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Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992–1004. ©2017 AACR.

Supplementary Figure 1: Radiotherapy suppresses tumor growth. Supplementary Figure 2: Characterization of the TME of B16/F10 tumors post-RT. Supplementary Figure 3: Stereotactic radiation does not change Treg in draining lymph nodes nor in spleens of treated mice. Supplementary Figure 4: Persistently increased Treg Post-RT. Supplementary Figure 5: Stereotactic radiation increases the suppressive markers of Treg in the RENCA tumor model. Supplementary Figure 6: Stereotactic radiation increases the suppressive markers of Treg in the MC38 tumor model. Supplementary Figure 7: Stereotactic radiation does not change the expression of the suppressive markers of Treg in DLNs. Supplementary Figure 8: Stereotactic radiation does not change the expression of the suppressive markers of Treg in spleens. Supplementary Figure 9: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the B16/F10 model. Supplementary Figure 10: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the RENCA model. Supplementary Figure 11: Expression of the selected markers of TIL-CD4+Foxp3- cells (Tconv) in the MC38 model. Supplementary Figure 12: Expression of 4-1BB on TIL-CD8+ cells. Supplementary Figure 13: The effect of TGF-beta blockade on different T cell subsets. Supplementary Figure 14: TGF-beta expression in the tumor microenvironment. Supplementary Figure 15: The effect of Fingolimod (FTY720) on peripheral blood lymphocytes counts. Supplementary Figure 16: Chemokine/cytokine expression of a selected panel in the tumor microenvironment post-radiation.

In vascularized composite allotransplantation (VCA), invasive tissue biopsies remain the gold standard in diagnosing rejection carrying significant morbidity. We aimed to show feasibility of tape-stripping for noninvasive immune monitoring in VCA. Tape-stripping was performed on allografts and native skin of upper extremity transplant recipients. Healthy nontransplanted individuals served as controls. The technique was also used in swine on naive skin in nontransplanted animals, native skin of treated, transplanted swine, nonrejecting VCAs, and rejecting VCAs. Extracted protein was analyzed for differences in cytokine expression using Luminex technology. Significantly decreased levels of INFγ and IL-1Ra were seen between human allograft samples and native skin. In swine, rejecting grafts had increased IL-1Ra compared to naive and native skin, decreased levels of GM-CSF compared to native skin, and decreased IL-10 compared to nonrejecting grafts. Unsupervised hierarchical clustering revealed rejecting grafts separated from the nonrejecting (P = 0.021). Variable importance in projection scores identified GM-CSF, IL-1Ra, and IL-2 as the most important profiles for group discrimination. Differences in cytokine expression are detectable in human VCA patient native skin and VCA graft skin using a noninvasive tape-stripping method. Swine studies suggest that differences in cytokines between rejecting and nonrejecting grafts are discernable.

Pancreatic cysts are common and often pose a management dilemma, because some cysts are precancerous, whereas others have little risk of developing into invasive cancers. We used supervised machine learning techniques to develop a comprehensive test, CompCyst, to guide the management of patients with pancreatic cysts. The test is based on selected clinical features, imaging characteristics, and cyst fluid genetic and biochemical markers. Using data from 436 patients with pancreatic cysts, we trained CompCyst to classify patients as those who required surgery, those who should be routinely monitored, and those who did not require further surveillance. We then tested CompCyst in an independent cohort of 426 patients, with histopathology used as the gold standard. We found that clinical management informed by the CompCyst test was more accurate than the management dictated by conventional clinical and imaging criteria alone. Application of the CompCyst test would have spared surgery in more than half of the patients who underwent unnecessary resection of their cysts. CompCyst therefore has the potential to reduce the patient morbidity and economic costs associated with current standard-of-care pancreatic cyst management practices.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies. Currently, approved drugs are given with non-curative intent as the only known cure is allogeneic bone marrow transplantation, which relies on the donor's immune system driving an allogeneic effect. Previous efforts to harness the endogenous immune system have been less successful. We present the results of a pilot study of K562/GM-CSF (GVAX) whole-cell vaccination in MDS patients. The primary objective of safety was met as there were no serious adverse events. One patient had a decrease in transfusion requirements and another demonstrated hematologic improvement suggesting a signal for clinical activity. In vitro correlative studies indicated biological effects on immune cells following vaccination. Although only a pilot study, results are encouraging that an immunotherapeutic approach with a whole-cell vaccine may be feasible in MDS patients.

SEEK and you may find cancer earlier Many cancers can be cured by surgery and/or systemic therapies when detected before they have metastasized. This clinical reality, coupled with the growing appreciation that cancer's rapid genetic evolution limits its response to drugs, have fueled interest in methodologies for earlier detection of the disease. Cohen et al. developed a noninvasive blood test, called CancerSEEK that can detect eight common human cancer types (see the Perspective by Kalinich and Haber). The test assesses eight circulating protein biomarkers and tumor-specific mutations in circulating DNA. In a study of 1000 patients previously diagnosed with cancer and 850 healthy control individuals, CancerSEEK detected cancer with a sensitivity of 69 to 98% (depending on cancer type) and 99% specificity. Science , this issue p. 926 ; see also p. 866

The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient's primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types.

BACKGROUND: The mechanisms that underlie allergic transfusion reactions (ATRs) are not well characterized, but likely involve recipient, donor, and product factors. To assess product factors associated with ATRs, we investigated candidate mediators in apheresis platelet (PLT) products associated with ATRs and controls. STUDY DESIGN AND METHODS: Using bead-based and standard enzyme-linked immunosorbent assays, we tested supernatants from 20 consecutive apheresis PLT transfusions associated with ATRs and 30 control products for concentrations of mediators in three categories: acute inflammatory mediators, direct agonists of basophils and mast cells, and growth and/or priming factors of basophils and mast cells. RESULTS: Median concentrations of the direct allergic agonists C5a, brain-derived neurotrophic factor (BDNF), and CCL5 (RANTES) were 16.6, 41.8, and 13.9% higher, respectively, in the supernatant of apheresis PLT products that were most strongly associated with ATRs (p 0.2 for all associations). CONCLUSION: The allergic agonists C5a, BDNF, and CCL5 may be mediators of ATRs in apheresis PLT products. Acute inflammatory proteins and basophil and/or mast cell growth and priming factors do not appear to be associated with apheresis PLT products that cause ATRs.

BACKGROUND We retrospectively explored changes in immunological parameters in men with biochemically recurrent prostate cancer treated with either 5 or 25 mg of lenalidomide in a randomized phase 2 trial, and determined whether those changes correlated with disease progression. METHODS Cytokine levels were compared for each patient at baseline and after 6 months of treatment with lenalidomide. Regression models for correlated data were used to assess associations of cytokine levels with lenalidomide treatment effect. Estimates were obtained using generalized estimating equations. Changes in circulating anti-prostate antibodies were evaluated using a high-throughput immunoblot technique. RESULTS Treatment with lenalidomide was associated with global changes in immunoreactivity to a number of prostate-associated antigens, as well as with changes in circulating levels of the TH2 cytokines IL-4, IL-5, IL-10, and IL-13. Disease progression in treated patients was associated with an increase in circulating IL-8 levels, while IL-8 levels decreased significantly in non-progressors. CONCLUSIONS Lenalidomide demonstrates immunomodulatory properties in patients with biochemically recurrent prostate cancer. The induction of novel anti-prostate antibodies is a potential mechanism for lenalidomide response. Changes in serum IL-8 levels may serve as a potential biomarker in treated patients. These hypotheses require formal testing in future prospective trials. Prostate 72:487–498, 2012. © 2011 Wiley Periodicals, Inc.

