The Immune System Strikes Back: Cellular Immune Responses against Indoleamine 2,3-dioxygenase

The enzyme indoleamine 2,3-dioxygenase (IDO) exerts an well established immunosuppressive function in cancer. IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. In the present study, we tested the notion whether IDO itself may be subject to immune responses.The presence of naturally occurring IDO-specific CD8 T cells in cancer patients was determined by MHC/peptide stainings as well as ELISPOT. Antigen specific cytotoxic T lymphocytes (CTL) from the peripheral blood of cancer patients were cloned and expanded. The functional capacity of the established CTL clones was examined by chrome release assays. The study unveiled spontaneous cytotoxic T-cell reactivity against IDO in peripheral blood as well as in the tumor microenvironment of different cancer patients. We demonstrate that these IDO reactive T cells are indeed peptide specific, cytotoxic effector cells. Hence, IDO reactive T cells are able to recognize and kill tumor cells including directly isolated AML blasts as well as IDO-expressing dendritic cells, i.e. one of the major immune suppressive cell populations.IDO may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies. Furthermore, as emerging evidence suggests that IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, IDO-based immunotherapy holds the promise to boost anti-cancer immunotherapy in general

HLA-A2-restricted T-cell responses against IDO as measured by IFN-γ ELISPOT.

<p>PBMC from healthy donors, breast cancer patients, melanoma patients, and renal cell carcinoma patients were analyzed. All individuals were HLA-A2⁺. The peptides IDO5 (IDO_199-207; ALLEIASCL) <i>(</i><i>a</i><i>)</i>, IDO2 (IDO_164-172; FLVSLLVEI) <i>(</i><i>b</i><i>)</i>, and IDO6 (IDO_320-328; VLSKGDGL) <i>(</i><i>c</i><i>),</i> were examined. T cells were stimulated once <i>in vitro</i> with peptide before being plated at 4×10⁵ cells per well in duplicates either without or with the relevant IDO peptide. The average number of IDO-specific spots (after subtraction of spots without added peptide) was calculated per 4×10⁵ PBMC for each patient (white triangle). <i>(</i><i>a</i><i>), Top,</i> Example of an ELISPOT response against IDO5 (IDO_199-207; ALLEIASCL) in PBMC from a breast cancer patient.</p

Functional capacity of IDO5-specific T cells to kill cancer cell lines treated with IFN-γ for up-regulation of IDO, or treated with IDO ShRNA for down-regulation of IDO.

<p><i>(</i><i>a</i><i>),</i> Lysis by the IDO5-specific T-cell clone (RBS35) of the HLA-A2⁺ breast cancer cell lines CAMA-1 <i>(right)</i> and MDA-MB-231 <i>(left)</i> before and after IFN-γ treatment. <i>(</i><i>b</i><i>)</i>, Histograms showing intracellular IDO expression in CAMA-1 before and after IFN-γ treatment. Data are representative of 3 experiments. Intracellular IDO expression was given by a one-tailed two sampled t-test comparing MFI_IDO (dark histograms) and MFI_{Isotype control} (light histograms), where MFI is the Mean Fluorescence Intensity. The fold of expression was defined as MFI_IDO/MFI_{Isotype control}. <i>Top:</i> CAMA-1 (<i>p</i> = 0.020 and MFI_IDO/MFI_{Isotype control}  = 2.3). <i>Bottom:</i> CAMA-1 + IFN-γ treatment (<i>p</i> = 0.004 and MFI_IDO/MFI_{Isotype control}  = 3.5). <i>(</i><i>c</i><i>),</i> Histograms showing HLA-A2 expression in CAMA-1 before and after IFN-γ treatment. Data are representative of 3 experiments. HLA-A2 expression was given by a one-tailed two sampled t-test comparing MFI_HLA-A2 (dark histograms) and MFI_{Isotype control} (light histograms). The fold of expression was defined as MFI_HLA-A2/MFI_{Isotype control}. <i>Top:</i> CAMA-1 (<i>p</i> = 0.004 and MFI_HLA-A2/MFI_{Isotype control}  = 43.7). <i>Bottom:</i> CAMA-1 + IFN-γ treatment (<i>p</i> = 0.002 and MFI_IDO/MFI_HLA-A2  = 141.2). <i>(</i><i>d</i><i>)</i>, Lysis of the colon cancer cell line SW480 transfected with IDO ShRNA for down-regulation of IDO protein expression by an IDO5-specific T-cell bulk culture. As a positive control, SW480 cells transfected with control ShRNA were used as target cells. All assays were performed in different E∶T ratios. <i>(</i><i>e</i><i>)</i>, Histograms showing intracellular IDO expression in SW480 transfected with control ShRNA (<i>p</i> = 0.001 and MFI_IDO/MFI_{Isotype control}  = 4.8) <i>(top)</i> and IDO ShRNA (<i>p</i> = 0.040 and MFI_IDO/MFI_{Isotype control}  = 2.1) <i>(bottom)</i>.</p

