Fuliang Chu

Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions

Foxp3+ regulatory T (Treg) cells suppress different types of immune responses to help maintain homeostasis in the body. How Treg cells regulate humoral immunity, including germinal center reactions, is unclear. Here we identify a subset of Treg cells expressing CXCR5 and Bcl-6 that localize to the germinal centers in mice and humans. The expression of CXCR5 on Treg cells depends on Bcl-6. These CXCR5+Bcl-6+ Treg cells are absent in the thymus but can be generated de novo from CXCR5−Foxp3+ natural Treg precursors. A lack of CXCR5+ Treg cells leads to greater germinal center reactions including germinal center B cells, affinity maturation of antibodies and the differentiation of plasma cells. These results unveil a Bcl-6-CXCR5 axis in Treg cells that drives the development of follicular regulatory T (TFR) cells that function to inhibit the germinal center reactions.

Safety and Activity of PD1 Blockade by Pidilizumab in Combination with Rituximab in Patients with Relapsed Follicular Lymphoma: a Single Group, Open-label, Phase 2 Trial

BACKGROUND Endogenous or iatrogenic antitumour immune responses can improve the course of follicular lymphoma, but might be diminished by immune checkpoints in the tumour microenvironment. These checkpoints might include effects of programmed cell death 1 (PD1), a co-inhibitory receptor that impairs T-cell function and is highly expressed on intratumoral T cells. We did this phase 2 trial to investigate the activity of pidilizumab, a humanised anti-PD1 monoclonal antibody, with rituximab in patients with relapsed follicular lymphoma. METHODS We did this open-label, non-randomised trial at the University of Texas MD Anderson Cancer Center (Houston, TX, USA). Adult (≥18 years) patients with rituximab-sensitive follicular lymphoma relapsing after one to four previous therapies were eligible. Pidilizumab was administered at 3 mg/kg intravenously every 4 weeks for four infusions, plus eight optional infusions every 4 weeks for patients with stable disease or better. Starting 17 days after the first infusion of pidilizumab, rituximab was given at 375 mg/m(2) intravenously weekly for 4 weeks. The primary endpoint was the proportion of patients who achieved an objective response (complete response plus partial response according to Revised Response Criteria for Malignant Lymphoma). Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00904722. FINDINGS We enrolled 32 patients between Jan 13, 2010, and Jan 20, 2012. Median follow-up was 15.4 months (IQR 10.1-21.0). The combination of pidilizumab and rituximab was well tolerated, with no autoimmune or treatment-related adverse events of grade 3 or 4. The most common adverse events of grade 1 were anaemia (14 patients) and fatigue (13 patients), and the most common adverse event of grade 2 was respiratory infection (five patients). Of the 29 patients evaluable for activity, 19 (66%) achieved an objective response: complete responses were noted in 15 (52%) patients and partial responses in four (14%). INTERPRETATION The combination of pidilizumab plus rituximab is well tolerated and active in patients with relapsed follicular lymphoma. Our results suggest that immune checkpoint blockade is worthy of further study in follicular lymphoma. FUNDING National Institutes of Health, Leukemia and Lymphoma Society, Cure Tech, and University of Texas MD Anderson Cancer Center.

Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development

In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4+ T cells specialized in providing help to B lymphocytes in the induction of germinal centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)—a basic helix–loop–helix (bHLH) transcription factor—is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4+ T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.

Cross talk between follicular Th cells and tumor cells in human follicular lymphoma promotes immune evasion in the tumor microenvironment.

The microenvironment of human follicular lymphoma (FL), an incurable B cell non-Hodgkin’s lymphoma, is thought to play a major role in its pathogenesis and course. Microenvironmental cells of likely importance include follicular Th cells (TFH) and regulatory T cells (Tregs), and understanding their interactions with FL tumor cells is necessary to develop novel therapeutic strategies. We found that IL-4 and CD40L are expressed by intratumoral TFH and induce production of CCL17 and CCL22 by FL tumor cells. IL-4 alone induces only CCL17 but enhances stimulation by CD40L of both CCL17 and CCL22. Consistent with our in vitro results, mRNA transcripts of IL-4 correlated with CCL17, but not CCL22, in gene expression profiling studies of FL biopsies, whereas CD40L correlated with both CCL17 and CCL22. Tumor supernatants induced preferential migration of Tregs and IL-4–producing T cells rather than IFN-γ–producing T cells, and Abs to CCR4 significantly abrogated the migration of Tregs. Our results suggest that through two distinct mechanisms, intratumoral TFH induce production of CCL17 and CCL22 by FL tumor cells and facilitate active recruitment of Tregs and IL-4–producing T cells, which, in turn, may stimulate more chemokine production in a feed-forward cycle. Thus, TFH appear to play a major role in generating an immunosuppressive tumor microenvironment that promotes immune escape and tumor survival and growth. Our results provide novel insights into the cross talk among TFH, tumor cells, and Tregs in FL, and offer potential targets for development of therapeutic strategies to overcome immune evasion.

TCL1: a shared tumor-associated antigen for immunotherapy against B-cell lymphomas

Immunotherapy with therapeutic idiotype vaccines offers promise for treatment of B-cell malignancies. However, identification of novel immunogenic lymphoma-associated antigens that are universally expressed is necessary to overcome the barriers of patient-specific idiotype vaccines. Here, we determined whether T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene could be a target for immunotherapy of B-cell malignancies. We show that TCL1 mRNA and protein are selectively expressed in normal B cells but markedly hyperexpressed in multiple human B-cell lymphomas, including follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, and splenic marginal zone B-cell lymphoma. We demonstrated that TCL1-specific CD8(+) T cells can be generated from HLA-A*0201 (HLA-A2)(+) normal donors and identified TCL1(71-78) (LLPIMWQL) as the minimal epitope recognized by these T cells. More importantly, TCL1(71-78) peptide-specific T cells were present in the peripheral blood and tumor-infiltrating lymphocytes of lymphoma patients, could be expanded in vitro, and lysed autologous tumor cells but not normal B cells in an HLA-A2-restricted manner. Our results suggest that TCL1 is naturally processed and presented on the surface of lymphoma cells for recognition by cytotoxic T cells and can serve as a novel target for development of immunotherapeutic strategies against common B-cell lymphomas.

Selective targeting of Toll-like receptors and OX40 inhibit regulatory T cell function in follicular lymphoma

Immunotherapeutic strategies are promising approaches for the treatment of follicular lymphoma (FL). However, their efficacy may be limited by immunosuppressive elements in the immune system and tumor microenvironment. Therefore, strategies to reverse the effects of the immunosuppressive elements are needed. We observed that regulatory T cells (Tregs) were increased in the peripheral blood at diagnosis and persisted in high numbers after induction of clinical remission with a cyclophosphamide and doxorubicin‐containing chemotherapy regimen in FL patients. High levels of peripheral blood Tregs prior to therapy were associated with decreased progression‐free survival in FL patients treated with either chemotherapy or combination immunotherapy that targeted CD20 and PD‐1 with monoclonal antibodies rituximab and pidilizumab, respectively. Intratumoral and peripheral blood Tregs potently suppressed autologous antitumor effector T cells in FL. However, the effects of FL Tregs could be reversed by triggering Toll‐like receptors (TLR) with TLR ligands Pam3CSK4 (TLR 1/2), flagellin (TLR 5), and CpG‐B (TLR 9), and/or OX40. The TLR ligands synergized with each other as well as OX40 signaling to inhibit Tregs. Furthermore, they restored the function of FL tumor‐specific effector T cells. Our results suggest that a state of tolerance exists in FL patients at diagnosis and after induction of clinical remission, and agents that activate TLRs 1/2, 5, and 9, and OX40 may serve as adjuvants to enhance the efficacy of antitumor immunotherapeutic strategies and preventive vaccines against infectious diseases in these patients.

Bypassing STAT3-mediated inhibition of the transcriptional regulator ID2 improves the antitumor efficacy of dendritic cells

Blocking tumor-derived cytokine signaling in dendritic cells enables engineered dendritic cells to function as an antitumor vaccine. Engineering antitumor activity Dendritic cells are critical mediators of the immune response to infection. Despite their potential, antitumor vaccines based on injecting dendritic cells have not been effective. Using mouse models of melanoma, Li et al. found that tumor cells release cytokines that suppress the ability of tumor-infiltrating dendritic cells to mount an antitumor response. The cytokines released from melanoma cells activated the transcription factor STAT3, which repressed the expression of the gene encoding the transcription regulator ID2. Constitutively expressing ID2 dampened production of proinflammatory cytokines in dendritic cells and promoted dendritic cell–mediated immunostimulatory lymphocyte responses. Vaccination experiments in tumor-bearing mice suggested that engineering dendritic cells to overcome or prevent the tumor-derived cytokine response in the injected dendritic cells, by either deleting STAT3 or overexpressing ID2, might prove an effective immunotherapy strategy in cancer patients. Despite the potent ability of dendritic cells (DCs) to stimulate lymphocyte responses and host immunity, granulocyte-macrophage colony-stimulating factor–derived DCs (GM-DCs) used as antitumor vaccines have demonstrated relatively modest success in cancer immunotherapy. We found that injecting GM-DCs into melanoma tumors in mice, or culturing GM-DCs with melanoma-secreted cytokines or melanoma-conditioned medium, rapidly suppressed DC-intrinsic expression of the gene encoding inhibitor of differentiation 2 (ID2), a transcriptional regulator. Melanoma-associated cytokines repressed Id2 transcription in murine DCs through the activation of signal transducer and activator of transcription 3 (STAT3). Enforced expression of ID2 in GM-DCs (ID2–GM-DCs) suppressed their production of the proinflammatory cytokine tumor necrosis factor–α (TNF-α). Vaccination with ID2–GM-DCs slowed the progression of melanoma tumors and enhanced animal survival, which was associated with an increased abundance of tumor-infiltrating interferon-γ–positive CD4+ effector and CD8+ cytotoxic T cells and a decreased number of tumor-infiltrating regulatory CD4+ T cells. The efficacy of the ID2–GM-DC vaccine was improved by combinatorial treatment with a blocking antibody to programmed cell death protein–1 (PD-1), a current immunotherapy that overcomes suppressive immune checkpoint signaling. Collectively, our data reveal a previously unrecognized STAT3-mediated immunosuppressive mechanism in DCs and indicate that DC-intrinsic ID2 promotes tumor immunity by modulating tumor-associated CD4+ T cell responses. Thus, inhibiting STAT3 or overexpressing ID2 selectively in DCs may improve the efficiency of DC vaccines in cancer therapy.

