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Keywords:

  • immunophenotype;
  • T cells;
  • intracellular cytokine staining;
  • melanoma

Purpose and Appropriate Sample Types

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Literature Cited
  7. Supporting Information

This panel was optimized to assess CD4+ and CD8+ T cell responses to various tumor antigens from melanoma patients. The panel was tested on single-cell derived T cell isolates (SCD-T) and T cell lines derived from peripheral blood mononuclear cells (PBMC) from melanoma patients, T cell lines from the tumor environment of melanoma patients, and fresh and cryopreserved PBMC (healthy donors). Staining can be performed in 96-well plates for high-throughput.

Background

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Literature Cited
  7. Supporting Information

The T cell response to human melanoma is broad, encompassing both effector and regulatory T cell populations with diverse functional profiles (1). Tumor specific T cells with cytokine profiles that do not follow the standard Th1/Th2 dichotomy have been demonstrated (2, 3). These descriptions include tumor antigen-specific effector and regulatory T cells that co-produce Th1 and Th2 cytokines. Thus, immunological monitoring of T cells from melanoma patients needs to assess a wide spectrum of functions and often has access to only a limited number of cells. To provide a tool for this immunological monitoring, we developed a panel of Ab-conjugates for assessing cytokine profiles that cross “traditional” T cell subset boundaries. As such, this panel would be useful for those interested in human T cells with a mixed cytokine profile, such as induced regulatory T cells, IL-10+ CD8+ T cells, T cells involved in allergic responses, etc. Further, it should be useful for samples with limited cell numbers, such as tumor biopsies, fine-needle aspirations, or cerebral spinal fluid, as well as samples from which multiple separate samples for Th1 versus Th2 intracellular cytokine staining are not possible. This panel was developed following guidelines described previously (4). To maximize sensitivity, the brightest fluorochromes were reserved for IL-2, IL-4, IL-10, IFN-γ, and TNF-α. A dump channel was used to exclude dead cells, B cells, and monocytes/macrophages. Next, testing a collection of Ab-conjugates optimized the differentiation of T cell subsets. APC-Cy7 and Quantum dot (QD) 605 were selected for discrimination of CD4 and CD8, respectively, because of their stability in this panel (Fig. 1 and Table 1).

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Figure 1. Gating strategy and example staining. A: Cells in the singlet gate are selected, live CD3+ CD14 CD19 cells identified, followed by exclusion of dye aggregates (gray box) and after gating on small lymphocytes, CD4+ and CD8+ T cells are selected for further analysis. CD4+ and CD8+ T cells positive for individual cytokines are separately gated and subsequently, a Boolean gate encompassing all combinations (25) of cytokine+/− T cells is created. The median fluorescence intensity (MFI) of cytokine+ T cells is evaluated, providing a measure of the magnitude of the response. B and C: Determination of cytokine+ T cells following gating described in (A). B: Shown is a CD4+ T cell line derived from the tumor of a melanoma patient stimulated with an allogeneic melanoma tumor cell line. The frequency of T cells positive for IFN-γ, TNF-α, IL-2, or IL-10 following antigenic stimulation was significantly greater than unstimulated T cells. C: Shown is a CD4+ T cell line derived from the tumor of a melanoma patient stimulated with an autologous melanoma tumor cell line. The frequency of T cells positive for TNF-α, IL-10, or IL-4 following antigenic stimulation was significantly greater than unstimulated T cells. The T cell lines in B and C are from two different patients stimulated with the same melanoma cell line. Data in B and C are presented as low-resolution dot plots. Robust cytokine responses from mitogen-stimulated PBMC are presented in Supporting Information Figure S7.