Specificity and functional capacity of IDO5-specific T-cells assayed by ⁵¹Cr-release assays.

<p><i>(</i><i>a</i><i>),</i> Lysis of T2-cells pulsed with IDO5 peptide or an irrelevant peptide (the HLA-A2 high affinity binding epitope HIV-1 pol_476-484 (ILKEPVHGV)) by a T-cell clone (RBS35). <i>(</i><i>b</i><i>)</i>, Specific lysis by RBS35 of the HLA-A2⁺/IDO⁺ colon cancer cell line SW480 without or with the addition of the HLA-class I specific antibody W6/32, and lysis of the HLA-A2⁺/IDO^-colon cancer cell line HCT-116. All assays were performed in different E∶T ratios. <i>(</i><i>c</i><i>)</i> Histograms showing intracellular IDO expression in cancer cell lines. Data are representative of 3 experiments. Intracellular IDO expression was given by a one-tailed two sampled t-test comparing MFI_IDO (dark histograms) and MFI_{Isotype control} (light histograms), where MFI is the Mean Fluorescence Intensity. <i>Left:</i> HCT-116 (<i>p</i> = 0.300). <i>Right:</i> SW480 (<i>p</i> = 0.002).</p

Specificity and functional capacity of IDO5-specific T cells assayed by ⁵¹Cr-release assays.

<p><i>(</i><i>a</i><i>),</i> Lysis by the IDO5-specific T-cell clone (RBS35) of the HLA-A2⁺/IDO⁺ melanoma cell line FM55M without and with the addition of cold T2-cells pulsed with IDO5 peptide or an irrelevant peptide (HIV-1 pol_476-484) (inhibitor to target ratio  = 20∶1), and NK cell activity of RBS35 examined using the natural killer cell line K562 as target cells. <i>(</i><i>b</i><i>),</i> Lysis by RBS35 of AML-blasts enriched from 5 HLA-A2⁺ AML patients and 1 HLA-A2^- AML patient. B cells and T cells were depleted from the bone marrow of the AML patients using CD19⁺ and CD3⁺ microbeads, respectively. The highly enriched AML-blasts were used as target cells with or without the addition of the HLA-class I specific antibody W6/32. <i>(</i><i>c</i><i>),</i> Lysis of T2-cells pulsed with IDO5 peptide or an irrelevant peptide (HIV-1 pol_476-484), and lysis of the HLA-A2⁺/IDO⁺ colon cancer cell line SW480 by an IDO5-specific T-cell bulk culture. <i>(</i><i>d</i><i>),</i> Lysis of the HLA-A2⁺/IDO⁺ colon cancer cell line SW480 and HLA-A2⁺/IDO^- colon cancer cell line HCT-116 by three different IDO5-specific T-cell clones (RBS26 (<i>white triangle</i>), RBS31 (<i>black triangle</i>), RBS46 (<i>grey triangle</i>)) assayed by ⁵¹Cr-release assay. All assays were performed in different E∶T ratios.</p

Tetramer analysis of IDO5-specific T cells.

