Trans-vivo Delayed-type Hypersensitivity Research Articles

Extracellular Vesicle-associated GARP/TGFβ:LAP Mediates "Infectious" Allo-tolerance.

C57BL/6 mice were tolerized by i.p. injection of CBA/J splenocytes followed by anti-CD40L/CD154 antibody treatment on days 0, 2, and 4. On day 35, spleen and lymph nodes were extracted and isolated lymphocytes were restimulated with sonicates of CBA splenocytes overnight. sEVs were extracted from culture supernatants by ultracentrifugation (100 000g) and assayed for (a) the presence of TGFβ:LAP associated with tetraspanins CD81,CD63, and CD9 by enzyme-linked immunosorbent assay; (b) GARP, critical to membrane association of TGFβ:LAP and to activation from its latent form, as well as various TGFβ receptors; and (c) TGFβ-dependent function in 1° and 2° immunosuppression of tetanus toxoid-immunized B6 splenocytes using trans-vivo delayed-type hypersensitivity assay. After tolerization, CBA-restimulated lymphocytes secreted GARP/TGFβ:LAP-coated extracellular vesicles. Like IL35 subunits, but unlike IL10, which was absent from ultracentrifuge pellets, GARP/TGFβ:LAP was mainly associated with CD81+ exosomes. sEV-bound GARP/TGFβ:LAP became active in both 1° and 2° immunosuppression, the latter requiring sEV uptake by "bystander" T cells and reexpression on the cell surface. Like other immune-suppressive components of the Treg exosome, which are produced in a latent form, exosomal GARP/TGFβ:LAP produced by allo-specific regulatory T cells undergoes either immediate activation (1° suppression) or internalization by naive T cells, followed by surface reexpression and subsequent activation (2°), to become suppressive. Our results imply a membrane-associated form of TGFβ:LAP that, like exosomal IL35, can target "bystander" lymphocytes. This new finding implicates exosomal TGFβ:LAP along with Treg-derived GARP as part of the infectious tolerance network.

Abstract 127: The Lung-Associated Self-Antigen Type V Collagen is Regulated by Nuclear Factor of Activated T Cells Isoform c3 in Chronic Hypoxia

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) is a progressive and often fatal consequence of chronic lung diseases, chronic exposure to high altitude and sleep apnea. PH results from arterial remodeling, enhanced vasoreactivity, and Th17 cell-mediated perivascular inflammation. Naturally occurring “nTH17” cells, can be detected in fetal life and are in homeostatic equilibrium with natural T regulatory cells. nTH17 cells are specific for a limited range of self-antigens, including type V collagen (colV). ColV is normally hidden from the immune system within type I col in the extracellular matrix of the lungs. We have shown that type V collagen is a self-antigen uncovered by CH. On the other hand, we have shown that mice lacking nuclear factor of activated T cells isoform c3 (NFATc3) globally and specifically in smooth muscle (SMC-NFATc3 KO) are protected from CH-induced PH. No differences in cardiac function were detected between SMC-NFATc3 KO and control mice using a Vevo LAZR-X ultrasound system before CH. In silico analysis of Col5A1 promoter shows NFATc3 binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates Col5A1 expression contributing to the development of acute autoreactivity to collagen V in response to CH, representing an upstream mechanism in PH development. Col5A1 was one of the 1792 genes differentially affected by both CH and smooth muscle NFATc3 in isolated pulmonary arteries (RNA Illumina Sequencing). Col5A1 mRNA increased significantly after 5 days of CH in control mice (fold change from normoxia±SD=2.2±0.8, n=5) vs. SMC-NFATc3 KO (0.8±0.3, n=3, p<0.03). Cellular autoimmunity to colV was determined using a trans-vivo delayed-type hypersensitivity assay (TV-DTH). Splenocytes from 5 days CH-exposed control mice displayed a TV-DTH colV response significantly higher than that from CH-exposed SMC-NFATc3 KO mice (footpad swelling [10 -4 ”] ± SD of naïve recipient mice injected with cells from CH control mice 25.6±4.2, n=8 vs. CH SMC-NFATc3 KO mice 5.3±0.5, n=7; p<0.0001). Our results suggest that smooth muscle NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen ColV contributing to pulmonary vascular inflammation.

