Monitoring PD-1 Phosphorylation to Evaluate PD-1 Signaling during Antitumor Immune Responses.

PD-1 expression marks activated T cells susceptible to PD-1-mediated inhibition but not whether a PD-1-mediated signal is being delivered. Molecular predictors of response to PD-1 immune checkpoint blockade (ICB) are needed. We describe a monoclonal antibody (mAb) that detects PD-1 signaling through the detection of phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of mouse and human PD-1 (phospho-PD-1). We showed PD-1+ tumor-infiltrating lymphocytes (TILs) in MC38 murine tumors had high phosphorylated PD-1, particularly in PD-1+TIM-3+ TILs. Upon PD-1 blockade, PD-1 phosphorylation was decreased in CD8+ TILs. Phospho-PD-1 increased in T cells from healthy human donors after PD-1 engagement and decreased in patients with Hodgkin lymphoma following ICB. These data demonstrate that phosphorylation of the ITSM motif of PD-1 marks dysfunctional T cells that may be rescued with PD-1 blockade. Detection of phospho-PD-1 in TILs is a potential biomarker for PD-1 immunotherapy responses.

KIR3DL3 Is an Inhibitory Receptor for HHLA2 that Mediates an Alternative Immunoinhibitory Pathway to PD1.

Blockade of the PD1 pathway is a broadly effective cancer therapy, but additional immune-inhibitory pathways contribute to tumor immune evasion. HERV-H LTR-associating 2 (HHLA2; also known as B7H5 and B7H7) is a member of the B7 family of immunoregulatory ligands that mediates costimulatory effects through its interaction with the CD28 family member transmembrane and immunoglobulin domain containing 2 (TMIGD2). However, HHLA2 has also been known to have inhibitory effects on T cells. Here, we report that we have identified killer cell immunoglobulin-like receptor, three immunoglobulin domains and long cytoplasmic tail 3 (KIR3DL3) as an inhibitory receptor for HHLA2 in T cells and natural killer (NK) cells and have generated HHLA2 and KIR3DL3 antibodies that block the immune-inhibitory activity of HHLA2, preserving the costimulatory signal. It is known that HHLA2 is frequently expressed in several tumor types, including clear cell renal cell carcinoma (ccRCC). We found that HHLA2 expression was nonoverlapping with PDL1 expression in ccRCC, suggesting that HHLA2 mediates a mechanism of tumor immune evasion that is independent from PDL1. Blockade of both the PD1 and KIR3DL3 pathways may be a more effective way to reverse tumor immune evasion.See related Spotlight on p. 128.

A secreted PD-L1 splice variant that covalently dimerizes and mediates immunosuppression.

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.

PD-L1 Binds to B7-1 Only In Cis on the Same Cell Surface.

Programmed death ligand 1 (PD-L1)-mediated immunosuppression regulates peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but also binds to B7-1 (CD80) to regulate T-cell function. The binding interaction of PD-L1 with B7-1 and its functional role need further investigation to understand differences between PD-1 and PD-L1 tumor immunotherapy. We examined the molecular orientation of PD-L1 binding to B7-1 using cell-to-cell binding assays, ELISA, and flow cytometry. As expected, PD-L1-transfected cells bound to PD-1-transfected cells, and B7-1 cells bound to CD28 or CTLA-4-transfected cells; however, PD-L1 cells did not bind to B7-1 cells. By ELISA and flow cytometry with purified proteins, we found PD-L1 and B7-1 had a strong binding interaction only when PD-L1 was flexible. Soluble PD-1 and B7-1 competed for binding to PD-L1. Binding of native PD-L1 and B7-1 in cis on the same cell surface was demonstrated with NanoBiT proximity assays. Thus, PD-L1-B7-1 interaction can occur in cis on the same cell but not in trans between two cells, which suggests a model in which PD-L1 can bend via its 11-amino acid, flexible stalk to bind to B7-1 in cis, in a manner that can competitively block the binding of PD-L1 to PD-1 or of B7-1 to CD28. This binding orientation emphasizes the functional importance of coexpression of PD-L1 and B7-1 on the same cell. We found such coexpression on tumor-infiltrating myeloid cells. Our findings may help better utilize these pathways in cancer immunotherapy. Cancer Immunol Res; 6(8); 921-9. ©2018 AACR.

RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance.

We report that programmed death ligand 2 (PD-L2), a known ligand of PD-1, also binds to repulsive guidance molecule b (RGMb), which was originally identified in the nervous system as a co-receptor for bone morphogenetic proteins (BMPs). PD-L2 and BMP-2/4 bind to distinct sites on RGMb. Normal resting lung interstitial macrophages and alveolar epithelial cells express high levels of RGMb mRNA, whereas lung dendritic cells express PD-L2. Blockade of the RGMb-PD-L2 interaction markedly impaired the development of respiratory tolerance by interfering with the initial T cell expansion required for respiratory tolerance. Experiments with PD-L2-deficient mice showed that PD-L2 expression on non-T cells was critical for respiratory tolerance, but expression on T cells was not required. Because PD-L2 binds to both PD-1, which inhibits antitumor immunity, and to RGMb, which regulates respiratory immunity, targeting the PD-L2 pathway has therapeutic potential for asthma, cancer, and other immune-mediated disorders. Understanding this pathway may provide insights into how to optimally modulate the PD-1 pathway in cancer immunotherapy while minimizing adverse events.

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1).

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti-PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti-PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

CD160Ig fusion protein targets a novel costimulatory pathway and prolongs allograft survival.