<p><i>(</i><i>a</i><i>),</i> An example of IDO5-specific, CD8 T-cell enriched PBMC from a renal cell carcinoma patient visualised by flow cytometry staining using the tetramer complex HLA-A2/IDO5-PE, and CD8-allophycocyanin after one <i>in vitro</i> stimulation with IDO5 peptide. As a negative control, cells were stained with the tetramer complex HLA-A2/HIV-1 pol_476–484-PE, and CD8-allophycocyanin. <i>(</i><i>b</i><i>),</i> PBMC from healthy donors or from patients with breast cancer, melanoma cancer or renal cell carcinoma were stained with the tetramer complex HLA-A2/IDO5 or HLA-A2/HIV-1 pol_476–484 and analyzed by flow cytometry either <i>ex vivo</i> or after one <i>in vitro</i> peptide stimulation. The dotted lines illustrate that IDO5-tetramer positive cells are detectable both <i>ex vivo</i> and <i>in vitro</i> in the same patients. <i>(</i><i>c</i><i>),</i> An example of CD45RA and CD28 phenotype analysis of IDO5-tetramer/CD8 gated cells from CD8 T-cell enriched PBMC from a renal cell carcinoma patient visualised <i>ex vivo</i> by flow cytometry. For comparison, the cells were stained with isotype matched controls <i>(</i><i>d</i><i>),</i> An example of an IL-2 expanded TIL culture from a melanoma patient visualised by flow cytometry staining using the tetramer complex HLA-A2/IDO5-PE, and CD8-APC-Cy7. As a negative control, the TIL were stained with the tetramer complex HLA-A2/HIV-1 pol_476–484-PE, and CD8-APC-Cy7. <i>(</i><i>e</i><i>),</i> As a positive control of the IDO5 tetramer, an IDO5-specific T-cell clone (RBS35) was stained with the HLA-A2/HIV-1 pol_476–484-PE and HLA-A2/IDO5-PE tetramers.</p

HLA-A2 binding affinity of selected IDO-derived peptides.

*<p>The C5 value is the peptide concentration required for half maximal stabilization of HLA-A2.</p

Functional capacity of an IDO5-specific T-cell clone (RBS35) to kill immune cells assayed by ⁵¹Cr-release assays.

<p><i>(</i><i>a</i><i>)</i>, <i>Left:</i> Lysis of autologous <i>in vitro</i> immatured and matured DC. <i>Right:</i> Lysis of allogeneic HLA-A2⁺<i>in vitro</i> immatured and matured DC. All assays were performed in different E∶T ratios. <i>(</i><i>b</i><i>),</i> Histograms showing intracellular IDO expression in DC. Data are representative of 3 experiments. Intracellular IDO expression was given by a one-tailed two sampled t-test comparing MFI_IDO (dark histograms) and MFI_{Isotype control} (light histograms), where MFI is the Mean Fluorescence Intensity. <i>Left: In vitro</i> immatured DC (<i>p</i> = 0.100). <i>Right: In vitro</i> matured DC (<i>p</i> = 0.001). <i>(</i><i>c</i><i>)</i>, Lysis of autologous CD14⁺ monocytes, CD3⁺ T cells and CD19⁺ B cells isolated directly <i>ex vivo</i> from IDO⁺ PBMC, and lysis of autologous CD14⁺ monocytes, CD3⁺ T cells and CD19⁺ B cells after IFN-γ treatment. As a control, we used <i>in vitro</i> generated autologous IDO^- immatured DC and IDO⁺ matured DC. <i>(</i><i>d</i><i>)</i>, Examples of HLA-A2 restricted T-cell responses against EBV BMLF1_280-288 (GLCTLVAML) as measured by ELISPOT in PBMC from a breast cancer patient. Cultures of PBMC were treated with IFN-γ for 5 days with autologuous freshly isolated CD8 T-cells <i>(left)</i> or with autologous IDO5 specific T-cells (at a PBMC:IDO5 specific T-cell ratio of 300∶1) <i>(right)</i> before examination for reactivity against the HLA-A2 restricted epitope from EBV BMLF1_280-288 (GLCTLVAML). Three different PBMC concentrations were examined; 1,5×10⁵ cells, 5×10⁴ cells (<i>two top rows</i>) and 10⁴ cells (<i>bottom two rows</i>).</p

