Inhibition of the alloantibody response by CD95 ligand.

We investigated the effect of Fas/APO1-ligand (CD95L) gene transfer on allogeneic immune responses in vivo. A colon carcinoma cell line from BALB/c mice, CT26, was stably transfected with a vector encoding mouse CD95L and was inoculated into C57BL/6 mice. CD95L expression markedly reduced allogeneic cytotoxic T lymphocyte and helper T lymphocyte activity directed toward CT26. Strikingly, expression of CD95L on these allogeneic tumors completely inhibited the generation of alloantibodies of both IgM and IgG subclasses. Thus, CD95L inhibited alloantibody production and conferred localized immune suppression through this mechanism. These results provide insight into the role of CD95L in regulating the alloantibody response and the generation of local immune responses.

Tissue-specific consequences of the anti-adenoviral immune response: implications for cardiac transplants.

The immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. This would limit the use of adenovirus-mediated gene transfer in disease states in which long-term gene expression is required. While studying the effect of the anti-adenoviral immune response in transplantation, we found that transgene expression persisted in cardiac isografts transfected with an adenovirus encoding beta-galactosidase. Transfected grafts remained free of inflammation, despite the presence of an immune response to the vector. Thus, adenovirus-mediated gene transfer may have therapeutic value in cardiac transplantation and heart diseases. Furthermore, immunological limitations of adenoviral vectors for gene therapy are not universal for all tissue types.

Connective tissue growth factor promotes fibrosis downstream of TGFbeta and IL-6 in chronic cardiac allograft rejection.

Cardiac transplantation is an effective treatment for multiple types of heart failure refractive to therapy. Although immunosuppressive therapeutics have increased survival rates within the first year posttransplant, chronic rejection (CR) remains a significant barrier to long-term graft survival. Indicators of CR include patchy interstitial fibrosis, vascular occlusion and progressive loss of graft function. Multiple factors have been implicated in the onset and progression of CR, including TGFbeta, IL-6 and connective tissue growth factor (CTGF). While associated with CR, the role of CTGF in CR and the factors necessary for CTGF induction in vivo are not understood. To this end, we utilized forced expression and neutralizing antibody approaches. Transduction of allografts with CTGF significantly increased fibrotic tissue development, though not to levels observed with TGFbeta transduction. Further, intragraft CTGF expression was inhibited by IL-6 neutralization whereas TGFbeta expression remained unchanged, indicating that IL-6 effects may potentiate TGFbeta-mediated induction of CTGF. Finally, neutralizing CTGF significantly reduced graft fibrosis without reducing TGFbeta and IL-6 expression levels. These findings indicate that CTGF functions as a downstream mediator of fibrosis in CR, and that CTGF neutralization may ameliorate fibrosis and hypertrophy associated with CR.

Critical role for IL-6 in hypertrophy and fibrosis in chronic cardiac allograft rejection.

Chronic cardiac allograft rejection is the major barrier to long term graft survival. There is currently no effective treatment for chronic rejection except re-transplantation. Though neointimal development, fibrosis, and progressive deterioration of graft function are hallmarks of chronic rejection, the immunologic mechanisms driving this process are poorly understood. These experiments tested a functional role for IL-6 in chronic rejection by utilizing serial echocardiography to assess the progression of chronic rejection in vascularized mouse cardiac allografts. Cardiac allografts in mice transiently depleted of CD4+ cells that develop chronic rejection were compared with those receiving anti-CD40L therapy that do not develop chronic rejection. Echocardiography revealed the development of hypertrophy in grafts undergoing chronic rejection. Histologic analysis confirmed hypertrophy that coincided with graft fibrosis and elevated intragraft expression of IL-6. To elucidate the role of IL-6 in chronic rejection, cardiac allograft recipients depleted of CD4+ cells were treated with neutralizing anti-IL-6 mAb. IL-6 neutralization ameliorated cardiomyocyte hypertrophy, graft fibrosis, and prevented deterioration of graft contractility associated with chronic rejection. These observations reveal a new paradigm in which IL-6 drives development of pathologic hypertrophy and fibrosis in chronic cardiac allograft rejection and suggest that IL-6 could be a therapeutic target to prevent this disease.

