Differential representations of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases
Dipendra K. Mitra1,3,
Stephen C. De Rosa1,
Andrew Luke2,
Arumugam Balamurugan3,
Binod K. Khaitan4,
James Tung1,
Narinder K. Mehra3,
Abba I. Terr2,
Anne O'Garra5,
Leonore A. Herzenberg1,
Leonard A. Herzenberg1 and
Mario Roederer6
1 Department of Genetics and
2 Allergy Clinic, Immunology Division, Department of Medicine, Stanford University, Stanford, CA 94305, USA
3 Histocompatibility & Immunogenetics Department and
4 Department of Dermatology, All India Institute of Medical Sciences, New Delhi 110029, India
5 Department of Immunobiology, DNAX Research Institute, Palo Alto, CA 94304, USA
6 Department of Stomatology, San Francisco, CA 94143-0422, USA
Correspondence to:
M. Roederer, Department of Stomatology, 521 Parnassus Ave C634C, San Francisco, CA 94143-0422, USA
 |
Abstract
|
---|
We identified functionally polarized subsets of CD4 memory T cells on the basis of the expression of CD11a, CD45RA and CD62L. Within the several phenotypically distinct subsets of CD4 memory cells are two that, upon stimulation, produce primarily IL-4 (MT2, CD45RACD62L+CD11adim) or primarily IFN-
(MT1, CD45RACD62LCD11abright). In addition, four other phenotypically distinct subsets of CD4 cells have unique cytokine profiles. To determine the clinical relevance of the representation of these cell types, we analyzed blood from patients with the chronic diseases leprosy and atopy. These diseases are characterized as immunologically polarized, since T cell responses in affected individuals are often strongly biased towards Th1 (dominated by IFN-
production) or Th2 (IL-4 production). We show here that this polarization reflects homeostatic or differentiation mechanisms affecting the representation of the functionally distinct subsets of memory CD4 T cells, MT1 and MT2. Significantly, the representation of the MT1 and MT2 subsets differs dramatically between subjects with tuberculoid leprosy (a Th1 disease), or lepromatous leprosy or atopic disease (Th2 diseases). However, there was no difference in the cytokine profiles of these or any of the other finely resolved CD4 subsets, when compared between individuals across all disease states. Thus, it is the representation of these subsets in peripheral blood that is diagnostic of the polarized state of the immune system.
Keywords: cytokine profile, flow cytometry, intracellular cytokine staining, Th1, Th2
 |
Introduction
|
---|
It has recently become apparent that phenotypically-distinct subsets of memory CD4 T cells have distinct functional capacities. Indeed, selective expression of IFN-
is associated with high CD11a expression in murine CD4 T cells (1) and selective expression of IL-4 is associated with CD62L expression in human CD4 T cells (2,3). More detailed definition of cytokine profiles of the many identifiable subsets of CD4 subsets has been nearly impossible because of the inability to simultaneously measure the expression of cytokine as well as the four or more surface molecules that uniquely identify functionally distinct subsets.
We have recently extended the capabilities of flow cytometric analysis to routinely perform eight-color analysis or higher (4). Using this technology, we can devote five colors to discriminate unique CD4 subsets while simultaneously measuring the production of cytokines such as IL-2, IL-4 and IFN-
. In this report, we confirm the previous associations of biased cytokine production with phenotype and extend these analyses to demonstrate that four-color immunophenotyping identifies highly polarized (i.e. towards IL-4 or IFN-
production) cell types. We also analyzed whether or not this phenotypic identification had clinical relevance using peripheral blood samples from humans with immunologically polarized disease.
Certain chronic human diseases can be characterized as immunologically polarized (5). For example, atopic disease manifestations to antigens such as pollen or mold spores include asthma, eczema and hay fever, and are characterized by allergen-specific IgE in the peripheral blood and activation of mast cells and eosinophils (6,7). Such responses have been shown to be associated with overproduction of IL-4, IL-5 and IL-13 , which are characteristic of the Th2 subset of T lymphocytes (810).
Leprosy is a spectral disease associated with two polar forms. Tuberculoid leprosy (TT) is characterized by clinico-immunologically localized paucibacillary lesions and a Th1-like cytokine profile (increased IFN-
, decreased IL-4 and IL-5). Lepromatous leprosy (LL) exhibits diffuse, disseminated multibacillary lesions, with essentially no cell-mediated immunity against Mycobacterium leprae and a Th2-like cytokine profile (1116). Between these polar forms lie a number of other forms of the spectrum such as borderline tuberculoid (BT), borderline (BB) and borderline lepromatous (BL) showing a mixture of the distinctive characteristics found in each pole.