Few published studies characterize early lymphocyte recovery after intensive chemotherapy for acute myelogenous leukemia (AML). To test the hypothesis that lymphocyte recovery mirrors ontogeny, we characterized early lymphocyte recovery in 20 consecutive patients undergoing induction timed sequential chemotherapy for newly diagnosed AML. Recovering T lymphocytes were predominantly CD4+ and included a greatly expanded population of CD3+CD4+CD25+Foxp3+ T cells. Recovering CD3+CD4+CD25+Foxp3+ T cells were phenotypically activated regulatory T cells and showed suppressive activity on cytokine production in a mixed lymphocyte reaction. Despite an initial burst of thymopoiesis, most recovering regulatory T cells were peripherally derived. Furthermore, regulatory T cells showed marked oligoclonal skewing, suggesting that their peripheral expansion was antigen-driven. Overall, lymphocyte recovery after chemotherapy differs from ontogeny, specifically identifying a peripherally expanded oligoclonal population of activated regulatory T lymphocytes. These differences suggest a stereotyped immunologic recovery shared by patients with newly diagnosed AML after induction timed sequential chemotherapy. Further insight into this oligoclonal regulatory T-cell population will be fundamental toward developing effective immunomodulatory techniques to improve survival for patients with AML.

Initially developed by Edelson in the 1970s, extracorporeal photopheresis (ECP) is a process by which peripheral blood mononuclear cells (PBMC) are isolated from the circulation by discontinuous leukapheresis, exposed ex vivo to pro-apoptotic doses of 8-methoxypsoralen and ultraviolet A radiation, and then reinfused to the patient (1). ECP was approved by the Food and Drug Administration for advanced cutaneous T-cell lymphoma (CTCL) in 1988 (1), and is now utilized for the treatment of various lymphocyte-mediated diseases, including graft-versus-host disease (GvHD) and solid organ transplant rejection (2–4). Mycosis fungoides (MF) and Sezary syndrome (SS), the major variants of CTCL, are malignancies of clonal T lymphocytes that preferentially infiltrate the skin (5–7). Overt blood involvement is uncommon in clinically early MF, but may be encountered in advanced MF and in SS, which is currently defined as an erythrodermic and leukemic expression of CTCL (8). ECP is often used as a first-line therapy for advanced MF with blood involvement and SS, and the initial pivotal study in which two ECP treatments were administered on consecutive days at 4-week intervals demonstrated that ECP monotherapy induced a substantial improvement in 83% of erythrodermic patients, with 21% of patients eventually achieving a complete response (CR) (1, 9). Of note, in responding patients, the mean time to a positive response was 22.4±9.6 weeks, meaning that ECP must be administered for 32 weeks or more before its effectiveness can be properly assessed. Subsequent studies have shown that the overall response rates to ECP range widely from 31% to 86%, depending on patient selection, treatment frequency, and use of adjunct therapies (10, 11). Despite its utilization for more than two decades, the precise mechanism(s) of action by which ECP exerts its therapeutic benefit on CTCL is not clearly understood nor have reliable predictors of response been identified (12–14). Possible and not necessarily mutually exclusive mechanisms of action include: Induction of an anti-clonotypic immune response against the malignant clone (15). Production of immunosuppressive or immunomodulatory cytokines [e.g. TNF-α, interleukin-10 (IL-10)] by treated mononuclear cells with normalization of the Th1/Th2 immune balance (16–20). Induction of regulatory T (Treg) cells that inhibit neoplastic T-cell growth directly or indirectly via their effect on dendritic cells (21–24). Clinical parameters that have been associated with sustained beneficial responses of CTCL to ECP include short disease durations (early treatment) and clinical improvement before 6 months (9, 25, 26). Laboratory parameters that have been reported to be correlated with favorable responses to ECP include (1) near-normal CD4/CD8 ratios or absolute number of CD8+ cells in the peripheral blood (9, 27), (2) the presence of modest numbers of Sezary cells (28), and (3) relatively low percentages of CD4+CD7− T cells, a phenotype that is often expressed by neoplastic T cells (25). The primary goal of this study was to determine whether the cytokine profile in serum samples obtained at baseline and after 3 months of ECP monotherapy could be used as an early predictor of a clinical response to ECP monotherapy. Previously, we reported that baseline levels of soluble interleukin-2 receptor (sIL-2R) and lactate dehydrogenase (LDH) were not useful in this regard (29). However, sIL-2R and LDH levels did correlate with clinical and other laboratory parameters used to evaluate the response to ECP. A second objective was to evaluate the effect of ECP monotherapy on Th1 and Th2 cytokine levels in the serum. It is widely accepted that the neoplastic T cells of most cases of MF/SS are polarized to secrete Th2 cytokines, and that in advanced CTCL, a Th2 immune profile dominates and contributes to immunosuppression, eosinophilia, and increased IgE levels (30, 31). Our hypothesis was that a shift toward Th1 might occur as an early event in patients responding to ECP as reported for clinically early MF (19). The third goal of this study was to correlate usual clinical and laboratory measures of clinical response to ECP with possible molecular ‘tumor-associated markers’ (STAT3, MICB, and T-plastin/PLS3) in patients with blood involvement. Our hypothesis was that response to ECP monotherapy will result in an early decrease in the level of proposed tumor-associated marker message (mRNA) in PBMC samples obtained during the first 3 months of ECP monotherapy. Lastly, we planned to evaluate clinical and laboratory data during the initial 3 months of ECP treatment in order to identify potential predictors of response.

Essentially all squamous cervical cancers and their precursor lesions, high grade cervical intraepithelial neoplasia (CIN2/3), are caused by persistent human papil-lomavirus (HPV) infection. However, not all CIN2/3 lesions progress to cancer. In a brief, observational study window monitoring subjects with CIN2/3 from protocol entry (biopsy diagnosis) to definitive therapy (cervical conization) at week 15, in a cohort of 50 subjects, we found that 26% of CIN2/3 lesions associated with HPV16, the genotype most commonly associated with disease, underwent complete histologic regression. Nonetheless, HPV16-specific T cell responses measured in peripheral blood obtained at the time of study entry and at the time of conization were marginally detectable directly ex vivo, and did not correlate with lesion regression. This finding suggests that, in the setting of natural infection, immune responses which are involved in elimination of cervical dysplastic epithelium are not represented to any great extent in the systemic circulation.

Purpose: Pathologic examination of prostate glands removed from patients with prostate cancer commonly reveals infiltrating CD4+ and CD8+ T cells. Little is known about the phenotype of these cells, despite accumulating evidence suggesting a potential role for chronic inflammation in the etiology of prostate cancer. Experimental Design: We developed a technique that samples the majority of the peripheral prostate through serial needle aspirates. CD4+ prostate-infiltrating lymphocytes (PIL) were isolated using magnetic beads and analyzed for subset skewing using both flow cytometry and quantitative reverse transcription-PCR. The transcriptional profile of fluorescence-activated cell sorted prostate-infiltrating regulatory T cells (CD4+, CD25+, GITR+) was compared with naïve, peripheral blood T cells using microarray analysis. Results: CD4+ PIL showed a paucity of TH2 (interleukin-4–secreting) cells, a surprising finding given the generally accepted association of these cells with chronic, smoldering inflammation. Instead, CD4+ PIL seemed to be skewed towards a regulatory Treg phenotype (FoxP3+) as well as towards the TH17 phenotype (interleukin-17+). We also found that a preponderance of TH17-mediated inflammation was associated with a lower pathologic Gleason score. These protein level data were reflected at the message level, as analyzed by quantitative reverse transcription-PCR. Microarray analysis of pooled prostate-infiltrating Treg revealed expected Treg-associated transcripts (FoxP3, CTLA-4, GITR, LAG-3) as well as a number of unique cell surface markers that may serve as additional Treg markers. Conclusion: Taken together, these data suggest that TH17 and/or Treg CD4+ T cells (rather than TH2 T cells) may be involved in the development or progression of prostate cancer.

