3 prior to and weekly after transplantation. Both CTLA4Ig and anti-CD40L were given at 500μg/day ip on days 0,2,4,6 relative to the transplant. TableIn the absence of immunosuppression rejection depending on the indirect pathway was at least as fast as rejection depending on direct recognition (gp 4 vs 3). Both strategies for immunosuppression were more effective for BALB/c recipients than for B6 recipients (gps 6 vs 5 and 10 vs 9). Compared to their controls, neither the direct nor indirect pathway was especially sensitive to CsA and anti-CD8 immunosuppression (gps 7 vs 5 and 8 vs 6). Costimulatory blockade was more effective in the absence of a direct response (gp 12 vs 10) than in the absence of an indirect response; and only gp 12 had longterm surviving grafts, although they eventually rejected at >100 days. These results emphasize the importance of strain combinations when testing immunosuppressive strategies. They indicate that T cell costimulatory blockade suppresses the indirect pathway better then the direct pathway. They also suggest that T cell costimulatory blockade will be most effective when combined with strategies to prevent direct allo-responses.