Clin Cancer Res-2001-Clay-1127-35.pdf

Embed Size (px)

Citation preview

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    1/10

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    2/10

    Review

    Assays for Monitoring Cellular Immune Responses to Active

    Immunotherapy of Cancer

    1

    Timothy M. Clay, Amy C. Hobeika,

    Paul J. Mosca, H. Kim Lyerly, and

    Michael A. Morse2

    Departments of Surgery [T. M. C., A. C. H., P. J. M., H. K. L.],Medicine [M. A. M.], Pathology [H. K. L.], and Immunology[H. K. L.], Duke University Medical Center, Durham, North Carolina27710

    Abstract

    Numerous cancer immunotherapy strategies are cur-

    rently being tested in clinical trials. Although clinical effi-

    cacy will be the final test of these approaches, the long and

    complicated developmental pathway for these agents neces-

    sitates evaluating immunological responses as intermediate

    markers of the most likely candidates for success. This has

    emphasized the need for assays that accurately detect and

    quantitate T cell-mediated, antigen-specific immune re-

    sponses. This review evaluates the currently used in vivo and

    in vitro methods of assessing T-cell number and function,

    including delayed-type hypersensitivity, tetramer analysis,

    ELISPOT, flow cytometry-based analysis of cytokine ex-

    pression, and PCR-based detection of T-cell receptor gene

    usage or cytokine production. We provide examples of how

    each has been used to monitor recent clinical trials and a

    discussion of how well each correlates with clinical outcome.

    Introduction

    The development of strategies for actively stimulating im-

    munological rejection of tumors, previously an elusive goal, has

    been accelerated by demonstration of the prerequisites for anti-

    gen-specific immunity that has revealed numerous avenues for

    delivering antigens for presentation to T cells. Whole tumor

    vaccines mixed with adjuvant, gene-modified tumors, tumor

    antigen-encoding viral vectors, protein and peptide antigen, and

    dendritic cells loaded with antigen are all being studied in

    clinical trials. To promote a candidate to an evaluation in a

    large-scale clinical trial, it is usually necessary to demonstrate

    that the treatment has a significant impact on an intermediate

    predictive of clinical outcome. For cytotoxic agents, this marker

    is typically tumor regression. For agents not expected to cause

    tumor regression but that still may have a beneficial effect, a

    biological marker is usually chosen based on the presumed

    mode of activity. For immunotherapy, such a marker would be

    stimulation of a tumor antigen-specific immune response detect-

    able by one or more immunological assays. Although effectors

    such as monocytes, natural killer cells, and antibodies may have

    an important role in antitumor immunity, most consider it vital

    to use assays that evaluate the number and function of CD8

    CTLs that directly recognize tumor peptides presented by MHC

    molecules on the surface of a tumor cell as a trigger for direct

    cytolysis, and CD4 helper T cells, particularly T helper type 1

    responses, that lead to CTL generation. A number of assays

    show promise as methods for quantifying and characterizing the

    T-cell response to immunizations and for serially monitoringthese responses. These tests of immunity include in vivo func-

    tional measures, in vitro phenotypic assays, and in vitro func-

    tional assays. In this review, we will initially discuss these

    assays and how they have been used thus far in clinical trials.

    Subsequently, we will compare their performance as intermedi-

    ate markers of clinical benefit and conclude by reviewing im-

    portant considerations for choosing immune assays.

    In Vivo Measures of Antigen-specific Immunity

    DTH.3 In the DTH test, antigen in the form of soluble

    protein alone or as antigen loaded onto antigen-presenting cells

    is injected intradermally, and the diameter of erythema or indu-

    ration after 4872 h is measured. CD4 T helper cells that

    recognize the antigen presented on local antigen-presenting cellsmediate the response by releasing cytokines that increase vas-

    cular permeability and recruit monocytes and other inflamma-

    tory cells to the site. Less frequently, a similar response may be

    mediated by CD8 T cells (1). The cutoff for a positive

    response has not been standardized nor has the dose for DTH

    testing, although protein antigens are generally administered as

    1050 g in 0.1 ml. This low dose is considered small enough

    that it does not induce a systemic immune response or cause

    excessive skin toxicity but is of a sufficient magnitude to induce

    a detectable local response.

    DTH remains one of the most frequent immune tests per-

    formed in immunotherapy studies (24), but several issues must

    be taken into account. The first is whether the DTH response is

    truly antigen specific. Thurner et al. (5) vaccinated patients with

    peptide-loaded DCs and detected induration and erythema at the

    injection site in 7 of 11 patients but also found similar results for

    DCs not loaded with any antigen. Conversely, in our own

    studies, some patients without obvious induration or erythema

    had infiltrates of T cells at the injection site in skin biopsies

    Received 10/13/00; revised 2/7/01; accepted 2/7/01.1 Supported by NIH Grants U01CA72162-03, 5-P01CA47741-08, and1-P01-CA78673-01A1 and the C. Douglas McFadyen Fund. M. A. M. isa recipient of an American Society of Clinical Oncology Career Devel-opment Award and is supported by NIH Grant M01RR00030.2 To whom requests for reprints should be addressed, at Box 3233,Durham, NC 27710. Phone: (919) 681-8350; Fax: (919) 681-7970; E-

    mail: [email protected].

    3 The abbreviations used are: DTH, delayed-type hypersensitivity; DC,dendritic cell; IL, interleukin; PBMC, peripheral blood mononuclearcell; TCR, T-cell receptor; CMV, cytomegalovirus; CDR, complemen-tarity determining region; V-D, variable-diversity; D-J, diversity-join-ing; ELISPOT, enzyme-linked immunospot; LDA, limiting dilution

    analysis.

    1127Vol. 7, 11271135, May 2001 Clinical Cancer Research

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    3/10

    taken after DTH testing with DCs loaded with carcinoembry-

    onic antigen peptide (6). Other components of the immunizing

    agent may also contribute to the DTH response. For example,

    intradermal granulocyte/macrophage-colony stimulating factor,

    a component of some vaccine strategies, by itself, may induce a

    granulocyte/macrophage-colony stimulating factor-specificDTH reaction (7). Some authors have grown the cells to be used

    for immunization in fetal bovine serum, which contains proteins

    that may serve as potential immunogens. The second issue is

    whether the DTH reaction should be measured as an all-or-none

    end point or whether it may possess a dose-response relation-

    ship with the immunizing agent. Schreiber (3), for example,

    observed that the diameter of erythema and induration at an

    autologous, unmodified tumor injection site increased with each

    immunization (except the last) and was greater with higher cell

    doses, suggesting the possibility of correlating dose and immune

    response. The third issue, the concordance of DTH with other

    immune assays, has not been clarified entirely. Disis et al. (8)

    observed that DTH induration of10 mm (but not 59 mm)

    correlated with a positive HER-2/neu-specific T-cell prolifera-

    tive response (stimulation index, 2.0) in patients immunized

    with HER2/neu peptides. Fourth, more data are needed to sup-

    port a correlation of clinical outcome with DTH response be-

    cause the number of patients studied in any one trial has been

    small. Nestle (9) immunized melanoma patients with DCs

    loaded with MAGE-1, MAGE-3, MART-1, gp-100, or tyrosin-

    ase peptides along with keyhole limpet hemocyanin by direct

    intra-lymph node injection. Nine of the 12 immunized patients

    developed DTH responses to peptide-loaded DCs, and 5 had

    diameters 10 mm; 4 of these patients had clinically detectable

    tumor regression. Clinical trials of autologous colon cancer plus

    bacillus Calmette Guerin have suggested that those who develop

    DTH reactivity have a greater chance of remaining disease free

    than those who do not (10). One obvious criticism, though, is

    that the ability to mount a DTH reaction to an antigen is merely

    a marker for a patient with a better performance status and

    greater likelihood of more favorable outcome than an individual

    who cannot mount a response. Critical to answering this criti-

    cism will be the inclusion of control antigens in immunizations

    strategies. Patients who respond to the control antigen but not

    the tumor antigen could then be compared with those who

    respond to both to determine whether outcome is more closely

    related to tumor antigen-specific response.