Characterizing the polarization of the immune response in chronic diseases such as leprosy can provide both diagnostic and mechanistic insights into the progression of the disease. However, identification and quantitation of cells mediating the polarized responses in such chronic diseases has proven quite difficult. Single-cell cytokine response assays to antigen are now becoming more common; for a majority of diseases, however, determining a functional Th1 or Th2 dominance requires cloning or selective expansion in the presence of antigena process usually requiring several weeksor isolation of cells from the site of pathology during the peak of the immune response (9,14,1720). Direct evaluation of the polarization in peripheral blood samples is preferable; however, even in patients with active chronic disease, there may be too few cells to readily define the cytokine profile of memory Th cells stimulated with mitogen (21) or even with specific antigen (if the antigen is known).
In this report, we use multiparameter flow cytometry to study the cytokine profiles of discrete subsets of T cells identified on the basis of a combination of cell surface markers. We show that in chronic atopic diseases and lepromatous leprosy, or in tuberculoid leprosy, increased Th2 or Th1 responses (respectively) are manifested in blood as an increase in the representation of specific populations defined by these cell surface markers. Importantly, however, the functional profile of the individual cells within any given subset is the same across individuals in all disease states. Thus we show that the polarized response of the memory T cell compartment reflects the differential representation of distinct subsets rather than altered functionality of the cells in the compartments. These findings introduce a new potential for clinical monitoring of disease progression, particularly during therapeutic intervention such as allergic desensitization and/or vaccination protocols.
 |
Methods
|
---|
Patient samples
Fourteen patients (non-smokers, seven female) with either allergic rhinitis or asthma seen at Stanford University Allergy Clinic between February and April 1997 were selected for this study. All had at least four positive wheal and flare epicutaneous skin prick test reactions for inhalant allergens including tree, grass, weed pollens and dust mite. None of the patients received systemic corticosteroid therapy. Control subjects were selected during the same time period and had no history of atopic manifestations and completely negative skin tests. All subjects gave informed consent. In addition, peripheral blood mononuclear cells (PBMC) from 11 TT leprosy and five LL leprosy patients from the Leprosy Clinic of the All India Institute of Medical Sciences, India, were obtained for this study. Patients were diagnosed by clinical and bacteriological criteria, and clinic-immunologically classified as polar TT or LL leprosy according to the Ridley and Jopling classification (22). All TT patients had well-demarcated single paucibacillary lesions that were histopathologically confirmed; all LL patients had multiple disseminated asymmetrical lesions. Patients were initially diagnosed within 46 weeks before collecting PBMC and none had received any form of corticosteroid since anti-leprosy therapy (dapsone and rifampicin) was initiated at diagnosis. All subjects gave informed consent.
To evaluate the effect of the long shipping period on the samples from India, we collected blood samples from control subjects at Stanford at the same time as the collection in India; these were left on the bench top until all samples were co-processed. In addition, control samples were also collected from the same endemic area and from the same ethnic group in India, and shipped with the TT and LL samples. Both of these sets of controls were indistinguishable (once EMA+ dead cells were excluded) in our assays from the fresh samples obtained from the Stanford control group and were therefore grouped together as a single control group.
Samples of whole blood were shipped by courier from India on two occasions. In one instance, shipment required 120 h; these five TT leprosy samples showed poor functional responses and were not included in the analysis of Fig. 5(C)
(however, these samples showed normal phenotypic properties indistinguishable from the other samples and were included in the phenotyping analyses). In the other case, shipment was accomplished in 60 h and full functional analysis was performed.
A majority of the control and atopy samples and all but five TT leprosy samples were coded; sample preparation and data analysis were concluded before the disease status was revealed. At least one control sample was nearly always included whenever leprosy or atopy samples were analyzed.
Preparation of cells
Cells were isolated from heparinized blood by Ficoll-Hypaque centrifugation, washed 3 times with RPMI 1640 (Gibco/BRL) and resuspended in medium at 1 x 106/ml for further processing. For stimulations or culture, cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bioproducts, Calabasas, CA), L-glutamine (290 µg/ml), penicillin (100 U/ml) and streptomycin (70 µg/ml) in a 37°C CO2 incubator.
Stimulation and immunofluorescence staining
CD4 is rapidly internalized after stimulation of protein kinase C [phorbol myristate acetate (PMA)]; CD62L is proteolytically removed from the cell surface by metalloproteinases (MMP) after even mild stimulation. To overcome these problems, we pre-stain cells with fluorescently conjugated antibodies to these two molecules. Thus, when CD4 is internalized, the fluorochrome is internalized with it; CD4+ T cells can be easily identified and discriminated from CD8+ or CD4CD8 T cells. The inclusion of monensin to cause accumulation of cytokines in the Golgi also prevents degradation of the internalized fluorochromes. Identification of CD62L+ cells is accomplished by the inclusion of a specific inhibitor of MMP, KB-R8301 (generous gift of K. Okumura), which, in combination with the presence of the anti-CD62L Ig, significantly inhibits the down-modulation of CD62L (23). Pre-staining with antibodies did not affect the responsiveness of the cells (24).
All antibodies were either obtained from DNAX (Palo Alto, CA) or PharMingen (San Diego, CA) and conjugated in our laboratory. PBMC (2 x 106) were pre-stained in 100 µl of culture medium at room temperature with Cy7phycoerythrin (PE) CD4 and Texas RedCD62L for 15 min and then washed twice with room temperature culture medium. PBMC were then cultured in 1 ml medium in a 24-well plate. The following were added to the cultures for stimulation: PMA (1 ng/ml), ionomycin (2 µM), monensin (Calbiochem, CA; 2 µM) and KB-8301 (1 µM). After 6 h, which we determined to be optimal for simultaneous detection of IL-2, IL-4 and IFN-
(25), cells were washed twice with PBS supplemented with 1% BSA and 0.1% sodium azide (PBS/BSA/Azide). Cells were then surface stained with ethidium monoazide bromide (EMA), CD8, CD11a and CD45RA for 15 min at room temperature, and washed in PBS twice. Cells were fixed in 2% formaldehyde for 20 min at room temperature in the dark and then washed twice with PBS/BSA/Azide. For permeabilization, cells were resuspended in 150 µl 0.5% saponin (in PBS/BSA/Azide) and incubated at room temperature for 10 min in the dark. Cells were pelleted and resuspended gently in 25 µl of 0.5% saponin buffer containing optimal amounts of fluorochrome-conjugated anti-cytokine mAb. Cells were further incubated for 30 min at room temperature in the dark; then washed twice in 0.5% saponin buffer, washed 3 times in PBS/BSA/Azide and finally resuspended in 200 µl PBS/BSA/Azide at 4°C. FACS analysis was done within 12 h; 13 x 105 events were collected for analysis. Eight-color FACS analysis was performed as described (4).
In most experiments, we stained with 5 µg/ml EMA (Molecular Probes, Eugene, OR) simultaneously with the antibody stains, to exclude dead cells which can non-specifically bind antibodies. After washing and before fixation, cells were exposed to fluorescent light for 10 min and then washed. EMA diffuses into dead cells and intercalates into DNA; upon exposure to fluorescent light it is photo-cross-linked and is covalently bound.
The discrimination between CD11ahigh and CD11alow cells is often difficult to determine in the CD45RA population. We adopted the following procedure to set the gate position objectively. The CD11a histogram for the total lymphocyte population was examined. This histogram is always bimodal, segregating into CD11ahigh and CD11alow populations. The CD11a gate was set to the histogram minimum between these two peaks and applied to the CD45RA population as described in the text.
RNA quantitation
Quantitative PCR was performed as previously described (26), with minor modifications. For CD3 stimulation, we used Leu4 in the presence of P815 cells (no co-stimulation), or P815 cells stably transfected with human CD80 or CD86 (kind gift of L. Lanier) (27) at final ratio of 5:1 T cells:P815.
Data analysis
Compensation for spectral overlap as well as all gating, statistical analysis and graphical presentation of flow cytometric data was performed using FlowJo (Tree Star, San Carlos, CA). Statistical comparison and presentation of clinical data was performed using JMP (SAS Institute, Cary, NC). Comparisons of distributions were performed by the non-parametric two-sample Wilcoxon rank test.
 |
Results
|
---|
Cytokine profiles of T cell subsets
In healthy adults, roughly half of the mature T cells in blood are naive T cells, which arise from positive and negative selection in the thymus but have not yet encountered their cognate antigenic peptide. Naive T cells are identified by the expression of CD45RA and CD62L, low-level expression of CD11a, and the lack of expression of activation antigens (28,29). When stimulated by antigen presentation, they proliferate rapidly and produce IL2; however, they do not produce effector cytokines such as IL-4, IL-5, IL-10 or IFN-
.
The remaining peripheral T cells are a heterogeneous population of memory T cells that have undergone proliferative expansion in response to antigen, and have differentiated to phenotypically and functionally distinct subsets. Three subsets in peripheral blood of healthy subjects are identified by the expression of a combination of T cell lineage markers, CD4 and CD8, and the CD45RA and CD62L differentiation markers (Fig. 1
). One of these subsets, CD45RACD62L+, includes the only cells that can provide effective help for B cells (2). A second subset, CD45RACD62L, is equally well represented on average. Cells expressing CD45RA but not CD62L are relatively rare in healthy people (averaging 5% of CD4 T cells); however, this population can be significantly expanded in pathogenic states such as HIV infection (30).

