Characterization of immune responses to anti-PD-1 mono and combination immunotherapy in hematopoietic humanized mice implanted with tumor xenografts.

BACKGROUND: The success of agents that reverse T-cell inhibitory signals, such as anti-PD-1/PD-L1 therapies, has reinvigorated cancer immunotherapy research. However, since only a minority of patients respond to single-agent therapies, methods to test the potential anti-tumor activity of rational combination therapies are still needed. Conventional murine xenograft models have been hampered by their immune-compromised status; thus, we developed a hematopoietic humanized mouse model, hu-CB-BRGS, and used it to study anti-tumor human immune responses to triple-negative breast cancer (TNBC) cell line and patient-derived colorectal cancer (CRC) xenografts (PDX). METHODS: BALB/c-Rag2nullIl2rγnullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. Mice were evaluated for human chimerism in the blood and assigned into experimental untreated or nivolumab groups based on chimerism. TNBC cell lines or tumor tissue from established CRC PDX models were implanted into both flanks of humanized mice and treatments ensued once tumors reached a volume of ~150mm3. Tumors were measured twice weekly. At end of study, immune organs and tumors were collected for immunological assessment. RESULTS: Humanized PDX models were successfully established with a high frequency of tumor engraftment. Humanized mice treated with anti-PD-1 exhibited increased anti-tumor human T-cell responses coupled with decreased Treg and myeloid populations that correlated with tumor growth inhibition. Combination therapies with anti-PD-1 treatment in TNBC-bearing mice reduced tumor growth in multi-drug cohorts. Finally, as observed in human colorectal patients, anti-PD-1 therapy had a strong response to a microsatellite-high CRC PDX that correlated with a higher number of human CD8+ IFNγ+ T cells in the tumor. CONCLUSION: Hu-CB-BRGS mice represent an in vivo model to study immune checkpoint blockade to human tumors. The human immune system in the mice is inherently suppressed, similar to a tumor microenvironment, and thus allows growth of human tumors. However, the suppression can be released by anti-PD-1 therapies and inhibit tumor growth of some tumors. The model offers ample access to lymph and tumor cells for in-depth immunological analysis. The tumor growth inhibition correlates with increased CD8 IFNγ+ tumor infiltrating T cells. These hu-CB-BRGS mice provide a relevant preclinical animal model to facilitate prioritization of hypothesis-driven combination immunotherapies.

Induction of remission results in spontaneous enhancement of anti-tumor cytotoxic T-lymphocyte activity in dogs with B cell lymphoma.

Characterization of the tumor microenvironment, particularly the immune cells that infiltrate tumors, provides important predictive and prognostic information in humans with lymphoma and other types of cancer. Tumor associated T lymphocytes have not been previously described in dogs with lymphoma. Therefore, we investigated the phenotype and function of T cells in the lymph nodes of dogs with B cell Non-Hodgkin's lymphoma (NHL), as well as the function of T cells in circulation of these dogs. We found that CD4+ and CD8+ T lymphocytes were few in number and minimally responsive to mitogenic stimuli compared to T cells in lymph nodes of normal dogs. Additionally, regulatory T cells (Treg) were significantly increased in tumor tissues compared to lymph nodes of healthy dogs. To better understand cell mediated antitumor immune responses we developed a non-radioactive assay to measure cytotoxic T lymphocyte (CTL) mediated killing of autologous tumor cells. Using this assay, we found that spontaneous CTL activity in the blood of dogs with lymphoma improved significantly following induction of tumor remission using doxorubicin. Coincident with the improvement in CTL activity, circulating Treg numbers were significantly decreased compared to pretreatment levels. We conclude from these studies that CTL activity in dogs with lymphoma can be significantly improved following induction of tumor remission using chemotherapy, as assessed using a new non-radioactive CTL assay.

Antigen-specific blockade of T cells in vivo using dimeric MHC peptide.

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR zeta-chain and recruitment and phosphorylation of zeta-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.

B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells.

Dendritic cells (DCs), unique antigen-presenting cells (APCs) with potent T cell stimulatory capacity, direct the activation and differentiation of T cells by providing costimulatory signals. As such, they are critical regulators of both natural and vaccine-induced immune responses. A new B7 family member, B7-DC, whose expression is highly restricted to DCs, was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC fails to bind the B7.1/2 receptors CD28 and cytotoxic T lymphocyte-associated antigen (CTLA)-4, but does bind PD-1, a receptor for B7-H1/PD-L1. B7-DC costimulates T cell proliferation more efficiently than B7.1 and induces a distinct pattern of lymphokine secretion. In particular, B7-DC strongly costimulates interferon gamma but not interleukin (IL)-4 or IL-10 production from isolated naive T cells. These properties of B7-DC may account for some of the unique activity of DCs, such as their ability to initiate potent T helper cell type 1 responses.

Monitoring antigen-specific T cells using MHC-Ig dimers.

The lack of high affinity reagents has made distinguishing T cells on the basis of antigen specificity difficult to accomplish. This unit provides protocols that utilize innovations in molecular design to permit construction of soluble multivalent MHC complexes (MHC-Ig dimers) with high avidity for cognate T cell receptors. MHC-Ig dimers display stable binding properties when they interact with antigen-specific T cells thus allowing their use in the staining of antigen-specific T cells by flow cytometry. Methods for constructing and detecting these MHC-Ig dimers are included along with protocols for applying their use for the quantitation of antigen-specific T cells.

Enhanced antigen-specific antitumor immunity with altered peptide ligands that stabilize the MHC-peptide-TCR complex.

T cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I-restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells.

Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Position-dependent transcriptional regulation of the murine dihydrofolate reductase promoter by the E2F transactivation domain.

Activity of the dihydrofolate reductase (dhfr) promoter increases at the G1-S-phase boundary of the cell cycle. Mutations that abolish protein binding to an E2F element in the dhfr promoter also abolish the G1-S-phase increase in dhfr transcription, indicating that transcriptional regulation is mediated by the E2F family of proteins. To investigate the mechanism by which E2F regulates dhfr transcription, we moved the E2F element upstream and downstream of its natural position in the promoter. We found that the E2F element confers growth regulation to the dhfr promoter only when it is proximal to the transcription start site. Using a heterologous E2F element, we showed that position-dependent regulation is a property that is promoter specific, not E2F element specific. We demonstrated that E2F-mediated growth regulation of dhfr transcription requires activation of the dhfr promoter in S phase and that the C-terminal activation domains of E2F1, E2F4, and E2F5, when fused to the Gal4 DNA binding domain, are sufficient to specify position-dependent activation. To further investigate the role of activation in dhfr regulation, we tested other transactivation domains for their ability to activate the dhfr promoter. We found that the N-terminal transactivation domain of VP16 cannot activate the dhfr promoter. We propose that, unlike other E2F-regulated promoters, robust transcription from the dhfr promoter requires an E2F transactivation domain close to the transcription start site.

Transcriptional regulation of the dihydrofolate reductase gene.

As cells approach S phase, many changes occur to create an environment conducive for DNA synthesis and commitment to cell division. The transcription rate of many genes encoding enzymes involved in DNA synthesis, including the dihydrofolate reductase (dhfr) gene, increases at the G1/S boundary of the cell cycle. Although a number of transcription factors interact to finely tune the levels of dhfr RNA produced, two families of transcription factors, Sp1 and E2F, play central roles in modulating dhfr levels. A region containing several Sp1-binding sites is required for both regulated and basal transcription levels. In contrast, the E2F-binding sites near the transcription start site are required only for regulated transcription. A model is presented for the regulation of the dhfr gene which may also pertain to other cell cycle-associated genes.

Introduction to the E2F family: protein structure and gene regulation.

E2F is a heterodimer composed of two partners, such as E2F1 and DP1. Although E2F1 can bind DNA as a homodimer and increase promoter activity, optimal DNA-binding and transcriptional activity occurs in the heterodimeric form. A model (Fig. 3) for the involvement of E2F activity in cell growth control that incorporates viral oncoproteins, positive regulators of cell growth (cyclins) and negative regulators of cell growth (tumor suppressor proteins) can now be advanced. Each aspect of this model is addressed in subsequent chapters of this book. It is likely that binding of growth-suppressing proteins, such as Rb, can inhibit the transactivation potential of E2F1, either by blocking the interaction of E2F1 with a separate component of the transcription complex or by bringing a repressor domain to the transcription complex (Flemington et al. 1993; Helin et al. 1993; Weintraub et al. 1992; Zamanian and La Thangue 1993; Zhu et al. 1993). Phosphorylation or sequestration of Rb by viral oncoproteins can free E2F. The influence of viral oncoproteins on E2F activity and the regulation of the different E2F complexes is the focus of the contributions by Cobrinik and by Cress and Nevens. The interaction of the free E2F induces a bend in the DNA that may also play a role in transactivation, perhaps by bringing proteins (such as an Sp1 or CCAAT family member) separated by distance on the promoter DNA into contact (Huber et al. 1994). Because E2F target genes encode proteins critical for cell growth, deregulation of E2F activity can have severe consequences, such as apoptosis or uncontrolled proliferation. The effect of altered expression of E2F activity on the cell cycle and on tumorigenicity is the focus of the contribution by Adams and Kaelin. Finally, a comparison of E2F to the genetically well-characterized factors that regulate G1/S phase transcription in yeast is the subject of the chapter by Breeden. This volume concludes with Farnham's summary of the rapid gains in knowledge concerning the E2F gene family that have been made in the past several years and provides a series of questions and lines of investigation that will be the focus of future studies.

Cloning, chromosomal location, and characterization of mouse E2F1.

E2F has been implicated in growth control because of its association with the retinoblastoma protein and the presence of E2F binding sites in the promoters of several growth-regulated genes. Proteins that bind to an E2F site have been cloned from human and mouse cells. However, these two proteins (human E2F1 and mouse DP-1) are quite different in sequence. We have now cloned a mouse cDNA encoding a protein 86% identical to the human E2F1 protein. The mouse E2F1 cDNA encodes a 430-amino-acid protein with a predicted molecular weight of 46,322 and detects mRNAs of 2.7 and 2.2 kb. Using primers complementary to sequences in the mouse E2F1 3' untranslated region, we mapped the mouse E2F1 gene to chromosome 2, near the Agouti and c-src loci. To understand the role of the different E2F family members in the growth of mouse NIH 3T3 cells, we examined the levels of E2F1 and DP-1 mRNAs in different stages of the cell cycle. Since the levels of E2F1 but not DP-1 mRNA correlated with changes in transcription from the dhfr promoter, we examined whether E2F1 could activate various growth-regulated promoters. We found that E2F1 could activate some (dhfr, thymidine kinase, and DNA polymerase alpha) but not all (thymidylate synthase, cad, and c-myc) of these promoters. On the basis of changes in levels of E2F1 and its ability to transactivate growth-regulated promoters, we propose that E2F1 may mediate growth factor-initiated signal transduction.

A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter.

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary.