Histocompatibility Complex Antigens Research Articles

Abstract 5632: Combinatorial epigenetic targeting of PRC2 complexes upregulates MHC antigen presentation components in melanoma and gastrointestinal cancer cells

Abstract Low tumor immunogenicity is associated with immune escape, immunotherapy resistance, and poor patient survival [1-3]. Major histocompatibility complex (MHC) antigen presentation pathways (APPs) play a crucial role in promoting tumor T-cell recognition and initiating antitumoral immune responses. However, epigenetic silencing of APPs is a common mechanism in various cancers, including melanoma and gastrointestinal malignancies [3,4]. In the current investigation, mouse melanoma (B16/OVA) and human colon cancer (LOVO and HCT116), small intestine cancer (HUTU80), and normal colonic epithelial (CCD841) cell lines were treated with epigenetic drug candidates. As reported [5], a decrease in histone H3K27 methylation marks via polycomb repressive complex 2 (PRC2) inhibitors, as well as altered histone acetylation status from histone deacetylase 3 (HDAC3)-specific inhibition, increased the expression of APP components, based on immunoblotting and RT-qPCR experiments. Normal colonic epithelial cells showed resistance to the treatments, indicating cancer cell-specific effects of the test agents. In B16/OVA cells, flow cytometry and enzyme linked immunosorbent assays corroborated that HDAC3 and/or PRC2 inhibitor combinations increased cell surface occupancy of MHC-I complexes and increased CD8+ T-cell activation. These findings have implications for the clinical application of next-generation combinatorial epigenetic agents [6,7], targeted towards increased tumor immunogenicity and enhanced efficacy of new immunoepigenetic strategies. Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

RETRACTED ARTICLE: A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function

Overcoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present an innovative ultra-high-content assay platform for interrogating T-cell-mediated killing of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1800 annotated compounds enabled identification of small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function by increasing cytolytic granule and type II interferon secretion in T-cells in addition to upregulating major histocompatibility complex (MHC) expression and antigen presentation in tumor cells. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of first-in-class IO therapeutic agents.

Antibody-Drug Conjugates Targeting CD45 Plus Janus Kinase (JAK) Inhibitors As Conditioning for Allogeneic Hematopoietic Stem Cell Transplantation

<h3>Introduction</h3> Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative therapy for the treatment of hematologic diseases. AlloHSCT is associated with significant treatment-related morbidity and mortality from conditioning-related toxicities and graft-versus-host disease (GvHD), which limits its use to the most life-threatening indications, such as acute myeloid leukemia (AML). However, for conditions like AML that are primarily diagnosed in elderly patients, alloHSCT may be contraindicated due to poor patient functional status or medical comorbidities. Thus, an unmet clinical need exists for novel, highly-effective alloHSCT conditioning regimens with greater tolerability and minimal toxicity. <h3>Objectives</h3> Previously, antibody-drug conjugates (ADCs) composed of a CD45.2 antibody bound to the ribosome-inactivating protein saporin (CD45-SAP) were shown to allow high-level engraftment of syngeneic murine hematopoietic stem cells (HSCs). Here, we describe ADC-based conditioning regimens for murine alloHSCT which overcome immune barriers to engraftment imposed by minor histocompatibility antigen (miHA) and major histocompatibility complex (MHC) mismatches. <h3>Methods</h3> Mice were conditioned for HSCT with 3 mg/kg CD45-SAP 7 days prior to infusion of 10 × 10⁶ bone marrow cells. Haploidentical F1-to-parent (CB6F1→B6) and miHA-mismatched (BALB/c→DBA/2) HSCT models were used. For GvHD studies, a parent-to-F1 (B6→CB6F1) model was used in which recipients were infused with 25 × 10⁶ donor splenocytes after CD45-SAP treatment or sublethal irradiation. <h3>Results</h3> CD45-SAP combined with T cell depletion was sufficient for high-level engraftment in our alloHSCT models (Fig 1). The selective Janus kinase (JAK) inhibitor baricitinib, previously shown by our lab to prevent GvHD, also permitted HSC engraftment when mice received the drug during the peri-transplant period (Fig 2A-B). Graft stability in baricitinib-treated mice was optimized by either combining daily baricitinib treatment with pre-transplant T cell depletion, or by delivering baricitinib via continuous infusion (Fig 2C-D). Notably, CD45-SAP conditioned mice showed minimal morbidity or mortality from GvHD when infused with allogeneic splenocytes, whereas mice conditioned with sublethal irradiation developed acute GvHD. (Fig 3). <h3>Conclusion</h3> CD45-SAP combined with T cell depletion and/or pharmacologic immunomodulation with baricitinib effectively conditioned mice for engraftment of mismatched HSCs. Strikingly, mice conditioned with CD45-SAP did not develop GvHD even when infused with high doses of allogeneic splenocytes. These studies provide proof-of-principle evidence that targeted immunotherapeutics (ADCs plus JAK inhibitors) can prepare HSCT recipients for engraftment of allogeneic donor HSCs with minimal toxicities and without development of GvHD.