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Table 1. Summary table for application of OMIP-008
PurposeT cell cytokine production following in vitro stimulation
SpeciesHuman
Cell typesPBMC, T cell clones/lines, tumor-resident T cell lines
Cross referencesOMIP-001

CD3 was labeled after fixing and permeabilizing the cells to ensure that activated T cells having down-regulated their TCR/CD3 complex were included in the analysis (4). To permit resolution of CD4 following activation of T cells with PMA and ionomycin (used as a mitogenic control), CD4 was also labeled post-fixation/permeabilization for all samples. Specificity of the cytokine staining is demonstrated by reduced staining following blocking steps prior to intracellular staining with the fluorochrome-conjugated anticytokine reagents: (i) the surface-stained, fixed, and permeabilized cells are blocked with purified anticytokine antibodies of the identical clones and (ii) the fluorochrome-conjugated reagents are blocked with recombinant human cytokine (Table 2). These blocking steps typically reduce cytokine+ staining to background levels, or to a level allowing unambiguous gate setting, and as such these fully stained and blocked samples are used to determine placement of cytokine+ gates. This provides an alternative to individual FMO controls for each cytokine, which may not be feasible due to limited cell numbers from patient samples. The level of autofluorescence of the T cell lines has not been a substantial confounder in these experiments and we have not noted marked differences between the autofluorescence of cultured as compared with uncultured T cells from healthy donors.

Table 2. Reagents used for OMIP-008
SpecificityCloneFluorochromePurpose
  1. APC, allophycocyanin; Ax, Alexa; Bi, biotin; Cy, cyanine; PacBlu, pacific blue; PE, R-phycoerythrin; PerCP, peridinin chlorophyll protein; QD, quantum dot; ViViD, LIVE/DEAD fixable violet dead cell stain.

IL-2MQ1-17H12PerCP-Cy5.5Function
IL-48D4-8Ax488
IL-10JES3-9D7PE
IFN-γB27APC
TNF-αMAb11PE-Cy7
CD3UCHT1Ax700Lineage
CD4RPA-T4APC-Cy7
CD83B5QD605
CD14M5E2PacBluDump
CD19HIB19eFluor450
Dead cellsViViD

Human Subjects

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Literature Cited
  7. Supporting Information

The Health Sciences Institutional Review Board that serves both the William S. Middleton Memorial Veterans Hospital and the University of Wisconsin-Madison approved this study (Human Subjects Protocol # 1992-031). Written informed consent was obtained from the participants in this study.

Similarity to Published OMIPs

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Literature Cited
  7. Supporting Information

OMIP-001 was designed to detect cytokine-producing T cells, including IL-2, IFN-γ, and TNF-α (5). Our panel extends OMIP-001 with the addition of IL-4 and IL-10, thus broadening its potential application for the discrimination of T cells exhibiting traditional Th1 and Th2 responses, as well as those with unconventional cytokine profiles.