Abstract P280: Type V Collagen is a Lung-Associated Self-Antigen Uncovered by Chronic Hypoxia

Hypoxic pulmonary hypertension (PH) is a progressive and often fatal consequence of chronic hypoxia (CH) exposure (chronic lung diseases, high altitude and sleep apnea). We recently demonstrated that T helper 17 (T H 17) cells are localized in the perivascular region of pulmonary arteries and contribute to CH-induced PH, at least in part, by causing pulmonary arterial (PA) remodeling. Naturally occurring “nT H 17” cells, can be detected as early as week 17 of fetal life in humans, and are in homeostatic equilibrium with natural T regulatory (nTregs) cells. nT H 17 cells are specific for a limited range of self-antigens, including type V collagen (colV). ColV is normally sequestered within type I col in the extracellular matrix of the lungs hidden from the immune system. Activation of matrix metalloproteinase-2, which is induced by CH, can expose colV. Therefore, we tested the hypothesis that ColV is a lung-associated self-antigen uncovered by CH. We found colV immunoreactivity only in lungs from mice exposed to hypobaric CH for 5 days vs. controls and colV mRNA expression is significantly increased in isolated PA (log fold change mean±SEM, normoxia= 0.22±0.08 vs. CH=0.48±0.02, n=3, p=0.02) suggesting that CH uncovers hidden colV. We then, determined cellular autoimmunity to colV using a trans-vivo delayed-type hypersensitivity assay (TV-DTH). Splenocytes obtained from 5 days CH-exposed mice displayed a TV-DTH colV response significantly higher than that from normoxic mice (footpad swelling [10 -4 ”] normoxia=11.8±1.1 vs. CH= 35.6±3.0, n=14, p=0.0001), while there was minimal response to col I. To determine that reactivity to colV contributes to CH-induced PH, we induced peripheral tolerance to colV by administering 25 μl of 0.08 μg/μl of bovine colV (Sigma) in PBS in each nare or PBS every two days for two weeks prior and during CH (21 days). Right ventricular systolic pressure (RVSP) was higher in vehicle-treated compared to colV-treated mice (mmHg CH vehicle= 32.4±0.3 vs. CH colV= 27.8±0.9, n=4, p=0.0085) suggesting that oral tolerance to col V attenuated PH. These results are ground-breaking suggesting that acute autoreactivity to colV develops after exposure to hypoxia. Next, we will determine whether a disrupted balance of nTh17/nTreg mediates this response.

Identification of mechanisms of immune regulation and exhaustion generated by tumor-derived exosomes in patients with advanced non-small cell lung cancer using the trans-vivo DTH assay.

175 Background: The success of PD-1/PD-L1 inhibition is clearly dependent on optimal patient selection. Developing validated biomarkers that identify patients with an increased probability of response to these antibodies remains a challenge. DTH responses are a standard method to evaluate cellular immune responses in animal and human immunotherapy trials. The trans-vivo delayed-type hypersensitivity (tvDTH) methodology, using the SCID mouse footpad swelling response to human PBMC (peripheral blood mononuclear cells) + antigen injection, and using antibodies to block the regulatory pathways, was developed in our laboratory and has been used to characterize regulation systems in tolerant transplant recipients and more recently in prostate cancer. Methods: In this pilot project we aim to investigate the mechanisms of regulatory immune responses in patients with advanced NSCLC receiving anti-cancer therapies using the tvDTH, and to correlate tvDTH results with responses to checkpoint inhibitor blockade therapy. So far we have enrolled 4 NSCLC patients. PBMCs are obtained by blood draw and lung cancer antigen (LCA) is prepared from malignant pleural effusion cellular pellet or fresh frozen tumor. 1 of 4 patients’ sample was negative for malignancy. Results: Our preliminary data suggests that all NSCLC patients with pleural effusions positive for malignancy had a PD-1-dependent exhausted immune response towards their LCA. Remarkably, 1 of these patients not only showed PD-1-dependent exhaustion, but also PD-1-dependent bystander suppression, whereby a positive control immune response was reduced by co-injection of LCA, suggesting the existence of immunosuppressive mechanisms. Unlike our previous results with prostate cancer, no patient showed CTLA-4-dependent exhaustion or suppression. Importantly, an antigen preparation from a NSCLC that showed no malignant cells by cytology did not induce a swelling response. Conclusions: We plan to expand our study patient population and follow up our patients in order to correlate tvDTH results with clinical outcomes.

