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.

Highly conserved CDR3 region in circulating CD4(+)Vß5(+) T cells may be associated with cytotoxic activity in Chagas disease.

Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4(+) T cells from chagasic patients preferentially express T cell receptor (TCR) ß-chain variable region (Vß) 5. The aim of this work was to determine whether T cells expressing this particular Vß region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vß5·1 expressing CD4(+) T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jß region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4(+)Vß5·1(+) cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy.

Prevalence of donor-specific anti-HLA antibodies during episodes of renal allograft rejection.

BACKGROUND: Recent studies suggest that the appearance of anti-HLA antibodies in the early posttransplant period is associated with an increased incidence of acute and chronic rejection months later. However, very little is known about the prevalence of anti-HLA antibodies at the time that the rejection episodes are diagnosed. The purpose of this study was to analyze retrospectively 420 sera from 263 renal allograft recipients who were readmitted to the hospital for any reason between 1989 and 1998 in order to determine if a correlation existed between the presence of donor-specific anti-HLA antibodies and graft rejection. METHODS: Sera were assayed for IgG HLA class I and II antibodies by ELISA. The ELISA results were analyzed using contingency tables with Fisher's exact test and compared with mismatched antigens in the donor. RESULTS: Antibodies to donor HLA class I molecules in the posttransplant sera were extremely rare, occurring in only 6 of the 420 sera (1.4%) analyzed. Antibodies to donor class II antigens were slightly more common, occurring in 25 of the 420 sera (6%). In 21 of these 25 cases (84%), the presence of donor-specific HLA class II antibodies was associated with episodes of either acute (n=14) or chronic rejection (n=7). Five patients had antibodies to both class I and class II donor antigens, and all five of them lost their grafts to rejection. CONCLUSION: Although the presence of donor-specific HLA antibodies presented a significant risk for acute or chronic rejection, 77% of all acute and chronic rejections occurred in patients without detectable HLA antibodies.

Mechanisms of altered transcription by cigarette smoke.

The article highlighted in this issue is "The Activity of NF-kappaB in Swiss 3T3 Cells Exposed to Aqueous Extracts of Cigarette Smoke Is Dependent on Thioredoxin," by Stephan Gebel and Thomas Müller (pp. 75-81).

Exposure to cigarette tar inhibits ribonucleotide reductase and blocks lymphocyte proliferation.

Cigarette smoking causes profound suppression of pulmonary T cell responses, which has been associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. Exposure of human T cells to cigarette tar or its major phenolic components, hydroquinone and catechol, causes an immediate cessation of DNA synthesis without cytotoxicity. However, little is known of the mechanisms by which this phenomenon occurs. In this report we demonstrate that hydroquinone and catechol inhibit lymphocyte proliferation by quenching the essential tyrosyl radical in the M2 subunit of ribonucleotide reductase.

DR- and DQ-associated protection from type 1A diabetes: comparison of DRB1*1401 and DQA1*0102-DQB1*0602*.

The transmission disequilibrium test was used to analyze haplotypes for association and linkage to diabetes within families from the Human Biological Data Interchange type 1 diabetes repository (n = 1371 subjects) and from the Norwegian Type 1 Diabetes Simplex Families study (n = 2441 subjects). DQA1*0102-DQB1*0602 was transmitted to 2 of 313 (0.6%) affected offspring (P < 0.001, vs. the expected 50% transmission). Protection was associated with the DQ alleles rather than DRB1*1501 in linkage disequilibrium with DQA1*0102-DQB1*0602: rare DRB1*1501 haplotypes without DQA1*0102-DQB1*0602 were transmitted to 5 of 11 affected offspring, whereas DQA1*0102-DQB1*0602 was transmitted to 2 of 313 affected offspring (P < 0.0001). Rare DQA1*0102-DQB1*0602 haplotypes without DRB1*1501 were never transmitted to affected offspring (n = 6). The DQA1*0101-DQB1*0503 haplotype was transmitted to 2 of 42 (4.8%) affected offspring (P < 0.001, vs. 50% expected transmission). Although DRB1*1401 is in linkage disequilibrium with DQB1*0503, neither of the two affected children who carried DQA1*0101-DQB1*0503 had DRB1*1401. However, all 13 nonaffected children who inherited DQA1*0101-DQB1*0503 had DRB1*1401. In a case-control comparison of patients from the Barbara Davis Center, DQA1*0101-DQB1*0503 was found in 5 of 110 (4.5%) controls compared with 3 of 728 (0.4%) patients (P < 0.005). Of the three patients with DQB1*0503, only one had DRB1*1401. Our data suggest that both DR and DQ molecules (the DRB1*1401 and DQA1*0102-DQB1*0602 alleles) can provide protection from type 1A diabetes.