Literature Cited

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Literature Cited
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Purpose and Appropriate Sample Types
  3. Background
  4. Human Subjects
  5. Similarity to Published OMIPs
  6. Literature Cited
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22035_sm_SuppInfo.doc135KSupporting Information
MIFlowCyt_Item_Location.doc45KSupporting Information
CYTO_22035_sm_SuppInfoFig1A.tif337KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by serial 2-fold dilutions. A Plots represent concatenated .fcs files, with fluorescent intensity on the y-axis and Ab concentration on the x-axis. Titers used in OMIP-xxx are highlighted in red. B Titration curves of the plots shown in A with the ratio of the median fluorescence intensity (MFI; positive MFI/negative MFI) on the y-axis and Ab concentration on the y-axis. Titers used in OMIP-xxx are represented as open symbols. Ab concentrations are “μl Ab / 100 μl buffer” for all Abs except IL-4, IL-10, and IFN-γ, which are “μg Ab / ml”.
CYTO_22035_sm_SuppInfoFig1B.tif100KOnline Fig.1 Reagent titrations. Reagents were titrated on healthy donor PBMC by serial 2-fold dilutions. A Plots represent concatenated .fcs files, with fluorescent intensity on the y-axis and Ab concentration on the x-axis. Titers used in OMIP-xxx are highlighted in red. B Titration curves of the plots shown in A with the ratio of the median fluorescence intensity (MFI; positive MFI/negative MFI) on the y-axis and Ab concentration on the y-axis. Titers used in OMIP-xxx are represented as open symbols. Ab concentrations are “μl Ab / 100 μl buffer” for all Abs except IL-4, IL-10, and IFN-γ, which are “μg Ab / ml”.
CYTO_22035_sm_SuppInfoFig2A.tif61KOnline Fig.2 Total CD4+ T cells were isolated from healthy donors and stained for Th2 cytokines. A CD4+ T cells were cultured under Th2-biasing conditions using plate-bound anti-CD3 (10 μg/ml, clone OKT3), soluble anti-CD28 (2 μg/ml, clone CD28.2), IL-2 (40 IU/ml), IL-4 (12.5 ng/ml), anti-IFNγ (5 μg/ml, clone B27), and anti-IL-10 (5 μg/ml, clone JES3-9D7). After 4 days, cells were transferred to new plates and expanded for 3 days in the above media without anti-CD3 or anti-CD28 antibodies. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 4 hrs in the presence of BFA and monensin. Surface-bound IL-4 was blocked with unconjugated anti-IL-4 Ab (clone 8D4-8) prior to permeabilization, and was followed by staining with Ax488-conjugated 8D4-8. B CD4+ T cells were activated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 (at concentrations used in A) for 4 days. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 6 hrs in the presence of BFA and monensin. Data presented are not those in Online Fig.1. Titers used in OMIP-xxx are highlighted in red.
CYTO_22035_sm_SuppInfoFig2B.tif49KOnline Fig.2 Total CD4+ T cells were isolated from healthy donors and stained for Th2 cytokines. A CD4+ T cells were cultured under Th2-biasing conditions using plate-bound anti-CD3 (10 μg/ml, clone OKT3), soluble anti-CD28 (2 μg/ml, clone CD28.2), IL-2 (40 IU/ml), IL-4 (12.5 ng/ml), anti-IFNγ (5 μg/ml, clone B27), and anti-IL-10 (5 μg/ml, clone JES3-9D7). After 4 days, cells were transferred to new plates and expanded for 3 days in the above media without anti-CD3 or anti-CD28 antibodies. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 4 hrs in the presence of BFA and monensin. Surface-bound IL-4 was blocked with unconjugated anti-IL-4 Ab (clone 8D4-8) prior to permeabilization, and was followed by staining with Ax488-conjugated 8D4-8. B CD4+ T cells were activated with plate-bound anti-CD3, soluble anti-CD28, and IL-2 (at concentrations used in A) for 4 days. Cells were subsequently cultured in the presence of PMA (20 ng/ml) and Ionomycin (1 μM) (left plot) or its absence (right plot) for 6 hrs in the presence of BFA and monensin. Data presented are not those in Online Fig.1. Titers used in OMIP-xxx are highlighted in red.
CYTO_22035_sm_SuppInfoFig3A.tif33KOnline Fig.3 Cytokine response from a T cell line from the blood of a melanoma patient stimulated with PMA and Ionomycin or unstimulated. To emphasize the Th2 response, IL-10 is displayed on the y-axis and IL-4 on the x-axis. A Shown are the CD4+ T cells stimulated (left panel) and unstimulated (right panel). B Shown are the CD8+ T cells stimulated (left panel) and unstimulated (right panel). In all panels, the cells are gated as described in Figure 1.