    In our opinion, DTH testing is useful as a preliminary

    screen for whether more detailed immune analysis is likely to be

    fruitful, and because it is relatively straightforward to perform

    and may possibly serve as an in vivo measure of the traffickingof lymphocytes to sites of tumor antigen, DTH testing will likely

    remain part of the immune analysis of cancer vaccines. The

    ability to isolate, expand, and assess the phenotype or function

    of antigen-specific T cells from skin biopsies of DTH sites may

    serve as an additional strategy to evaluate antigen-specific im-

    mune responses (11).

    In Vitro Measures of Immune Response

    Sources of Lymphocytes for in Vitro Immune Analysis.

    In vitro immune analyses require an adequate source of T cells

    with a frequency and functional activity reflective of the true

    immune response to the immunization. Clearly, peripheral blood

    is the most convenient source of T cells, but at least one study

    has questioned whether peripheral blood T-cell activity corre-

    lates with clinical response (12). Lee et al. (12) vaccinated

    patients with gp100 peptide with or without IL-2 and observed

    that despite detecting antigen-specific T cells in peripheral

    blood of some individuals immunized with gp100 alone, nonehad clinical signs of tumor regression. Conversely, although no

    antigen-specific T cells could be cultured ex vivo from the

    PBMCs of gp100 plus IL-2-treated patients, these were the only

    individuals in whom tumor regressions occurred. One possible

    explanation is that the antigen-specific T cells had migrated out

    of the peripheral blood, perhaps into tumor or other tissues.

    Although tumors may contain antigen-specific T cells (13), the

    detection of a lymphocytic infiltrate in a tumor has not uni-

    formly correlated with an improved prognosis in cancer patients,

    and in one study, tumor-infiltrating lymphocytes were shown to

    have defects in the expression of the TCR-associated molecule

    CD3, specifically the chain (14). Regional lymph nodes drain-

    ing the immunization site may contain the most recently stim-

    ulated T cells, but it has been shown that even healthy, non-

    tumor-bearing individuals may have lymph nodes harboring

    MART-1-specific T cells (15). Finally, T cells specific for the

    antigen of interest have been cloned from DTH sites, and

    although this may serve as a surrogate for tumor infiltration, the

    conditions at a skin injection site not infiltrated with tumor are

    likely to be different from tumor tissue itself. Therefore, despite

    the theoretical concerns, sampling of peripheral blood lympho-

    cytes has remained the standard. Important considerations for

    peripheral blood sampling include the timing of collection be-

    fore and after immunization and whether to perform the analy-

    ses real-time on fresh specimens or simultaneously on cryo-

    preserved cells.

    In Vitro Phenotypic Measures of Antigen-specific Cellular

    Immune Responses

    Analysis of T-Cell Receptor V Region Usage. The

    magnitude of an antigen-specific immune response may be

    determined by enumerating T cells according to a phenotypic

    marker such as the TCR using flow cytometric or PCR-based

    techniques. Expansions of TCRs expressing particular variable

    (V) region- or V- chains may be detected by flow cytometry

    using antibodies that recognize different variable or joining

    region subfamilies of the TCR or chains. An increase in the

    number of cells expressing a particular J-, J-, V- ,or V-

    chain would indicate development of oligoclonality, a possible

    sign of induction of a specific immune response (16). This

    approach has limited value for a number of reasons: (a) only aminority of T cells expressing a particular J-, J-, V-, or V-

    combination will be specific for a particular antigen; (b) the

    response to most antigens is quite diverse and uses many dif-

    ferent joining and variable regions; (c) monospecific antibodies

    are not available for all J region or V region gene subfamilies,

    and therefore, this analysis is incomplete at best. Nonetheless, if

    the antigens that are the target of the immune response are

    unknown, this method may still have some usefulness.

    Peptide MHC Tetramers. More recently, it has become

    possible to visualize antigen-specific T cells under flow cytom-

    etry by using soluble, fluorescently labeled, multimeric MHC-

    peptide complexes (17) that bind stably, specifically, and avidly

    1128 Immune Responses to Immunotherapy

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    4/10

    to antigen-specific T cells. During flow cytometric analysis, one

    can gate on the CD8 T cells and look for expression of

    antigen-specific TCRs. The largest body of data regarding the

    usefulness of tetramers is derived from studies of viral epitope-

    specific CTLs. Analysis of peripheral blood T cells specific for

    CMV and EBV demonstrated that between 0.2 and 2.5% ofcirculating CD8 cells were specific for peptides representing

    these antigens. Some authors have found correlation of MHC

    tetramer positivity and cytotoxicity in traditional microcytotox-

    icity assays (17), and the intensity of staining of CD8 T cells

    with peptide MHC tetramers appears to correlate with T-cell

    avidity for the antigen (18), but tetramer positive cells occasion-

    ally fail to kill targets expressing the specific epitope (16).

    Several recent studies have demonstrated the utility of flow

    cytometric analysis using peptide MHC tetramers to quantitate

    CD8 T cells specific for tumor antigens or control antigens

    used frequently in immunotherapy protocols (12, 19, 20). Dun-

    bar et al. (21) have used peptide MHC tetramers to allow the

    selection of antigen-specific T cells from peripheral blood or

    lymph nodes by cell sorting. These selected T cells were cloned

    for further analysis and were shown to respond to specific

    antigen by cytokine production.

    Although peptide MHC tetramers are powerful tools, they

    have certain limitations. They can only be used to detect im-

    mune responses to known antigens, because the peptide of

    interest must be loaded into the peptide MHC tetramer and thus

    must already be known and synthesized. Additionally, only

    class I MHC tetramers have been available routinely for wide-

    spread use, although class II tetramers have been described.

    Finally, because of the exquisite sensitivity of peptide/MHC

    tetramers for quantitating antigen-specific T cells, an interesting

    question has been raised regarding whether CD8() cells that

    bind to peptide/MHC tetramers are nave or antigen-experienced

    (memory T cells). Pittet et al. (15) observed that 10 of 13

    melanoma patients and 6 of 10 healthy individuals had high

    frequencies (1 of 2500 CD8 T cells) of Melan-A-specific

    cells in the peripheral blood. All of these Melan-A-specific cells

    from the healthy individuals and seven of the patients displayed

    a naive CD45RA(hi)/RO() phenotype. In three of the patients,

    memory CD45RA(lo)/RO() Melan-A-specific cells were

    observed. In contrast, influenza matrix-specific CTLs from all

    individuals exhibited a CD45RA(lo)/RO() memory pheno-

    type. One patient was observed to have an evolution of the

    Melan-A-specific cell phenotype over time. This suggests that in

    addition to simply detecting peptide MHC-positive cells, it may

    be important to assess whether antigen-specific cells are nave

    or memory T cells to determine whether the detected antigen-specific T cells have been stimulated by the immunization

    strategy.

    TCR Complementarity Determining Region 3. Anti-

    gen-specific T cells may also be phenotypically detected by

    PCR techniques for detecting a restricted TCR repertoire (22) by

    sequencing the third CDR (CDR3) of the TCR. The CDR3

    region encodes the highly polymorphic portion of the TCR

    responsible for recognizing peptide-MHC complexes. For the

    chain, the CDR3 region encodes the V-D segment and D-J

    segment junctions, whereas for the -chain, it encodes the V-J

    junction. Using V, D, or J region subfamily-specific PCR prim-

    ers, PCR may be performed to detect the development of re-

    stricted TCR gene usage (23, 24). Some studies in melanoma

    patients (25, 26) and renal cell carcinoma patients (27) have

    detected a restricted TCR gene usage. However, other studies in

    melanoma have found unrestricted TCR gene usage (28, 29).4 It

    is too soon to determine the role of this technology in monitor-

    ing immune responses in clinical trials, and more studies areneeded. In particular, it has been used primarily as a qualitative

    measure of skewing of the T-cell repertoire, and it may not be

    possible to easily correlate its results with clinical outcome.

    Nonetheless, its advantages include the small amount of speci-

    men required, the ability to perform the analysis from cells

    directly isolated from the blood to avoid introducing biases

    caused by ex vivo expansion, and the reproducibility and internal

    controls that permit analysis of samples collected at different

    times. Recently, a more automated and rapid fluorescence-based

    method for CDR3 length analysis of expressed TCR gene fam-

    ilies that was able to distinguish between polyclonal, oligo-

    clonal, and monoclonal CDR3 distributions has been developed

    (30).