CD160 is a cell surface molecule expressed by most NK cells and approximately 50% of CD8(+) cytotoxic T lymphocytes. Engagement of CD160 by MHC class-I directly triggers a costimulatory signal to TCR-induced proliferation, cytokine production and cytotoxic effector functions. The role of CD160 in alloimmunity is unknown. Using a newly generated CD160 fusion protein (CD160Ig) we examined the role of the novel costimulatory molecule CD160 in mediating CD4(+) or CD8(+) T cell driven allograft rejection. CD160Ig inhibits alloreactive CD8(+) T cell proliferation and IFN-γ production in vitro, in particular in the absence of CD28 costimulation. Consequently CD160Ig prolongs fully mismatched cardiac allograft survival in CD4(-/-), CD28(-/-) knockout and CTLA4Ig treated WT recipients, but not in WT or CD8(-/-) knockout recipients. The prolonged cardiac allograft survival is associated with reduced alloreactive CD8(+) T cell proliferation, effector/memory responses and alloreactive IFN-γ production. Thus, CD160 signaling is particularly important in CD28-independent effector/memory CD8(+) alloreactive T cell activation in vivo and therefore may serve as a novel target for prevention of allograft rejection.

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells.

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.

Enhancing SIV-specific immunity in vivo by PD-1 blockade.

Chronic immunodeficiency virus infections are characterized by dysfunctional cellular and humoral antiviral immune responses. As such, immune modulatory therapies that enhance and/or restore the function of virus-specific immunity may protect from disease progression. Here we investigate the safety and immune restoration potential of blockade of the co-inhibitory receptor programmed death 1 (PD-1) during chronic simian immunodeficiency virus (SIV) infection in macaques. We demonstrate that PD-1 blockade using an antibody to PD-1 is well tolerated and results in rapid expansion of virus-specific CD8 T cells with improved functional quality. This enhanced T-cell immunity was seen in the blood and also in the gut, a major reservoir of SIV infection. PD-1 blockade also resulted in proliferation of memory B cells and increases in SIV envelope-specific antibody. These improved immune responses were associated with significant reductions in plasma viral load and also prolonged the survival of SIV-infected macaques. Blockade was effective during the early (week 10) as well as late ( approximately week 90) phases of chronic infection even under conditions of severe lymphopenia. These results demonstrate enhancement of both cellular and humoral immune responses during a pathogenic immunodeficiency virus infection by blocking a single inhibitory pathway and identify a novel therapeutic approach for control of human immunodeficiency virus infections.

CD160 inhibits activation of human CD4+ T cells through interaction with herpesvirus entry mediator.

CD160, a glycosylphosphatidylinositol-anchored member of the immunoglobulin superfamily, is expressed on both cytolytic lymphocytes and some unstimulated CD4+ T cells. Here we show that CD160 expression was increased after activation of human CD4+ T cells and that crosslinking CD160 with monoclonal antibody strongly inhibited CD3- and CD28-mediated activation. We found that herpesvirus entry mediator (HVEM) was a ligand of CD160 that acted as a 'bidirectional switch' for T cell activation, producing a positive or negative outcome depending on the engagement of HVEM by CD160 and known HVEM ligands such as B and T lymphocyte attenuator (BTLA) and the T lymphocyte receptor LIGHT. Inhibition of CD4+ T cell activation by HVEM-transfected cells was dependent on CD160 and BTLA; when the cysteine-rich domain 1 of HVEM was deleted, this inhibition was lost, resulting in strong T cell activation. CD160 thus serves as a negative regulator of CD4+ T cell activation through its interaction with HVEM.

TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells.

The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Here we showed that TIM-4 is expressed on human and mouse macrophages and dendritic cells, and both TIM-4 and TIM-1 specifically bound phosphatidylserine (PS) on the surface of apoptotic cells but not any other phospholipid tested. TIM-4(+) peritoneal macrophages, TIM-1(+) kidney cells, and TIM-4- or TIM-1-transfected cells efficiently phagocytosed apoptotic cells, and phagocytosis could be blocked by TIM-4 or TIM-1 monoclonal antibodies. Mutations in the unique cavity of TIM-4 eliminated PS binding and phagocytosis. TIM-4 mAbs that blocked PS binding and phagocytosis mapped to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors whose recognition of PS is critical for the efficient clearance of apoptotic cells and prevention of autoimmunity.

In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations.

In-Fusion can join any two pieces of DNA that have a 15-bp overlap at their ends. The result is equivalent to a recombination event at the ends of the DNAs. The 15-bp overlap may be engineered by inclusion in primers used to PCR amplify a segment of DNA. Originally described for inserting one piece of DNA into a restriction enzyme-digested plasmid, we have found In-Fusion can join four or more pieces of DNA in a single reaction. We used this insight to construct seamless fusion proteins, modular vectors with readily interchangeable segments, and novel mutagenesis strategies. Replacement In-Fusion can be used to delete any desired DNA segment in a plasmid and replace it with any desired new DNA segment without limitations on position or size.

Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction.

In progressive viral infection, antiviral T cell function is impaired by poorly understood mechanisms. Here we report that the inhibitory immunoregulatory receptor CTLA-4 was selectively upregulated in human immunodeficiency virus (HIV)-specific CD4(+) T cells but not CD8(+) T cells in all categories of HIV-infected subjects evaluated, with the exception of rare people able to control viremia in the absence of antiretroviral therapy. CTLA-4 expression correlated positively with disease progression and negatively with the capacity of CD4(+) T cells to produce interleukin 2 in response to viral antigen. Most HIV-specific CD4(+) T cells coexpressed CTLA-4 and another inhibitory immunoregulatory receptor, PD-1. In vitro blockade of CTLA-4 augmented HIV-specific CD4(+) T cell function. These data, indicating a reversible immunoregulatory pathway selectively associated with CD4(+) T cell dysfunction, provide a potential target for immunotherapy in HIV-infected patients.

PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression.

Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.

Restoring function in exhausted CD8 T cells during chronic viral infection.

Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 'helpless' CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.