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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.

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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

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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.

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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



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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.



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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.



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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.

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