The immune system undergoes rapid reconstitution after autologous or syngeneic bone marrow transplantation with the re-establishment of tolerance to self-antigens. Administration of drugs such as cyclosporine that inhibit thymic-dependent clonal deletion disrupts the reconstitution of the immune system. In the absence of a peripheral regulatory T cells eliminated by the preparative regimen, systemic autoimmunity with pathology similar to graft-versus-host disease often develops. Moreover, the resolution of autoaggression is dependent on the reconstitution of CD4+ regulatory T cells. This study examined the specificity and function of this regulatory population assessed ex vivo that plays a critical role in down-regulating the autoreactive T lymphocyte response in cyclosporine-induced syngeneic graft-versus-host disease. The results suggest that both the antigen-specific regulatory and pathogenic effector T cells recognize a common peptide antigen framework (CLIP, a peptide derived from the invariant chain) presented by major histocompatibility complex class II molecules. Analysis of the CD4+ T-cell compartment revealed two subsets of CLIP-reactive T cells that differentially require the N- and C-terminal flanking domain of this peptide. Regulatory function is associated with the cells that require the C-terminal flanking domain. This population expresses the Foxp3 nuclear transcription factor and plays a critical role in re-establishing tolerance to self-major histocompatibility complex class II antigens. In addition to suppressing the production of type 1 cytokines, these regulatory Tcells can direct the apoptotic death of the pathogenic autoreactive lymphocytes. This study also suggests that the development of functional regulatory activity is an active response initiated by the presence of autoreactive lymphocytes that can present the target antigen (major histocompatibility complex class II CLIP) to the regulatory T cells. Moreover, this process can be mimicked by peptide antigen in the absence of the pathogenic effector lymphocytes leading to the development of functional regulatory T-cell activity.

Several clinical trials evaluating the induction of autoimmune graft-versus-host disease (GVHD) after autologous bone marrow transplantation (BMT) as antitumor immunotherapy have shown that autologous GVHD is associated with increased production of interleukin (IL)-10. The induction of autologous GVHD also segregated with single nucleotide polymorphisms in the IL-10 promoter region (IL-10 -592 and IL-10 -1082 ) and with CA repeats in the first intron of the interferon (IFN)-gamma gene. Polymorphisms within these promoter regions can significantly modify the cytokine response because of differential transcription factor efficiency. This study evaluated the relationship between inheritance of polymorphisms within the IL-10 promoter and in the IFN-gamma gene and the overall survival of patients who received autologous BMT for metastatic breast cancer. Peripheral mononuclear cells from 87 women enrolled in 3 autologous BMT (plus induction of autologous GVHD) clinical trials were examined. By using a Cox proportional hazard model, trends in survival after autologous BMT were analyzed. The model included inheritance polymorphisms of IL-10 -592 , IL-10 -1082 , CA repeats within the first intron of the IFN-gamma gene, estrogen and progesterone receptor status, and stage of disease. Increased survival was significantly associated with patients having the IL-10 -592 promoter allele associated with high IL-10 production (hazard ratio, 0.23; 95% confidence interval, 0.09-0.55; P = .001). The effect of the strong IL-10 promoter allele on survival seems to be independent of the development of clinical autologous GVHD. However, decreased survival was significantly associated with patients having CA repeats associated with higher IFN-gamma transcription (hazard ratio, 2.34; 95% confidence interval, 1.21-4.54; P = .011). Inheritance of specific alleles that modify IL-10 and IFN-gamma production may have unexpected effects on the efficacy of immune-based strategies after autologous BMT. Additional studies are necessary to further define the influence of IL-10 and IFN-gamma on the immune response after BMT.

Administration of cyclosporine (CyA) following autologous bone marrow transplantation elicits a T-lymphocyte autoaggression syndrome with remarkable similarity to acute and chronic graft-versus-host disease (GVHD). This syndrome termed syngeneic (S) GVHD is mediated by a restricted repertoire of autoreactive T cells that promiscuously recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain (CLIP). The present studies evaluated and compared antigen-specific autoreactive T cells during acute and chronic SGVHD isolated ex vivo with a soluble MHC class II-immunoglobulin (sMHC class II-Ig) molecular construct. Two major subsets were detected that had overlapping specificity recognizing the MHC class II binding domain of CLIP but were differentially dependent on the N- and C-terminal flanking domains of this peptide. Both subsets were detected in acute and chronic SGVHD. Interestingly, the cytokine profiles of the CLIP-reactive T cells closely correlated with the onset and progression of disease. Levels of type 1 cytokines, particularly IFN-γ mRNA production assessed by quantitative polymerase chain reaction (PCR), were dominant during acute SGVHD, whereas chronic SGVHD was associated with type 2 cytokine mRNA production. Although there was a dramatic polarization with respect to cytokine production, only subtle changes in antigen specificity were detected. Of additional interest, autoreactive T cells producing IL-10 mRNA were detected in both acute and chronic SGVHD, suggesting that this cytokine may play an important but perhaps paradoxical role in both the onset and progression of this experimental autoaggression syndrome.

Graft-versus-host disease (GVHD) is characterized by an impairment of mechanisms that underlie the development of immunologic tolerance. Although the cytokine storm associated with GVHD leads to expression of cell surface markers on both effector and regulatory T cells, regulatory CD4+ T cells that play an instrumental role in the maintenance of tolerance appear to uniquely express the Foxp3 transcriptional repressor. Foxp3 mRNA expression was significantly decreased in peripheral blood mononuclear cells from patients with either allogeneic GVHD or autologous GVHD compared with patients without GVHD. Expression of Foxp3 negatively correlated with the severity of GVHD but positively correlated with recent thymic emigrants. The results suggest that defective thymic function contributes to the impaired reconstitution of immune regulatory mechanisms following transplantation. The decrease in regulatory mechanisms after transplantation appears to provide an environment permissive to the development of GVHD. (Blood. 2004;104:2187-2193)

BACKGROUND Administration of cyclosporine after syngeneic bone marrow transplantation elicits a systemic autoaggression syndrome termed syngeneic graft-versus-host disease (SGVHD). The effector T cells recognize a peptide from the invariant chain termed CLIP (MHC class II invariant chain peptide) presented on MHC class II molecules. Moreover, the N-terminal flanking region of CLIP interacts with the T-cell receptor (TcR) beta chain. METHODS The current study uses a novel approach to isolate and examine the responding T cells ex vivo. A soluble MHC class II molecule using immunoglobulin (Ig)G (MHC class II-Ig) as a scaffold was constructed. The MHC class II-Ig molecule loaded with different peptide variants of CLIP (lacking the N-terminal or C-terminal flanking regions of CLIP) was used to isolate antigen-specific T cells from animals with SGVHD by panning or by flow cytometric sorting. RESULTS Two subsets of antigen specific T cells restricted by the N- and C-terminal flanking regions of CLIP can be isolated during acute SGVHD that express the Vbeta8.5 TcR determinant but secrete different cytokines (interferon [IFN]-gamma, interleukin [IL]-10) as detected by real-time quantitative polymerase chain reaction (PCR). Spectratyping of the complementarity-determining region 3 regions reveals that the N-terminal CLIP reactive population has greater diversity in both the CDR3 and J regions than the C-terminal CLIP reactive subset. Interestingly, the N-terminal CLIP reactive cells can be preferentially detected in the target tissues during acute SGVHD. However, only IFN-gamma messenger (m)RNA was detected in the tissues. CONCLUSION The ability to isolate and examine antigen specific T cells ex vivo has revealed unexpected differences in TcR diversity and cytokine production in SGVHD.

Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992–1004. ©2017 AACR.

Administration of cyclosporine A (CsA) after autologous stem cell transplantation elicits an autoimmune syndrome with pathology similar to graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with the appearance of autoreactive T cells directed at major histocompatibility class (MHC) class II antigens. In the rat model of autologous GVHD, clonal analysis reveals that the effector T cells are highly conserved and recognize a peptide from the invariant chain peptide presented by MHC class II. Although human autologous GVHD effector T cells share a similar phenotypic specificity, clonality of the response in humans has not been determined. To examine the human effector T-cell response, the T-cell repertoire of peripheral blood lymphocytes was assessed by complementarity-determining region 3 (CDR3) size distribution analysis and T-cell clonotype analysis in 26 patients treated with CsA after transplantation. Autologous GVHD developed in 3 of 4 patients with human leukocyte antigen (HLA)-DRB1*0701, and clonal expansions of β-chain variable region (BV)16+ T cells were shared. Clonal expansions within BV15+ and BV22+ T cells were also detected in 4 of 6 patients with HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively. Sequencing of BV16 cDNA for which the CDR3 size pattern exhibited apparent clone predominance revealed an identical CDR3 peptide sequence in 2 different patients, one with HLA-DRB1*0701 and the other with HLA-DRB1*1502. These findings indicate that the discrete antigen-driven expansion of T cells is involved in autologous GVHD.

Administration of the immunosuppressive drug cyclosporine after syngeneic or autologous bone marrow transplanta- tion elicits a T-lymphocyte-dependent autoimmune syndrome similar to graft-versus-host disease (GVHD). The onset of this autoaggression syndrome, termed syngeneic GVHD, is associated with the development of a highly restricted re p e rt o i re of CD8 + a u t o reactive T cells that recognize a peptide from the invariant chain, termed CLIP, p resented by major histocompatibility complex (MHC) class II molecules. Clonal analysis reveals 2 distinct subsets of a u t o reactive T cells defined by their activation re q u i rement for either the N-terminal or the C-terminal fla n k i n g regions of CLIP and by their cytokine pro file. The studies here reveal that the autoreactive T-cell clones re q u i r i n g the N-terminal flanking region of CLIP produce type 1 cytokines (interf e ron (IFN)-γ , interleukin (IL)-2, and tumor n e c rosis factor-α). In contrast, the autoreactive T-cell clones that re q u i re the C-terminal flanking region of CLIP p roduce type 2 cytokines (IL-4, IL-10, transforming growth factor-β). As assessed in a local graft-versus-host re a c- tion assay, the N-terminal fla n k i n g - restricted clones mediate changes consistent with acute GVHD, whereas the clones responsive to the C-terminal flanking region do not. More o v e r, the autoreactive T-cell clones restricted by the C - t e rminal flanking region of CLIP ameliorate the pathogenic potential of the cells responsive to the N-term i n a l flanking region of CLIP. The mechanism accounting for this re g u l a t o ry affect appears to be the downregulation of mRNA message for type 1 cytokines (IFN-γ and IL-2). The C-term i n a l - restricted autoreactive T-cell clones, how- e v e r, could manifest disease with dermal changes similar to those seen in chronic syngeneic GVHD, provided that I F N -γ was present. Consistent with these observations was the demonstration that type 1 cytokines are pre f e re n t i a l l y detected during the acute phase of syngeneic GVHD, whereas type 2 cytokines dominate during the chronic phase. The results suggest that acute and chronic syngeneic GVHD is mediated by distinct autoreactive T cells, which are separated by their fine specificity for the CLIP-MHC class II complex and by their cytokine pro fil e s .

Background. Administration of the immunosuppressive drug cyclosporine after syngeneic/autologous bone marrow transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous or syngeneic GVHD, is associated with the development of a highly restricted repertoire of cytolytic T lymphocytes that promiscuously recognizes major histocompatibility complex class II determinants, including self. Methods. Vβ8.5+CD8+ effector lymphocytes and T-cell clones were isolated from Lewis rats with cylosporine-induced syngeneic GVHD. The specificity of the effector T cells and T-cell clones was examined in vitro. The pathogenicity of the T-cell clones was confirmed in vivo using a local graft-versus-host reaction assay. Results. Clonal analysis reveals that the pathogenic effector T cells recognize a peptide from the invariant chain termed CLIP in association with major histocompatibility complex class II determinants. Moreover, there appears to be an additional interaction between the N-terminal flanking region of CLIP and the Vβ segment of the T cell receptor. Conclusion. The results suggest that recognition of this highly conserved peptide along with the additional interaction between the flanking region and the T cell receptor may account for the promiscuous activity of the autologous/syngeneic GVHD autoreactive T cells.

Administration of the immunosuppressive drug cyclosporine (CsA) after syngeneic/autologous bone marrow transplantation (BMT) elicits an autoimmune syndrome with pathology virtually identical to graft-vs-host disease (GVHD). The induction of this syndrome, termed syngeneic/autologous GVHD, is a two-tiered process requiring both the active inhibition of thymic-dependent clonal deletion and the elimination of mature T cells that have an immunoregulatory effect. Eradication of the peripheral immunoregulatory compartment by the preparative regimen provides a permissive environment for the activation of the syngeneic/autologous GVHD effector T cells. Although the repertoire of autoreactive effector T lymphocytes is highly conserved, these T cells promiscuously recognize MHC class II determinants. This novel specificity of the autoreactive lymphocytes appears to be dependent on the peptide derived from the MHC class II invariant chain. Recent studies also suggest that these promiscuous autoreactive T cells can effectively target and eliminate MHC class II-expressing tumor cells. Administration of cytokines that upregulate the target antigen or expand the effector population can potentiate the antitumor activity of syngeneic/autologous GVHD. Although the induction of syngeneic/autologous GVHD is an untoward effect of CsA immunosuppression, mobilization of these autoimmune mechanisms provides a promising immunotherapeutic approach for certain neoplastic diseases.

Administration of the immunosuppressive drug cyclosporine after autologous bone marrow transplantation induces a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome termed autologous GVHD has significant antitumor activity. Associated with autologous GVHD is the development of T lymphocytes that recognize major histocompatibility complex (MHC) class II determinants, including self. The present studies attempted to characterize and define the molecular specificity of the effector T lymphocytes in autologous GVHD induced in patients with metastatic breast cancer. The results suggest that the effector cells associated with human autologous GVHD are CD8+ T lymphocytes expressing the α/β T-cell receptor. Additional studies show that the effector T cells recognize MHC class II antigens in association with a peptide from the invariant chain (CLIP). Pretreatment of autologous lymphoblast target cells with anti-CLIP antibody completely blocked lysis mediated by autologous GVHD effector T cells. On the other hand, force loading this peptide markedly enhanced the susceptibility of the target cells to recognition by the autoreactive T cells. The recognition of the MHC class II CLIP complex may account for the novel specificity of the effector T cells associated with human autologous GVHD. Moreover, identification of the target peptide may allow for the development of novel immunotherapeutic strategies to enhance the antitumor efficacy of autologous GVHD.

Cancer Immunosurveillance Gone Bad? A subset of patients who develop scleroderma, a debilitating autoimmune disease, have an elevated risk of developing cancer. These patients harbor autoantibodies to RPC1, an RNA polymerase subunit encoded by the POLR3A gene. Joseph et al. (p. 152 , published online December 5; see the Perspective by Teng and Smyth ) explored whether the RPC1 autoantibodies target a “foreign” antigen derived from a mutated POLR3A gene. Sequence analysis revealed that POLR3A mutations were present in tumors from six of eight patients with RPC1 autoantibodies but in no tumors from eight control patients who lacked RPC1 autoantibodies. Cell culture data suggested that the POLR3A mutations triggered cellular and humoral immune responses in the patients. These results provide support for the “immunosurveillance” hypothesis, which posits the continual eradication of nascent tumor cells via immune responses.

High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4(+) T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4(+) T cells with relative preservation of memory CD4(+)Foxp3(+) T cells. In particular, CD4(+)CD45RA(-)Foxp3(+hi) effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4(+)Foxp3(+) T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4(+) T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy's GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD.

In this study, we compared the traditional murine model with renal pedicle clamp with models that clamped the renal artery or vein alone as well as to a whole body ischemia-reperfusion injury (WBIRI) model. Male C57BL/6J mice underwent either clamping of the renal artery, vein, or both (whole pedicle) for 30 or 45 min followed by reperfusion, or 10 min of cardiac arrest followed by resuscitation up to 24 h. After 30 min of ischemia, the mice with renal vein clamping showed the mostly increased serum creatinine and the most severe renal tubule injury. After 45 min of ischemia, all mice with renal vasculature clamping had a comparable increase in serum creatinine but the renal tubule injury was most severe in renal artery-clamped mice. Renal arterial blood flow was most decreased in mice with a renal vein clamp compared with a renal artery or pedicle clamp. A 30-or 45-min renal ischemia time led to a significant increase in the protein level of interleukin-6, keratinocyte-derived chemokine (KC), and granular colony-stimulating factor in the ischemic kidney, but the KC was the highest in the renal pedicle-clamped kidney and the lowest in the renal vein-clamped kidney. Of note, 10 min of WBIRI led to kidney dysfunction and structural injury, although less than longer time clamping of isolated renal vasculature. Our data demonstrate important differences in ischemic AKI models. Understanding these differences is important in designing future experimental studies in mice as well as clinical trials in humans.

