Raewyn Broady

CD4+ T‐regulatory cells: toward therapy for human diseases

T‐regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function of different types of Tregs can lead to autoimmunity, allergy, and graft rejection, whereas an over‐abundance of Tregs can inhibit anti‐tumor and anti‐pathogen immunity. Experimental models in mice have demonstrated that manipulating the numbers and/or function of Tregs can decrease pathology in a wide range of contexts, including transplantation, autoimmunity, and cancer, and it is widely assumed that similar approaches will be possible in humans. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4+ Tregs: forkhead box protein 3 (FOXP3)+ Tregs and interleukin‐10‐producing type 1 Tregs (Tr1 cells). The aim of this review is to highlight current information on the characteristics of human FOXP3+ Tregs and Tr1 cells that make them an attractive therapeutic target. We discuss the progress and limitations that must be overcome to develop methods to enhance Tregs in vivo, expand or induce them in vitro for adoptive transfer, and/or inhibit their function in vivo. Although many technical and theoretical challenges remain, the next decade will see the first clinical trials testing whether Treg‐based therapies are effective in humans.

Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor

Adoptive immunotherapy with regulatory T cells (Tregs) is a promising treatment for allograft rejection and graft-versus-host disease (GVHD). Emerging data indicate that, compared with polyclonal Tregs, disease-relevant antigen-specific Tregs may have numerous advantages, such as a need for fewer cells and reduced risk of nonspecific immune suppression. Current methods to generate alloantigen-specific Tregs rely on expansion with allogeneic antigen-presenting cells, which requires access to donor and recipient cells and multiple MHC mismatches. The successful use of chimeric antigen receptors (CARs) for the generation of antigen-specific effector T cells suggests that a similar approach could be used to generate alloantigen-specific Tregs. Here, we have described the creation of an HLA-A2-specific CAR (A2-CAR) and its application in the generation of alloantigen-specific human Tregs. In vitro, A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function before, during, and after A2-CAR-mediated stimulation. In mouse models, human A2-CAR-expressing Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic GVHD caused by HLA-A2+ T cells. Together, our results demonstrate that use of CAR technology to generate potent, functional, and stable alloantigen-specific human Tregs markedly enhances their therapeutic potential in transplantation and sets the stage for using this approach for making antigen-specific Tregs for therapy of multiple diseases.

Inflammatory Effects of Ex Vivo Human Th17 Cells Are Suppressed by Regulatory T Cells

Th17 cells are proinflammatory cells associated with many immune-mediated diseases. Major factors limiting the study of human Th17 cells are the lack of an accepted method for their in vitro differentiation or for isolation of a homogenous population of Th17 cells that do not cosecrete IFN-γ. To overcome these hurdles, we established a novel method to isolate in vivo differentiated Th17 cells from peripheral blood by sorting CD161+CCR4+CCR6+CXCR3−CD4+ T cells. The resulting cells produce high levels of IL-17 but not IFN-γ, express high levels of retinoic acid-related orphan receptor variant 2, and maintain this phenotype upon expansion. Ex vivo Th17 cells exhibit a low cytotoxic potential and are hyporesponsive to polyclonal anti-CD3/anti-CD28 stimulation. Importantly, ex vivo Th17 cells were susceptible to suppression by both naive and memory regulatory T cells (Tregs), which inhibited production of IL-17, IL-22, and CXCL8. Moreover, Tregs suppressed the antifibrotic effects of Th17 cells in a wound-healing model. These findings provide new tools for the study of normal and pathological functions of bona fide Th17 cells in humans. They also provide new insight into the cross-talk between Th17 cells and immune and nonimmune cells, and they establish the paradigm that adoptive Treg-based therapies may effectively limit Th17-mediated inflammation.

Cutaneous GVHD is associated with the expansion of tissue-localized Th1 and not Th17 cells

Studies in mice have shown that proinflammatory Th17 cells can cause acute graft-versus-host disease (aGVHD) related tissue damage; however, whether they play a role in human aGVHD remains unclear. In a prospective study, we measured the proportion of Th17 cells in the blood and skin of patients at the onset of aGVHD. We found no difference in the proportion or amount of IL-17 produced by T cells in the blood of patients with aGVHD (n = 20) compared with time-matched patients without GVHD (n = 14). Moreover, Th17 cells were not increased in the skin of patients with cutaneous aGVHD (n = 7) compared with healthy controls (n = 10). In contrast, we found significantly more interferon-γ-producing T cells in the skin of patients with aGVHD compared with controls. These data support the long-standing paradigm that tissue localized interferon-γ-producing cells are the perpetrators of aGVHD.

Control of tissue-localized immune responses by human regulatory T cells

Treg cells control immune responses to self and nonharmful foreign antigens. Emerging data from animal models indicate that Treg cells function in both secondary lymphoid organs and tissues, and that these different microenvironments may contain specialized subsets of Treg cells with distinct mechanisms of action. The design of therapies for the restoration of tissue‐localized immune homeostasis is dependent upon understanding how local immune responses are influenced by Treg cells in health versus disease. Here we review the current state of knowledge about human Treg cells in four locations: the skin, lung, intestine, and joint. Despite the distinct biology of these tissues, there are commonalities in the biology of their resident Treg cells, including phenotypic and functional differences from circulating Treg cells, and the presence of cytokine‐producing (e.g. IL‐17+) FOXP3+ cells. We also highlight the challenges to studying tissue Treg cells in humans, and opportunities to use new technologies for the detailed analysis of Treg cells at the single‐cell level. As emerging biological therapies are increasingly targeted toward tissue‐specific effects, it is critical to understand their potential impact on local immune regulation.

Transcriptome Analysis Reveals Markers of Aberrantly Activated Innate Immunity in Vitiligo Lesional and Non-Lesional Skin

Background Vitiligo is characterized by the death of melanocytes in the skin. This is associated with the presence of T cell infiltrates in the lesional borders. However, at present, there is no detailed and systematic characterization on whether additional cellular or molecular changes are present inside vitiligo lesions. Further, it is unknown if the normal appearing non-lesional skin of vitiligo patients is in fact normal. The purpose of this study is to systematically characterize the molecular and cellular characteristics of the lesional and non-lesional skin of vitiligo patients. Methods and Materials Paired lesional and non-lesional skin biopsies from twenty-three vitiligo patients and normal skin biopsies from sixteen healthy volunteers were obtained with informed consent. The following aspects were analyzed: (1) transcriptome changes present in vitiligo skin using DNA microarrays and qRT-PCR; (2) abnormal cellular infiltrates in vitiligo skin explant cultures using flow cytometry; and (3) distribution of the abnormal cellular infiltrates in vitiligo skin using immunofluorescence microscopy. Results Compared with normal skin, vitiligo lesional skin contained 17 genes (mostly melanocyte-specific genes) whose expression was decreased or absent. In contrast, the relative expression of 13 genes was up-regulated. The up-regulated genes point to aberrant activity of the innate immune system, especially natural killer cells in vitiligo. Strikingly, the markers of heightened innate immune responses were also found to be up-regulated in the non-lesional skin of vitiligo patients. Conclusions and Clinical Implications As the first systematic transcriptome characterization of the skin in vitiligo patients, this study revealed previously unknown molecular markers that strongly suggest aberrant innate immune activation in the microenvironment of vitiligo skin. Since these changes involve both lesional and non-lesional skin, our results suggest that therapies targeting the entire skin surface may improve treatment outcomes. Finally, this study revealed novel mediators that may facilitate future development of vitiligo therapies.

Novel agents improve survival of transplant patients with multiple myeloma including those with high-risk disease defined by early relapse (<12 months)

The treatment of multiple myeloma (MM) has changed with the advent of thalidomide, bortezomib, and lenalidomide, the so-called novel agents (NAs). Given the complexity of MM therapy in the NA era we pursued a population based study to assess for improvements in survival as well as to characterize the relevance of early relapse (within 12 months) and the International Staging System in this clinical setting. We reviewed our experience with 460 patients with MM treated with autologous stem cell transplant (ASCT) between 1988 and 2008, of whom 306 had relapsed. The cohort was divided into two groups based upon relapse pre-2004 and relapse during/after 2004 (2004+), which correlated to availability of bortezomib and lenalidomide. Improvements in both overall survival (OS) (median 32.0 months vs. 71.8 months; p < 0.001) and post-relapse survival (PRS) (median 15.2 months vs. 42.8 months; p < 0.001) correlated with the NA era. Exposure to NAs conferred a better PRS (median 35.7 months vs. 9.1 months; p < 0.001). Although all patients had improvements in survival, those who relapsed late continued to do better. Lastly, in the NA era, the ISS remains an important prognostic tool in relapse, but only in the late relapsing cohort.