    In Vitro Functional Measures of Antigen-specific

    Immune Responses

    T-cell number and function may be monitored by assays

    that detect T cells by an activity such as cytokine production,

    proliferation, or cytotoxicity.

    Lymphoproliferation Assay. The ability of T cells to

    proliferate in response to antigen has traditionally been used as

    an indicator of the presence of antigen-specific CD4 helper T

    cells. Typically, the specimen of purified T cells or PBMCs is

    mixed with various dilutions of antigen or antigen in the pres-

    ence of stimulator cells (irradiated autologous or HLA matched

    antigen-presenting cells). After 72120 h, [3H]thymidine was

    added, and DNA synthesis (as a measure of proliferation) wasquantified by using a gamma counter to measure the amount of

    radiolabeled thymidine incorporated into the DNA. A stimula-

    tion index can be calculated by dividing the number of cpm for

    the specimen by the number of cpm in cells incubated without

    antigen as a control.

    The proliferation assay has been used frequently in clinical

    trials to compare T-cell responses before and after immunization

    (3, 4, 3134). Depending on the immunization strategy, a small

    percentage of patients (31, 32) to as many as half (33) or all (34)

    patients have been found to respond by proliferation assays. Its

    major advantage is the ability to perform the assay directly on

    peripheral blood samples, giving a picture of the T-cell activity

    present in vivo (although the in vitro culture period can intro-

    duce artifacts in the results.) Its drawbacks are that it does notmeasure activity with direct mechanistic relevance to tumor

    rejection, it has not yet been convincingly correlated with clin-

    ical outcome (3, 31), it can be influenced by the nonspecific

    immune function of the patients, and the stimulation index does

    not necessarily correlate with the number of antigen-specific T

    cells present in vivo. High levels of proliferation by a few cells

    or low levels of proliferation by many cells would give a similar

    stimulation index. A recent flow cytometric assay measuring

    4 T. Clay, unpublished observation.

    1129Clinical Cancer Research

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    5/10

    distribution of cell membrane dyes into daughter cells produced

    during proliferation permits the number of antigen-responsive

    cells in a stimulation assay to be determined (35).

    Detection of Secreted Cytokines by ELISA and ELIS-

    POT Assays. Cytokine secretion by T cells in response to

    antigen may be detected by measuring either bulk cytokineproduction (by an ELISA) or enumerating individual cytokine

    producing T cells (by an ELISPOT assay). In the ELISA assay,

    PBMC specimens are incubated with antigen (with or without

    antigen-presenting cells), and after a defined period of time, the

    supernatant from the culture is harvested and added to microtiter

    plates coated with antibody for cytokines of interest such as

    IFN-, TNF-, or IL-2. Antibodies ultimately linked to a de-

    tectable label or reporter molecule are added, and the plates are

    washed and read. Generally, a single cytokine is measured in

    each well, although a recently described modification permits up

    to 15 cytokines to be measured in a single sample (36). In this

    procedure, antibodies to cytokines of interest are covalently

    bound to microspheres with uniform, distinctive proportions of

    red and orange fluorescent dyes. Detection antibodies conju-

    gated to a green fluorescent reporter dye are added, and flow

    cytometry is performed. By gating on a particular orange/red

    fluorescence indicating a particular cytokine of interest, it is

    possible to quantify the amount of cytokine that is proportional

    to the amount of green fluorescence. ELISA has been used for

    monitoring in several clinical trials (3, 4, 33), although the

    definition of a positive result differs (e.g., an amount of IFN-/

    well that is two times greater than control wells). Because this

    is an assay of the cytokine production of a population of cells,

    it does not give information about individual cells and cannot be

    used to enumerate the antigen-specific T cells. Furthermore, it

    does not measure the actual cytokine profile of these cells in

    vivo but rather, the ability of the cells to secrete cytokine when

    exposed to an antigenic stimulus. The ELISA assay can also be

    used to determine the levels of cytokines in serum or other body

    fluids. Although this may give a broad picture of the inflamma-

    tory state of a patient, it cannot be used to evaluate the cytokine

    profile in the milieu of the actual tumor cells. Flow cytometry-

    based assays (as will be discussed later) have also been found to

    be more reliable by some authors (37).

    The basic steps of an ELISPOT assay (38) are: (a) coating

    a 96-well microtiter plate with purified cytokine-specific anti-

    body; (b) blocking the plate to prevent nonspecific absorption of

    random proteins; (c) incubating the cytokine-secreting T cells

    with stimulator cells at several different dilutions; (d) lysing the

    cells with detergent; (e) adding a labeled second antibody; and

    (f) detecting the antibody-cytokine complex. The product of thefinal step is usually an enzyme/substrate reaction producing a

    colored product that can be quantitated microscopically, visu-

    ally, or electronically. Each spot represents one single cell

    secreting the cytokine of interest. The antigen-specific T-cell

    precursor frequency is determined by dividing the number of

    spots (cytokine-secreting cells) by the number of cells placed

    into the well. The ELISPOT assay has been shown to reliably

    detect the number of antigen-specific T cells in experiments in

    which known quantities of antigen-specific T cells were added

    to bulk PBMC preparations (39). Miyahira et al. (40) have

    reported that the CTL precursor frequency provided by ELIS-

    POT assay is comparable with that obtained by the limiting

    dilution analysis. Although rigorous statistical analysis has not

    been performed yet, there is interest in determining whether the

    ELISPOT assay correlates with survival (41). In a retrospective

    analysis of melanoma patients immunized with a polyvalent

    vaccine (42), MAGE-3 and Melan-A/MART-1-specific, IFN--

    secreting T cells were enumerated by ELISPOT. Those whodemonstrated antigen-specific T-cell secretion of IFN- had a

    longer recurrence free survival (12 months) than nonre-

    sponders (35 months).

    Because the task of counting the number of spots visually

    becomes difficult and time consuming with large numbers of

    spots (100), computerized plate readers using digital cameras

    have been developed (43). In our opinion, the computerized

    methods provide superlative discrimination of antigen-specific

    responses from background and make the ELISPOT an excel-

    lent choice for an immune monitoring assay in a large-scale

    study. Further modifications that may increase the usefulness of

    the ELISPOT include a dual color method for evaluating two

    different cytokine release patterns at a time (44) and the use of

    PBMCs loaded with poxvirus vectors encoding the antigen of

    interest as stimulators so that individuals of any HLA type

    (instead of just well-known HLA types) may be included in

    analyses (45).

    Detection of Intracellular Cytokines by Multiparameter

    Flow Cytometry. It was first demonstrated in murine models

    that different patterns of cytokine secretion could be used to

    differentiate between memory/effector T cells with different

    immune functions (46). The two patterns, T helper 1 with

    secretion of IL-2, IFN-, and TNF-, and T helper 2 with

    secretion of IL-4, IL-5, IL-6, IL-10, and IL-13, also appear to

    have human counterparts. Thus, it is possible to monitor im-

    mune responses in humans by characterizing the cytokine se-

    cretion pattern of T cells in peripheral blood, lymph nodes, or

    tissues by flow cytometry (reviewed in Ref. 47). Most methods

    involve a short period (4 6 h) ofin vitro T-cell activation (using

    antigen or mitogens) and source of stimulator cells (autologous

    antigen-presenting cells or PBMCs). For the last 3 4 h of

    stimulation, cytokine secretion is prevented by the addition of

    brefeldin A. After this stimulation period, most protocols rec-

    ommend staining with fluorochrome-conjugated anti-CD4 and

    anti-CD8 antibodies to allow gating on T cells and anti-CD69 to

    monitor activation of T cells. The cells are then fixed and

    permeabilized and stained with an antibody to the cytokine of

    interest (e.g., IFN- or IL-2). We have found that it is also

    possible to fix and permeabilize the cells followed by staining

    for surface and intracellular proteins. Three- or four-color flow

    cytometry is performed to enumerate the percentage of CD4()or CD8() CD69() cytokine() T cells. This assay has been

    modified so that it may be performed on PBMCs (48) or whole

    blood (49). Typical T-cell percentages for various antigens

    range from 0.1% (e.g., measles or mumps antigen) to 5% (CMV

    antigen; Ref. 47) or more.