We retrospectively explored changes in immunological parameters in men with biochemically recurrent prostate cancer treated with either 5 or 25 mg of lenalidomide in a randomized phase 2 trial, and determined whether those changes correlated with disease progression.Cytokine levels were compared for each patient at baseline and after 6 months of treatment with lenalidomide. Regression models for correlated data were used to assess associations of cytokine levels with lenalidomide treatment effect. Estimates were obtained using generalized estimating equations. Changes in circulating anti-prostate antibodies were evaluated using a high-throughput immunoblot technique.Treatment with lenalidomide was associated with global changes in immunoreactivity to a number of prostate-associated antigens, as well as with changes in circulating levels of the T(H) 2 cytokines IL-4, IL-5, IL-10, and IL-13. Disease progression in treated patients was associated with an increase in circulating IL-8 levels, while IL-8 levels decreased significantly in non-progressors.Lenalidomide demonstrates immunomodulatory properties in patients with biochemically recurrent prostate cancer. The induction of novel anti-prostate antibodies is a potential mechanism for lenalidomide response. Changes in serum IL-8 levels may serve as a potential biomarker in treated patients. These hypotheses require formal testing in future prospective trials.

Graft-versus-host disease is the leading cause of non-relapse mortality after allogeneic bone marrow transplantation. The cell-mediated immune mechanisms that underlie the pathogenesis of graft-versus-host disease remain unclear. In this study, 47 skin biopsies representing graft-versus-host disease grades 0-III, lichenoid, and sclerodermoid were included from 31 allogeneic bone marrow transplantation recipients. RNA from paraffin-embedded tissue was harvested. Transcript levels of the following markers were assessed and correlated with grade and survival: CD3, CD20, FoxP3, IL-17, gamma-interferon (IFN-gamma), transforming growth factor-beta (TGF-beta), IL-6, connective tissue growth factor (CTGF), allograft inflammatory factor-1(AIF-1), and IL-13. Levels of three markers significantly correlated with the length of survival (TGF-beta, correlation coefficient -20.8, P=0.016; AIF-1, 13.2, P=0.016; and CD20, 66, P=0.027). CD20 expression was limited to lichenoid cases. Levels of TGF-beta, AIF-1, and IFN-gamma appeared to correlate with histological progression, but did not reach statistical significance. Expression of FoxP3 correlated with worse survival, and approached statistical significance (P=0.053). Two potential mechanistic pathways were identified: the 'scleroderma' group (AIF-1 and TGF-beta) and the 'B-cell' group (CD20). Transcript levels of these markers were implicated in the progression from acute to chronic disease, and also correlated significantly with the duration of survival. Identification of these three markers may direct therapy selection with targeted agents, including the use of rituximab when B-lymphocytes are implicated.

BackgroundOvarian carcinoma is the fourth most common cause of death from cancer in women. Limited progress has been made toward improving the survival rate of patients with this disease in part because of the lack of a good animal model. We present here a model of spontaneous ovarian carcinoma arising in a normal Lewis rat.MethodsA spontaneously occurring tumor of the left ovary was found in a normal Lewis rat during necropsy, which was sectioned for histological examination and placed into single cell suspension. Tumor cells were passagedin vivoby intraperitoneal injection into immunocompetent Lewis rats, andin vitroculture resulted in generation of a cell line. Tumor cells were examined by flow cytometry for expression of estrogen receptor α, progesterone receptor, androgen receptor, her-2/neu, epithelial cell adhesion molecule, and CA125. β-catenin expression and cellular localization was assessed by immunocytochemistry. RNA was harvested for gene expression profiling and studying the expression of cytokines.ResultsThe tumor, designated FNAR, could be serially transplanted into Lewis rats and propagated as a cell linein vitro, maintaining the properties of the original tumor. The FNAR cells displayed striking morphologic similarities to human ovarian carcinoma, resembling the endometrioid carcinoma subtype of surface epithelial neoplasms. The cells expressed estrogen receptor α, progesterone receptor, androgen receptor, her-2/neu, epithelial cell adhesion molecule, CA125, and nuclear β-catenin. A gene expression profile showed upregulation of a number of genes that are also upregulated in human ovarian carcinoma.ConclusionThis reliable model of ovarian carcinoma should be helpful in better understanding the biology of the disease as well as the development of novel treatment strategies.

Peripheral blood cells from 28 patients with leukemic cutaneous T-cell lymphoma including 25 patients with Sezary syndrome were evaluated for expression of regulatory T-cell-associated markers (FoxP3, CD25, CTLA-4, neurophilin-1), T-cell activation markers (CD28 and its ligands B7.1 and B7.2) and NK cell-associated markers (NKG2D and its ligands Mic-A and Mic-B) using real-time quantitative polymerase chain reaction. T-plastin served as a positive genetic marker, and its expression correlated to blood tumor burden. More than 90% of samples had transcripts for CD28 and Mic-B, but less than 30% of samples expressed FoxP3, CTLA-4 and CD25. Expression of Mic-B by neoplastic cells could provide another mechanism to inhibit anti-tumor immune responses. FoxP3 expression correlated with a poor prognosis. Although the underlying mechanisms accounting for this correlation remain unclear, the expression of the Foxp3 and CTLA-4 regulatory elements indicates that a subset of leukemic cases displays a regulatory T-cell phenotype.

OX40 (CD134), a member of the tumor necrosis factor receptor superfamily, is expressed on activated T cells, including CD4 + CD25 + T regulatory (Treg) cells. To investigate the kinetics of OX40–OX40L in graft-versus-host disease (GVHD), OX40 mRNA transcript levels were temporally examined in peripheral blood mononuclear cells (PBMCs) from patients undergoing either allogeneic (allo) bone marrow transplantation (alloBMT) or autologous (auto) BMT with the induction of autoGVHD by cyclosporine (CsA) treatment posttransplant. Real-time quantitative PCR analysis revealed that OX40 mRNA expression decreased significantly in PBMCs from patients with either alloGVHD or autoGVHD compared with healthy individuals. No differences were detected between patients developing alloGVHD and those who did not develop this posttransplant complication. On the other hand, a decrease in OX40 mRNA levels correlated directly with the development of autoGVHD. Moreover, the upregulation of OX40 gene expression coincided with the resolution of autoGVHD. Interestingly, expression of OX40 by CD4 + T lymphocytes after stimulation with autoantigen (Ag) was significantly (>700-fold) increased with a concomitant increase in expression of the Foxp3 regulatory gene. Expression of OX40 was increased (maximum 11-fold) after allo-Ag via mixed-lymphocyte reaction response. CsA suppressed the upregulation of OX40 expression after allo-Ag in a dose-dependent manner. Taken together, these results suggest that the decrease in OX40 expression posttransplant includes the defective reconstitution of Treg cells, and the active inhibition of gene transcription by CsA.