Structural relationship of bacterial RecA proteins to recombination proteins from bacteriophage T4 and yeast.

RecA protein is essential in eubacteria for homologous recombination and promotes the homologous pairing and strand exchange of DNA molecules in vitro. Recombination proteins with weak sequence similarity to bacterial RecA proteins have been identified in bacteriophage T4, yeast, and other higher organisms. Analysis of the primary sequence relationships of DMC1 from Saccharomyces cerevisiae and UvsX of T4 relative to the three-dimensional structure of RecA from Escherichia coli suggests that both proteins are structural homologs of bacterial RecA proteins. This analysis argues that proteins in this group are members of a single family that diverged from a common ancestor that existed prior to the divergence of prokaryotes and eukaryotes.

Transplant acceptance following anti-CD4 versus anti-CD40L therapy: evidence for differential maintenance of graft-reactive T cells.

Inductive therapy with anti-CD4 or anti-CD40L monoclonal antibodies (mAb) leads to long-term allograft acceptance but the immune parameters responsible for graft maintenance are not well understood. This study employed an adoptive transfer system in which cells from mice bearing long-term cardiac allografts following inductive anti-CD4 or anti-CD40L therapy were transferred into severe combined immunodeficiency (SCID) allograft recipients. SCID recipients of cells from anti-CD4-treated mice (anti-CD4 cells) did not reject allografts while those receiving cells from anti-CD40L-treated mice (anti-CD40L cells) did reject allografts. Carboxyfluorescein succinimidyl ester (CFSE) labeling of transferred cells revealed that this difference was not associated with differential proliferative capacities of these cells in SCID recipients. Like cells from naïve mice, anti-CD40L cells mounted a Th1 response following transfer while anti-CD4 cells mounted a dominant Th2 response. Early (day 10) T-cell priming was detectable in both groups of primary allograft recipients but persisted to day 30 only in recipients treated with anti-CD4 mAb. Thus, anti-CD40L therapy appears to result in graft-reactive T cells with a naïve phenotype while anti-CD4 therapy allows progression to an altered state of differentiation. Additional data herein support the notion that anti-CD40L mAb targets activated, but not memory, cells for removal or functional silencing.

The classical complement pathway in transplantation: unanticipated protective effects of C1q and role in inductive antibody therapy.

Though complement (C) deposition within the transplant is associated with allograft rejection, the pathways employed have not been established. In addition, evidence suggests that C-mediated cytolysis may be necessary for the tolerance-inducing activities of mAb therapies. Hence, we assessed the role of the classical C pathway in acute allograft rejection and its requirement for experimental mAb therapies. C1q-deficient (C1q-/-) recipients rejected allografts at a faster rate than wild-type (WT) recipients. This rejection was associated with exacerbated graft pathology but not with enhanced T-cell responses in C1q-/- recipients. However, the humoral response to donor alloantigens was accelerated in C1q-/- mice, as an early IgG response and IgG deposition within the graft were observed. Furthermore, deposition of C3d, but not C4d was observed in grafts isolated from C1q-/- recipients. To assess the role of the classical C pathway in inductive mAb therapies, C1q-/- recipients were treated with anti-CD4 or anti-CD40L mAb. The protective effects of anti-CD4 mAb were reduced in C1q-/- recipients, however, this effect did not correlate with ineffective depletion of CD4+ cells. In contrast, the protective effects of anti-CD40L mAb were less compromised in C1q-/- recipients. Hence, this study reveals unanticipated roles for C1q in the rejection process.

Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae.

Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae. Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time. This suggested that repair tracts were frequently shorter than 1 kilobase. The two insertion mismatches were repaired with different efficiencies. Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches. When two different plasmids containing single-insertion mismatches were transformed into S. cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.

Nine novel conserved motifs in BRCA1 identified by the chicken orthologue.