HLA-C promotes proliferation and cell cycle progression in trophoblast cells

Objectives: The development of maternal-fetal immune tolerance and adequate trophoblast function are essential for the establishment and maintenance of pregnancy. Human leukocyte antigen (HLA), the major histocompatibility complex (MHC) antigen specific to humans, plays an important role in placentation and is involved in many pregnancy-associated diseases. HLA-C is the only classical MHC I gene expressed at the maternal-fetal interface. To investigate whether HLA-C plays an independent role in regulating the development of trophoblasts, we explored the effect of HLA-C expression on placental development.Methods: The role of HLA-C in the growth and migration of trophoblast JAR and HTR-8/Svneo cell lines was investigated after HLA-C-expressing lentivirus transfection.Results: The MTT assay and colony formation assay showed that HLA-C promoted cell proliferation. Furthermore, cell cycle analysis showed that HLA-C overexpression accelerated the transition of trophoblast cells from the G0/G1 phase to the S phase. However, FACS analysis and migration assay indicated that HLA-C had no significant influence on trophoblast apoptosis and migration.Conclusion: Our study demonstrated for the first time that besides being involved in immune tolerance, HLA-C can directly promote placental growth without interacting with immune cells, which could provide a new insight into studying the functions of HLA-C at the maternal-fetal interface.

Prognostic importance of human leukocyte antigen DRβ1 gene and protein tyrosine phosphatase nonreceptor Type 22 gene polymorphism in rheumatoid arthritis

Background: Major histocompatibility complex (MHC) or human leukocyte antigen (HLA) gene and some of the non-MHC genes are associated with genetic predisposition in rheumatoid arthritis (RA). The aim of the present study was to find the prevalence of HLA DRβ1 alleles and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) gene single-nucleotide polymorphism (SNP) in RA patients and also assessed their correlation with laboratory and clinical parameters. Materials and Methods: One hundred and fifty cases of RA and 100 healthy controls were studied. relation to RA patients. HLA DRβ1 typing was done by low-resolution sequence-specific primer polymerase chain reaction and auto-antibodies (Abs) (rheumatoid factor [RF], anti-cyclic citrullinated peptide 2 (CCP2) Ab, anti-nuclear Ab, double-stranded deoxyribose nucleic acid, anticardiolipin Ab) were done by enzyme-linked immunosorbent assay. Results: In patients with RA, the most common HLA DRβ1 allele was DRβ1*04 (23.3%), followed by DRβ1*10 (20.7%), DRβ1*03 (9.3%), and DRβ1*01 (8.7%). Contrary to this, some alleles of DRβ1 were significantly less expressed in RA such as DRβ1*07 (P = 0.000) and DRβ1*14 (P = 0.010). DRβ1*04 positive patients had significantly more RF positivity while DRβ1*10 positive patients had increased RF and anti-CCP2 Ab. DRβ1*04 positive cases had positive correlation with disease activity score. DRβ1*10 positive patients had negative correlation with the age of the RA patients. These patients have shown early onset of disease. PTPN22 C1858T SNP was found in only 4% cases of RA. No correlation was established with PTPN22 SNP positivity and clinical parameters. Conclusion: In our study, RA patients have less DRβ1*04 positivity as compared to the Western literature. In our area, DRβ1*10 is more common than DRβ1*01. HLA DRβ1*07 and *14 are found to be protective for RA. PTPN22 SNP has not shown any diagnostic or prognostic significance while a follow-up study may be useful for prognostic importance of HLA DRβ1 typing in RA.

