Maria-Luisa Alegre

Modulation of tryptophan catabolism by regulatory T cells

Regulatory T (TR) cells manifest constitutive expression of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), but the function of CTLA-4 in mediating the regulatory function of TR cells is unclear. We show here that mouse CD4+CD25+ cells, either resting or induced to overexpress CTLA-4 by treatment with antibody to CD3, initiated tryptophan catabolism in dendritic cells through a CTLA-4-dependent mechanism. This process required B7 expression and cytokine production by the dendritic cells. In contrast, TR cells cultured in the presence of bacterial lipopolysaccharide induced tryptophan catabolism by dendritic cells in a CTLA-4-independent but cytokine-dependent way. Thus, regulation of immunosuppressive tryptophan catabolism in dendritic cells might represent a major mechanism of action of TR cells.

T-cell regulation by CD28 and CTLA-4.

Activation of T lymphocytes is thought to require at least two signals, one delivered by the T-cell receptor complex after antigen recognition, and one provided on engagement of co-stimulatory receptors, such as CD28. Recent studies are providing clues as to the specific signalling roles of co-stimulatory receptors. Furthermore, superimposition of inhibitory signals, such as those delivered by cytotoxic T-lymphocyte antigen 4 (CTLA-4), leads to a complex network of positive and negative co-stimulatory signals, the integration of which modulates immune responses.

Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy.

Gut microbes affect immunotherapy The unleashing of antitumor T cell responses has ushered in a new era of cancer treatment. Although these therapies can cause dramatic tumor regressions in some patients, many patients inexplicably see no benefit. Mice have been used in two studies to investigate what might be happening. Specific members of the gut microbiota influence the efficacy of this type of immunotherapy (see the Perspective by Snyder et al.). Vétizou et al. found that optimal responses to anticytotoxic T lymphocyte antigen blockade required specific Bacteroides spp. Similarly, Sivan et al. discovered that Bifidobacterium spp. enhanced the efficacy of antiprogrammed cell death ligand 1 therapy. Science, this issue, p. 1079 and p. 1084; see also p. 1031 Gut microbes modulate the effectiveness of cancer immunotherapies in mice. [Also see Perspective by Snyder et al.] T cell infiltration of solid tumors is associated with favorable patient outcomes, yet the mechanisms underlying variable immune responses between individuals are not well understood. One possible modulator could be the intestinal microbiota. We compared melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing or after fecal transfer. Sequencing of the 16S ribosomal RNA identified Bifidobacterium as associated with the antitumor effects. Oral administration of Bifidobacterium alone improved tumor control to the same degree as programmed cell death protein 1 ligand 1 (PD-L1)–specific antibody therapy (checkpoint blockade), and combination treatment nearly abolished tumor outgrowth. Augmented dendritic cell function leading to enhanced CD8+ T cell priming and accumulation in the tumor microenvironment mediated the effect. Our data suggest that manipulating the microbiota may modulate cancer immunotherapy.

CTLA-4 Gene Polymorphism at Position 49 in Exon 1 Reduces the Inhibitory Function of CTLA-4 and Contributes to the Pathogenesis of Graves’ Disease

Activation of T cells requires at least two signals transduced by the Ag-specific TCR and a costimulatory ligand such as CD28. CTLA-4, expressed on activated T cells, binds to B7 present on APCs and functions as a negative regulator of T cell activation. Our laboratory previously reported the association of Graves’ disease (GD) with a specific CTLA-4 gene polymorphism. In theory, reduced expression or function of CTLA-4 might augment autoimmunity. In the present study, we categorized autoimmune thyroid disease patients and normal controls (NC) by genotyping a CTLA-4 exon 1 polymorphism and investigated the function of CTLA-4 in all subjects. PBMCs and DNA were prepared from GD (n = 45), Hashimoto’s thyroiditis (HT) (n = 18), and NC (n = 43). There were more GD patients with the G/G or A/G alleles (82.2% vs 65.1% in NC), and significantly fewer patients with the A/A allele (17.8% vs 34.9% in NC). In the presence of soluble blocking anti-human CTLA-4 mAb, T cell proliferation following incubation with allogeneic EBV-transformed B cells was augmented in a dose-dependent manner. Augmentation induced by CTLA-4 mAb was similar in GD and NC (GD, HT, NC = 156%, 164%, 175%, respectively). We related CTLA-4 polymorphism to mAb augmentation of T cell proliferation in each subgroup (GD, HT, NC). Although PBMC from individuals with the G/G alleles showed 132% augmentation, those with the A/A alleles showed 193% augmentation (p = 0.019). CTLA-4 polymorphism affects the inhibitory function of CTLA-4. The G allele is associated with reduced control of T cell proliferation and thus contributes to the pathogenesis of GD and presumably of other autoimmune diseases.

Targeting the NF-κB signaling pathway in Notch1-induced T-cell leukemia

T-cell acute lymphoblastic leukemia (T-ALL), unlike other ALL types, is only infrequently associated with chromosomal aberrations, but it was recently shown that most individuals with T-ALL carry activating mutations in the NOTCH1 gene. However, the signaling pathways and target genes responsible for Notch1-induced neoplastic transformation remain undefined. We report here that constitutively active Notch1 activates the NF-κB pathway transcriptionally and via the IκB kinase (IKK) complex, thereby causing increased expression of several well characterized target genes of NF-κB in bone marrow hematopoietic stem cells and progenitors. Our observations demonstrate that the NF-κB pathway is highly active in established human T-ALL and that inhibition of the pathway can efficiently restrict tumor growth both in vitro and in vivo. These findings identify NF-κB as one of the major mediators of Notch1-induced transformation and suggest that the NF-κB pathway is a potential target of future therapies of T-ALL.

TLR Engagement Prevents Transplantation Tolerance

In many experimental models, heart, pancreas and kidney allografts are accepted long‐term following costimulation‐targeting therapies, whereas skin, lung and intestine resist the induction of tolerance under the same regimens. We noted that a common feature of the resistant organs is their constant exposure to commensal microbes and hypothesized that these microorganisms may stimulate Toll‐like receptors (TLRs), promote alloresponses and prevent tolerance induction. This hypothesis prompts the predictions that TLR engagement at the time of transplantation should avert tolerance to heart allografts in animals treated with costimulation‐targeting therapies, whereas inhibition of TLR signaling should promote tolerance to skin allografts under the same conditions. Indeed, engagement of a single TLR was sufficient to prevent anti‐CD154‐mediated long‐term cardiac allograft acceptance and correlated with abolished intragraft recruitment of CD4+/FoxP3+ regulatory T cells and the development of linked‐suppression. Conversely, a lack of donor and recipient MyD88‐dependent signaling led to successful skin allograft acceptance in anti‐CD154‐treated animals. Thus, the status of TLR signaling contributes to the resistance versus susceptibility of organs to transplantation tolerance.

A non-activating humanized anti-CD3 monoclonal antibody retains immunosuppressive properties in vivo

OKT3, a mouse anti-human CD3 mAb, is a potent immunosuppressive agent used in clinical transplantation to prevent or treat allograft rejection. Associated with this therapy is the systemic release of several cytokines that result in a series of adverse side effects. This release of cytokines is dependent on the cross-linking mediated by OKT3 between T cells and the FcγR-bearing cells. To generate an anti-human CD3 mAb with reduced activating properties as compared with OKT3, we have transferred the complementary determining regions of OKT3 onto human IgG frameworks and then performed point mutations that reduce the affinity of the “humanized” anti-CD3 mAbs for FcγRs. Initial, in vitro, studies showed that whereas OKT3 and the parental humanized anti-CD3 mAbs activated T cells similarly, a humanized Fc variant failed to do so. Both the Fc variant and the activating anti-CD3 mAbs induced comparable modulation of the TCR and suppression of cytolytic T cell activity, in vitro. In the current study, we exploited an experimental model in which human splenocytes from cadaveric organ donors were inoculated into severe combined immunodeficient mice (hu-SPL-SCID mice) to test the activating and immunosuppressive properties of these anti-human CD3 mAbs in vivo. Unlike injection of OKT3 or of the parental humanized mAb, administration of the Fc variant did not result in T cell activation in vivo, as evidenced by the lack of induction of surface markers of activation, and of systemic human cytokines, including IL-2. Importantly, similar prolongation of human allograft survival was achieved with all anti-CD3 mAbs, indicating that the nonactivating anti-CD3 mAbs retained significant immunosuppressive properties in vivo. Thus, the use of an Fc variant in clinical transplantation should result in fewer side effects than observed with OKT3, while maintaining its clinical efficacy.

Local expression of B7-H1 promotes organ-specific autoimmunity and transplant rejection

A number of studies have suggested B7-H1, a B7 family member, inhibits T cell responses. Therefore, its expression on nonlymphoid tissues has been proposed to prevent T cell-mediated tissue destruction. To test this hypothesis, we generated transgenic mice that expressed B7-H1 on pancreatic islet beta cells. Surprisingly, we observed accelerated rejection of transplanted allogeneic B7-H1-expressing islet beta cells. Furthermore, transgenic B7-H1 expression broke immune tolerance, as some of the mice spontaneously developed T cell-dependent autoimmune diabetes. In addition, B7-H1 expression increased CD8+ T cell proliferation and promoted autoimmunity induction in a T cell adoptive transfer model of diabetes. Consistent with these findings, B7-H1.Ig fusion protein augmented naive T cell priming both in vitro and in vivo. Our results demonstrate that B7-H1 can provide positive costimulation for naive T cells to promote allograft rejection and autoimmune disease pathogenesis.

Role of natural killer cell subsets in cardiac allograft rejection

To achieve donor‐specific immune tolerance to allogeneic organ transplants, it is imperative to understand the cell types involved in acute allograft rejection. In wild‐type mice, CD4+ T cells are necessary and sufficient for acute rejection of cardiac allografts. However, when T‐cell responses are suboptimal, such as in mice treated with costimulation‐targeting agents or in CD28‐deficient mice, and perhaps in transplanted patients taking immunosuppressive drugs, the participation of other lymphocytes such as CD8+ T cells and NK1.1+ cells becomes apparent. We found that host NK but not NKT cells were required for cardiac rejection. Ly49G2+ NK cells suppressed rejection, whereas a subset of NK cells lacking inhibitory Ly49 receptors for donor MHC class I molecules was sufficient to promote rejection. Notably, rejection was independent of the activating receptors Ly49D and NKG2D. Finally, our experiments supported a mechanism by which NK cells promote expansion and effector function of alloreactive T cells. Thus, therapies aimed at specific subsets of NK cells may facilitate transplantation tolerance in settings of impaired T‐cell function.

The commensal microbiome is associated with anti–PD-1 efficacy in metastatic melanoma patients

Good bacteria help fight cancer Resident gut bacteria can affect patient responses to cancer immunotherapy (see the Perspective by Jobin). Routy et al. show that antibiotic consumption is associated with poor response to immunotherapeutic PD-1 blockade. They profiled samples from patients with lung and kidney cancers and found that nonresponding patients had low levels of the bacterium Akkermansia muciniphila. Oral supplementation of the bacteria to antibiotic-treated mice restored the response to immunotherapy. Matson et al. and Gopalakrishnan et al. studied melanoma patients receiving PD-1 blockade and found a greater abundance of “good” bacteria in the guts of responding patients. Nonresponders had an imbalance in gut flora composition, which correlated with impaired immune cell activity. Thus, maintaining healthy gut flora could help patients combat cancer. Science, this issue p. 91, p. 104, p. 97; see also p. 32 Gut bacteria influence patient response to cancer therapy. Anti–PD-1–based immunotherapy has had a major impact on cancer treatment but has only benefited a subset of patients. Among the variables that could contribute to interpatient heterogeneity is differential composition of the patients’ microbiome, which has been shown to affect antitumor immunity and immunotherapy efficacy in preclinical mouse models. We analyzed baseline stool samples from metastatic melanoma patients before immunotherapy treatment, through an integration of 16S ribosomal RNA gene sequencing, metagenomic shotgun sequencing, and quantitative polymerase chain reaction for selected bacteria. A significant association was observed between commensal microbial composition and clinical response. Bacterial species more abundant in responders included Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium. Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti–PD-L1 therapy. Our results suggest that the commensal microbiome may have a mechanistic impact on antitumor immunity in human cancer patients.