The breast cancer susceptibility gene BRCA1 encodes a multifunctional protein that is mutated in many hereditary breast and ovarian cancers. We have cloned a homologue of the human BRCA1 gene from chicken; the gene encodes a 1749 amino acid protein that is 33% identical to human BRCA1. Phylogenetic analyses of the chicken and mammalian proteins indicate that the chicken gene is a bona fide BRCA1 orthologue, the first to be described from a non-mammal. Most of the chicken protein has diverged considerably from its mammalian orthologues, although the RING and BRCT repeat regions are highly conserved. This marked overall sequence divergence has allowed us to identify nine additional highly-conserved motifs (ranging from 8--56 amino acids in length) which are likely important for BRCA1 function.

Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice.

Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1. Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination. We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects. All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant. Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay. rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers. Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type. Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling. These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination.

Regulation of cardiac allograft rejection by T lymphocytes.

Cardiac allograft rejection is diagnosed by routine, surveillance endomyocardial biopsy (EMB). EMB is an invasive and expensive procedure that detects ongoing, rather than impending, rejection, and may be influenced by sampling error due to nonuniform distribution of myocardial lesions. T cells are the principal regulators of allograft rejection; therefore, identifying T cell functions that are associated with cardiac allograft rejection should provide insight for the development of noninvasive monitoring techniques that may augment and reduce the frequency of EMB. Experiments that define the protective and deleterious activities of Th1 and Th2 T cell subsets are clearly warranted, and will direct the potential use of cytokine manipulating therapies aimed at inducing tolerogenic T cell responses. (Trends Cardiovasc Med 1996;6:168-173).

Genetic vaccination-induced immune responses to the human immunodeficiency virus protein Rev: emergence of the interleukin 2-producing helper T lymphocyte.

Rev M10 is a trans-dominant negative inhibitor of HIV replication. Hence, stable transduction of CD4+ T cells with Rev M10 represents a novel gene therapy aimed at inhibiting HIV replication within these cells, thereby slowing the progression of AIDS. However, the immune system may recognize Rev M10 as foreign and target transduced cells for elimination. In the current study, mice were genetically immunized with a plasmid encoding Rev M10, to (1) identify immune parameters that may be induced by Rev M10 gene transfer, (2) determine the impact of repeated introduction of the Rev M10-encoding plasmid on the immune response to the transgene product, and (3) determine if cotransfection with a plasmid encoding TGFbeta1 would suppress the response. Kinetic studies revealed that Rev-specific IL-2-producing helper T lymphocytes (HTLs) appeared following the second genetic immunization, peaked after the third, and persisted at peak levels for at least 6 weeks. Rev-specific HTLs were CD4+, and the development of these cells was ablated by cotransfection with TGFbeta1. Other cytokines were not readily detectable when immune splenocytes were restimulated with Rev in vitro, and Rev-specific IgG antibodies were not present in the sera of these mice. To our knowledge, this represents the first report that genetic immunization with Rev M10 induces an immune response that is dominated by IL-2-producing HTLs. Further, this study demonstrates the potential utility of introducing immunosuppressive genes as a means to control the immune response to foreign transgene products.

Immunogenetic therapy of human melanoma utilizing autologous tumor cells transduced to secrete granulocyte-macrophage colony-stimulating factor.

We performed a clinical study of five patients with melanoma to evaluate the immunobiological effects of retrovirally transduced autologous tumor cells given as a vaccine to prime draining lymph nodes. Patients were inoculated with both wild-type (WT) and GM-CSF gene-transduced tumor cells in different extremities. Approximately 7 days later, vaccine-primed lymph nodes (VPLNs) were removed. There was an increased infiltration of dendritic cells (DCs) in the GM-CSF-secreting vaccine sites compared with the WT vaccine sites. This resulted in a greater number of cells harvested from the GM-CSF-VPLNs compared with the WT-VPLNs at a time when serum levels of GM-CSF were not detectable. Four of five patients proceeded to have the adoptive transfer of GM-CSF-VPLN cells secondarily activated and expanded ex vivo with anti-CD3 MAb and IL-2. One patient had a durable complete remission of metastatic tumor. Utilizing cytokine (IFN-gamma, GM-CSF, IL-10) release assays, GM-CSF-VPLN T cells manifested diverse responses when exposed to tumor antigen in vitro. In two of two patients, GM-CSF-VPLN T cell responses were different from those of matched WT-VPLN cells. This study documents measurable immunobiologic differences of GM-CSF-transduced tumor cells given as a vaccine compared with WT tumor cells. The complete tumor remission in one patient provides a rationale to pursue this approach further.