IL-15 enhances the antitumor effect of human antigen-specific CD8+ T cells by cellular senescence delay

ABSTRACT Optimal expansion protocols for adoptive human T-cell therapy often include interleukin (IL)-15; however, the mechanism by which IL-15 improves the in vivo antitumor effect of T cells remains to be elucidated. Using human T cells generated from HLA-A2+ donors against novel T-cell epitopes derived from the human U266 myeloma cell line Ig light chain V-region (idiotype) as a model, we found that T cells cultured with IL-15 provided superior resistance to tumor growth in vivo, compared with IL-2, after adoptive transfer into immunodeficient hosts. This effect of IL-15 was associated with delayed/reversed senescence in tumor antigen-specific memory CD8+ T cells mediated through downregulation of P21WAF1, P16INK4a, and P53 expression. Compared to IL-2, IL-15 stimulation dramatically activated JAK3-STAT5 signaling and inhibited the expression of DNA damage genes. Thus, our study elucidates a new mechanism for IL-15 in the regulation of STAT signaling pathways and CD8+ T-cell senescence.

CXCR5+CD8+ T cells are localized in B cell follicles and germinal centers and exhibit regulatory and anti-tumor function

Germinal centers (GC) are specialized anatomic sites where B cells undergo somatic hypermutation, clonal expansion, and affinity-based selection. Stringent regulation of GC reactions is critical for homeostatic B cell development, as well as T-dependent humoral immunity against self and foreign antigens. Follicular helper T cells (Tfh, CXCR5hiPDhiBcl-6+CD4+) that reside in GC serve as specialized B helper T cells and provide survival and selection signals to GC B cells. Despite recent understandings of Tfh function in autoimmunity, immunodeficiency, and B-cell lymphoma, mechanisms that regulate Tfh development and function remain limited. In the current study, we determined the presence of CD45RA-CXCR5+CD8+ T cells in human peripheral blood, tonsillar tissues, and follicular lymphoma (FL) tumor samples by FACS and confocal microscopy. We found that CXCR5+CD8+ T cells were present in high numbers and localized to GCs and T cell zones in the tonsillar tissues and FL, but present in low numbers in the peripheral blood. Surface marker analysis suggested that these cells displayed an activated status. Importantly, FL tumor samples had significantly more CXCR5+CD8+ T cells than tonsillar tissues. Further analysis using intracellular cytokine staining showed that CXCR5+CD8+ T cells produced higher levels of IFN-γ and TNF-α compared to CXCR5-CD8+ subset. Next, we performed T-B cell co-culture experiments to evaluate whether CXCR5+CD8+ T cells could regulate Tfh function in humans. FACS-sorted Tfh cells were co-cultured with naive or memory B cells in the presence or absence of CXCR5+CD8+ T cells. These experiments indicated that CXCR5+CD8+ T cells suppressed Tfh function, as demonstrated by reduced differentiation of naive and/or memory B cells into plasmablasts cells (CD19intCD38+). To test CXCR5+CD8+ T cell function in vivo, we employed the EG7 lymphoma model in TCR-/- mice. Briefly, ovabulmin-primed OT1-specific CXCR5+CD8+ T cells were adoptively transferred into EG7 tumor-bearing TCR-/- mice, and tumor size and survival rate were determined. In summary, our data suggest that CXCR5+CD8+ T cells may have multiple roles, as they not only inhibited Tfh function, but also exhibited strong anti-tumor immunity.

Anti-PD-1 antibodies for the treatment of B-cell lymphoma: Importance of PD-1+ T-cell subsets

Monoclonal antibodies specific for programmed cell death 1 (PDCD1, best known as PD-1) have been shown to mediate antineoplastic effects in follicular lymphoma patients. However, the relative proportion of intratumoral PD-1+ T-cell subsets, in particular follicular helper T cells (which exert pro-tumor functions) and effector T cells (which have anticancer activity), may impact clinical outcome, and should therefore be carefully considered for patient selection in this setting.