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 1. T cell subsets have unique cytokine profiles. (A) Flow cytometric determination of cytokine profiles. Eight-color, 10-parameter FACS(4) was used to determine the cytokine profile of fine T cell subsets stimulated with PMA/ionomycin. The left panels show progressive gating of the data: by scatter for lymphocytes, by CD4 or CD8 for lineage and then by CD45RA and CD62L to distinguish naive (CD45RA+CD62L+) cells from three types of memory cells. EMA fluorescence could be used to exclude dead cells, which non-specifically bind some antibodies including anti-CD4 and anti-IL4 (in general, <5% of CD4 and up to 20% of CD8 became EMA+ after stimulation with PMA and ionomycin). On the right, the distribution of expression for IL-4 versus IFN- and for IL-2 are shown for each of the differentiation stages of CD4 or CD8 T cells. Note that the apparent division of the `negative' populations into a main population and one on the axis is a mathematical and visual artefact of the data compensation process. All events in the first decade of fluorescence are essentially equivalent regardless of their position within that decade. (B) Quantitative PCR determination of cytokine profiles. CD4 subsets were sorted to >98% purity by FACS, based on the expression of CD4, CD45RA and CD62L. In most healthy individuals, CD45RA+CD62L CD4 cells are too rare to sort enough cells for quantitative PCR analysis. Sorted naive and memory cells were stimulated either with PHA, anti-CD3 or anti-CD3 with co-stimulation provided by B7-1 or B7-2. Shown are example amplifications of mRNA with dilutions of specific cytokine competitor RNA for the three cell types stimulated with anti-CD3+B7-1. No IL-4 production could be detected in the naive or CD45RACD62L cells. Arrows point at the expected locations of the cell-derived amplicon (wild-type length, upper band) and artificially constructed competitor-derived amplicon (lower band) for each cytokine message. (C) Densitometric scanning of the gels such as shown in (B) was used to quantitate the absolute number of mRNA molecules for each cytokine; results are presented as cytokine mRNA molecules per cell. Error bars represent ± SD for results from four different experiments on T cells sorted from four different individuals. Unstimulated T cells were also analyzed, but showed no detectable cytokine message. Naive T cells have ~10-fold less message per cell than do memory T cells; however, this amount is easily detectable and is still several orders of magnitude above the signal for unstimulated T cell.
|
|
Each of these subsets has a unique cytokine profile after stimulation with PMA and ionomycin (Fig. 1
). The cytokine profiles, identified by FACS analysis after stimulation with PMA and ionomycin, are essentially identical to those seen in purified populations stimulated with other mitogens including phytohemagglutinin, anti-CD3 and anti-CD3 with co-stimulation, and assessed by quantitative PCR (Fig. 1
). As indicated above, naive (CD45RA+CD62L+) T cells produce IL-2 but little IL-4 or IFN-
. The CD45RA+CD62L T cells, in contrast, have a differentiated effector cytokine profile. Furthermore, although like naive cells they do not express CD45R0, they do express high levels of CD11a, and often express CD38 and CD57 (data not shown), supporting the designation as a memory/effector subset. Finally, consistent with previous findings showing that CD45RACD62L+ provide B cell help for antibody production and isotype switching (2), cells in this subset are the main producers of IL-4.
In these experiments, we used EMA staining to discriminate cells which died during the stimulation from live cells. Dead cells will bind some (but not all) antibody reagents non-specifically, leading to potentially artefactual results. As shown in Fig. 1(a)
, for example, most apparently CD4, CD8 double-positive cells are dead cells that non-specifically bind antibodies to CD4, CD8, and IL-4 (but not IFN-
). Without the ability to exclude dead cells, the IL-4-producing cells in the CD4 compartment would be significantly over-reported. For our experiments, the average fraction of dead cells after PMA/ionomycin stimulation was 6% (CD4), 8% (CD8), 16% (CD4CD8) and 88% (CD4+CD8+).
Identification of functionally polarized CD4 subsets
By further subdividing the two CD45RA memory cell subsets on the basis of expression of CD11a, we noted that IFN-
was associated with high CD11a expression in these cells, in keeping with recent results from the mouse (1). This combination of CD62L with CD11a allows the identification of four subsets of CD4+CD45RA cells with unique cytokine profiles (Fig. 2
).