Limiting dilution analysis for alloreactive, TCGF-secretory T cells. Two related LDA methods that discriminate between unstimulated precursor T cells and in vivo-alloactivated T cells.

We have developed a rapid limiting dilution analysis technique for quantitating alloantigen-specific TCGF-secretory T lymphocytes (operationally defined as helper T lymphocytes or HTL) in murine lymphoid populations. A simple permutation of this technique allows the distinction between HTL that have encountered antigen in vivo and the large population of naive precursor HTL (pHTL) with the same alloantigen specificity. We present evidence to validate these LDA techniques, and show that lymphoid tissue from mice bearing sponge matrix allografts, but not isografts, contain HTL that have been activated in vivo. In addition, we demonstrate a requirement for restimulation with alloantigen for secretion of lymphokine by the in vivo-activated cells. We suggest that this differential LDA technique could serve as a valuable tool in evaluating in vivo immunity and/or the in vivo efficacy of immunosuppressive, as well as immunopotentiating, therapies.

Mobilization of T lymphocytes following cardiac transplantation. Evidence that CD4-positive cells are required for cytotoxic T lymphocyte activation, inflammatory endothelial development, graft infiltration, and acute allograft rejection.

Modified limiting dilution analysis (LDA) techniques were used to evaluate the mobilization of antigen-stimulated helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) following allogeneic heterotopic cardiac transplantation. These modified LDA techniques allow a quantitative comparison of T cells that have been stimulated by antigen in vivo versus unstimulated precursor T cells of the same antigen specificity. Endothelial changes associated with mononuclear cell infiltration of the transplant were studied using endothelia-specific monoclonal antibodies and immunohistochemistry. Early (day 3) infiltration of cardiac allografts was characterized by a prevalence of donor alloantigen-specific HTL over CTL. Immunohistology revealed that the day-3 infiltrate was associated with areas of differentiated vascular endothelium, located primarily in the subepicardial region. Though donor-specific precursor HTL and CTL were present in the peripheral lymphoid tissues and blood, very few of them had been stimulated at this early time. During the latter phases of the response (days 6-9), antigen-stimulated HTL and CTL were present in the rejecting heart with CTL dominating the response. Accumulation of large numbers of donor-specific CTL in the allograft correlated with extensive inflammatory endothelial development, myocyte destruction, and loss of graft function by day 9. Stimulated HTL and CTL were detectable in peripheral lymphoid tissues at days 6 and 9. In addition, a marked increase in the number of donor-specific precursor CTL, but not precursor HTL, was observed in the lymphoid tissues at the peak of the response. Depletion of class II MHC-restricted T cells by in vivo treatment with anti-CD4 mAb eliminated HTL activity in all lymphoid compartments assessed and markedly reduced the number of CTL infiltrating the allograft. In addition, no stimulated CTL were detectable in lymphoid tissues, and the number of precursor CTL was not increased. In anti-CD4-treated recipients, cardiac allografts remained functional with minimal histological evidence of rejection for at least 21 days. Though graft-associated inflammatory endothelia were absent in anti-CD4-treated recipients at day 6, endothelial differentiation was observed in day 21 allografts in anti-CD4-treated recipients. These observations indicate that inflammatory endothelial development may precede T cell infiltration and subsequent loss of the cardiac allograft function. Thus, CD4-positive HTL are required for (1) graft-associated inflammatory endothelial development; (2) CTL activation in peripheral lymphoid tissues; (3) CTL accumulation in allografted tissues; and (4) acute cardiac allograft rejection.

Development and evaluation of a limiting dilution analysis technique that can discriminate in vivo alloactivated cytotoxic T lymphocytes from their naive CTL precursors.