Evolving Approaches in the Identification of Allograft-Reactive T and B Cells in Mice and Humans.

Whether a transplanted allograft is stably accepted, rejected, or achieves immunological tolerance is dependent on the frequency and function of alloreactive lymphocytes, making the identification and analysis of alloreactive T and B cells in transplant recipients critical for understanding mechanisms, and the prediction of allograft outcome. In animal models, tracking the fate of graft-reactive T and B cells allows investigators to uncover their biology and develop new therapeutic strategies to protect the graft. In the clinic, identification and quantification of graft-reactive T and B cells allows for the early diagnosis of immune reactivity and therapeutic intervention to prevent graft loss. In addition to rejection, probing of T and B cell fate in vivo provides insights into the underlying mechanisms of alloimmunity or tolerance that may lead to biomarkers predicting graft fate. In this review, we discuss existing and developing approaches to track and analyze alloreactive T and B cells in mice and humans and provide examples of discoveries made utilizing these techniques. These approaches include mixed lymphocyte reactions, trans-vivo delayed-type hypersensitivity, enzyme-linked immunospot assays, the use of antigen receptor transgenic lymphocytes, and utilization of peptide-major histocompatibility multimers, along with imaging techniques for static multiparameter analysis or dynamic in vivo tracking. Such approaches have already refined our understanding of the alloimmune response and are pointing to new ways to improve allograft outcomes in the clinic.

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness.Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells.The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

Microchimerism and regulation in living related kidney transplant families.

Long-term harmful effects of immunosuppressive drugs and chronic rejection are a persistent impetus to establish methods to induce immunological tolerance to allografts. PCR-based studies have found evidence that humans and other placental mammals can have prolonged extremely low levels of maternal cells as well as other non-self cells, referred to as microchimerism. The persistence of these cells suggests a mechanism for the maintenance of the regulatory T-cell (Treg) responses frequently detected in offspring to non-inherited maternal antigens. We test the hypothesis that the detection of very low copy levels of insertion/deletion (Indel) alleles consistent with non-inherited maternal genes, will correlate with immune regulation to non-inherited maternal antigens as detected by a trans-vivo Delayed-Type Hypersensitivity (tvDTH) assay in kidney transplant recipients, normal donors and their immediate biological family members. Preliminary data reported here compares qPCR amplification of rare DNA templates in the peripheral blood polymorphonuclear (PMN) fraction of cells, with the results of tvDTH assays for linked suppression of recall antigen responses in the presence of non-inherited maternal antigens [NIMA]. The two assays do not show a definitive correlation.