The CD11abrightCD62L subset, which we designate MT1, is the most `Th1-like' in that it has the highest ratio of IFN-
-producing to IL4-producing cells (i.e. it is the most IFN-
-biased). Similarly, the CD11adimCD62L+ (MT2) subset is the most `Th2-like', in that it has the highest ratio of IL4 producers to IFN-
producers (it is the most IL4-biased). The CD11abrightCD62L+ (MT0) subset is `Th0-like'; finally, few cells in the CD11adimCD62L (MTn) subset make either IL-4 or IFN-
. Interestingly, this functional division is also seen in the CD8 subsets (although the relative proportion of IL-4-producing subsets is lower than in the CD4 lineage; furthermore, IL-4 production by CD8 cells is highly donor dependent). Thus, the CD4 and CD8 lineages have differentiated phenotypes with similar cytokine profiles, consistent with studies showing that differentiation of Tc1 and Tc2 can be achieved using the same signals that drive Th1 and Th2 development (31).
While the apparent differences in IL-4 production between different CD4 subsets is small in absolute magnitude, it is highly statistically significant. The sensitivity of the FACS assay for IL-4 production is much lower than that for IFN-
and may underestimate the proportion of IL-4-producing cells. In Table 1
, we computed the fraction of each of the six CD4 subsets that produce IL-4 or IFN-
and, for each subset, the ratio of cells producing these cytokines. Note that naive T cells and MT1 T cells, which do not make IL-4 (Fig. 1B
), show about 12% positive cells by FACS: this value can be viewed as the background for the FACS IL-4 assay, making the relatively small increment in IL-4-producing cells in, for example, the MT2 subset, substantially larger by comparison. Even without taking this background into account, MT2 cells have nearly 4 times as many IL-4-producing cells as MT1 in all disease states we examined [average ratio of IL-4+ cells in MT2 to MT1 is 3.7 (IQR 1.35.4) for individuals in all disease states; this value is different from 1.0 with p < 104].
Clinical correlates of functionally polarized subsets
Since atopic disease or TT leprosy are disorders previously characterized as Th2- or Th1-dominated, respectively, we used the phenotypic analyses described above to examine differences in peripheral blood T cell subsets of subjects with these diseases. Because the precise antigen(s) dominating the immune responses in these individuals is unknown, it was necessary to use a generic stimulus (i.e. PMA/ionomycin) to determine the functional state of the composite immune system. As shown in Fig. 1
, the analysis of cytokine profile using the flow cytometric assay (with PMA/ionomycin stimulation) is essentially the same as using more physiological stimuli such as CD3/CD28 and is most likely representative of the profile obtained for any given fine T cell subset after antigen stimulation.
We found no differences in the cytokine profiles of the phenotypically defined subsets (MTn, MT0, MT1 or MT2) in atopy or TT leprosy compared to controls (Fig. 3
). However, we found that atopic subjects have a significant increase in the numbers of MT2 cells (CD62L+CD11adim: IL4 biased) and a significant decrease in MT1 cells (CD62LCD11abright: IFN-
biased) in the peripheral blood (Fig. 4
). Conversely, subjects with TT leprosy have a significant decrease in MT2 cells and a significant increase in MT1 cells. The number of IL4+IFN-
+ (double-producing) cells within all subsets and in all subjects tested was exactly predicted by the proportions of single-positive T cells (data analysis not shown).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 4. Representation of CD45RA T cells in leprosy or atopy. Five representative examples of T cell profiles of healthy, atopic or TT leprosy individuals are shown. Samples were gated for CD4+CD45RA lymphocytes as shown in Fig. 1(A) . The bottom-most panel for the control group is a control sample obtained from India, representing the same ethnic group and endemic area as the leprosy samples. Different combinations of fluorochromeantibody conjugations were used in some of the experiments shown here; hence, the absolute fluorescence between different individuals is not comparable. The relative position of the CD62L gate was based on negatively versus positively stained cells; the relative position of the CD11a was chosen to split the two major peaks that are always easily discerned in the total lymphocyte population.
|
|
Unlike the case for CD4 T cells, we found no statistically significant differences in the representations or cytokine profiles of the fine subsets of CD8 T cells between individuals with either TT or LL leprosy, atopy or no disease. This is not inconsistent with our study design, selecting subjects with known functional polarization in the CD4 compartment (i.e. Th1- or Th2-biased diseases).
Note that enumeration of these subsets can be performed using commercially available four-color flow cytometers by measuring CD4, CD11a, CD45RA and CD62L. The eight-color analyses we performed was required to demonstrate the correlation between phenotype (i.e. surface markers) and function (i.e. intracellular cytokine production).