CYTO_22035_sm_SuppInfoFig3B.tif30KOnline Fig.3 Cytokine response from a T cell line from the blood of a melanoma patient stimulated with PMA and Ionomycin or unstimulated. To emphasize the Th2 response, IL-10 is displayed on the y-axis and IL-4 on the x-axis. A Shown are the CD4+ T cells stimulated (left panel) and unstimulated (right panel). B Shown are the CD8+ T cells stimulated (left panel) and unstimulated (right panel). In all panels, the cells are gated as described in Figure 1.
CYTO_22035_sm_SuppInfoFig4A.tif246KOnline Fig.4 Cytokine response from Hut78, a cutaneous CD4+ T cell lymphoma cell line, following various in vitro stimulations. A Hut78 was stimulated with PMA (60 ng/ml) for 6 hours (upper panel) or unstimulated (lower panel). B Hut78 was stimulated with plate-bound anti-CD3 (5 μg/ml, clone OKT3), plate-bound anti-CD3 + 10 ng/ml IL-2, or soluble anti-CD3 (1 μg/ml, clone OKT3) + soluble anti-CD28 (1 μg/ml, clone CD28.2), or were unstimulated. The gates from the unstimulated sample were copied onto the stimulated samples. Shown are the IFN-γ vs IL-4 and the IL-10 vs IL-2 responses as overlays.
CYTO_22035_sm_SuppInfoFig4B.tif81KOnline Fig.4 Cytokine response from Hut78, a cutaneous CD4+ T cell lymphoma cell line, following various in vitro stimulations. A Hut78 was stimulated with PMA (60 ng/ml) for 6 hours (upper panel) or unstimulated (lower panel). B Hut78 was stimulated with plate-bound anti-CD3 (5 μg/ml, clone OKT3), plate-bound anti-CD3 + 10 ng/ml IL-2, or soluble anti-CD3 (1 μg/ml, clone OKT3) + soluble anti-CD28 (1 μg/ml, clone CD28.2), or were unstimulated. The gates from the unstimulated sample were copied onto the stimulated samples. Shown are the IFN-γ vs IL-4 and the IL-10 vs IL-2 responses as overlays.
CYTO_22035_sm_SuppInfoFig5.tif150KOnline Fig.5 Selection of CD4 reagent. Healthy donor PBMC were stimulated with PMA + ionomycin for 4 hrs. The cells were fixed and permeabilized followed by staining with anti-CD4 antibody conjugates. Shown are CD4+ T cells from the live lymphocyte gate. The antibody conjugate (APC-Cy7) selected for use in the OMIP is indicated with an asterisk. Clone RPA-T4 was conjugated to FITC, PE-Cy7, APC-H7, or APC-Cy7; clone L200 was conjugated to PerCP; clone S3.5 was conjugated to Pacific Orange or QD605; clone OKT4 was conjugated to A×700.
CYTO_22035_sm_SuppInfoFig6A.tif262KOnline Fig.6 Staining and biological controls. A T cell line from a human melanoma tumor was stimulated with allogeneic tumor. The intracellular cytokines were blocked using purified Ab (IL-4, IL-10, TNF, IFN-γ) prior to staining with fluorochrome-conjugated Ab or the fluorochrome-conjugated Ab was blocked with recombinant human cytokine (IL-2) prior to addition to the cells (lower panels). The cytokine+ gates from the blocked sample are copied onto the unblocked sample for determination of the frequency of cytokine+ cells induced by the stimulation (upper panels). Shown are the CD4+ T cells from the live lymphocyte gate. B T cell line with known IFN-γ response to autologous tumor included as a biological control of the stimulation condition (left panel). The unstimulated T cell line is shown in the right panel. Shown are the CD8+ T cells from the live lymphocyte gate.
CYTO_22035_sm_SuppInfoFig6B.tif59KOnline Fig.6 Staining and biological controls. A T cell line from a human melanoma tumor was stimulated with allogeneic tumor. The intracellular cytokines were blocked using purified Ab (IL-4, IL-10, TNF, IFN-γ) prior to staining with fluorochrome-conjugated Ab or the fluorochrome-conjugated Ab was blocked with recombinant human cytokine (IL-2) prior to addition to the cells (lower panels). The cytokine+ gates from the blocked sample are copied onto the unblocked sample for determination of the frequency of cytokine+ cells induced by the stimulation (upper panels). Shown are the CD4+ T cells from the live lymphocyte gate. B T cell line with known IFN-γ response to autologous tumor included as a biological control of the stimulation condition (left panel). The unstimulated T cell line is shown in the right panel. Shown are the CD8+ T cells from the live lymphocyte gate.
CYTO_22035_sm_SuppInfoFig7.tif450KOnline Fig.7 48-hr stability of staining. PBMC from a healthy donor were stimulated with PMA and Ionomycin for 4 hours followed by staining as outlined in the OMIP. Samples were acquired fresh (Day 1), or were stored at 4°C for either 24 hours (Day 2) or 48 hours (Day 3) before acquisition. Upper panels show the stability of the APC-Cy7 CD4+ and QD605 CD8+ T cell staining. Bottom panels show the stability of the different cytokine staining.

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