Eduardo Huarte

Aim: Graft-versus-host disease (GvHD) is a major complication arising in patients undergoing allogenic hematopoietic stem cell transplantation. Material & methods: We tested ruxolitinib (a selectiv...

Background: Allogeneic hematopoietic cell transplantation (alloHCT) is potentially curative for a variety of hematologic malignancies. However, rather than targeting tumor cells, alloreactive donor T cells may attack healthy recipient tissue triggering graft-versus-host disease (GVHD) resulting in morbidity and mortality. Acute GVHD (aGVHD) clinically presents with gastrointestinal (GI) pathology and is primarily T cell mediated. In contrast, chronic GVHD commonly demonstrates skin inflammation and fibrosis with an autoimmune-like phenotype. Pharmacological inhibition of JAK/STAT signaling has the potential to regulate aberrant immune cell functions involved in the pathogenesis of acute and chronic GVHD. Aim: Characterize the effectiveness of ruxolitinib to modulate in vitro and in vivo immunological mechanisms that are described to be critical in acute and chronic GVHD pathogenesis. Materials and Methods: MHC-mismatch aGVHD was induced in recipient BALB/c mice receiving C57BL/6 donor transplant. Sclerodermatous cGVHD was induced in C57BL/6 mice using LP/J strain donor splenocytes and bone marrow cells. Ruxolitinib or vehicle was orally administered twice daily. Efficacy was assessed by GVHD symptom scores, cachexia, and histological analysis of end organ damage. Mixed lymphocyte reactions (MLR) were cultured for five days in the presence of ruxolitinib for changes in activation markers and cytokine release. Results: Ruxolitinib treatment ameliorated daily aGVHD severity scores with concomitant reduction of GI tract infiltration and tissue damage. Overall aGVHD mice survival was significantly improved with JAK1/2 inhibition. Therapeutic ruxolitinib treatment initiated after cGVHD symptom onset significantly inhibited the chronic-progressive symptom phenotype and reduced tissue markers of inflammation and fibrosis. Ruxolitinib demonstrated concentration-dependent inhibition of activated T cells and reduced JAK-STAT signaling cytokines considered relevant in GVHD inflammation. Conclusion: Ruxolitinib, a potent and selective JAK1/2 inhibitor, significantly ameliorated pro-inflammatory pathways in vitro and in vivo which are thought to be highly relevant for acute and chronic GVHD pathogenesis. On May 24, 2019, the FDA approved ruxolitinib for steroid-refractory acute graft-versus-host disease (aGVHD) in adult and pediatric patients 12 years and older. Ruxolitinib is currently being evaluated in clinical trial (REACH3: NCT03112603) for patients with steroid-refractory cGVHD.

Regulatory T cells (Tregs) induced during autoimmunity often become quiescent and unable to resolve disease, suggesting inadequate activation. Resolution of established experimental autoimmune encephalomyelitis (EAE) can be achieved with myelin oligodendrocyte glycoprotein (MOG) fused to reovirus protein σ1 (MOG-pσ1), which activates Tregs, restoring protection, but requiring other regulatory cells to revitalize them. B cells have a dichotomous role in both the pathogenesis and recovery from EAE. Although inflammatory B cells contribute to EAE’s pathogenesis, treatment of EAE mice with MOG-pσ1, but not OVA-pσ1, resulted in an influx of IL-10–producing B220⁺CD5⁺ B regulatory cells (Bregs) enabling Tregs to recover their inhibitory activity, and in turn, leading to the rapid amelioration of EAE. These findings implicate direct interactions between Bregs and Tregs to facilitate this recovery. Adoptive transfer of B220⁺CD5⁻ B cells from MOG-pσ1–treated EAE or Bregs from PBS-treated EAE mice did not resolve disease, whereas the adoptive transfer of MOG-pσ1–induced B220⁺CD5⁺ Bregs greatly ameliorated EAE. MOG-pσ1–, but not OVA-pσ1–induced IL-10–producing Bregs, expressed elevated levels of B and T lymphocyte attenuator (BTLA) relative to CD5⁻ B cells, as opposed to Tregs or effector T (T_eff) cells, whose BTLA expression was not affected. These induced Bregs restored EAE Treg function in a BTLA-dependent manner. BTLA^−/− mice showed more pronounced EAE with fewer Tregs, but upon adoptive transfer of MOG-pσ1–induced BTLA⁺ Bregs, BTLA^−/− mice were protected against EAE. Hence, this evidence shows the importance of BTLA in activating Tregs to facilitate recovery from EAE.

Mammaglobin-A (MAM-A) is a secretory protein that is overexpressed in 80 % of human breast cancers. Its near-universal expression in breast cancer as well as its exquisite tissue specificity makes it an attractive target for a breast cancer prevention vaccine, and we recently initiated a phase 1 clinical trial of a MAM-A DNA vaccine. Previously, we have identified multiple MAM-A CD8 T cell epitopes using a reverse immunology candidate epitope approach based on predicted binding, but to date no attempt has been made to identify epitopes using an unbiased approach. In this study, we used human T cells primed in vitro with autologous dendritic cells expressing MAM-A to systematically identify MAM-A CD8 T cell epitopes. Using this unbiased approach, we identified three novel HLA-A2-restricted MAM-A epitopes. CD8 T cells specific for these epitopes are able to recognize and lyse human breast cancer cells in a MAM-A-specific, HLA-A2-dependent fashion. HLA-A2⁺/MAM-A⁺ breast cancer patients have an increased prevalence of CD8 T cells specific for these novel MAM-A epitopes, and vaccination with a MAM-A DNA vaccine significantly increases the number of these CD8 T cells. The identification and translational validation of novel MAM-A epitopes has important implications for the ongoing clinical development of vaccine strategies targeting MAM-A. The novel MAM-A epitopes represent attractive targets for epitope-based vaccination strategies, and can also be used to monitor immune responses. Taken together these studies provide additional support for MAM-A as an important therapeutic target for the prevention and treatment of breast cancer.

Peptide vaccines derived from CD8+ T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8+ T-cell responses, even in the absence ...

Lymphocytes are a key component of the immune system and their differentiation and function are directly influenced by cancer. We examined peripheral blood lymphocyte (PBL) gene expression as a biomarker of illness and treatment effect using the Affymetrix Human Gene ST1 platform in patients with metastatic renal cell carcinoma (mRCC) who received combined treatment with IL-2, interferon-?-2a and dendritic cell vaccine. We examined gene expression, cytokine levels in patient serum and lymphocyte subsets as determined by flow cytometry (FCM). Pre-treatment PBLs from patients with mRCC exhibit a gene expression profile and serum cytokine profile consistent with inflammation and proliferation not found in healthy donors (HD). PBL gene expression from patients with mRCC showed increased mRNA of genes involved with T-cell and T_REG-cell activation pathways, which was also reflected in lymphocyte subset distribution. Overall, PBL gene expression post-treatment (POST) was not significantly different than pre-treatment (PRE). Nevertheless, treatment related changes in gene expression (post-treatment minus pre-treatment) revealed an increased expression of T-cell and B-cell receptor signaling pathways in responding (R) patients compared to non-responding (NR) patients. In addition, we observed down-regulation of T_REG-cell pathways post-treatment in R vs. NR patients. While exploratory in nature, this study supports the hypothesis that enhanced inflammatory cytotoxic pathways coupled with blunting of the regulatory pathways is necessary for effective anti-cancer activity associated with immune therapy. This type of analysis can potentially identify additional immune therapeutic targets in patients with mRCC.

γδ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human γδ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of γδ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRδ^−/− mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRγδ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. γδ T cells were the major source of IL-17 following infection and also produced IFN-γ. Depletion of γδ T cells from C57BL/6, IL-17Rα^−/−, and GMCSF^−/− mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when γδ T cells were depleted from IFN-γ^−/− mice, enhanced susceptibility was observed. Neutralization of γδ T cells in the absence of TNF-α did not further impair immunity. In the absence of TNF-α or γδ T cells, B. abortus-infected mice showed enhanced IFN-γ, suggesting that they augmented production to compensate for the loss of γδ T cells and/or TNF-α. While the protective role of γδ T cells was TNF-α-dependent, γδ T cells were not the major source of TNF-α and activation of γδ T cells following B. abortus infection was TNF-α-independent. Additionally, bovine TCRγδ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-γ. Collectively, these results demonstrate γδ T cells are important for early protection to B. abortus infections.

Human IBD, including UC and Crohn's disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti-inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL-1β, TNF-α, IL-6, IL-17, IL-22, CXCL9, CXCL10, CXCL11, and IFN-γ in the colons of mice with colitis. APP-mediated protection requires T cells, as protection was abated in Rag-1−/− or TCRα−/− mice but not in IL-10−/−, IRF-1−/−, μMT, or TCRδ−/− mice. Administration of APP during colitis to TCRα−/− mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCRαβ cells in APP-mediated protection. APP treatment also inhibited CXCR3 expression by TCRαβ cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4+ or CD8+ T cells alone did not abolish APP-mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.

Stimulation of Ag‐specific inducible Treg can enhance resolution of autoimmune disease. Conventional methods to induce Treg often require induction of autoimmune disease or subjection to infection. Reovirus adhesin, protein σ1 (pσ1), can successfully facilitate tolerance when fused to a tolerogen. We tested whether myelin oligodendrocyte glycoprotein (MOG) fused to pσ1 (MOG‐pσ1) can stimulate Ag‐specific Treg. We show that C57BL/6 mice treated nasally with MOG‐pσ1 fail to induce MOG‐specific Abs and delayed‐type hypersensitivity (DTH) responses and resist EAE. Such resistance was attributed to stimulation of Foxp3⁺ Treg, as well as Th2 cells. MOG‐pσ1's protective capacity was abrogated in IL‐10^−/− mice, but restored when adoptively transferred with MOG‐pσ1‐induced Treg. As a therapeutic, MOG‐pσ1 diminished EAE within 24 h of nasal application, unlike recombinant MOG (rMOG), pσ1, or pσ1+rMOG, implicating the importance of Ag specificity by pσ1‐based therapeutics. MOG‐pσ1‐treated mice showed elevated IL‐4, IL‐10, and IL‐28 production by CD4⁺ T cells, unlike rMOG treated or control mice that produced elevated IFN‐γ or IL‐17, respectively. These data show the feasibility of using pσ1 as a tolerogen platform for Ag‐specific tolerance induction and highlight its potential use as an immunotherapeutic for autoimmunity.