Performance of Expanded Newborn Screening in Norway Supported by Post-Analytical Bioinformatics Tools and Rapid Second-Tier DNA Analyses

In 2012, the Norwegian newborn screening program (NBS) was expanded (eNBS) from screening for two diseases to that for 23 diseases (20 inborn errors of metabolism, IEMs) and again in 2018, to include a total of 25 conditions (21 IEMs). Between 1 March 2012 and 29 February 2020, 461,369 newborns were screened for 20 IEMs in addition to phenylketonuria (PKU). Excluding PKU, there were 75 true-positive (TP) (1:6151) and 107 (1:4311) false-positive IEM cases. Twenty-one percent of the TP cases were symptomatic at the time of the NBS results, but in two-thirds, the screening result directed the exact diagnosis. Eighty-two percent of the TP cases had good health outcomes, evaluated in 2020. The yearly positive predictive value was increased from 26% to 54% by the use of the Region 4 Stork post-analytical interpretive tool (R4S)/Collaborative Laboratory Integrated Reports 2.0 (CLIR), second-tier biochemical testing and genetic confirmation using DNA extracted from the original dried blood spots. The incidence of IEMs increased by 46% after eNBS was introduced, predominantly due to the finding of attenuated phenotypes. The next step is defining which newborns would truly benefit from screening at the milder end of the disease spectrum. This will require coordinated international collaboration, including proper case definitions and outcome studies

RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation

Chikungunya virus (CHIKV) is an arthritogenic alphavirus causing epidemics of acute and chronic arthritic disease. Herein we describe a comprehensive RNA-Seq analysis of feet and lymph nodes at peak viraemia (day 2 post infection), acute arthritis (day 7) and chronic disease (day 30) in the CHIKV adult wild-type mouse model. Genes previously shown to be up-regulated in CHIKV patients were also up-regulated in the mouse model. CHIKV sequence information was also obtained with up to ≈8% of the reads mapping to the viral genome; however, no adaptive viral genome changes were apparent. Although day 2, 7 and 30 represent distinct stages of infection and disease, there was a pronounced overlap in up-regulated host genes and pathways. Type I interferon response genes (IRGs) represented up to ≈50% of up-regulated genes, even after loss of type I interferon induction on days 7 and 30. Bioinformatic analyses suggested a number of interferon response factors were primarily responsible for maintaining type I IRG induction. A group of genes prominent in the RNA-Seq analysis and hitherto unexplored in viral arthropathies were granzymes A, B and K. Granzyme Aand to a lesser extent granzyme K, but not granzyme B, mice showed a pronounced reduction in foot swelling and arthritis, with analysis of granzyme Amice showing no reductions in viral loads but reduced NK and T cell infiltrates post CHIKV infection. Treatment with Serpinb6b, a granzyme A inhibitor, also reduced arthritic inflammation in wild-type mice. In non-human primates circulating granzyme A levels were elevated after CHIKV infection, with the increase correlating with viral load. Elevated granzyme A levels were also seen in a small cohort of human CHIKV patients. Taken together these results suggest granzyme A is an important driver of arthritic inflammation and a potential target for therapy. Trial Registration: ClinicalTrials.gov NCT0028129