The N- and C-terminal flanking domains of the invariant chain peptide, CLIP, have remarkable immunological properties. Addition of these flanking domains to a foreign peptide antigen increases its immunologic potency. The present studies evaluated whether altering a peptide ligand from the tumor-associated antigen c-neu with the flanking domains of CLIP could modify the systemic immune response. The results indicate that the immunogenicity of an MHC class II restricted peptide (NEU) derived from c-neu was significantly altered by addition of the flanking domains from CLIP. Interestingly, selective modification of the peptide with either the N- or the C-terminal flanking domains resulted in functionally divergent systemic immune responses. Immunization of normal F344 rats with the NEU peptide modified with the N-terminal domain of CLIP (N-NEU) resulted in an immune response primarily consisting of type 1 (IL-2, IFNgamma) cytokine producing T cells. On the other hand, type 2 (IL-4) cytokine responses were largely predominant following immunization with the self-peptide modified with the C-terminal flanking domain (NEU-C). The functionally divergent responses elicited by the modified self-peptides were accompanied by significant changes in the expression of the CD28/CTLA4/B7 family of co-stimulatory molecules. Immunization with the N-NEU peptide led to enhanced expression of CD28 in the antigen-specific, CD4+ T cell compartment while expression of B7.1 was dramatically reduced in antigen-specific CD8+ T cells. Comparatively, expression of CTLA4 was down-regulated in the antigen-specific CD4+ T cell compartment following immunization with NEU-C peptide. The N-NEU peptide also had a direct effect on dendritic cells leading to the up-regulation of B7.1 expression. Taken together, functionally divergent systemic immune responses can be elicited by strategically altering a self-peptide ligand with the N- and C-terminal flanking domains of CLIP. Moreover, changes in expression of co-stimulatory molecules that are required for T cell activation and T cell-T cell communication may account for the polarization of the immune response elicited by the chimeric peptides.

Administration of cyclosporine after autologous bone marrow transplantation elicits a T-lymphocyte autoaggression syndrome with remarkable similarity to graft-versus-host disease (GVHD). This syndrome, termed syngeneic GVHD (SGVHD), with both acute and chronic phases, is mediated by a restricted repertoire of autoreactive T cells that promiscuously recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain (MHC class II-invariant chain peptide; CLIP). This study evaluated and compared antigen-specific autoreactive T cells during acute and chronic SGVHD ex vivo isolated with a soluble MHC class II immunoglobulin molecular construct. This approach allows for the direct assessment of the functional behavior of the effector T cells without potential modification by in vitro culture. Two major subsets were detected that had overlapping specificity recognizing the MHC class II-binding domain of CLIP but that were differentially dependent on the N- and C-terminal flanking domains of this peptide. The N- and C-terminal subsets were primarily associated with acute and chronic SGVHD, respectively. The cytokine profiles of the CLIP-reactive T cells, however, were most informative and closely correlated with the onset and progression of disease. Levels of type 1 cytokine, particularly interferon-γ, messenger RNA (mRNA) production, assessed by quantitative polymerase chain reaction, were dominant during acute SGVHD, whereas chronic SGVHD was associated with type 2 cytokine mRNA production. Although there was a dramatic polarization with respect to cytokine production, only subtle changes in antigen specificity could be detected. Unexpectedly, the functional behavior within the antigen-specific effector cell populations is not fixed and seems to change as the disease progresses to the chronic phase. Concordant with the evolution of the effector T-cell response is a differential loss in B7.1 mRNA expression in the N-terminal CLIP-reactive T-cell subset that may reflect the regulation of this autoimmune response. Of additional interest, autoreactive T cells producing interleukin-10 mRNA were detected in both acute and chronic SGVHD, suggesting that this cytokine may play an important but perhaps paradoxical role in both the onset and progression of this experimental autoaggression syndrome.

Administration of cyclosporine after syngeneic bone marrow transplantation elicits a systemic autoaggression syndrome termed syngeneic graft-versus-host disease (SGVHD). The effector T cells recognize a peptide from the invariant chain termed CLIP (MHC class II invariant chain peptide) presented on MHC class II molecules. Moreover, the N-terminal flanking region of CLIP interacts with the T-cell receptor (TcR) beta chain.The current study uses a novel approach to isolate and examine the responding T cells ex vivo. A soluble MHC class II molecule using immunoglobulin (Ig)G (MHC class II-Ig) as a scaffold was constructed. The MHC class II-Ig molecule loaded with different peptide variants of CLIP (lacking the N-terminal or C-terminal flanking regions of CLIP) was used to isolate antigen-specific T cells from animals with SGVHD by panning or by flow cytometric sorting.Two subsets of antigen specific T cells restricted by the N- and C-terminal flanking regions of CLIP can be isolated during acute SGVHD that express the Vbeta8.5 TcR determinant but secrete different cytokines (interferon [IFN]-gamma, interleukin [IL]-10) as detected by real-time quantitative polymerase chain reaction (PCR). Spectratyping of the complementarity-determining region 3 regions reveals that the N-terminal CLIP reactive population has greater diversity in both the CDR3 and J regions than the C-terminal CLIP reactive subset. Interestingly, the N-terminal CLIP reactive cells can be preferentially detected in the target tissues during acute SGVHD. However, only IFN-gamma messenger (m)RNA was detected in the tissues.The ability to isolate and examine antigen specific T cells ex vivo has revealed unexpected differences in TcR diversity and cytokine production in SGVHD.

Administration of the immunosuppressive drug cyclosporine A (CsA) following autologous stem cell transplantation paradoxically elicits a systemic autoimmune syndrome resembling graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with autoreactive CD8(+) T cells that recognize major histocompatibility complex (MHC) class II determinants in association with a peptide from the invariant chain. To investigate the potential role of cytokines and chemokines in autologous GVHD, interleukin 2 (IL-2), IL-4, IL-10, interferon gamma (IFN-gamma), and macrophage inflammatory protein-1alpha (MIP-1alpha) gene expression in peripheral blood mononuclear cells (PBMCs) was determined in 36 patients treated with CsA following transplantation and correlated with the induction of cytolytic activity against autologous phytohemagglutinin-stimulated lymphocytes (PHA-blasts) and the breast cancer cell line (T47D). The determination of gene expression by real-time polymerase chain reaction (PCR) revealed that IL-10 mRNA levels by PBMCs in patients with autologous GVHD were 29-fold higher than in healthy individuals. IFN-gamma (4-fold), IL-2 (3-fold), and MIP-1alpha (44-fold) mRNA levels were also increased in GVHD-induced patients compared with healthy individuals. The ability of PBMCs to lyse autologous PHA-blasts and T47D tumor cells exhibited an identical temporal relationship with expression of IL-10 and IFN-gamma during autologous GVHD. Moreover, the susceptibility to autologous GVHD as assessed in 75 patients was significantly associated with the IL-10(-1082) G/G polymorphic alleles, allelic variants in the promoter region that govern IL-10 production. These findings indicate that IL-10 may play an unexpected but critical role in autologous GVHD and could be utilized to enhance a graft-versus-tumor effect after transplantation. Interestingly, polymorphisms in the IL-10 promoter region may also explain differences in the susceptibility of patients to autologous GVHD induction.

The use of the immunosuppressive drug cyclosporine A (CsA) in solid organ transplantation can be associated with the development of vasculopathy as part of the complex immune response involved in chronic rejection, including autoimmune recognition. Although CsA can directly affect endothelial cells, this drug alters the T cell repertoire promoting autoimmune recognition. The present studies evaluated the ability of CsA-induced autoreactive T cells to mediate vascular lesions in syngeneic heart grafts. Graft vasculopathy developed in syngeneic heart grafts following either the primary induction of autoimmunity with CsA or the adoptive transfer of CsA-induced autoreactive T cells. Initially, an inflammatory response occurred in the medial wall of the small arterial vessels, accompanied by a perivascular lymphocytic infiltrate (including a lymphocytic infiltrate into the myocardium), followed by progression of vascular disease with endothelial cell proliferation. The development and progression of vascular disease correlated with the cytokine profile of the infiltrating lymphocytes with type 1 cytokines detected early and type 2 cytokines detected as the disease progressed. Initiation of this response correlated with upregulation of the target antigen recognized by the CsA-induced autoreactive T cells, the MHC class II-invariant chain peptide complex. This antigen complex, when upregulated on endothelial cells by interferon, allowed effective targeting by the autoreactive T lymphocytes. Strategies to inhibit the upregulation of MHC class II antigens by treatment of the recipients with chloroquine truncated the disease process. The results of these studies suggest that CsA-induced autoreactive mechanisms can contribute to the development of graft vasculopathy.