The statistical summary in Figs 5 and 6
further demonstrates that changes in the cytokine profiles of individual subsets, or in the representation of the MTn and MT0 subsets, do not occur in atopic diseases or polar leprosyeither tuberculoid or lepromatous (Fig. 5C
). In contrast, there is an inverse effect on the numbers of MT1 and MT2 cells in these polarized diseases (Fig. 5A
), i.e. MT1 is preferentially increased in TT leprosy, a Th1 disease, whereas MT2 is preferentially increased in atopic disorders or LL, both considered as Th2 diseases. Figure 6
shows the significant difference in the CD4 memory representations of these individuals. Whether this remodeling occurred prior to disease (and possibly contributed to the clinical course of disease) or as a result of disease remains to be determined. In any case, the relative representations of the functionally distinct immune subsets can be taken to be an index of the degree of polarization of the peripheral immune system (Fig. 5B
).
 |
Discussion
|
---|
Studies presented here show that the dominance of the Th1 and Th2 response pattern by peripheral blood T cells from patients with atopic disease or polar leprosy is manifested as a change in T cell subset representation rather than in the proportion of cytokine-producing cells within any given subset (Table 1
and Fig. 5
). These findings extend the initial observation made by Field et al. (32), who found that the CD45RACD62L+ subset (MT2+MT0) was expanded in a single atopic individual. In essence, we show that simultaneous measurement of CD4, CD11a, CD45RA and CD62L allows enumeration of the MT1 (IFN-
-biased) and MT2 (IL4-biased) subsets, the ratio of which reflects the balance of cytokine production characteristic of the chronic disease state.
It is important to note that the most important conclusion of our report, i.e. that polarized disease states have characteristic alterations in subsets of memory CD4 T cells, does not depend on the functional measurements (intracellular cytokine staining) that we performed. Four-color immunophenotyping is sufficient to quantitate the degree of functional polarization in the immune system.
Previously identification of Th1 and Th2 functional dominance in a number of diseases including leprosy, leishmaniasis, schistosomiasis and atopic manifestations (5,7,3336) could only be achieved by selective expansion of antigen-specific effector clones or by visualization of effector T cells at the site of inflammation (19). In contrast, we show that this dominance can be rapidly identified by flow cytometry on fresh blood samples. This can be performed by simultaneous measurement of CD4, CD45RA, CD62L and CD11aon newer, four-color flow cytometers; it does not require a stimulation assay nor an intracellular staining assay. Thus, the methodology we describe could facilitate clinical monitoring of changes in chronic disease that may lead to pathology or immunosuppression as a result of imbalance of Th1 and Th2 responses (5). Furthermore, this approach may be especially valuable for monitoring changes in cytokine profiles during immunotherapy in allergic disease or drug therapy of other diseases such as leprosy, leishamaniasis or even in vaccination protocols.
A number of studies have attempted to define phenotypic markers for Th1 or Th2 cells. Preferential IL-12Rß2 mRNA expression in Th1 cells was observed in mice and in humans (37,38). Selective expression of receptors for P-selectin and E-selectin was noted for Th2 and Th1 cells respectively, and hypothesized to mediate the recruitment of these cells to the site of inflammation (39). On the other hand, CD62L (L-selectin), necessary for homing to lymphoid tissues, is preferentially expressed on IL-4-producing T cells (Fig. 2
) (3,32). Our findings extend these observations by showing that enumeration of clinically relevant polarized T cell subsets requires the measurement of CD11a, CD62L as well as CD45RA on CD4 T cells.
The differential expression of the selectins or selectin receptors may be related to the selective recruitment of cytokine-polarized effector T cells at the pathologic sites. However, many factors may influence the selective differentiation and expansion of Th1 and Th2 cells, including the antigen-presenting cell, hormones, adjuvant, dose of antigen, co-stimulatory molecules, affinity of the immunogenic peptide for MHC, regulatory cytokines and the genetic background of the individual. Among these, cytokines have been shown most consistently to be an important driving factor (4043). The altered levels of defined subsets such as those we have demonstrated here may also represent antigen-driven or cytokine-driven expansion. Alternatively, or in addition, such changes may be influenced by the genetic background of the individual as well as environmental factors. In any case, the representation of these subsets is highly stable over a period of several years in any given individual, implying a strong homeostatic control mechanism.
It is important to note that the measurements we report here are based on the cells found in the peripheral blood. As such, it is expected that these measurements may not accurately reflect the T cells in either secondary lymphoid tissue or, perhaps more importantly, at the site of the lesion itself. Preliminary analysis of cells isolated from biopsies of leprosy patients confirm that the local effector cells show a similar phenotypic bias as we found for the peripheral blood: in lesions from Th1 disease (TT leprosy), T cells expressed high levels of CD11a and CCR5; in lesions from Th2 disease (LL leprosy), T cells expressed low levels of CD11a and high levels of CCR3 (D. K. Mitra and N. K. Mehra, unpublished). Thus, the imbalance that we measure in the peripheral blood may herald a much larger imbalance in the antigen-specific-immune response carried out at the lesion itself.