    Some studies with serial analysis of intracellular cytokine

    induction have demonstrated correlation with clinical outcome.

    In a Phase I/II study of immunization with SRL 172 in patients

    with stage IV malignant melanoma, lymphocyte activation was

    assayed prior to each vaccine administration using a fluores-

    cence-activated cell sorter-based intracellular cytokine assay

    (50). Induction of intracellular IL-2 production was associated

    1130 Immune Responses to Immunotherapy

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    6/10

    with improved survival. Surprisingly, induction of IFN- or

    both IL-2 plus IFN- was not associated with improved sur-

    vival, demonstrating the complexities in choosing surrogate

    markers. Reinartz et al. (51) followed intracellular cytokine

    production in T cells obtained at various time points during

    immunization of ovarian cancer patients with the anti-idiotypevaccine ACA125. Early in the immunizations, predominantly

    IL-2 and IFN- were observed, but later a T helper 2 pattern was

    observed. This correlated with generation of anti-anti-idiotype

    antibodies and prolonged survival.

    The major drawback of this approach is that the cells

    detected are no longer viable, and thus cells cannot be sorted and

    cultured to produce clones. Recently, a novel method that uses

    flow cytometry to detect surface-expressed cytokines was de-

    scribed (52). Magnetofluorescent liposomes containing several

    thousand fluorescein molecules and colloidal magnetic particles

    and conjugated to antibodies specific for IFN- and IL-10 were

    shown capable of detecting surface expression of these mole-

    cules on 12.5 and 34.8% of T cells. Most of these cells were

    shown to have intracellular cytokine when they were permeabi-

    lized for analysis. The cells remain viable so they may be sorted

    for use in other assays. This method would not be applicable for

    all cytokines because some, such as Il-2, IL-4, and IL-5, cannot

    be detected on cell surfaces.

    Measurement of Cytokine mRNA Levels by Real-Time

    Quantitative RT-PCR

    Quantitative RT-PCR is a highly accurate molecular

    method for measuring the levels of transcripts of a gene or genes

    of interest in sample RNA (53). Kruse et al. (54) applied the

    technique to the analysis of cytokine mRNA from cryopreserved

    normal donor blood samples. Recently, Kammula et al. (55)

    used the technique in clinical trials of melanoma peptide-based

    vaccines to detect antigen-specific T-cell responses by compar-

    ing pre- and postvaccine samples from melanoma patients.

    Peripheral blood samples and tumor tissues obtained by fine

    needle aspiration were evaluated. For PBMC samples, the

    method entails thawing cells into fresh medium, allowing them

    to recover from thawing for 10 h, and then incubating the cells

    for an additional 2 h with either the peptide used in the vaccine

    or an irrelevant peptide, followed by total RNA isolation. Quan-

    titative RT-PCR was then used to measure cytokine mRNA

    levels in the samples. Data were normalized to expression of a

    control gene, such as CD8. This study showed that quantitative

    RT-PCR can be used to detect antigen-specific T-cell responses

    in peripheral blood samples. Additionally, localization of anti-

    gen-specific T cells to tumor sites was demonstrated by analysisof biopsy samples without any in vitro stimulation step. Further

    studies are needed, in part to determine the relative sensitivity of

    this methodology, including comparative studies against other

    assay techniques, and to provide additional studies so that the

    reliability of the method may be assessed.

    Direct Cytotoxicity Assays. Cytotoxicity assays are ap-

    pealing because measurement of the ability of CD8 CTLs to

    lyse tumor is thought to be a relevant marker for in vivo

    antitumor activity. The microcytotoxicity assay involves mixing

    the specimen containing T cells or PBMCs with antigen-

    expressing targets loaded with 51Cr or europium and measuring

    the release of the chromium or europium after target cell lysis.

    Because autologous tumor is often difficult to obtain, surrogate

    targets are often used, such as HLA-matched allogeneic tumor

    cell lines, and targets that can be loaded with the antigen of

    interest (such as autologous DCs loaded with peptide or genetic

    material encoding the antigen, or T2 cells loaded with peptide).

    Targets sensitive to natural killer cell lysis (K562 and Daudi) arealso included to determine the level of nonspecific lytic activity.

    The percentage of lysis of the targets after incubation for 4 h is

    calculated by comparison with the maximum achievable lysis of

    the target. Using different E:T ratios, it is possible to derive a

    value for the potency of cytotoxicity measured in lytic units,

    the number of T cells needed to achieve a stated amount of lysis.

    Theoretically, lytic units can be used to compare various CTL

    preparations. Cytotoxicity assays have been used for immune

    monitoring in studies of passively delivered T cells (56) and

    active immunotherapy approaches (31, 57).

    One drawback to the microcytotoxicity assay is its relative

    insensitivity. Although bulk CTL assays represent a useful tech-

    nique to give high versus low or versus readouts, they are

    not particularly quantitative. Furthermore, there is a need to

    stimulate the CTLs multiple times before testing their lytic

    activity (31), because it is unusual to find antigen-specific lysis

    by cells directly isolated from the peripheral blood, even in

    vaccinated patients (58). These multiple stimulations may dis-

    tort the composition and activity of the T-cell population from

    its original state. Also, as discussed above, because autologous

    tumor is difficult to obtain, other targets must be used that may

    not reflect the actual ability to lyse autologous tumor cells in

    vivo. For example, tumor cells may down-regulate their MHC

    molecules or up-regulate their own Fas ligand, causing T-cell

    apoptosis. It has also been shown that the CTL response is

    heterogeneous with different levels of avidity for the antigen

    (59). Because targets used for in vitro testing usually express

    high levels of antigen, lysis may not reflect the ability to lyse

    tumor in vivo if the in situ tumor expresses low levels of the

    antigen. Finally, questions as to the correlation with clinical

    response have been raised. In at least one study, clinical regres-

    sions were observed in two patients in the absence of CTL

    activity (57). Modifications to the cytotoxicity assay that make

    it simpler and more reproducible are being developed including

    flow cytometric techniques for separating dead (lysed) target

    cells stained with propidium iodide from live (unlysed) cells

    stained with a cyanine membrane dye.

    Quantifying CTL Precursors by LDA. LDA, a cum-

    bersome but more quantitative assay for CTL precursor fre-

    quency, correlates T-cell number from a functional activity.

    LDA analyses involve the serial dilution of T cells in a verylarge number of wells, followed by an in vitro stimulation phase

    and target lysis phase. Poisson distribution analysis is applied to

    the results to determine the proportion of wells at a particular

    T-cell dilution that have 1 antigen-specific precursor at the

    start of the stimulation. Analysis of the frequencies of positive

    wells in successively higher dilutions as a function of log (T

    cells/well) generates a line, the slope of which is proportional to

    the precursor frequency. In addition to being cumbersome, labor

    intensive, and extremely operator dependent, the LDA is also

    flawed by the intrinsic assumption that a single antigen-specific

    T cell can be expanded during the stimulation phase to generate

    a signal above a mathematically determined threshold.

    1131Clinical Cancer Research

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    7/10

    Because the LDA assay is somewhat complicated and

    because a large number of cells are required for testing antigen

    specificity by LDA, it has been used in few published studies.

    Moller et al. (60) evaluated the T-cell response to immuniza-

    tions of melanoma patients with IL-7 gene-modified autologous

    tumor cells using LDA and found that after vaccination, PBMCscontained an increased number of tumor-reactive proliferative

    as well as cytolytic cells. In three of six patients, the frequencies

    of antimelanoma cytolytic precursor cells increased between

    2.6- and 28-fold. Two of these patients showed a minor clinical

    response. The same group demonstrated similar results with

    IL-12 gene-modified melanoma cells (61). DSouza et al. (62)

    evaluated Melan-A/Mart-1-specific CTL precursors in mela-

    noma patients using LDA and further demonstrated that they

    expressed a memory phenotype. More recently, Romero et al.