Background Myelodysplatic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies, characterized by the ineffective hematopoiesis, and risk of progression to acute myeloid leukemia. Allogeneic stem cell transplant (SCT) remains the only potentially curative therapy, but toxicities limit its application in older adults. Vaccination strategies have been developed to modulate the immune system, ideally with less toxicity. We present results from a pilot study of single agent K562/GM-CSF whole cell vaccination in MDS patients (pts). Methods Poor-risk or transfusion-dependent MDS pts (n=5) were enrolled and received K562/GM-CSF whole cell vaccine (1 X 108 cells) every 3 weeks for 4 cycles followed by a booster vaccination 8 weeks later. Eligible pts could receive supportive care only for the 2 months preceding study entry, have no history of SCT, and not be on immunosuppressants. Blood, bone marrow, and skin biopsies were taken prior to vaccination, day 4 following first vaccine, and at 4 weeks after the booster vaccination. Diversity of the T cell compartment was assessed in the blood, marrow and skin using the TCRExpress Quantitative Analysis Kit (Biomed Immunotech, Tampa FL). Fragment length analysis was performed by the DNA Analysis Facility. In vitro stimulation studies for proliferation and cytokine production were performed using peripheral blood monocytes (PBMCs) for the pt with the best clinical response (>4 years). Proliferation of banked PBMCs, collected prior to every vaccination and stimulated with the vaccine, was assessed using 3H-Thymidine. The cytokines generated were measured using a multiplexed bead based immunoassay (Human 17-plex bioplex pro-panel) (Biorad, Hercules CA). Results All pts tolerated the vaccinations well with localized skin reactions being common. Clinically, 2 pts normalized their blood counts and became transfusion independent following the immunotherapy. One responding pt (CMML) remained stable without need for medical intervention for 4 yrs and only recently showed progression. T cell spectratyping indicated oligocolonal populations of T cells in post-vaccination skin, blood and marrow samples. T cells infiltrating the skin were tracked to the marrow. Interestingly, the best clinical responder demonstrated the most restricted skewed repertoire with a significant number of oligoclonal T cells tracking from skin to marrow (n=5). The marrow also had infiltrates of oligoclonal T cells not detected in the post-vaccination skin. Further, this skewed repertoire was absent when the pt relapsed. Proliferation assays measuring this pt’s T cell cytokine production in response to the vaccine cells in vitro, showed increased levels of IL-2, IL-13 and IL-5 that were suppressed or not produced by the time of the 4th vaccination. Inversely, IL-6, IL-10, IL-17, IL-1beta, MIP-1beta and TNF alpha levels increased throughout. The expected proliferative “boost” was seen with the initiation of the booster vaccine series at the time of progression, and co-culture of the pt’s lymphocytes with the vaccine cells suppressed the ability of the vaccine cells to produce GM-CSF in vitro. The ability to suppress GM-CSF production decreased during therapy and the pt’s lymphocytes had no effect on GM-CSF production by the vaccine at the end of the immunotherapy. Conclusions All pts showed T cell skewing by spectratyping analysis, suggesting that each had a change in T cell proliferation patterns in response to the vaccine. One pt had a significant clinical response and the most specific T cell response by spectratyping to the original vaccine, followed by the absence of these cells in the marrow at the time of progression. This suggests that an immune response may have stabilized his disease and progression was associated with loss of this T cell population. Proliferation studies suggest that the lymphocytes recognized the vaccine. Lastly, GM-CSF levels produced by the vaccine were decreased during the vaccination cycles suggesting that the pt’s lymphocytes and/or tumor had a suppressive effect on the vaccine cells. It is unclear if the GM-CSF suppression was essential, detrimental, or unrelated to the pt’s clinical response. Further study of the T cells patterns in these pts may elucidate details of the immune response that are integral to clinical responses. Disclosures: No relevant conflicts of interest to declare.

Abstract 3426 Background: Allergic transfusion reactions (ATRs) occur in ∼2–3% of platelet transfusions, but their etiology remains largely unclear. Both donor and recipient factors have been shown to contribute to ATR risk. An in vitro model of ATRs would enable mechanistic studies. Objective: To develop a basophil activation test as a model of ATRs to apheresis platelets (AP) by measuring histamine release (HR) and leukotriene C4 (LTC4) production after exposing basophils to AP supernatants. Methods: Basophil-enriched suspensions (∼5–15% basophils) from healthy donors were prepared using double-Percoll density centrifugation. After washing and counting basophil numbers using Alcian blue staining, cells were resuspended in buffer containing 2 ng/mL IL-3 and 5mM Ca++/Mg++ such that 20–30×104 basophils were added in 0.025mL aliquots. IL-3 is a potent basophil priming agent and was used to model the increased recipient susceptibility to allergic reactions that has been observed clinically. Reaction tubes (1.5 mL) were set-up containing 0.025mL of 2× (twice the final conc.) of ATR/control supernatant (final titer 1:2–1:8), negative (buffer alone), and positive control reagents (100 ng/mL anti-IgE antibody, 50 ng/mL C5a, 1 μM FMLP). Cells and reaction tubes were pre-warmed separately to 37°C for 15' before aliquoting 0.025mL cell suspension to reaction tubes for a final volume of 0.050mL. After 30′ incubation at 37°C, 0.950mL of cold buffer (without IL-3 or Ca++/Mg++) was added to each reaction tube. Tubes were centrifuged 10" at ∼300g (4°C) before removing 0.900mL for histamine detection by automated fluorimetry and LTC4 detection by ELISA. AP supernatants that were (n=10) or were not (n=10) associated with ATRs were tested. HR values were determined as a percentage of the total histamine content by lysis with perchloric acid (1.6%). Results used in analysis are net HR or LTC4 production (AP sample minus IL-3 primed negative control) normalized to HR or LTC4 production observed with positive control. Results: IL-3 priming of basophils was necessary in order to evoke a response after exposure to AP supernatant. HR in response to AP incubation with unprimed basophils was HR and LTC4 production within individual reactions were correlated (Spearman rho=0.83, P No statistically significant differences were observed in HR or LTC4 production when ATR and control groups were compared (P>0.1). ATRs were stratified into clinical categories of mild (pruritus/urticaria, n=3), moderate (angioedema/dyspnea, n=5), and severe (any symptom with hypotension, n=2). There were no differences or trends in HR and LTC4 production among the categories (P>0.2). There was no correlation between pre/post transfusion tryptase elevations in recipient plasma (n=4) and HR or LTC4 production. HR and LTC4 production was similar among blood types of AP products (A: n=12, O: n=5, AB: n=3), (P>0.7). We previously reported that concentrations of C5a are higher in AP products associated with ATRs than controls. C5a content in AP supernatant modestly correlated with basophil HR release (rho: 0.47, P=0.04), but less with LTC4 production (rho: 0.27, P=0.3). Summary: An in vitro basophil activation test that measures HR and LTC4 production was developed to study ATRs. We find that 1) IL-3 priming of basophils was necessary to elicit responses to AP supernatants; 2) LTC4 and HR are correlated within each reaction; 3) there are no differences in HR and LTC4 production between ATR and control AP supernatants were observed, regardless of clinical ATR severity; and 4) HR is associated with C5a content in the AP supernatant. Conclusions: The observations of 1) an overall requirement for basophil priming and 2) the lack of correlation between the clinical severity of ATRs and basophil HR and LTC4 release implicate recipient atopic priming as a risk factor for ATRs. However, the association of AP C5a content with HR suggests a donor/product role in ATRs, as well. Studies focusing on the nature of recipient susceptibility may further elucidate mechanisms of ATRs. Disclosures: No relevant conflicts of interest to declare.