Therapeutic drug monitoring for triazoles: A needs assessment review and recommendations from a Canadian perspective

Therapeutic drug monitoring (TDM) is necessary for certain drugs to ensure that the levels are sufficient to be effective, but not so high as to cause adverse effects. This review summarizes the literature regarding TDM for newer-generation extended-spectrum triazoles, including when TDM may be necessary for each drug and why, and laboratory techniques used for the measurement of levels of these drugs. The document includes recommendations for the use of TDM for each triazole that is discussed.

Allogeneic haematopoietic stem cell transplantation for chronic lymphocytic leukaemia: outcome in a 20‐year cohort

The curative potential of allogeneic haematopoietic stem cell transplant (allo HSCT) in chronic lymphocytic leukaemia CLL is established, with a demonstrated role for graft‐versus‐leukaemia and less certainty for other factors in determining outcome. The first two decades of CLL patients proceeding to allo HSCT at the Leukaemia/Bone Marrow Transplant Program of British Columbia (n = 49 consecutive, 1991–2009) were studied to clarify factors predicting outcome. The donor was related in 29 (59%) and unrelated in 20 (41%). Conditioning was reduced‐intensity in 27 (55%) and myeloablative in 22 (45%). Thirty‐one of 49 patients survive with median follow‐up of 5 years (0·2–15). Cumulative incidence of non‐relapse mortality; complete remission (CR); clearance of fluorescence in situ hybridization (FISH) abnormality and progression at 10 years was 36%; 69%; 55% and 22%. Overall survival (OS) was 63% at 2 years; 55% at 5 years and beyond. Factors predicting OS (P value by log rank <0·05) were: comorbidity index <3, FISH rank (Dohner) and 17p deletion, alemtuzumab pre‐HSCT, achievement of CR post‐HSCT, donor chimerism >90%, clearance of FISH abnormality post‐HSCT and absence of high‐grade (3–4) graft‐versus‐host disease. Results from this province‐wide, two‐decade cohort demonstrated that a substantial proportion of patients with high‐risk CLL become long term disease‐free survivors.

Outcome of Patients With Non-Hodgkin Lymphomas With Concurrent MYC and BCL2 Rearrangements Treated With CODOX-M/IVAC With Rituximab Followed by Hematopoietic Stem Cell Transplantation

BACKGROUND Double-hit lymphoma is characterized by the presence of concurrent MYC (myelocytomatosis oncogene) and BCL2 (B-cell lymphoma 2) gene rearrangements. Prognosis is poor with standard chemoimmunotherapy. Since 2003, the British Columbia Cancer Agency has used CODOX-M/IVAC+R (cyclophosphamide, vincristine, doxorubicin, methotrexate, cytarabine, ifosfamide, and etoposide, combined with rituximab) followed by consolidative hematopoietic cell transplantation as definitive treatment for double-hit lymphoma. PATIENTS AND METHODS A retrospective review of the survival outcomes of patients with double-hit lymphoma treated at our institution was conducted. Thirty-two patients diagnosed with non-Hodgkin lymphoma with concurrent MYC and BCL2 translocations from 2003 to 2013 were identified. Cases with MYC or BCL2 amplification and those with overexpression in immunohistochemistry analysis were excluded. RESULTS Median age at diagnosis was 53.0 years (range, 35.5-70.9 years), 23 (72%) were male, and 30 (94%) had stage III to IV disease. CODOX-M/IVAC+R was administered in 25 (78%) patients and 20 (80%) achieved a partial remission or better, of which 9 (36%) had a complete remission. Nineteen of the 32 (59%) patients underwent upfront hematopoietic cell transplantation. At a median follow-up of living patients of 26.4 months, 14 (44%) were alive in remission, 15 (47%) died, and 3 (9%) were alive in relapse. The 2-year progression-free survival (PFS) and overall survival (OS) of all patients were 41% and 53%, respectively. The sixteen patients treated with CODOX-M/IVAC+R followed by hematopoietic cell transplantation had a 2-year PFS of 60% and 2-year OS of 82%. CONCLUSION Patients with double-hit lymphoma treated with CODOX-M/IVAC+R followed by hematopoietic cell transplantation can achieve durable remissions, although disease progression before transplantation remains a significant problem.