Development of the Intestinal Transplantation Model With Major Histocompatibility Complex Inbred CLAWN Miniature Swine

BackgroundClinical intestinal transplantation (Int-Tx) is associated with some problems such as rejection, infection, graft-versus-host disease, and ischemia-reperfusion injury (IRI). To determine mechanisms of rejection as well as to develop treatment strategies for Int-Tx, this study was designed to establish both heterotopic and orthotropic Int-Tx models using major histocompatibility antigen complex (MHC) inbred CLAWN miniature swine. Materials and MethodsEleven CLAWN miniature swine received MHC matched but minor antigen mismatched allogenic intestinal grafts. Four animals received intestinal grafts heterotopically and kept host intestine intact. The remaining 7 animals received intestinal grafts orthotopically and resected host small intestine. Continuous infusion of tacrolimus was given from day 0 for 12 days. ResultsHeterotopically transplanted small intestine were well perfused after revascularization; however, grafts easily underwent ischemic changes during or soon after abdomen closure due to oppression of the grafts in the limited abdominal space. In contrast, all of 7 orthotopically transplanted intestinal grafts in which recipients' small intestine was removed from the jejunum to the ileum had no signs of severe ischemia associated with compartment syndrome. Elevation of the serum concentration of inflammatory cytokines and the progression of lethal acidosis seen in recipients of heterotipic transplantation were markedly less in the case of orthotopic transplantation. Two recipients survived more than 30 days, and 1 long-term survivor showed no evidence of rejection at day 90 despite the fact that tacrolimus was stopped at day 12. ConclusionsIn this study, we demonstrated the establishment of a clinically relevant orthotopic Int-Tx model with long survival in MHC inbred CLAWN miniature swine. We believe that this unique MHC inbred swine Int-Tx model is useful for developing treatment strategies for clinical Int-Tx.

Abstract 4401: Tumor-associated antigen gene-loading polyplex micelle is a promising platform for anti-cancer DNA vaccine

Abstract Backgrounds: Polyplex micelle attracts an increasing attention as a gene carrier, because of efficient gene transduction and biocompatibility in vivo. In this study, we examined the potential of polyplex micelle carrying genes encoding tumor-associated antigen (TAA): squamous cell carcinoma antigen recognized by T cells-3, CD40L and GM-CSF as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Methods and Results: Intraperitoneal administration of polyplex micelles were predominantly distributed and expressed in the lymph nodes, spleen and liver. The triple DNA vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer compared with mock controls, of which long-survivor mice induced complete rejection of re-challenged CT26 tumors. The DNA vaccine also inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung cancers in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+-DC, CD4+ and CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ cells using their neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. Conclusions: TAA/CD40L+GM-CSF gene-loaded polyplex micelle is a novel vaccine platform to elicit tumor rejection immunity regardless of recipient's MHC-haplotype. Note: This abstract was not presented at the meeting. Citation Format: Lin Cui, Kouichi Furugaki, Kensuke Osada, Kazunori Kataoka, Kenji Nakano. Tumor-associated antigen gene-loading polyplex micelle is a promising platform for anti-cancer DNA vaccine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2015-4401

A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response.

Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2′-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2′-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-015-1329-z) contains supplementary material, which is available to authorized users.

The level of viral infection of antigen-presenting cells correlates with the level of development of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in susceptible SJL/J mice but not in resistant C57BL/6 mice. Previous studies have indicated that the major histocompatibility complex (MHC) genes play the most prominent role in the development of TMEV-induced demyelinating disease. In this study, we used C57BL/6.S (B6.S) congenic mice, which carry H-2(s) MHC genes instead of H-2(b) MHC genes in conjunction with the C57BL/6 (B6) background genes. Our data show that virus-infected B6.S mice are free from disease and have significantly lower viral loads than susceptible SJL mice, particularly in the spinal cord. A strong protective Th1-type T helper response with virtually no pathogenic Th17 response was detected in B6.S mice, in contrast to the reduced Th1- and robust Th17-type responses in SJL mice. Notably, lower levels of viral infectivity in B6.S antigen-presenting cells (APCs) correlated with the disease resistance and T-cell-type response. In vitro studies using APCs from B6.S and SJL mice show that TLR2, -3, -4, and -7, but not TLR9, signaling can replace viral infection and augment the effect of viral infection in the differentiation of the pathogenic Th17 cell type. Taken together, these results strongly suggest that the viral replication levels in APCs critically affect the induction of protective versus pathogenic Th cell types via the signaling of pattern recognition receptors for innate immune responses. Our current findings further imply that the levels of viral infectivity/replication and TLR-mediated signaling play critical roles in the pathogenesis of chronic viral diseases. This study indicates that innate immune cytokines produced in antigen-presenting cells stimulating the T cell immune responses during early viral infection play a critical role in determining the susceptibility of mice to the development of demyelinating disease. The level of innate immune cytokines reflects the level of initial viral infection in the antigen-presenting cells, and the level determines the development of T cell types, which are either protective or pathogenic. The level of initial viral infection to the cells is controlled by a gene or genes that are not associated with the major histocompatibility antigen complex genes. This finding has an important implication in controlling not only chronic viral infections but also infection-induced autoimmune-like diseases, which are closely associated with the pathogenic type of T cell responses.