A subset imbalance and the resulting cytokine imbalance may dictate resistance or susceptibility to pathogenic organism, or susceptibility to normally non-pathogenic stimuli such as allergens or self-antigens, or, in the case of leprosy, affect the clinical course of disease (i.e. TT versus LL). Recent data supports the hypothesis that the pre-existing balance of these cells can affect the course of disease. For example, in a mouse model, animals predisposed to IL-4 production developed less delayed-type hypersensitivity and were protected from Th1-mediated autoimmune inflammatory diseases (44). The hypothesis is also supported by recent clinical investigations in humans (45,46) showing inverse associations between tuberculin reactivity (Th1) and atopic disorders, and between multiple sclerosis (Th1 like) and atopy.
These findings raise the question as to whether, upon exposure to M. leprae, individuals with pre-existing high levels of MT1 (IFN-
-biased) cells are more likely to develop a Th1 response and thus tuberculoid leprosy, whereas those with high levels of MT2 (IL-4-biased) are more likely to develop a Th2 response leading to the LL form. Our preliminary studies on a small number of untreated individuals with LL leprosy confirm that they have elevated numbers of MT2 cells and decreased numbers of MT1 cellssimilar to that found in individuals with atopic diseases. However, we cannot rule out that the imbalance in subset representation is a consequence of other factors that determine the Th1/Th2 polarization after exposure to the causative agents. In any event, studies presented here provide a method for testing this hypothesis, and for determining the relationship between subset representation and the progress and nature of chronic diseases characterized by polarized immune responses.
Finally, we find no evidence that there is any alteration in the functional capacity of any given T cell in chronic diseasesrather, the functionality of a T cell from such an individual can be predicted by examining the functionality of a T cell from the same subset in a healthy individual. Rather, our data show that the functional polarization of the immune system is most likely due to homeostatic and/or differentiation mechanisms that regulate the number of these functionally distinct T cell subsets.
 |
Acknowledgments
|
---|
We thank Drs Lewis Lanier, Douglas Robinson, Robert Coffman and Anne Kelso for critical review of the manuscript and helpful discussion; and Drs Lawrence Steinman, Peter Katsikis, Nicole Baumgarth and members of the Herzenberg laboratory for helpful discussions. DNAX is supported by ScheringPlough Research Institute. This work was supported by National Institutes of Health grants LM-04836, AI-31770 and CA-42509.
 |
Abbreviations
|
---|
EMA ethidium monoazide bromide |
LL lepromatous leprosy |
MMP metalloproteinases |
PBMC peripheral blood mononuclear cell |
PE phycoerythrin |
PHA phytohemagglutinin |
PMA phorbol myristate acetate |
TT tuberculoid leprosy |
 |
Notes
|
---|
Transmitting editor: R. Hardy
Received 4 February 1999,
accepted 21 July 1999.
 |
References
|
---|
-
Baumgarth, N., Egerton, M. and Kelso, A. 1997. Activated T cells from draining lymph nodes and an effector site differ in their responses to TCR stimulation. J. Immunol., in press.
-
Tsuji, T., Nibu, R., Iwai, K., Kanegane, H., Yache, A., Seki, H., Miyawaki, T. and Taniguchi, N. 1994. Efficient induction of immunoglobulin production in neonatal naive B cells by memory CD4+ T cell subset expressing homing receptor L-selectin. J. Immunol. 152:4417.[Abstract/Free Full Text]
-
Kanegane, H., Kasahara, Y., Niida, Y., Yachie, A., Sughii, S., Takatsu, K., Taniguchi, N. and Miyawaki, T. 1996. Expression of L-selectin (CD62L) discriminates Th1- and Th2-like cytokine-producing memory CD4+ T cells. Immunology 87:186.[ISI][Medline]
-
Roederer, M., Rosa, S. D., Gerstein, R., Anderson, M., Bigos, M., Stovel, R., Nozaki, T., Parks, D., Herzenberg, L. and Herzenberg, L. 1997. 8-Color, 10-parameter flow cytometry: elucidation of complex leukocyte heterogeneity. Cytometry, in press.