Immunological Tolerance Monitoring in Solid Organ Transplantation

In organ transplantation, achieving an operational tolerance is the ultimate goal. However, inducing this tolerance with a minimal dose of anti-rejection drugs can only be safely achieved when guided by biomarkers reflecting the immune reactivity in patients. We review recently described biomarkers and assays for the identification of patients for their risk of organ rejection and their suitability for drug weaning. The clinical assessment of tolerance has been attempted with immunological tools, including the analysis of immune cell subsets, regulatory T cells, ELISPOT, and trans-vivo delayed-type hypersensitivity assays. These methods are promising tools for diagnosis; however, their ability to determine the presence and persistence of responsiveness to their transplant is not available. As the omics technologies advance, comprehensive and high-throughput biomarker studies have become more accessible, more affordable, and more customizable. Validating the use of microarray analysis has important implications for monitoring patients for the development of tolerance following transplantation as well as for determining the mechanisms by which tolerance is induced and maintained. Finally, collaborative efforts are needed to design and perform prospective multicenter trials to validate the identified biomarkers across different laboratories.

Human Prostate Tumor Antigen–Specific CD8+ Regulatory T Cells Are Inhibited by CTLA-4 or IL-35 Blockade

Regulatory T cells play important roles in cancer development and progression by limiting the generation of innate and adaptive anti-tumor immunity. We hypothesized that in addition to natural CD4(+)CD25(+) regulatory T cells (Tregs) and myeloid-derived suppressor cells, tumor Ag-specific Tregs interfere with the detection of anti-tumor immunity after immunotherapy. Using samples from prostate cancer patients immunized with a DNA vaccine encoding prostatic acid phosphatase (PAP) and a trans-vivo delayed-type hypersensitivity (tvDTH) assay, we found that the detection of PAP-specific effector responses after immunization was prevented by the activity of PAP-specific regulatory cells. These regulatory cells were CD8(+)CTLA-4(+), and their suppression was relieved by blockade of CTLA-4, but not IL-10 or TGF-β. Moreover, Ag-specific CD8(+) Tregs were detected prior to immunization in the absence of PAP-specific effector responses. These PAP-specific CD8(+)CTLA-4(+) suppressor T cells expressed IL-35, which was decreased after blockade of CTLA-4, and inhibition of either CTLA-4 or IL-35 reversed PAP-specific suppression of tvDTH response. PAP-specific CD8(+)CTLA-4(+) T cells also suppressed T cell proliferation in an IL-35-dependent, contact-independent fashion. Taken together, these findings suggest a novel population of CD8(+)CTLA-4(+) IL-35-secreting tumor Ag-specific Tregs arise spontaneously in some prostate cancer patients, persist during immunization, and can prevent the detection of Ag-specific effector responses by an IL-35-dependent mechanism.

Donor-Specific Indirect Pathway Analysis Reveals a B-Cell-Independent Signature which Reflects Outcomes in Kidney Transplant Recipients

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < MONO < SI < CR; p < 0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOL > MONO = SI > CR; p < 0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Abstract 5581: Antigen-specific CTLA-4-regulated T-cell responses elicited in patients with prostate cancer treated by DNA immunization

Abstract Background: We and others have been interested in plasmid DNA vaccines as a means of eliciting tumor antigen-specific CD4+ and CD8+ T-cell responses. We have investigated plasmid DNA vaccines encoding prostatic acid phosphatase (PAP), a well-characterized prostate tumor antigen, in rodent models and found these to be effective in eliciting antigen-specific CD8+ T cells with evidence of anti-tumor efficacy. Multiple immunizations at high plasmid doses, however, elicited antigen-specific IL-10 secretion. We have recently completed a phase I clinical trial evaluating a DNA vaccine encoding PAP in patients with minimal residual prostate cancer. In that trial, while in vitro PAP-specific T-cell responses were elicited in multiple patients following six biweekly immunizations, others did not have evidence of an immune response, and some responses were not detectable until months later. The current study was conducted to determine whether immunization might have elicited an antigen-specific regulatory T-cell response in some individuals. Methods: Peripheral blood mononuclear cells obtained from 21 patients prior to immunization, and two weeks after six biweekly immunizations, were evaluated for PAP-specific immune responses in a SCID mouse trans-vivo delayed-type hypersensitivity (DTH) assay. Specifically, DTH responses to PAP in PBMC were evaluated in the presence or absence of antibodies to regulatory molecules (IL-10, TGFß, CTLA-4, PD-1) to “uncover” antigen-specific regulatory mechanisms. Results: 8/21 individuals had a PAP-specific T-cell response following immunization that was not detectable pre-immunization and which was “uncovered” by CTLA-4 blockade. This response was found to persist in at least one individual for several months. IL-10- and TGFß-regulated responses were not detected. Baseline regulatory PAP-specific T cells could be detected that suppressed concurrent tetanus-specific DTH responses. 6/8 individuals with a detectable CTLA-4-regulated response experienced a favorable increase in PSA doubling time. Conclusions: Patients with prostate cancer can have PAP-specific regulatory T cells. Plasmid DNA immunization can elicit effector T cells specific for PAP that in many individuals is regulated by CTLA-4. Further studies are underway to characterize the regulatory T cell population(s), however these findings suggest that the efficacy of DNA vaccines might be further augmented by combination with anti-CTLA-4 blockade. In addition, these studies suggest that these trans-vivo DTH methods, in use for evaluating mechanisms of antigen-specific tolerance, could be more generally applied to the immune monitoring of anti-tumor vaccine clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5581.

Early and Limited Use of Tacrolimus to Avoid Rejection in an Alemtuzumab and Sirolimus Regimen for Kidney Transplantation: Clinical Results and Immune Monitoring

Alemtuzumab induction with 60 days of tacrolimus treatment and continuous sirolimus treatment prevented acute rejection in nine of 10 consecutive renal allograft recipients. All patients are alive with a functioning kidney graft at 27-39 months of follow-up. Extensive immune monitoring was performed in all patients. Alloantibody detection, cytokine kinetics assay (CKA), and trans vivo delayed-type hypersensitivity (DTH) assay were performed every 6 months showing correlation with clinical evolution. Despite alloantibody presence in five patients, eight patients remain without the need for specific treatment and only sirolimus monotherapy in decreasing dosage. Four patients take only 1 mg sirolimus daily with levels of 3-4 ng/mL. One patient showed clinical signs of rejection at month 9 post-transplant, with slow increase in serum creatinine and histological signs of mixed cellular (endarteritis) and humoral rejection (C4d positivity in peritubular capillaries and donor-specific antibody (DSA)). In summary, the addition of tacrolimus therapy for 2 months to a steroid-free, alemtuzumab induction and sirolimus maintenance protocol limited the previously shown acute rejection development. Nevertheless, alloantibody was present in serum and/or C4d present on 1-year biopsy in half the patients. The combination of CKA and DSA monitoring or the performance of transvivo DTH correlated with immune status of the patients.

Operational tolerance after liver transplantation

The achievement of an immunosuppression (IS)-free state after transplantation represents the ultimate goal of any immunosuppressive regimen. While clinical operational tolerance (COT) remains the exception after other types of solid organ transplantation, several cases of COT have been described after liver transplantation (LT). Overall, the experience gained so far worldwide demonstrates that COT can be achieved safely in one quarter of selected individuals, irrespective of the immunological background of donor and recipient, patient age, indication for LT, study endpoint, length of the weaning period and of pre/post-weaning follow-up, presence or not of chimerism. However, most transplant physicians still believe that the achievement of COT is still out of reach for the majority of LT recipients because of the potential risk for transplant survival, the non-randomized nature of most of the studies reported so far, and the selective nature of the patients enrolled in such studies, making them non-representative of the whole population of LT recipients. Despite these concerns, the present article demonstrates that this attitude is potentially no longer justified, given the growing evidence that a permanent and stable IS-free state can be achieved in a proportion of individuals who have received a LT for non-immune mediated liver diseases.