Identification of microRNAs involved in acute rejection and spontaneous tolerance in murine hepatic allografts.

Graft acceptance without the need for immunosuppressive drugs is the ultimate goal of transplantation therapy. In murine liver transplantation, allografts are accepted across major histocompatibility antigen complex barriers without the use of immunosuppressive drugs and constitute a suitable model for research on immunological rejection and tolerance. MicroRNA (miRNA) has been known to be involved in the immunological responses. In order to identify mRNAs in spontaneous liver allograft tolerance, miRNA expression in hepatic allografts was examined using this transplantation model. According to the graft pathological score and function, miR-146a, 15b, 223, 23a, 27a, 34a and 451 were upregulated compared with the expression observed in the syngeneic grafts. In contrast, miR-101a, 101b and 148a were downregulated. Our results demonstrated the alteration of miRNAs in the allografts and may indicate the role of miRNAs in the induction of tolerance after transplantation. Furthermore, our data suggest that monitoring the graft expression of novel miRNAs may allow clinicians to differentiate between rejection and tolerance. A better understanding of the tolerance inducing mechanism observed in murine hepatic allografts may provide a therapeutic strategy for attenuating allograft rejection.

Evaluation of an immune-privileged scaffold for In vivo implantation of tissue-engineered trachea

Forty tracheas were harvested from donor New Zealand rabbits. Thirty of the tracheas were randomly divided into four treatment groups corresponding to 4, 5, 6, or 7% NaClO4 and one untreated group (n = 6 each group). Scanning electron microscopy distinctly revealed the cilium of epithelial cells in the fresh trachea. The internal surface of the trachea was rough in the 4% treatment group and smooth in the 5% treatment group, whereas the matrix was fractured in the 6% treatment group and highly fractured in the 7% treatment group. We observed that the number of nuclei in the cells of the 4, 5, 6, and 7% treatment groups decreased compared to the cells of the untreated group (p 0.05) in the 5% treatment group. In addition, histopathological analysis showed that the fiber component and basement membrane of the matrix in the 5% treatment group were retained after optimal decellularization. Despite the preserved cartilage, in vitro immunohistochemical analysis revealed that the matrix did not show the presence of major histocompatibility complex (MHC) antigens. The remaining ten donor tracheas, which were divided into a positive control group and an optimal decellularized group, were used for allogeneic transplantation. Blood samples were taken regularly, and histologic examinations were performed at 30 days postimplantation, which showed no significant immune rejection. In conclusion, we surveyed the structural integrity through morphological observation and compared the biomechanical and immunogenic changes in the tracheal matrix under the different treatments. The optimal decellularized tracheal matrix with preserved cartilage, which was acquired via 5% NaClO4 treatment, exhibited structural integrity, antigen cell removal and immune privilege and would be suitable for use as a tissue-engineered trachea for in vivo transplantation in rabbit models.

Intraperitoneal administration of a tumor-associated antigen SART3, CD40L, and GM-CSF gene-loaded polyplex micelle elicits a vaccine effect in mouse tumor models.

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+ DCs and CD4+/CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.

