Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein responsemediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDPcholine pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis. The endoplasmic reticulum (ER)3 is a multifunctional organelle responsible for the folding and assembly of all proteins targeted to the secretory pathway (1). As such, the ER can adapt to accommodate an increased load of nascent polypeptides. For example, when B-lymphocytes differentiate into antibody-secreting plasma cells, an elaborate network of rough ER develops to facilitate immunoglobulin production (2-4). Likewise, the rough ER is highly developed in other specialized secretory cell types such as pancreatic acinar cells that secrete copious amounts of digestive enzymes (5). In contrast, the ER is sparse in non-secretory cells, such as reticulocytes (6). ER abundance, therefore, is regulated according to the demands on the secretory pathway. However, the mechanisms that regulate ER biogenesis are incompletely defined (7).A key regulator of ER homeostasis is the unfolded protein response (UPR) pathway, a complex signaling system emanating from the ER membrane (8). When the protein folding capacity of the ER is challenged, the UPR relieves the resulting stress by repressing translation, increasing expression of ER chaperones and folding enzymes, and enhancing ER-associated degradation (8). In addition, recent studies have uncovered a connection between the UPR and ER abundance (9, 10). The UPR-regulated transcription fa...

A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6α (ATF6α), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6α drives ER expansion and can do so in the absence of XBP1(S). Overexpression of active ATF6α induces PtdCho biosynthesis and modulates the CDP-choline pathway differently than does enforced expression of XBP1(S). These data indicate that ATF6α and XBP1(S) have the ability to regulate lipid biosynthesis and ER expansion by mechanisms that are at least partially distinct. These studies reveal further complexity in the potential relationships between UPR pathways, lipid production and ER biogenesis.

The trigger initiating an autoimmune response against melanocytes in vitiligo remains unclear. Patients frequently experience stress to the skin prior to depigmentation. 4-tertiary butyl phenol (4-TBP) was used as a model compound to study the effects of stress on melanocytes. Heat shock protein (HSP)70 generated and secreted in response to 4-TBP was quantified. The protective potential of stress proteins generated following 4-TBP exposure was examined. It was studied whether HSP70 favors dendritic cell (DC) effector functions as well. Melanocytes were more sensitive to 4-TBP than fibroblasts, and HSP70 generated in response to 4-TBP exposure was partially released into the medium by immortalized vitiligo melanocyte cell line PIG3V. Stress protein HSP70 in turn induced membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and activation of DC effector functions towards stressed melanocytes. Melanocytes exposed to 4-TBP demonstrated elevated TRAIL death receptor expression. DC effector functions were partially inhibited by blocking antibodies to TRAIL. TRAIL expression and infiltration by CD11c+ cells was abundant in perilesional vitiligo skin. Stressed melanocytes may mediate DC activation through release of HSP70, and DC effector functions appear to play a previously unappreciated role in progressive vitiligo.

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CAR T therapy targeting solid tumors is restrained by limited infiltration and persistence of those cells in the tumor microenvironment (TME). Here, we developed approaches to enhance the activity of CAR T cells using an orthotopic model of locally advanced breast cancer. CAR T cells generated from Th/Tc17 cells given with the STING agonists DMXAA or cGAMP greatly enhanced tumor control, which was associated with enhanced CAR T cell persistence in the TME. Using single-cell RNA sequencing, we demonstrate that DMXAA promoted CAR T cell trafficking and persistence, supported by the generation of a chemokine milieu that promoted CAR T cell recruitment and modulation of the immunosuppressive TME through alterations in the balance of immune-stimulatory and suppressive myeloid cells. However, sustained tumor regression was accomplished only with the addition of anti–PD-1 and anti–GR-1 mAb to Th/Tc17 CAR T cell therapy given with STING agonists. This study provides new approaches to enhance adoptive T cell therapy in solid tumors.

Graft-versus-host disease (GVHD) is the major cause of nonrelapse morbidity and mortality after allogeneic stem cell transplantation (allo-SCT). Prevention and treatment of GVHD remain inadequate and commonly lead to end-organ dysfunction and opportunistic infection. The role of interleukin (IL)-17 and IL-22 in GVHD remains uncertain, due to an apparent lack of lineage fidelity and variable and contextually determined protective and pathogenic effects. We demonstrate that donor T cell-derived IL-22 significantly exacerbates cutaneous chronic GVHD and that IL-22 is produced by highly inflammatory donor CD4 T cells posttransplantation. IL-22 and IL-17A derive from both independent and overlapping lineages, defined as T helper (Th)22 and IL-22 Th17 cells. Donor Th22 and IL-22 Th17 cells share a similar IL-6-dependent developmental pathway, and while Th22 cells arise independently of the IL-22 Th17 lineage, IL-17 signaling to donor Th22 directly promotes their development in allo-SCT. Importantly, while both IL-22 and IL-17 mediate skin GVHD, Th17-induced chronic GVHD can be attenuated by IL-22 inhibition in preclinical systems. In the clinic, high levels of both IL-17A and IL-22 expression are present in the skin of patients with GVHD after allo-SCT. Together, these data demonstrate a key role for donor-derived IL-22 in patients with chronic skin GVHD and confirm parallel but symbiotic developmental pathways of Th22 and Th17 differentiation.

Chronic graft-versus-host disease (cGVHD) causes significant morbidity and mortality in patients after allogeneic bone marrow (BM) or stem cell transplantation (allo-SCT). Recent work has indicated that both T and B lymphocytes play an important role in the pathophysiology of cGVHD. Previously, our group showed a critical role for the germinal center response in the function of B cells using a bronchiolitis obliterans (BO) model of cGVHD. Here, we demonstrated for the first time that cGVHD is associated with severe defects in the generation of BM B lymphoid and uncommitted common lymphoid progenitor cells. We found an increase in the number of donor CD4 T cells in the BM of mice with cGVHD that was negatively correlated with B-cell development and the frequency of osteoblasts and Prrx-1-expressing perivascular stromal cells, which are present in the B-cell niche. Use of anti-DR3 monoclonal antibodies to enhance the number of donor regulatory T cells (T) in the donor T-cell inoculum ameliorated the pathology associated with BO in this model. This correlated with an increased number of endosteal osteoblastic cells and significantly improved the generation of B-cell precursors in the BM after allo-SCT. Our work indicates that donor T play a critical role in preserving the generation of B-cell precursors in the BM after allo-SCT. Approaches to enhance the number and/or function of donor T that do not enhance conventional T-cell activity may be important to decrease the incidence and severity of cGVHD in part through normal B-cell lymphopoiesis.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that affects the function and development of immune cells. Here, we show that recipient mice receiving AhR T cells have improved survival and decreased acute graft-versus-host disease (aGVHD) in 2 different murine allogeneic bone marrow transplant (BMT) models. We also show that CD4 T cells lacking AhR demonstrate reduced accumulation in secondary lymphoid tissue because of low levels of proliferation 4 days after BMT. Additionally, we found a significant increase in the quantity of peripherally induced regulatory donor T (pT) cells in the colon of recipients transplanted with AhR T cells 14 days after transplant. Blockade of AhR using a clinically available AhR antagonist greatly enhanced the in vitro generation of inducible T (iT) cells from naïve CD4 human T cells. We have identified AhR as a novel target on donor T cells that is critical to the pathogenesis of aGVHD.