Abstract 1009 Delayed immune reconstitution with increased risk of opportunistic infection is a major complication of HLA-haploidentical stem cell transplantation, especially in protocols employing extensive T cell depletion of the graft. Previous studies at our institution with high-dose, post-transplantation Cy (PT/Cy) have reported low rates of non-relapse mortality and serious opportunistic infections. Here we characterize immune reconstitution in fifty-three consecutive hematologic malignancies patients receiving nonmyeloablative conditioning, T cell-replete, HLA-haploidentical bone marrow transplantation (BMT), and graft versus host disease prophylaxis including PT/Cy. Patients with advanced hematologic malignancies (median age 51, range 14–71; 5 AML, 2 ALL, 4 MDS, 2 CML, 4 CLL, 1 CMML, 25 NHL, 7 Hodgkins, 3 mantle cell) received Cy 14.5 mg/kg/day IV on days −6 and −5, fludarabine 30 mg/m2/day IV on days −6 to −2, 200 cGy of TBI on day -1 and T cell replete bone marrow from donors with a median age of 44 (range 14–68). GVHD prophylaxis consisted of Cy (50 mg/kg/day) on days 3 and 4, mycophenolate mofetil for 30 days, and tacrolimus for 6 months. Grafts contained an infused median TNC/kg of 4.1 e8 (range 2.6–6.6 e8), CD3+/kg 3.6 e7 (range 1.7–6.7e7) and a CD34+/kg of 3.5e6 (range 1.4–7.0e6). Sustained engraftment of donor cells occurred in 86% of evaluable patients (44/51).The median times to neutrophil (>500/μL) and platelet recovery (>20,000/μL) were 17 days (range, 13–92 days) and 28 days (range, 13–580 days), respectively. Post-transplantation recovery of lymphocyte subsets is shown in Table 1 and Figure 1 and is notable for the following: 1) The median lymphocyte count at day 30 after transplantation is >180/ml and recovers to over 800/ml by day 60; 2) CD4+ T cell counts recover to a median >120/ml by day 60 and >220/ml by day 180 after transplantation; and 3) recovery of CD31+ recent thymic emigrants and CD45RA+ naïve T cells is delayed compared to recovery of memory T cells. T cell receptor spectratyping analysis on a subset of 10 patient/donor pairs chosen specifically for having no relapse/no GVHD (n=4), GVHD and no relapse (n=3), or late relapse (n=3) revealed that patients without relapse, GVHD, or recent viral infection had excellent reconstitution of the T cell repertoire to the level of the pre-transplant donor, as early as 6 months post-transplant (Figure 2). CMV specific T cell response using ELISPOT measured on a subset of 17 patients whose donors were reactive to CMV, revealed that donor-derived immunity to CMV returns by Day 60 in about 70% of patients (12/17) (Figure 3). In conclusion, immune reconstitution after non-myeloablative haploidentical T cell replete BMT with PT/Cy compares favorably with other reduced intensity conditioning alternative donor regimens and suggests that PT/Cy selectively preserves pathogen-specific memory T cells necessary to protect against infection. Further correlations of immune reconstitution with specific infectious and overall outcomes are being analyzed.Figure 1T-, B-, and NK-cell ReconstitutionFigure 1. T-, B-, and NK-cell ReconstitutionFigure 2T cell receptor spectratypingFigure 2. T cell receptor spectratypingFigure 3CMV-specific T cell frequencyFigure 3. CMV-specific T cell frequencyTable 1.Post-transplantation Lymphocyte Subset RecoveryMedian (cells/μL) (N)Interquartile range (cells/μL)ALCDonor1765 (46)1480–2100Recipient pre-BMT840 (45)425–1295Day 30184 (49)54–402Day 60820 (38)470–1260Day 180915 (34)670–1560Day 3653060 (22)820–2030CD3+CD4+CD45RA+ (naïve)Donor119 (33)82–189Recipient pre-BMT22 (34)4–38Day 300.33 (35)0.07–1Day 603 (29)1–9Day 18011 (23)5–31Day 36523 (13)13–92CD3+CD4+CD45RA−CCR7+ (central memory)Donor135 (33)95–158Recipient pre-BMT54 (34)11–79Day 302 (35)0.5–11Day 6034 (29)10–79Day 18061 (23)35–117Day 36589 (13)60–122CD3+CD4+CD45RA−CCR7− (effector memory)Donor187 (33)130–245Recipient pre-BMT87 (34)14–134Day 303 (35)1–18Day 6059 (29)14–122Day 180102 (23)43–179Day 365142 (12)64–204CD3+CD4+CD45RA+CD31+ (recent thymic emigrants)Donor61 (33)30–97Recipient pre-BMT6 (34)1–16Day 300.9 (35)0.03–0.4Day 601 (29)0.5–2Day 1804 (23)1–11Day 3657 (13)2–12CD3+CD4+Foxp3+Donor28 (33)23–35Recipient pre-BMT13 (34)6–24Day 301 (35)0.1–5Day 608 (29)4–16Day 18013 (23)7–25Day 36514 (13)7–17ALC, absolute lymphocyte count; WBC, white blood cell count; Treg, regulatory T cell Disclosures: Jones: Aldagen: Patents & Royalties.

Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH).Primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin-V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune attack that can confound experiments using primary cells, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 cells (an interleukin-2-dependent cell line with the phenotype and function of activated natural killer [NK] cells), tumor necrosis factor (TNF)-alpha, and gamma-irradiation. Apoptosis was measured by annexin-V staining and caspase 3/7 activity.In PNH patients, CD34+ hematopoietic progenitors lacking glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP(-)) were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% vs 49%; p0.05) and allogeneic (28% vs 58%; p0.05) cell-mediated killing from the same patients. In the inducible PIG-A model, GPI-AP(-) TF-1 cells exhibited less apoptosis than induced, GPI-AP+ TF-1 cells in response to allogeneic cell-mediated killing, NK92-mediated killing, TNF-alpha, and gamma-irradiation. GPI-AP(-) TF-1 cells maintained resistance to apoptosis when effectors were raised against GPI-AP(-) cells, arguing against a GPI-AP being the target of immune attack in PNH. NK92-mediated killing was partially inhibited with blockade by specific antibodies to the stress-inducible GPI-AP ULBP1 and ULBP2 that activate immune effectors. Clonal competition experiments demonstrate that the mutant clone expands over time under proapoptotic conditions with TNF-alpha.PIG-A mutations contribute to clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under proapoptotic stresses.

OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). INTRODUCTION: Whether PNH cells are inherently less susceptible to apoptosis remains controversial. Studies using PNH mouse models and lymphoid cell lines have failed to find a difference in apoptosis, but reports using K562 cells have found a relative resistance. Studies using primary CD34+ cells from PNH patients have also found a relative resistance of PNH cells, but not in comparison to CD34+ cells from normal controls. We designed experiments 1) to control for genetic heterogeneity among PNH and control cell lines, 2) to address the potential methodological issues associated with studying lymphocytes, which are rarely affected in PNH, and 3) address inconsistency comparing normal and PNH progenitors that have experienced different in vivo environments. METHODS: GPI-anchored protein (GPI-AP) positive and negative primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune selection in vivo, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Using a doxycycline-inducible wild type PIG-A tet-SUPER transgene expression system, in which GPI-AP− and GPI-AP+ cells are isogenic, we assessed apoptosis resistance and clonal expansion in response to various pro-apoptotic stimuli. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 effectors, TNF-α, and γ-irradiation. Apoptosis was measured by annexin V/PI staining (effector experiments) or caspase 3/7 activity (TNF-α and γ-irradiation experiments). Blocking experiments of NK92-mediated killing utilized mAb to ULBP1 and ULBP2, as per Hanaoka et al. Clonal competition experiments tracked wild type and PIG-A mutant TF-1 cells using flow cytometry after exposure to TNF-α as a surrogate for immune attack. RESULTS: In PNH patients, GPI-AP− CD34+ hematopoietic progenitors were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% versus 49%, p CONCLUSIONS: PIG-A mutations contribute to the clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under pro-apoptotic stresses. Lack of GPI-anchored ULBP1 and ULBP2 expression contributes to the apoptosis resistance in a cell-mediated killing model but is not solely responsible for the apoptosis resistance. The global apoptosis resistance translated into expansion of the PNH clone in an in vitro model of immune attack.