Effects of continuous sedation with propofol on peripheral blood mononuclear cell and intercellular adhesion molecule in beagles with combined burn-blast injuries

To observe the effects of continuous sedation with propofol on peripheral blood mononuclear cell (PBMC) and intercellular adhesion molecule 1 (ICAM-1) in beagles with combined burn-blast injuries. A total of 32 male beagles were randomly divided into 4 groups of normal control (NC), combined injury control (CC), propofol 1 (P1) and propofol 2 (P2) (n = 8 each). Except for NC group, the other 3 groups were subject to severe combined burn-blast injury. And sodium lactate Ringer's solution was infused after trauma according to the Parkland formula, including NC group. At the same time, P1 and P2 groups received continuous intravenous infusions of 2 mg×kg(-1)×h(-1), 5 mg×kg(-1)×h(-1) doses of propofol respectively for 72 hours. The serum concentrations of ICAM-1 and lymphocyte function associated antigen-1 (LFA-1) were measured by enzyme-linked immunosorbent assay (ELISA) at 6, 24, 48, 72 h post-injury. Flow cytometry was used to detect the major histocompatibility complex (MHC) antigen expression on CD14(+) monocytes, CD4(+)/CD8(+) T lymphocyte rate and PBMC apoptosis rate. The level of ICAM-1 in CC group ((10.5 ± 1.1), (10.8 ± 1.3), (12.3 ± 1.4) ng/ml) was significantly higher than that in NC group ((7.4 ± 1.4), (7.4 ± 1.1), (7.4 ± 1.6) ng/ml) at 12, 24, 48 h post-injury (all P < 0.05). The level of ICAM-1 in P1 group was significantly lower than that in CC group ((10.7 ± 1.3) vs (12.3 ± 1.4) ng/ml) while the level of ICAM-1 in P2 group was significantly lower than that in P1 group at 72 h post-injury ((8.8 ± 1.4) vs (10.7 ± 1.3) ng/ml) (both P < 0.05). The level of LFA-1 in CC group ((7.3 ± 1.3), (8.4 ± 1.3), (9.6 ± 1.7) ng/ml) was significantly higher than that in NC group ((5.1 ± 1.2), (5.4 ± 1.3), (5.8 ± 1.2) ng/ml) at 24, 48, 72 h post-injury (all P < 0.05). MHC antigen expression on the CD14(+) monocytes of P2 group was obviously higher than that of CC and P1 groups ((46 ± 13)% vs (26 ± 15)% and (32 ± 12)%, both P < 0.05). The CD4/CD8 rate in P1 and P2 was significantly higher than that in CC group (1.71 ± 0.26, 1.82 ± 0.31 and 1.81 ± 0.24, 1.96 ± 0.24 vs 1.41 ± 0.34, 1.34 ± 0.26) at 48, 72 h post-injury (all P < 0.05). At 72 h post injury, the PBMC apoptosis rate in CC and P1 group was obviously higher than that of the NC group ((2.57 ± 0.21)% and (1.64 ± 0.10)% vs (0.81 ± 0.11)%) (both P < 0.01); the apoptosis rate in P2 group was significantly lower than that in P1 group ((1.09 ± 0.15)% vs (1.64 ± 0.10)%) (P < 0.01). Propofol may improve the immune function after combined burn-blast injuries through suppressing an excessive release of ICAM-1 and PBMC apoptosis in a concentration-dependent manner.

Antigen-Specific Priming is Dispensable in Depletion of Apoptosis-Sensitive T Cells for GvHD Prophylaxis.

Prophylactic approaches to graft versus host disease (GvHD) have employed both phenotypic reduction of T cells and selective elimination of host-primed donor T cells in vitro and in vivo. An additional approach to GvHD prophylaxis by functional depletion of apoptosis-sensitive donor T cells without host-specific sensitization ex vivo showed remarkable reduction in GHD incidence and severity. We address the role and significance of antigen-specific sensitization of donor T cells and discuss the mechanisms of functional T cell purging by apoptosis for GvHD prevention. Host-specific sensitization is dispensable because migration is antigen-independent and donor T cell sensitization is mediated by multiple and redundant mechanisms of presentation of major and minor histocompatibility complex and tissue antigens by donor and host antigen-presenting cells. Our data suggest that potential murine and human GvH effectors reside within subsets of preactivated T cells susceptible to negative regulation by apoptosis prior to encounter of and sensitization to specific antigens.