    (63) has made additional modifications of the assay to increase

    the ability to quantify the number of precursors. Thurner et al.

    (5) used this modification, which involves one cycle of in vitro

    stimulation with antigen before testing the cells for cytotoxicity,to measure MAGE-3A1 peptide-specific immune responses in a

    study of immunizations with DCs loaded with MAGE-3A1

    peptide. Eight of 11 patients were found to have increases in

    MAGE-3A1 CTL precursor frequency after immunization.

    Nonetheless, it is likely that this assay will be less frequently

    used in the future as newer assays that are more sensitive

    become accepted.

    Comparison of the Assays

    There is a paucity of studies that have directly compared

    the various assays for their performance in evaluating immune

    responses, and most of the data comes from studies of responses

    against viral antigens. Tan et al. (64) showed that estimates of

    CD8 T-cell frequencies specific for EBV-related antigens

    varied considerably according to the method used. Values ob-

    tained from MHC-peptide tetramer staining were, on average,

    4.4-fold higher than those obtained from ELISPOT assays,

    which were, in turn, on average, 5.3-fold higher than those

    obtained from LDA. Tetramer staining showed that as many as

    5.5% circulating CD8 T cells in a virus carrier were specific

    for a single EBV lytic protein epitope. Kuzushima et al. (65),

    using EBV-specific T-cell lines, confirmed that flow cytometric

    analysis is more sensitive than LDA for CTL precursors and

    ELISPOT in detecting IFN--producing T cells. The results of

    direct T-cell staining using multimeric peptide-MHC complexes

    raise important questions about the meaning of precursor fre-

    quencies estimated from LDA. One possible explanation for thisdiscrepancy is that only a fraction of cloned T cells are lytic;

    however, functional assays on sorted tetramer binding cells

    argue against this. Another major difference between the LDA

    and the direct detection assays, such as tetramer staining, is that

    the LDA depends on cell division. Greater than 10 divisions of

    a single precursor would be necessary during the stimulation

    phase of the LDA to register as a positive response. In cases of

    chronic viral infection, the precursor frequencies estimated by

    LDA appear to be closer to those estimated by direct staining

    with multimeric MHC-peptide. The LDA may therefore give a

    more meaningful figure of T cells with long-term growth po-

    tential.

    Considerations for Choosing Immune Assays

    Before choosing one or more immunological assays to

    monitor induction of antitumor immune responses, it is impor-

    tant to consider the performance characteristics of the assay in

    detecting immune responses, what magnitude of the immune

    response should be considered a positive response, and whetherthe assay results actually predict clinical outcome. Desirable

    performance characteristics of an assay for detecting T-cell

    responses would include: (a) adequate sensitivity, specificity,

    reliability, and reproducibility; (b) measurement of the true state

    of in vivo T-cell activity without introducing significant distor-

    tions; (c) simple and rapid to perform; and (d) requirement for

    only small quantities of specimens. As described above, tet-

    ramer analysis is highly sensitive, followed by ELISPOT. In our

    experience, the reliability of tetramers varies with some prepa-

    rations, yielding no staining of T-cell clones,5 and also, because

    not all peptides form functional tetramers, tetramers with an

    untried peptide must be tested empirically. ELISPOT assays

    digitally analyzed have considerable reliability in our hands, but

    there is little published data on interlaboratory reproducibility

    (66). Tetramer analysis is quick to perform, whereas the other

    flow cytometric assays of T-cell cytokine production take 8 h

    to perform and analyze. ELISPOT plates can be prepared in bulk

    in advance, and by using automated pipettes, plate washers, and

    plate readers, large numbers can be set up quickly and effi-

    ciently. The PCR-based techniques for detecting TCR gene

    usage or cytokine mRNA transcription require the smallest

    quantity of specimens.

    The cutoff that should be accepted as indicative of an

    effective level of immunological response is entirely unknown

    (and may vary for each assay), but it is necessary to make

    educated guesses. A reasonable starting point for trying to

    determine what constitutes a clinically relevant immune re-sponse is the experience in animal models. Cure of a murine

    sarcoma required infusion of 3 10E4 T cells with receptors

    specific for the rejection epitope, if the sarcoma had been

    established for 3 days. It is estimated that this represents 0.2

    0.5% of the circulating leukocytes (67). Because previous stud-

    ies of adoptive immunotherapy for malignancies in humans

    have used fairly nonspecific T cells, it is difficult to find similar

    human data. In one study of stem cell transplant recipients at

    risk for EBV-associated lymphoproliferative disorders, two to

    four infusions of as few as 10 (7) EBV-specific CTLs/m2,

    starting from the time of maximal virus load, resulted in a 2- to

    3-log decrease of virus titers (68). In patients who develop

    lymphoproliferative disorders, infusion of a similar number of

    EBV-specific CTLs can eradicate the tumors (69). If there areapproximately 1.5 4.5 109 CD8 T cells in the circulation

    (70), then at the time of infusion, the EBV-specific T cells

    would represent as many as 1 of every 100 CD8 T cells. Thus,

    we propose that a level of peripheral blood antigen-specific

    CD8 T cells in the range of 1% may be necessary to cause

    tumor remission. Of course, antiviral T-cell responses tend to be

    of greater frequency and avidity than antitumor responses, and

    5 A. Hobeika, unpublished observations.

    1132 Immune Responses to Immunotherapy

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    8/10

    thus, whether the T-cell response to viral antigens is relevant to

    antitumor responses is unknown.

    Correlation with clinical outcome is the most critical issue

    for any intermediate marker. Markers along the pathway to the

    ultimate mechanism for clinical benefit are the most desirable.

    Next best are markers that correlate very closely with outcome.Just as tumor regression after administration of a cytotoxic agent

    does not always result in survival benefit, development of an

    immunological response may not necessarily predict clinical

    outcome. For immunological assays, the most likely reason for

    this is that the immune responses that cause tumor regression are

    not known with certainty, and the available immunological

    assays may not measure these mechanisms. Tumors may be

    destroyed by CTLs through insertion of perforins and delivery

    of granzymes, by Fas-Fas ligand interactions, or by cytokine-

    mediated toxicity. Standard cytotoxicity assays only measure

    the direct lysis. Modifications of cytotoxicity assays to measure

    target cell apoptosis may be necessary to measure Fas-Fas

    ligand-mediated interactions. It is possible that none of the

    currently available assays actually measures a function with

    direct relevance to how tumors are actually attacked immuno-

    logically in the body. This demonstrates the importance of

    collecting data on correlation of immune response with clinical

    outcome whenever possible.

    Conclusions and Areas for Further Investigation

    The development of assays for detecting immunological

    responses to cancer vaccines is essential if these strategies are to

    be optimized. Standards are needed for performing the assays

    and interpreting the results. Agreement is needed on whether to

    analyze samples directly isolated from blood or lymph nodes or

    after a period ofin vitro stimulation. Because the various assays

    yield estimates of antigen-specific T cells that sometimes differin magnitude, it is critical to compare the immune response

    detected by a particular assay in a particular patient with the

    immune response specific for a well-established, immunogenic

    antigen, such as EBV or CMV peptide. Reproducibility between

    laboratories and correlation among immune assays requires rig-

    orous evaluation. Because most of the immune assays do not

    measure an activity with direct relevance to tumor cell killing by

    the immune system, the importance of rigorous statistical anal-

    ysis to determine the assay with the greatest correlation with

    outcome is necessary. Currently, several different assays are

    necessary until it can be established which correlate the best

    with clinical outcome. In our opinion, although tetramer analy-

    sis has high sensitivity, ELISPOT is more versatile for moni-

    toring clinical trials and is more readily performed, given thecurrent limited availability of tetramers.

    References

    1. Puccetti, P., Bianchi, R., Fioretti, M. C., Ayroldi, E., Uyttenhove, C.,Van Pel, A., Boon, T., and Grohmann, U. Use of a skin test assay todetermine tumor-specific CD8 T cell reactivity. Eur. J. Immunol., 24:

    14461452, 1994.