Suppression of human IL-1beta, IL-2, IFN-gamma, and TNF-alpha production by cigarette smoke extracts.

BACKGROUND: Although cigarette smoking is known to have detrimental effects on the immune system, the nature of the immunosuppressive agent or agents is poorly understood. OBJECTIVE: The purpose of the current study was to evaluate the effects of cigarette smoke extracts from high-tar (unfiltered Camel), medium-tar (Marlboro), and low-tar (Carlton) cigarettes on the in vitro production of IL-1beta, IL-2, IFN-gamma, and TNF-alpha. METHODS: The concentrations of hydroquinone and catechol in cigarette smoke extracts were determined by using HPLC. Human PBMCs were treated with cigarette smoke extracts, hydroquinone, or catechol, and stimulated with anti-CD3 and phorbol-12-myristate-13-acetate. Cytokine levels in the supernatants were quantified by ELISA. RESULTS: Pretreatment of PBMCs with cigarette smoke extracts derived from a single high- or low-tar cigarette suppressed the production of IL-1beta, IL-2, IFN-gamma, and TNF-alpha by greater than 90% without significant loss of cell viability. Nicotine, at a concentration comparable with that found in the highest-tar cigarettes (200 microg/mL), suppressed the production of IL-2, IFN-gamma, and TNF-alpha by only 21% to 38%. Catechol (50 micromol/L) inhibited production of IL-2 and IL-1beta by 62% to 73% but had little effect on TNF-alpha or IFN-gamma production. In contrast, hydroquinone inhibited the production of all 4 cytokines with IC(50) values ranging from 3 micromol/L(IL-1beta) to 29 micromol/L (IFN-gamma). However, HPLC determination of the hydroquinone concentrations in cigarette smoke extracts from single Camel (33+/-4 micromol/L), Marlboro (13+/-2 micromol/L), and Carlton (<1 micromol/L) cigarettes clearly demonstrated that the potent inhibitory effects of the low-tar cigarettes could not be accounted for by either hydroquinone or catechol. CONCLUSION: These studies indicate that cigarette smoke contains potent inhibitors of cytokine production, at least one of which is present even in low-tar cigarettes.

Identification of pathogenic T cells in patients with beryllium-induced lung disease.

Chronic beryllium disease (CBD) is caused by beryllium exposure and is characterized by granulomatous inflammation with accumulation of CD4+ T cells in the lung. We analyzed TCR beta-chain and alpha-chain genes expressed by these CD4+ T cells. In the lungs of individual patients, as well as among four of five CBD patients studied, different oligoclonal expansions within the Vbeta3 subset were found to express homologous or even identical CDR3 amino acid sequences. These related expansions were specific for CBD patients, were compartmentalized to lung, and persisted at high frequency in patients with active disease. Limiting dilution cloning and analysis of coexpressed TCR alpha-chain genes confirmed that these TCRs were selectively expanded by a common Ag involving beryllium. Overall, homologous TCR beta- and alpha-chains showed identical V regions and invariant charged residues within the CDR3 but considerable variability in TCRJ usage. Remarkably, CBD patients expressing nearly identical TCRs did not share common HLA-DRB1 or DQ alleles. These results implicate particular CD4+ cells in the pathogenesis of CBD and provide insight into how beryllium is recognized in human disease.

Symposium overview: alterations in cytokine receptors by xenobiotics.

A symposium entitled Alterations in Cytokine Receptors by Xenobiotics was held at the 37th Annual Meeting of the Society of Toxicology (SOT) in Seattle, Washington. The symposium was sponsored by the Immunotoxicology Specialty Section of SOT and was designed to present information on the effect of several different classes of xenobiotics on various aspects of receptor function (i.e., post-receptor signal transduction of receptor expression), or the involvement of cytokine receptors in the action of the toxicant under consideration. This symposium brought together scientists in the area of receptor immunobiology whose expertise in receptor modulation encompassed those major signaling agents involved in the normal immune response, i.e., proinflammatory cytokines, chemokines, interleukins, and interferons. The following is a summary of each of the individual presentations.

DRB1*04 and DQ alleles: expression of 21-hydroxylase autoantibodies and risk of progression to Addison's disease.