Assays on the Influence of Biomaterials on Allogeneic Rejection in Tissue Engineering

In tissue engineering, innate responses to biomaterial scaffolds will affect rejection of allogeneic cells. Biomaterials directly influence innate and adaptive immune cell adhesion, reactive oxygen intermediate production, cytokine secretion, nuclear factor-kappa B nuclear translocation, gene expression, and cell surface markers, all of which are likely to affect allogeneic rejection responses. A major goal in tissue engineering is to induce transplant tolerance, potentially by manipulating the biomaterial component. This review describes methods of measuring responses of macrophages, dendritic cells, and T cells stimulated in vitro and in vivo and addresses key factors in assay development. Such tests include mixed leukocyte reactions, enzyme-linked immunosorbent spot assays, trans-vivo delayed-type hypersensitivity assays, and measurement of dendritic cell subsets and anti-donor antibodies; we propose extending these studies to tissue engineering.

Lung transplant patients’ T cell responses to influenza vaccine viruses between seasons

We hypothesized that T cell responses to influenza viruses by lung transplant and healthy individuals would be similar. Twelve lung transplant patients and 12 healthy individuals received influenza vaccines during two seasons. Lymphocytes were isolated for the trans-vivo delayed-type hypersensitivity assay from blood samples following immunization. T cell responses to all three inactivated influenza antigens from each were similar between the groups. However, the response to the A/New Caledonia (H1N1) virus by transplant patients was greater than the healthy controls' response, but only in the first season. The magnitude of the T cell responses in lung transplant patients is similar to those of healthy control individuals. These responses may be important in T cell memory.

Dependency of the Trans Vivo Delayed Type Hypersensitivity Response on the Action of Regulatory T Cells: Implications for Monitoring Transplant Tolerance

The trans vivo delayed-type hypersensitivity (DTH) assay has been used for monitoring the immune status of clinical transplant recipients. Here we tested the hypothesis that the assay can reveal control of allograft rejection by CD25CD4 regulatory T cells (Treg). CBA.Ca (H2) recipients of heterotopic C57BL/10 (H2, B10) heart transplants were untreated or pretreated with anti-CD4 antibody and donor-specific blood. This protocol has been shown previously to induce operational tolerance to alloantigens that is dependent on CD25CD4 Treg. Four weeks after transplantation leukocytes were harvested and used for the trans vivo DTH assay. Cells were stimulated with irradiated B10 leukocytes or subcellular antigen and injected into ear pinnae of immune deficient CB17.SCID.beige hosts. Stimulation of leukocytes from recipients rejecting B10 cardiac allografts with recall alloantigen caused a "strong" swelling response, whereas similar stimulation of leukocytes from operationally tolerant mice resulted in significantly less swelling (n=17; P=0.003). When CD25 T cells were depleted from "tolerant" leukocytes, the swelling response triggered was similar to that obtained using cells from rejecting animals (P<0.001), while coinjection of purified CD25CD4 cells from tolerant recipients with rejecting cells suppressed the swelling response (P=0.007) to the level observed with cells from tolerant mice. Both the effector and the regulatory response were triggered by indirect allorecognition and blocking experiments showed that the regulation observed was dependent on cytoxic T lymphocyte antigen (CTLA) 4. The trans vivo DTH assay can be used to reveal regulation mediated by CD25CD4 T cells and is CTLA4 dependent.

Mouse Strain and Injection Site are Crucial for Detecting Linked Suppression in Transplant Recipients by Trans-Vivo DTH Assay

Chemokine-driven accumulation of lymphocytes, mononuclear and polymorphonuclear proinflammatory cells in antigenic tissue sites is a key feature of several types of T-cell-dependent autoimmunity and transplant rejection pathology. It is now clear that the immune system expends considerable energy to control this process, exemplified by the sequential layers of regulatory cell input, both innate and adaptive, designed to prevent a classical Type IV or 'delayed-type' hypersensitivity (DTH) reaction from occurring in the visual field of the eye. Yet, despite an abundance of in vitro assays currently available to the human T-cell immunologist, none of them adequately models the human DTH response and its various control features. The theme of this article is that it is relatively easy to model the effector side of the human DTH response with xenogeneic adoptive transfer models. However, we show that in order to detect inhibition of a recall DTH in response to colocalized donor antigen (linked suppression)--a characteristic feature of peripheral tolerance to an organ transplant--both the challenge site and the immunocompetence of the mouse adoptive host are critical factors limiting the sensitivity of the trans-vivo DTH test.