Gvhd Increases Apoptosis of CD8+, but Not CD4+, T Cells Expanded by Vaccines but Is Not Dependent on Fas Ligand

AbstractAbstract 2098Donor lymphocyte infusions (DLIs) administered following allogeneic blood and marrow transplantation (alloBMT) can enhance T cell reconstitution through homeostatic peripheral expansion (HPE), but can also cause graft-versus-host-disease (GVHD). Vaccines are a strategy to skew T cells toward a “third-party” tumor antigen, but it is not clear if the effects of GVHD on vaccine-responding T cells is equivalent to the effects on alloantigen-reactive polyclonal T cells. Previous work by others has demonstrated “bystander” effects of GVHD causing activation-induced cell death through the Fas pathway on polyclonal T cells; however, the impact of GVHD on T cells expanded by a vaccine expressing a third-party nonalloantigen remains unclear. T cell depleted (TCD) minor histocompatibility antigen (mHA)-mismatched alloBMT (CD45.1+ B6 –> CD45.1+/CD45.2+ B6 × C3H.SW) was followed by a CD45.1+ B6 DLI on day +14 to induce GVHD. CD45.2+ B6 Rag2-/- T cell receptor transgenic (TCRTg) CD4+ or CD8+ T cells specific for epitopes derived from the male histocompatibility antigen complex (HY) were administered on day +28 with an HY-expressing male dendritic cell vaccine to determine the impact of the allogeneic environment on the persistence, proliferation and survival of vaccine-responding T cells. Despite the fact that syngeneic and allogeneic recipients had similar degrees of lymphopenia, the absolute number of CD4+ and CD8+ TCRTg T cells was decreased 5 and 7 days respectively after adoptive transfer and vaccination in alloBMT recipients compared to syngeneic recipients (p < 0.05). To determine the mechanism of decreased persistence of the TCRTg T cells, proliferation was measured by assessing dilution of CFSE and apoptosis was measured by FACS analysis for Annexin V. Importantly, since both the CD4+ and CD8+ TCRTg T cells do not undergo HPE, all proliferation has to be antigen-driven. Neither CD4+ nor CD8+ TCRTg T cells proliferated in the absence of HY vaccine following alloBMT, confirming lack of bystander proliferation. Interestingly, both CD4+ (p < 0.01) and CD8+ (p < 0.05) TCRTg T cells undergo less vaccine-induced proliferation after alloBMT compared to syngeneic recipients. The amount of CD8+ TCRTg T cell apoptosis following vaccination was higher in allogeneic recipients (p < 0.05), but there was no difference in CD4+ TCRTg T cell apoptosis. Surprisingly, using a DLI from Fas ligand-deficient B6 (gld) donors on day +14 was sufficient to induce GVHD but did not prevent CD8+ TCRTg T cell apoptosis. Thus, diminished vaccine responses during GVHD appear to result in part from impaired CD8+ and CD4+ T cell vaccine-driven proliferation, but there is an additional contribution on CD8+ T cell apoptosis that is not dependent on recognition of alloantigen or the Fas-Fas ligand pathway. Disclosures:No relevant conflicts of interest to declare.

Pulmonary macrophages in rats after hemithoracic irradiation: analysis of morphology and expression of surface antigen

The pathogenesis of radiation pneumonitis is not completely understood. The long latent period and involvement of unirradiated lung tissue may indicate an immune reaction in the injurious process of irradiated lung. To investigate the role of pulmonary macrophages in radiation pneumonitis, morphology and membrane antigen expression of pulmonary macrophages were studied in irradiated rat lung tissue following 4000 R hemithoracic irradiation. Lungs were explanted at 2, 4, 6, 8, 16, and 28 weeks after irradiation. Cryosections of irradiated lung tissue were immunohistochemically studied, and alveolar macrophages in bronchoalveolar lavage fluid were also analyzed using monoclonal antibodies to rat major histocompatibility complex (MHC) antigens and macrophages with flow cytometry. Macrophage subpopulations were analyzed using a panel of monoclonal antibodies to MHC class I (HAM2), MHC class II (OX6) and macrophage differentiation antigens (ED1, ED2, ED3). Alveolar macrophages in bronchoalveolar lavage were morphologically studied by smear and flow cytometry of forward light scatter and 90 degrees light scatter. At 2 weeks after irradiation, when histological changes did not appear, small lymphocyte-like macrophages and large foamy macrophages were observed in both the smear and histograms by flow cytometry. At 4, 6, and 8 weeks after irradiation, these new populations had markedly increased. However, at 16 and 28 weeks after irradiation, the size and shape of alveolar macrophages had returned to normal. In the expression of macrophage membrane antigens, an increase in the frequency of MHC class II+ cells in lavaged cells appeared at 2 weeks after irradiation, and became significant at 4 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists

T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-γ1 and entry of extracellular Ca2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

Dipeptidyl Peptidase Inhibition Accelerates Dendritic Cell Cross Priming of Tumor-Reactive T Cells Resulting in Regression of Established Tumors

BACKGROUND: PT-100 is an aminoboronic dipeptide that competitively inhibits dipeptidyl peptidases. While PT-100 has no direct effect on tumor cells in vitro, it exhibits potent antitumor effects in vivo. We have shown that female C57BL/6 (B6) mice with MB49 tumors, which naturally express the male minor histocompatibility antigen complex (HY), are primed to HY, but the immune response is insufficient to control tumor growth. In this study, we used the well-characterized HY antigen system to examine the immunomodulatory effects of PT-100 during treatment-induced tumor regression.METHODS: B6 female mice were inoculated subcutaneously with MB49 (106 cells) on day 0 and treated daily with PT-100 by gavage. For re-challenge experiments mice received high dose MB49 (3×106 cells) three weeks after complete regression of primary tumors. IFN-g ELISPOT was used to measure HY antigen specific T cell responses in the spleen and lymph nodes (LNs) during tumor growth. For adoptive transfer experiments, T cells were magnetic-bead purified from LNs and spleens of tumor-bearing PT-100 treated, tumor-bearing sham treated, or naïve mice and injected intravenously into Rag1−/− recipients (1.2×106 cells) which were then inoculated with high dose MB49. T cells were depleted with monoclonal antibodies to CD4 and CD8. Dendritic cells (DCs) were depleted with diphtheria toxin (DT) in bone marrow chimeras expressing the DT receptor under the CD11c promoter. DC activation examined by flow cytometry. For vaccine experiments, HY-expressing DCs were cultured from male B6 bone marrow and injected intraperitoneally (1×105 cells).RESULTS: PT-100 treatment resulted in complete regression of MB49, even when limited to the first week (days 3–7) during tumor progression. Treatment started later than week 1 was insufficient to establish consistent, complete tumor regression. High-dose re-challenge of PT-100 treated mice resulted in initial growth followed by regression without additional PT-100. IFN-gELISPOT revealed a robust response against HY in spleens of controls on day 17. Interestingly, PT-100 treated mice had quantitatively similar priming, but the response peaked earlier (day 10), just prior to tumor regression. Purified T cells from PT-100 treated donors collected on day 17 mediated markedly enhanced tumor protection compared to recipients of T cells from sham treated tumor-bearing mice despite significantly more HY-reactive cells in the spleen and LNs of sham treated-tumor bearing mice by that time. T cell or DC depletion independently abrogated the anti-tumor effect of PT-100 and treatment with PT-100 increased CD80 and CD86 expression on LN DC populations in vivo. Although HY DC vaccination does not affect tumor growth, supplementation of the DC vaccine with PT-100 mediated a therapeutic effect resulting in regression of well-established tumors.CONCLUSIONS: PT-100 establishes a consistent and potent antitumor effect against MB49 dependent on T cells and DCs. Treatment results in a memory response that is protective against high dose MB49 re-challenge. PT-100-induced tumor regression is associated with enhanced early tumor priming, associated with increases in activated DCs. T cells from PT-100 treated mice elicit superior protection upon adoptive transfer compared to shams, despite quantitatively less tumor-primed T cells, suggesting the PT-100 antitumor effect may involve a qualitative difference in T cell function. PT-100 given as an adjuvant to a DC vaccine results in increased potency and regression of established tumors. Inhibition of dipeptidyl peptidases modulate naturally occurring anti-tumor immune responses and contribute to the generation of a therapeutic anti-cancer vaccine.

Extracorporeal Photopheresis Diminishes Graft-Versus-Host-Disease and Modulates Dendritic Cell Cytokine Production.