    2. Simons, J. W., Mikhak, B., Chang, J. F., DeMarzo, A. M., Carducci,M. A., Lim, M., Weber, C. E., Baccala, A. A., Goemann, M. A., Clift,S. M., Ando, D. G., Levitsky, H. I., Cohen, L. K., Sanda, M. G.,Mulligan, R. C., Partin, A. W., Carter, H. B., Piantadosi, S., Marshall,F. F., and Nelson, W. G. Induction of immunity to prostate cancerantigens: results of a clinical trial of vaccination with irradiated autol-

    ogous prostate tumor cells engineered to secrete granulocyte-macroph-age colony-stimulating factor using ex vivo gene transfer. Cancer Res.,59: 51605168, 1999.

    3. Schreiber, S., Kampgen, E., Wagner, E., Pirkhammer, D., Trcka, J.,Korschan, H., Lindemann, A., Dorffner, R., Kittler, H., Kasteliz, F.,Kupcu, Z., Sinski, A., Zatloukal, K., Buschle, M., Schmidt, W., Birn-

    stiel, M., Kempe, R. E., Voigt, T., Weber, H. A., Pehamberger, H.,Mertelsmann, R., Brocker, E. B., Wolff, K., and Stingl, G. Immuno-therapy of metastatic malignant melanoma by a vaccine consisting ofautologous interleukin-2-transfected cancer cells: outcome of a Phase Istudy. Hum. Gene Ther., 10: 983993, 1999.

    4. Salgaller, M. L., Lodge, P. A., McLean, J. G., Tjoa, B. A., Loftus,D. J., Ragde, H., Kenny, G. M., Rogers, M., Boynton, A. L., andMurphy, G. P. Report of immune monitoring of prostate cancer patientsundergoing T-cell therapy using dendritic cells pulsed with HLA-A2-specific peptides from prostate-specific membrane antigen (PSMA).Prostate, 35: 144151, 1998.

    5. Thurner, B., Haendle, I., Roder, C., Dieckmann, D., Keikavoussi, P.,Jonuleit, H., Bender, A., Maczek, C., Schreiner, D., von den Driesch, P.,Brocker, E. B., Steinman, R. M., Enk, A., Kampgen, E., and Schuler, G.Vaccination with Mage-3A1 peptide-pulsed mature, monocyte-deriveddendritic cells expands specific cytotoxic T cells and induces regression

    of some metastases in advanced stage IV melanoma. J. Exp. Med., 190:16691678, 1999.

    6. Morse, M. A., Deng,. Y, Coleman, D., Hull, S., Kitrell-Fisher, E.,Nair, S., Schlom, J., Ryback, M. E., and Lyerly, H. K. A Phase I studyof active immunotherapy with carcinoembryonic antigen peptide (CAP-1)-pulsed, autologous human cultured dendritic cells in patients withmetastatic malignancies expressing carcinoembryonic antigen. Clin.Cancer Res., 5: 13311338, 1999.

    7. McNeel, D. G., Schiffman, K., and Disis, M. L. Immunization withrecombinant human granulocyte-macrophage colony-stimulating factoras a vaccine adjuvant elicits both a cellular and humoral response torecombinant human granulocyte-macrophage colony-stimulating factor.Blood, 93: 26532659, 1999.

    8. Disis, M. L., Schiffman, K., Gooley, T. A., McNeel, D. G., Rinn, K.,and Knutson, K. L. Delayed-type hypersensitivity response is a predic-tor of peripheral blood T-cell immunity after HER-2/neu peptide im-

    munization. Clin. Cancer Res., 6: 13471350, 2000.9. Nestle, F. O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer,R., Burg, G., and Schadendorf, D. Vaccination of melanoma patientswith peptide- or tumor lysate-pulsed dendritic cells. Nat. Med., 4:

    328332, 1998.

    10. Hoover, H. C., Jr., Surdyke, M., Dangel, R., Peters, L. C., andHanna, M. G., Jr. Delayed cutaneous hypersensitivity to autologoustumor cells in colon cancer patients immunized with an autologous cell:Bacillus Calmette-Guerin vaccine. Cancer Res., 44: 16711676, 1984.

    11. Waanders, G. A., Rimoldi, D., Lienard, D., Carrel, S., Lejeune, F.,Dietrich, P. Y., Cerottini, J. C., and Romero, P. Melanoma-reactivehuman cytotoxic T lymphocytes derived from skin biopsies of delayed-type hypersensitivity reactions induced by injection of an autologousmelanoma cell line. Clin. Cancer Res., 3: 685696, 1997.

    12. Lee, K. H., Wang, E., Nielsen, M. B., Wunderlich, J., Migueles, S.,Connors, M., Steinberg, S. M., Rosenberg, S. A., and Marincola, F. M.

    Increased vaccine-specific T cell frequency after peptide-based vacci-nation correlates with increased susceptibility to in vitro stimulation butdoes not lead to tumor regression. J. Immunol., 163: 62926300, 1999.

    13. Panelli, M. C., Riker, A., Kammula, U., Wang, E., Lee, K-H.,Rosenberg, S. A., and Marincola, F. M. Expansion of tumor-T cell pairsfrom fine needle aspirates of melanoma metastases. J. Immunol., 164:495504, 2000.

    14. Whiteside, T. L. Signaling defects in T lymphocytes of patients withmalignancy. Cancer Immunol. Immunother., 48: 346352, 1999.

    15. Pittet, M. J., Valmori, D., Dunbar, P. R., Speiser, D. E., Lienard, D.,Lejeune, F., Fleischhauer, K., Cerundolo, V., Cerottini, J. C., andRomero, P. High frequencies of naive Melan-A/MART-1-specificCD8() T cells in a large proportion of human histocompatibilityleukocyte antigen (HLA)-A2 individuals. J. Exp. Med., 190: 705715,1999.

    1133Clinical Cancer Research

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    9/10

    16. Mackensen, A., Veelken, H., Lahn, M., Wittnebel, S., Becker, D.,

    Kohler, G., Kulmburg, P., Brennscheidt, U., Rosenthal, F., Franke, B.,

    Mertelsmann, R., and Lindemann, A. Induction of tumor-specific cyto-

    toxic T lymphocytes by immunization with autologous tumor cells and

    interleukin-2 gene transfected fibroblasts. J. Mol. Med., 75: 290296,

    1997.

    17. Altman, J. D., Moss, P. A. H., Goulder, P. J. R., Barouch, D. H.,

    McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J., and Davis,

    M. M. Phenotypic analysis of antigen-specific T lymphocytes. Science

    (Washington DC), 274: 9496, 1996.

    18. Yee, C., Savage, P. A., Lee, P. P., Davis, M. M., and Greenberg,

    P. D. Isolation of high avidity melanoma-reactive CTL from heteroge-

    neous populations using peptide-MHC tetramers. J. Immunol., 162:

    22272234, 1999.

    19. Romero, P., Dunbar, P. R., Valmori, D., Pittet, M., Ogg, G. S.,

    Rimoldi, D., Chen, J. L., Lienard, D., Cerottini, J. C., and Cerundolo, V.Ex vivo staining of metastatic lymph nodes by class I major histocom-

    patibility complex tetramers reveals high numbers of antigen-experi-

    enced tumor-specific cytolytic T lymphocytes. J. Exp. Med., 188: 1641

    1650, 1998.

    20. Dhodapkar, M. V., Steinman, R. M., Sapp, M., Desai, H., Fossella,

    C., Krasovsky, J., Donahoe, S. M., Dunbar, P. R., Cerundolo, V., Nixon,D. F., and Bhardwaj, N. Rapid generation of broad T-cell immunity in

    humans after a single injection of mature dendritic cells. J. Clin. Inves-

    tig., 104: 173180, 1999.

    21. Dunbar, P. R., Ogg, G. S., Chen, J., Rust, N., van der Bruggen, P.,

    and Cerundolo, V. Direct isolation, phenotyping, and cloning of low-

    frequency antigen-specific cytotoxic T lymphocytes from peripheral

    blood. Curr. Biol., 8: 413416, 1998.