The role of leukocyte function-associated antigen-1 in animal models of inflammation

Both preclinical and clinical data have identified leukocyte function-associated antigen-1 (LFA-1) as an important component of inflammatory disease states. We evaluated small molecule inhibitors of this glycoprotein in several animal models in which the inflammatory process is dependent on human or non-human primate LFA-1. ( R)-5(4-bromobenzyl)-3(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione, BIRT 377, effectively suppressed the production of human immunoglobulin (IgG) following reconstitution of severe combined immunodeficient (SCID) mice with human peripheral blood mononuclear cells. The BIRT 377 analog, BIX 642, inhibited the cellular infiltrate and increase in skin thickness associated with the delayed-type hypersensitivity reaction in previously immunized squirrel monkeys challenged with antigen. BIX 642 also inhibited the trans-vivo delayed-type hypersensitivity response in the footpads of SCID mice injected with human peripheral blood mononuclear cells and donor-sensitive antigen. These results demonstrate the efficacy of small molecule inhibitors of LFA-1 in preclinical models of inflammation dependent on human or non-human primate LFA-1.

Loss of tolerance to a maternal kidney transplant is selective for HLA class II: evidence from trans-vivo DTH and alloantibody analysis

We studied late graft rejection in a patient who had received a kidney transplant 9–10 years earlier from his mother and who had been off all immunosuppressive drugs for 7 years at the time of graft rejection onset. The mother differed for one HLA-A (A3) and one HLA-B (B62) antigen but had only a subtype mismatch at the HLA-DRβ1 locus (donor: DRβ1∗1104; recipient: DRβ1∗1102). A gradual rise in serum creatinine from 1.8 to 2.0 mg/dl at year 9 prompted a biopsy, which was negative for rejection (focal infiltrates but no tubulitis). Ten months later the patient’s creatinine had risen to > 3.4 mg/dl, and a second biopsy revealed extensive tubulitis, cellular rejection, and glomerular sclerosis. Sonicates of donor leukocytes triggered no delayed-type hypersensitivity (DTH) response above background (PBMC only) in the patient’s peripheral blood leukocytes obtained prior to year 9. A gradual recovery of antidonor DTH response between year 9 and 10 closely paralleled the change from tolerant to rejection status. Antidonor antibody was also undetectable in serum prior to year 9, but a donor-reactive antibody did develop at year 10.2 shortly after the peak of DTH response. The serum level of soluble donor HLA class I B62 antigen rose > 10-fold over prerejection level at the time of the biopsy-proven rejection, suggesting a possible trigger for both the cellular and humoral immune response. Nonetheless, we found no evidence for the de-velopment of humoral or cellular immunity to maternal HLA class I. Instead, DTH analysis of memory T cells of the patient obtained after rejection showed that a single maternal HLA DRβ1∗1104 allopeptide, differing by two amino acids in sequence from the peptide of the recipient (DRβ1∗1102), stimulated a strong memory DTH response. Similarly, we found an anti-HLA class II donor-specific antibody in serum that appeared to be crossreactive with DRβ1∗1104 and DRβ1∗1101 but not with the recipient DRβ1∗1102 antigen. The data support the idea of a profound unresponsive state at both the cellular (DTH) and humoral level toward maternal HLA class I antigens that was not reversed even during late cellular rejection, despite the release of high levels of soluble HLA class I. Furthermore, the data suggest that DTH recovery was a close correlate of the onset of rejection and this “indirect” alloresponse, like the anti-donor alloantibody response that followed, was directed not to noninherited maternal HLA-A,B antigens but to the maternal HLA DRβ1∗1104 subtype.