Background: Graft-versus-host disease (GVHD) remains one of the largest obstacles in allogeneic stem cell transplantation. Current therapies for GVHD generally involve systemic immunosuppression resulting in increased risk of infection and relapse. Extracorporeal Photopheresis (ECP) is an emerging therapy for chronic GVHD that offers the potential of selective immunomodulation. In ECP, patient leukocytes are treated with 8-methoxypsoralen (8-MOP) and UVA light, causing them to undergo apoptosis, and are re-infused into the patient. Because ECP has been studied mostly in the clinical setting, however, the mechanism by which this infusion of apoptotic cells impacts GVHD remains unclear. We hypothesized that ECP-treated cells interact with recipient dendritic cells (DCs) to induce tolerance. We applied ECP to a murine GVHD model and explored the effects of ECP-treated cells on DCs.Methods: ECP: Splenocytes were incubated with 8-MOP at 200 ng/ml and exposed to UVA light (5 J/cm2).Bone Marrow Transplant (BMT): C3H.SW mice were irradiated (1000 cGy) and given 10 × 106bone marrow and 5 ×106 lymph node C57BL/6 (B6) cells on day 0. 10 ×106 ECP-treated splenocytes were administered on day 1.Co-culture: Female B6 BM-derived DCs were cultured for 7 days with GM-SCF and IL-4 and incubated overnight with untreated, irradiated (3000 cGy) or ECP-treated splenocytes. Anti-CD40 was used to activate DCs. DC phenotype and cytokine production were examined using flow cytometry.Vaccine: Male B6 DCs were co-incubated with untreated, irradiated or ECP-treated female B6 splenocytes and injected into naive female B6 recipients on days 14 and 28. On day 42, responses against the male histocompatibility antigen complex (H-Y) were assayed using IFN-gamma ELISPOT.Results: 85–94% of ECP-treated cells were Annexin+/7AAD+ 24 hours after ECP treatment. Infusion of ECP-treated cells diminished GVHD-associated weight loss regardless of the strain of the ECP donor (Fig. 1) and restored responsiveness to H-Y vaccination. In co-culture experiments, the presence of ECP-treated cells did not alter phenotypic maturation of DCs. However, co-culture of DCs with ECP-treated cells did alter DC cytokine production resulting in less IL-12 production when compared to anti-CD40 alone (1.0% vs. 4.2% of CD11b+/CD11c+ cells). Finally, immunization of naive female mice with male DCs co-cultured with ECP-treated cells resulted in a modest but statistically significant (p=.0499) decrease in subdominant, class I H-Y responses compared to DCs incubated with untreated splenocytes.Conclusion: The administration of ECP-treated cells in vivo appears to lessen the severity of GVHD, allowing mice to maintain immunocompetence. DCs cultured in the presence of ECP-treated cells can mature phenotypically but exhibit altered cytokine production. Activated, co-incubated DCs are less potent when used as a vaccine. This data suggests that perhaps the alteration of recipient DCs may contribute to the beneficial effect of ECP in GVHD. Ongoing experiments continue to explore the role of recipient DCs in ECP treatment of GVHD. [Display omitted]

231 HISTOCOMPATIBLE PARTHENOGENETIC EMBRYONIC STEM CELLS

Organ or tissue transplantation is the preferred treatment for numerous diseases but is hindered by immunologic barriers. Genetically matched pluripotent embryonic stem cells generated via nuclear transfer (ntES cells) or parthenogenesis (pES cells) are possible sources of histocompatible cells and tissues. We have developed two ways of isolating pES cells that carry the full complement of major histocompatibility complex (MHC) antigens of the oocyte donors. One method entails activation of oocytes after blockade of karyokinesis in meiosis II, followed by selection of predominantly homozygous pES cells that have undergone recombination in their MHC antigen region to restore the heterozygous maternal MHC genotype (parthenote recombinant, or prES cells). The second method involves activation of immature oocytes after blockade of karyokinesis of meiosis I, followed by selection of predominantly heterozygous pES lines that retain the MHC genotype of the oocyte donor (parthenote clone recombinant, or pcrES cells). The cells are pluripotent by several criteria: teratoma formation, in vitro differentiation into hematopoietic elements, and high-level skin chimerism in blastocyst chimeras. Breeding of 8 founder females and examination of over 700 progeny failed to demonstrate germ line transmission of the pES cells. Injection of over 50 tetraploid embryos with these lines and embryo transfer have failed to support full gestational development. However, differentiated tissues from these pluripotent ES cells engraft when transplanted into genetically matched immunocompetent recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.