    22. Kalams, S. A., Johnson, R. P., Trocha, A. K., Dynan, M. J., Ngo,

    H. S., DAquila, R. T., Kurnick, J. T., and Walker, B. D. Longitudinal

    analysis of T-cell receptor (TCR) gene usage by human immunodefi-

    ciency virus 1 envelope-specific cytotoxic T lymphocyte clones reveals

    a limited TCR repertoire. J. Exp. Med., 179: 12611271, 1994.

    23. McKee, M. D., Clay, T. M., Rosenberg, S. A., and Nishimura, M. I.

    Quantitation of T-cell receptor frequencies by competitive PCR: gener-

    ation and evaluation of novel TCR subfamily and clone specific com-

    petitors. J. Immunother., 22: 93102, 1999.

    24. Kourilsky, P., Bousso, P., Calbo, S., and Gapin, L. Immunological

    issues in vaccine trials: T-cell responses. Dev. Biol. Stand., 95: 117

    124, 1998.

    25. Salvi, S., Segalla, F., Rao, S., Arienti, F., Sartori, M., Bratina, G.,

    Caronni, E., Anichini, A., Clemente, C., Parmiani, G., and Sensi, M.

    Overexpression of the T-cell receptor -chain variable region

    TCRBV14 in HLA-A2-matched primary human melanomas. Cancer

    Res., 55: 33743379, 1995.

    26. Sensi, M., Salvi, S., Castelli, C., Maccalli, C., Mazzocchi, A.,

    Mortarini, R., Nicolini, G., Herlyn, M., Parmiani, G., and Anichini, A.

    T-cell receptor (TCR) structure of autologous melanoma-reactive cyto-

    toxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes over-

    express in vivo the TCR chain sequence used by an HLA-A2-

    restricted and melanocyte-lineage-specific CTL clone. J. Exp. Med.,178: 12311246, 1993.

    27. Weidmann, E., Logan, T. F., Yasumura, S., Kirkwood, J. M.,

    Trucco, M., and Whiteside, T. L. Evidence for oligoclonal T-cell re-

    sponse in a metastasis of renal cell carcinoma responding to vaccination

    with autologous tumor cells and transfer of in vitro-sensitized vaccine-

    draining lymph node lymphocytes. Cancer Res., 53: 47454749, 1993.

    28. Romero, P., Gervois, N., Schneider, J., Escobar, P., Valmori, D.,

    Pannetier, C., Steinle, A., Wolfel, T., Lienard, D., Brichard, V., Van Pel,

    A., Jotereau, F., and Cerottini, J. C. Cytolytic T lymphocyte recognition

    of the immunodominant HLA-A*0201-restricted Melan-A/MART-1 an-

    tigenic peptide in melanoma. J. Immunol., 159: 23662372, 1997.

    29. Romero, P., Pannetier, C., Herman, J., Jongeneel, C. V., Cerottini,

    J. C., and Coulie, P. G. Multiple specificities in the repertoire of a

    melanoma patients cytolytic T lymphocytes directed against tumor

    antigen MAGE-1.A1. J. Exp. Med., 182: 10191028, 1995.

    30. Lue, C., Mitani, Y., Crew, M. D., George, J. F., Fink, L. M., and

    Schichman, S. A. An automated method for the analysis of T-cell

    receptor repertoires. Rapid RT-PCR fragment length analysis of the

    T-cell receptor chain complementarity-determining region 3. Am. J.

    Clin. Pathol., 111: 683690, 1999.

    31. Khleif, S. N., Abrams, S. I., Hamilton, J. M., Bergmann-Leitner, E.,

    Chen, A., Bastian, A., Bernstein, S., Chung, Y., Allegra, C. J., andSchlom, J. A Phase I vaccine trial with peptides reflecting ras oncogene

    mutations of solid tumors. J. Immunother., 22: 155165, 1999.

    32. Rucker, R., Bresler, H. S., Heffelfinger, M., Kim, J. A., Martin,

    E. W., Jr., and Triozzi, P. L. Low-dose monoclonal antibody CC49

    administered sequentially with granulocyte-macrophage colony-stimu-

    lating factor in patients with metastatic colorectal cancer. J. Immu-

    nother., 22: 8084, 1999.

    33. Sandmaier, B. M., Oparin, D. V., Holmberg, L. A., Reddish, M. A.,

    MacLean, G. D., and Longenecker, B. M. Evidence of a cellular immune

    response against sialyl-Tn in breast and ovarian cancer patients after

    high-dose chemotherapy, stem-cell rescue, and immunization with Ther-

    atope STn-KLH cancer vaccine. J. Immunother., 22: 54 66, 1999.

    34. Disis, M. L., Grabstein, K. H., Sleath, P. R., and Cheever, M. A.

    Generation of immunity to the HER-2/neu oncogenic protein in patients

    with breast and ovarian cancer using a peptide-based vaccine. Clin.Cancer Res., 5: 12891297, 1999.

    35. Givan, A. L., Fisher, J. L., Waugh, M., Ernstoff, M. S., and Wallace,

    P. K. A flow cytometric method to estimate the precursor frequencies of

    cells proliferating in response to specific antigens. J. Immunol. Methods,230: 99112, 1999.

    36. Carson, R. T., and Vignati, D. A. Simultaneous quantitation of 15

    cytokines using a multiplexed flow cytometric assay. J. Immunol. Meth-

    ods, 227: 4152, 1999.

    37. Tayebi, H., Lienard, A., Billot, M., Tiberghien, P., Herve, P., and

    Robinet, E. Detection of intracellular cytokines in citrated whole blood

    or marrow samples by flow cytometry. J. Immunol. Methods, 229:

    121130, 1999.

    38. Czerkinsky, C., Andersson, G., Ekre, H. P., Nilsson, L. A., Klares-

    kog, L., and Ouchterlony, O. Reverse ELISPOT assay for clonal anal-

    ysis of cytokine production. I. Enumeration of -interferon-secreting

    cells. J. Immunol. Methods, 110: 2936, 1988.

    39. Schmittel, A., Keilholz, U., and Scheibenbogen, C. Evaluation of

    the interferon- ELISPOT-assay for quantification of peptide specific T

    lymphocytes from peripheral blood. J. Immunol. Methods, 210: 167

    174, 1997.

    40. Miyahira, Y., Murata, K., Rodriguez, D., Rodriguez, J. R., Esteban,

    M., Rodrigues, M. M., and Zavala, F. Quantification of antigen specific

    CD8 T cells using an ELISPOT assay. J. Immunol. Methods, 181:

    4554, 1995.

    41. Enk, A. .H, Wolfel, T., and Knop, J. Decreased rate of progression

    and induction of tumor-specific immune response by adjuvant immu-

    notherapy in stage IV melanoma. Hautarzt, 50: 103108, 1999.

    42. Reynolds, S. R., Oratz, R., Shapiro, R. L., Hao, P., Yun, Z., Fotino,M., Vukmanovic, S., and Bystryn, J. C. Stimulation of CD8 T-cellresponses to MAGE-3 and Melan A/MART-1 by immunization to a

    polyvalent melanoma vaccine. Int. J. Cancer, 72: 972976, 1997.43. Vaquerano, J. E., Peng, M., Chang, J. W., Zhou, Y. M., and Leong,S. P. Digital quantification of the enzyme-linked immunospot (ELIS-POT). Biotechniques, 25: 830 834, 1998.

    44. Okamoto, Y., Abe, T., Niwa, T., Mizuhashi, S., and Nishida, M.Development of a dual color enzyme-linked immunospot assay forsimultaneous detection of murine T helper type 1- and T helper type2-cells. Immunopharmacology, 39: 107116, 1998.

    45. Larsson, M., Jin, X., Ramratnam, B., Ogg, G. S., Engelmayer, J.,Demoitie, M. A., McMichael, A. J., Cox, W. I., Steinman, R. M., Nixon,D., and Bhardwaj, N. A recombinant vaccinia virus-based ELISPOTassay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS, 13: 767777, 1999.

    46. Paul, W. E., and Seder, R. A. Lymphocyte responses and cytokines.Cell, 76: 241251, 1994.

    1134 Immune Responses to Immunotherapy

  • 7/27/2019 Clin Cancer Res-2001-Clay-1127-35.pdf

    10/10

    47. Maino, V. C., and Picker, L. J. Identification of functional subsetsby flow cytometry: intracellular detection of cytokine expression. Cy-tometry, 34: 207215, 1998.