Of 957 patients with type 1 diabetes without known Addison's disease 1.6% (n = 15) were positive for 21-hydroxylase autoantibodies. Among DQ8/DQ2 heterozygous patients, the percentage expressing 21-hydroxylase autoantibodies was 5% (10 of 208) vs. less than 0.5% of patients with neither DQ8 nor DQ2. Three of the diabetic patients found to have 21-hydroxylase autoantibodies on screening were subsequently diagnosed with Addison's disease. Overall, the genotype DQ8/DQ2, consisting of DRB1*0404/DQ8 with DRB1*0301/DQ2, was present in 14 of 21 patients with Addison's disease (8 of 12 with diabetes and 6 of 9 without diabetes or antiislet autoantibodies) vs. 0.7% of the general population (109 of 15,547; P < 10(-6)) and 11% of patients with DM without Addison's disease (62 of 578; P < 10(-6)). Among patients with diabetes with DQ8, Addison's disease was strongly associated with the specific DRB1 subtype, DRB1*0404 (8 of 9 patients from 8 families, in contrast to only 109 of 408 DQ8 DM patients with diabetes without Addison's disease having DRB1*0404; P < 0.001). Among 21-hydroxylase autoantibody-positive DQ8 patients, 80% with DRB1*0404 (12 of 15) had Addison's disease, in contrast to 1 of 10 autoantibody-positive patients with DRB1*0401 or DRB1*0402 (P < 0.001). We conclude that patients with DRB1*0404 and 21-hydroxylase autoantibodies are at high risk for Addison's disease. Patients with DRB1*0401 and DRB1*0402 have more limited progression to Addison's disease despite the presence of 21-hydroxylase autoantibodies.

Reversal of hydroquinone-mediated suppression of T cell proliferation by transfection of the M2 subunit of ribonucleotide reductase.

Hydroquinone (HQ) is a benzene derivative that is found in large quantities in cigarette tar as a result of the pyrolysis of tobacco flavinoids. HQ is a potent inhibitor of T cell proliferation, causing an immediate and complete cessation of DNA synthesis in IL-2-dependent human T lymphoblasts and Jurkat T cells without loss of cell viability. Previous studies from our laboratory demonstrated that the antiproliferative effects of RQ could be partially reversed by the addition of deoxyribonucleosides, but not by the corresponding ribonucleosides, suggesting that HQ might inhibit ribonucleotide reductase. In the present study, the molecular mechanism behind this observation was further investigated. Jurkat T cells were stably transfected with a pMEP4 expression vector containing the gene for the M2 subunit of ribonucleotide reductase under transcriptional control of the human metallothionein IIA promoter. M2-transfected Jurkat T cells exhibited a greater than three-fold increase in resistance to HQ compared to untransfected cells or cells transfected with the M2 gene in the reverse orientation. HQ resistance was associated with an increased level of M2 protein detected by Western blot. These results suggest that the benzene derivative inhibits lymphocyte proliferation by inhibiting ribonucleotide reductase.

Differential inhibition of DNA synthesis in human T cells by the cigarette tar components hydroquinone and catechol.

Hydroquinone (HQ), catechol, and phenol exist in microgram quantities in cigarette tar and represent the predominant form of human exposure to benzene. Exposure of human T lymphoblasts (HTL) in vitro to 50 microM HQ or 50 microM catechol decreased IL-2-dependent DNA synthesis and cell proliferation by >90% with no effect on cell viability. Phenol had no effect on HTL proliferation at concentrations up to 1 mm. The addition of HQ or catechol to proliferating HTL blocked 3H-TdR uptake by >90% within 2 hr without significantly affecting 3H-UR uptake, suggesting that both compounds inhibit a rate-limiting step in DNA synthesis. However, the effects of HQ and catechol appear to involve different mechanisms. Ferric chloride (FeCl3) reversed the inhibitory effect of catechol, but not HQ, corresponding with the known ability of catechol to chelate iron. HQ, but not catechol, caused a decrease in transferrin receptor (TfR, CD71) expression, comparable to the level observed in IL-2-starved cells. HQ also inhibited DNA synthesis in cultures of transformed Jurkat T lymphocytes, primary and transformed fibroblasts, and mink lung epithelial cells, indicating that its antiproliferative effect was not restricted to IL-2 mediated proliferation. However, DNA synthesis by primary lymphocytes was more sensitive to HQ (IC50 = 6 microM) than that of the transformed Jurkat T cell line (IC50 = 37 microM) or primary human fibroblasts (IC50 = 45 microM), suggesting that normal lymphocytes may be particularly sensitive to HQ. The effects of HQ and catechol on DNA synthesis could be partially reversed by a combination of adenosine deoxyribose and guanosine deoxyribose, suggesting that both compounds may inhibit ribonucleotide reductase.