We describe a permutation of the conventional limiting dilution analysis (LDA) technique that allows, for the 1st time, the differential enumeration of alloantigen-specific CTL that have been activated by alloantigens in vivo. This technique does not detect nonactivated CTL precursors, even those with similar alloantigen specificity. Data are presented to validate this limiting dilution analysis technique. Using this LDA technique, we demonstrate that large numbers of the donor-reactive CTL arrive in sponge matrix allografts (f = 1/3,599 cells), most or all of which are in an activated state (f = 1/4,385 cells). In contrast, alloactivated CTL constitute only a small fraction (f = 1/57,208 cells) of the donor-reactive CTL in the regional lymph node (f = 1/1,873 cells) of the same sponge allograft recipients. As expected, regional lymph nodes from sponge isograft recipients contain DBA/2-reactive CTL precursors (f = 1/1,873 cells), but no activated DBA/2-reactive CTL (f less than 1/385,529 cells). This LDA technique should be useful in studies regarding activation and redistribution of alloreactive CTL caused by allograft implantation.

Allogeneic class I MHC requirement for alloantigen-reactive helper T-lymphocyte responses in vivo. Evidence for indirect presentation of alloantigen.

The mechanisms by which host T cells recognize transplant-associated alloantigens in vivo have not been established. Two alloantigen presentation pathways may be used: (1) allogeneic class I and class II MHC molecules may be recognized directly by host CD8+ and CD4+ cells, respectively, or (2) allogeneic MHC molecules may be processed as foreign peptide and presented by host antigen-presenting cells to CD4+ cells in the context of self class II proteins. In this study, the sponge matrix allograft model was used to examine the relative contributions of these alloantigen presentation pathways to CD4+ T-cell activation in vivo. Limiting dilution analysis was used to quantify the localization of interleukin-2-producing helper T lymphocytes (HTL) following implantation of sponge allografts. Allografts either were disparate at both class I and class 11, or were derived from beta2-microglobulin knockout (beta2M-/-) mice, which express class II but are deficient in class I. Two measures of in vivo HTL function were monitored: (1) the accumulation of HTL within the allograft (a process that is dependent upon antigen-driven cytokine production), and (2) the development of cytolytic alloantibodies. After implantation of sponge allografts expressing both class I and class II, HTL were readily detectable in the allograft, and cytolytic alloantibodies were present in the serum. When mice were implanted with beta2M-/- sponge allografts, HTL failed to infiltrate these class I-deficient allografts, and alloantibodies were not detectable in the sera of recipients of beta2M-/- sponge allografts. This in vivo requirement for class I expression was not reflected by traditional in vitro measures of HTL function; cells obtained from lymphoid tissues mounted a mixed lymphocyte response and produced interleukin-2 when stimulated with beta2M-/- splenocytes in vitro. One possible interpretation of these data is that in vivo HTL functions are dependent upon the presence of class I-reactive CD8+ T cells. However, HTL readily infiltrated grafts expressing both class I and class II when recipients depleted of CD8+ T cells, and alloantibodies were produced. These observations support the idea that indirect presentation of allogeneic class I molecules plays a critical role in regulating CD4+ HTL functions associated with allograft rejection in vivo.

Evidence that human cardiac allograft acceptance is associated with a decrease in donor-reactive helper T lymphocytes.

We have reported that acute cardiac allograft rejection is associated with increased numbers of donor-reactive helper T lymphocytes (HTL) in the peripheral blood of patients. Further, increased frequencies of circulating donor-reactive HTL may predict allograft rejection episodes diagnosed by endomyocardial biopsy. The present study evaluates the relationship between donor-reactive HTL and allograft "acceptance" in cardiac transplant recipients bearing long-term allografts (> 1 year). Patients were categorized as either long-term acceptors or persistent rejecters based on the number of rejection episodes and the ability to withdraw steroid therapy. Limiting dilution analysis for IL-2-producing HTL was utilized, with cadaver donor splenocytes as a source of donor alloantigens. Donor-reactive HTL frequencies were determined from peripheral blood samples obtained before transplant, and at 1 month and 1 year after transplant. Individuals who accommodated their allografts and were withdrawn from steroid therapy had reduced numbers of donor-reactive HTL at 1 year after transplant as compared with earlier time points. Further, PBMC obtained from these individuals at 1 year after transplant responded weakly to donor alloantigens in a mixed lymphocyte response (MLR). This relationship between donor-reactive HTL and allograft accommodation was exemplified in a cardiac/liver transplant patient who was diagnosed with progressive multifocal leukoencephalopathy and removed from all immunosuppression. No subsequent rejection episodes were diagnosed. Donor-reactive HTL were not detectable and this individual failed to mount an MLR to donor alloantigens. However, a vigorous donor-reactive response was observed when MLR cultures were supplemented with exogenous IL-2. Therefore, nonresponsiveness to the allograft appeared to be due to a deficit in IL-2 production. In contrast, patients who experienced persistent rejection episodes and required continued steroid therapy maintained large numbers of donor-reactive HTL at 1 year after transplant. PBMC from these individuals responded vigorously to donor alloantigens in an MLR. Hence, monitoring donor-reactive HTL may identify individuals who have accommodated their graft and may tolerate a reduction in immunosuppression.