    48. Waldrop, S. L., Pitcher, C. J., Peterson, D. M., Maino, V. C., andPicker, L. J. Determination of antigen-specific memory/effector CD4T cell frequencies by flow cytometry: evidence for a novel, antigen-

    specific homeostatic mechanism in HIV-associated immunodeficiency.J. Clin. Investig., 99: 17391750, 1997.

    49. Suni, M. A., Picker, L. J., and Maino, V. C. Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry.J. Immunol. Methods, 212: 8998, 1998.

    50. Maraveyas, A., Baban, B., Kennard, D., Rook, G. A., Westby, M.,Grange, J. M., Lydyard, P., Stanford, J. L., Jones, M., Selby, P., andDalgleish, A. G. Possible improved survival of patients with stage IVAJCC melanoma receiving SRL 172 immunotherapy: correlation withinduction of increased of intracellular interleukin-2 in peripheral bloodlymphocytes. Ann. Oncol., 10: 817824, 1999.

    51. Reinartz, S., Boerner, H., Koehler, S., Von Ruecker, A., Schle-busch, H., and Wagner, U. Evaluation of immunological responses inpatients with ovarian cancer treated with the anti-idiotype vaccineACA125 by determination of intracellular cytokinesa preliminary re-port. Hybridoma, 18: 4145, 1999.

    52. Schefford, A., Assenmacher, M., Reiners-Schramm, L., Lauster, R.,and Radbruch, A. High-sensitivity immunofluorescence for detection ofthe pro- and anti-inflammatory cytokines interferon and interleukin-10on the surface of cytokine-secreting cells. Nat. Med., 6: 107110, 2000.

    53. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. Real timequantitative PCR. Genome Res., 6: 986994, 1996.

    54. Kruse, N., Pette, M., Toyka, K., and Riekmann, P. Quantification ofcytokine mRNA expression by RT-PCR in samples of previously frozenblood. J. Immunol. Methods, 210: 195203, 1997.

    55. Kammula, U. S., Lee, K-H., Riker, A. I., Wang, E., Ohnmacht,G. A., Rosenberg, S. A., and Marincola, F. M. Functional analysis ofantigen-specific T lymphocytes by serial measurement of gene expres-sion in peripheral blood mononuclear cells and tumor specimens. J. Im-munol., 163: 68676875, 1999.

    56. Schwartzentruber, D. J., Hom, S. S., Dadmarz, R., White, D. E.,Yannelli, J. R., Steinberg, S. M., Rosenberg, S. A., and Topalian, S. L.

    In vitro predictors of therapeutic response in melanoma patients receiv-ing tumor-infiltrating lymphocytes and interleukin-2. J. Clin. Oncol.,12: 14751483, 1994.

    57. Marchand, M., van Baren, N., Weynants, P., Brichard, V., Dreno,B., Tessier, M. H., Rankin, E., Parmiani, G., Arienti, F., Humblet, Y.,Bourlond, A., Vanwijck, R., Lienard, D., Beauduin, M., Dietrich, P. Y.,Russo, V., Kerger, J., Masucci, G., Jager, E., De Greve, J., Atzpodien,J., Brasseur, F., Coulie, P. G., van der Bruggen, P., and Boon T. Tumorregressions observed in patients with metastatic melanoma treated withan antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int. J. Cancer, 80: 219230, 1999.

    58. Tsang, K. Y., Zaremba, S., Nieroda, C. A., Zhu, M. Z., Hamilton,J. M., and Schlom, J. Generation of human cytotoxic T cells specific forhuman carcinoembryonic antigen epitopes from patients immunizedwith recombinant vaccinia-CEA vaccine. J. Natl. Cancer Inst., 87:982990, 1995.

    59. Dudley, M. E., Nishimura, M. I., Holt, A. K., and Rosenberg, S. A.

    Antitumor immunization with a minimal peptide epitope (G9209-2M)

    leads to a functionally heterogeneous CTL response. J. Immunother.,22: 288298, 1999.

    60. Moller, P., Sun, Y., Dorbic, T., Alijagic, S., Makki, A., Jurgovsky,

    K., Schroff, M., Henz, B. M., Wittig, B., and Schadendorf, D. Vacci-

    nation with IL-7gene-modified autologous melanoma cells can enhancethe antimelanoma lytic activity in peripheral blood of patients with a

    good clinical performance status: a clinical Phase I study. Br. J. Cancer,77: 19071916, 1998.

    61. Sun, Y., Jurgovsky, K., Moller, P., Alijagic, S., Dorbic, T., Geor-

    gieva, J., Wittig, B., and Schadendorf, D. Vaccination with IL-12

    gene-modified autologous melanoma cells: preclinical results and a first

    clinical Phase I study. Gene Ther., 5: 481490, 1998.

    62. DSouza, S., Rimoldi, D., Lienard, D., Lejeune, F., Cerottini, J. C.,

    and Romero, P. Circulating Melan-A/Mart-1 specific cytolytic T lym-

    phocyte precursors in HLA-A2 melanoma patients have a memory

    phenotype. Int. J. Cancer, 78: 699706, 1998.

    63. Romero, P., Cerottini, J. C., and Waanders, G. A. Novel methods to

    monitor antigen-specific cytotoxic T-cell responses in cancer immuno-

    therapy. Mol. Med. Today, 4: 305312, 1998.

    64. Tan, L. C., Gudgeon, N., Annels, N. E., Hansasuta, P., OCallaghan,C. A., Rowland-Jones, S., McMichael, A. J., Rickinson, A. B., and

    Callan, M. F. A re-evaluation of the frequency of CD8 T cells specific

    for EBV in healthy virus carriers. J. Immunol., 162: 18271835, 1999.

    65. Kuzushima, K., Hoshino, Y., Fujii, K., Yokoyama, N., Fujita, M.,

    Kiyono, T., Kimura, H., Morishima, T., Morishima, Y., and Tsurumi, T.

    Rapid determination of Epstein-Barr virus-specific CD8() T-cell fre-

    quencies by flow cytometry. Blood, 94: 30943100, 1999.

    66. Jager, E., Nagata, Y., Gnjatic, S., Wada, H., Stockert, E., Karbach,

    J., Dunbar, P. R., Lee, S. Y., Jungbluth, A., Jager, D., Arand, M., Ritter,

    G., Cerundolo, V., Dupont, B., Chen, Y. T., Old, L. J., and Knuth, A.

    Monitoring CD8 T-cell responses to NY-ESO-1: correlation of humoral

    and cellular immune responses. Proc. Natl. Acad. Sci. USA, 97: 4760

    4765, 2000.

    67. Hanson, H. L., Donermeyer, D. L., Ikeda, H., White, J. M., Shan-

    karan, V., Old, L. J., Shiku, H., Schreiber, R. D., and Allen, P. M.

    Eradication of established tumors by CD8 T cell adoptive immuno-therapy. Immunity, 13: 265276, 2000.

    68. Gustafsson, A., Levitsky, V., Zou, J. Z., Frisan, T., Dalianis, T.,Ljungman, P., Ringden, O., Winiarski, J., Ernberg, I., and Masucci,M.G. Epstein-Barr virus (EBV) load in bone marrow transplant recipi-ents at risk to develop post-transplant lymphoproliferative disease: pro-phylactic infusion of EBV-specific cytotoxic T cells. Blood, 95: 807814, 2000.

    69. Rooney, C. M., Smith, C. A., Ng, C. Y., Loftin, S. K., Sixbey, J. W.,Gan, Y., Srivastava, D. K., Bowman, L. C., Krance, R. A., Brenner,M. K., and Heslop, H. E. Infusion of cytotoxic T cells for the preventionand treatment of Epstein-Barr virus-induced lymphoma in allogeneictransplant recipients. Blood, 92: 15491555, 1998.

    70. Janeway, C. A., Jr., and Travers, P. Immunobiology: The ImmuneSystem in Health and Disease, pp. 2: 47. London: Current BiologyLimited, 1996.

    1135Clinical Cancer Research