The cigarette tar component p-benzoquinone blocks T-lymphocyte activation by inhibiting interleukin-2 production, but not CD25, ICAM-1, or LFA-1 expression.

Cigarette smoking has been shown to cause profound suppression of T-cell responses in the lungs, but the mechanism by which this phenomenon occurs is not known. We have shown that 10 microM p-benzoquinone (p-BQ), a thiol-reactive benzene derivative found in cigarette tar, inhibits mitogen-induced IL-2 production by human peripheral blood mononuclear cells by 76 +/- 7% without affecting lymphocyte/macrophage agglutination or blast transformation. The effect of p-BQ appeared to be specific for IL-2 production, since de novo induction of the IL-2 receptor alpha-chain (CD25) and ICAM-1 (CD54) and upregulation of LFA-1 alpha/beta (CD11a and CD18) were unaffected. In contrast, N-ethylmaleimide (NEM), another alpha,beta-unsaturated diketone with thiol-reactive properties similar to those of p-BQ, inhibited all of these Con A-induced activation events. These results suggest that p-BQ inhibits T-cell mitogenesis by blocking a thiol-dependent event that controls IL-2 production but not other T-cell activation events.

Inhibition of human T lymphoblast proliferation by hydroquinone.

Hydroquinone (HQ) is a major metabolite of benzene and is present in large quantities in cigarette tar as a result of the combustion of tobacco leaf pigments. We hypothesize that the immunosuppressive effects of cigarette smoking are due, in part, to the deposition of large quantities of HQ in the lungs. Exposure of primary human T lymphoblasts (HTL) in vitro to 50 microM HQ blocked IL-2-dependent proliferation by > 90% with no loss in viability. Inhibition of DNA synthesis was observed immediately after the addition of HQ to the cells. However, this effect could be reversed up to 6 hr later by simply washing the cells and reculturing them in the absence of HQ. HQ did not significantly alter intracellular glutathione levels up to 24 hr later, and the presence of 50 microM 2-mercaptoethanol or 500 microM dithiothreitol during the treatment did not prevent inhibition of DNA synthesis. HQ did not block binding of 125I-IL-2 to the cells, but inhibited the IL-2-dependent progression of HTL through S phase of the cell cycle. These observations demonstrate that HQ, in concentrations comparable to those found in cigarette tar, is a potent inhibitor of IL-2-dependent T cell proliferation and may therefore help to explain the potent immunosuppressive effects of cigarette smoke on lung T lymphocytes.

RO 31-8220, a novel protein kinase C inhibitor, inhibits early and late T cell activation events.

The improvement of graft survival over the past decade has mainly been due to the development of more highly specific immunosuppressive agents, such as cyclosporine (CsA) and FK506. CsA and FK506 inhibit T cell activation by interfering with the calcium-mediated pathway, one of two pathways needed for T cell activation. The other pathway, mediated by protein kinase C (PKC), is not currently a target of any clinically used immunosuppressive agent. The purpose of this study was to assess the immunosuppressive properties of Ro 31-8220, a member of a new family of potent and selective PKC inhibitors. Peripheral blood mononuclear cells were isolated from the blood of normal human donors and utilized in a series of standard immunological assays. Three discrete activation events were inhibited by Ro 31-8220: mitogen-induced interleukin (IL)-2 production (IC50 80 nM), IL-2-dependent T lymphoblast proliferation (IC50 350 nM), and IL-2Ralpha (CD25) expression (control cells were 83% CD25+, mean fluorescence intensity = 163 +/- 4, 400-nM-treated cells were 56% CD25+, mean fluorescence intensity = 130 +/- 7). Noninhibitory doses of CsA (8 nM) or FK506 (0.2 nM) suppressed mitogen-induced IL-2 production by 60-80% when combined with a noninhibitory dose (25 nM) of Ro 31-8220, indicating the potent synergy between these agents. The ability of Ro 31-8220 to inhibit both early and late activation events and to synergize with CsA/FK506 suggests that this family of compounds has great potential as immunosuppressive agents and as probes with which to elucidate the role of PKC in T cell activation.