In vivo mechanisms of alloreactivity. VI. Evidence that alloantigen deposition initiates both local and systemic mechanisms that influence CTL accumulation at a graft site.

We have used sponge matrix allografts to investigate how alloantigen influences the pattern of CTL accumulation at a graft site. These studies employed two limiting dilution analysis techniques to monitor CTL accumulation. One technique quantitates the subpopulation of CTL that show evidence of in vivo contact with graft alloantigens (alloantigen-conditioned CTL or cCTL). The other quantitates all CTL with specificity for graft alloantigens, regardless of their differentiative status. Using these techniques, we have demonstrated that sponge allografts acquire three types of CTL: (a) donor-reactive CTL precursors (pCTL), (b) donor-reactive cCTL, and (c) pCTL with irrelevant antigen specificity. Sponge isografts rarely acquire LDA-detectable CTL, unless they receive a concurrent allogeneic stimulus at a distant anatomic site. In that instance, sponge isografts acquired pCTL, but not cCTL. This indicates that an ongoing immune response to alloantigens can influence the immunologic characteristics of an unrelated inflammatory response occurring elsewhere. We further observed that sponge isografts can be made to acquire alloreactive cCTL only when specific alloantigens are placed in the isograft. This indicates that specific grant alloantigen present at the graft site plays a necessary but undefined role in the acquisition or development of cCTL in sponge allografts. Collectively, these data demonstrate that alloantigen deposition initiates both local and systemic mechanisms that influence alloreactive CTL accumulation at a graft site.

In vivo depletion of CD8+ T cells results in Th2 cytokine production and alternate mechanisms of allograft rejection.

A current hypothesis states that Th1 cytokines promote allograft rejection and that Th2 cytokines promote graft acceptance. We present data that question the tolerogenic activity of Th2 cytokines, and we suggest that Th2 cytokines may evoke allograft rejection by recruitment of alternate effector mechanisms. Unmodified rejection of mouse heterotopic cardiac allografts is associated with the accumulation of large numbers of donor-reactive CD8+ CTL within the allograft, which is indicative of a Th1-driven cellular response. However, when recipients are depleted of CD8+ CTL, rejection still occurs and is associated with an aggressive cellular infiltrate rich in eosinophils, large mononuclear cells, and fibroblast-like cells. Eosinophils, which are responsive to the Th2 cytokines IL-4 and IL-5, are not present in unmodified rejecting allografts. Differential production of Th1 versus Th2 cytokines was further suggested by altered levels of IgG2a (promoted by IFN gamma) and IgG1 (promoted by IL-4) alloantibody in the sera of these mice; IgG2a dominated the alloantibody response in unmodified allograft recipients, whereas IgG1 levels increased in recipients depleted of CD8+ CTL. Altered intragraft cytokine gene expression was verified by RT-PCR; Th1 (IL-2, IFN gamma), but not Th2 (IL-4, IL-5, IL-10), cytokine mRNAs were readily detectable in the allografts of unmodified recipients. In contrast, both Th1 and Th2 cytokine genes were expressed in the allografts of mice depleted of CD8+ CTL. These data suggest that donor-reactive CD8+ CTL inhibit intragraft production of Th2 cytokines, thereby promoting a Th1 dominated-rejection response. Elimination of CD8+ cells allows Th2 cytokine production, which may have deleterious, rather than protective, effects.