-
Sher, A. and Coffman, R. L. 1992. Regulation of immunity to parasites by T cells and T cell-derived cytokines. Annu. Rev. Immunol. 10:385.[ISI][Medline]
-
Romagnani, S. 1995. Atopic allergy and other hypersensitivities editorial overview: technological advances and new insights into pathogenesis prelude novel therapeutic strategies [editorial]. Curr. Opin. Immunol. 7:745.[ISI]
-
Corrigan, C. J. 1995. Elevated interleukin-4 secretion by T lymphocytes: a feature of atopy or of asthma? [editorial]. Clin. Exp. Allergy 25:485.[ISI][Medline]
-
Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A. and Coffman, R. L. 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136:2348.[Abstract/Free Full Text]
-
Romagnani, S. 1994. Lymphokine production by human T cells in disease states. Annu. Rev. Immunol. 12:227.[ISI][Medline]
-
Umetsu, D. T., Jabara, H. H., DeKruyff, R. H., Abbas, A. K., Abrams, J. S. and Geha, R. S. 1988. Functional heterogeneity among human inducer T cell clones. J. Immunol. 140:4211.[Abstract/Free Full Text]
-
Yamamura, M., Uyemura, K., Deans, R. J., Weinberg, K., Rea, T. H., Bloom, B. R. and Modlin, R. L. 1991. Definitive protective responses to pathogens: cytokine profiles in leprosy lesions [published erratum appears in Science 1992; 255(5040): 12]. Science 254:277.
-
Salgame, P., Abrams, J. S., Clayberger, C., Goldstein, H., Convit, J., Modlin, R. L. and Bloom, B. R. 1991. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science 254:279.[ISI][Medline]
-
Bloom, B., Salgame, P. and Diamond, B. 1992. Revisiting and revising suppressor T cells. Immunol. Today 13:131.[ISI][Medline]
-
Haanen, J. B., de Waal Malefijt, R., Res, P. C., Kraakman, E. M., Ottenhoff, T. H., de Vries, R. R. and Spits, H. 1991. Selection of a human T helper type 1-like T cell subset by mycobacteria. J. Exp. Med. 174:583.[Abstract]
-
Misra, N., Murtaza, A., Walker, B., Narayan, N. P., Misra, R. S., Ramesh, V., Singh, S., Colston, M. J. and Nath, I. 1995. Cytokine profile of circulating T cells of leprosy patients reflects both indiscriminate and polarized T-helper subsets: T-helper phenotype is stable and uninfluenced by relating antigens of Mycobacterium leprae. Immunology 86:97.[ISI][Medline]
-
Mitra, D., Rajalingam, R., Taneja, V., Bhattacharyya, B. C. and Mehra, N. K. 1997. HLA-DR polymorphism modulates the cytokine profile of Mycobacterium leprae HSP-reactive CD4+ T cells. Clin. Immunol. Immunopathol. 82:60.[ISI][Medline]
-
Del Prete, G. F., Der Carli, M., Mastromauro, C., Biagiotti, R., Macchia, D., Falagiani, P., Ricci, M. and Romagnani, S. 1991. Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J. Clin. Invest. 88:346.[ISI][Medline]
-
Parronchi, P., Macchia, D., Piccinni, M. P., Biswas, P., Simonelli, C., Maggi, E., Ricci, M., Ansari, A. A. and Romagnani, S. 1991. Allergen- and bacterial antigen-specific T-cell clones established from atopic donors show a different profile of cytokine production. Proc. Natl Acad. Sci. USA 88:4538.[Abstract]
-
Robinson, D. S., Hamid, Q., Ying, S., Tsicopoulos, A., Barkans, J., Bentley, A. M., Corrigan, C., Durham, S. R. and Kay, A. B. 1992. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326:298.[Abstract]
-
Yssel, H., Johnson, K. E., Schneider, P. V., Wideman, J., Terr, A., Kastelein, R. and De Vries, J. E. 1992. T cell activation-inducing epitopes of the house dust mite allergen Der p I. Proliferation and lymphokine production patterns by Der p I-specific CD4+ T cell clones. J. Immunol. 148:738.[Abstract/Free Full Text]
-
Romagnani, S., Maggi, E., Parronchi, P., Macchia, D., Piccinni, M. P. and Ricci, M. 1991. Increased numbers of Th2-like CD4+ T cells in target organs and in the allergen-specific repertoire of allergic patients. Possible role of IL-4 produced by non-T cells. Int. Arch. Allergy Appl. Immunol. 94:133.[ISI][Medline]
-
Ridley, D. and Jopling, W. 1966. Classification of leprosy according to immunity; a five-group system. Int. J. Lepr. 34:255.
-
Walcheck, B., Kahn, J., Fisher, J. M., Wang, B. B., Fisk, R. S., Payan, D. G., Feehan, C., Betageri, R., Darlak, K., Spatola, A. F. and Kishimoto, T. K. 1996. Neutrophil rolling altered by inhibition of L-selectin shedding in vitro. Nature 380:720.[ISI][Medline]
-
Roederer, M., Bigos, M., Nozaki, T., Stovel, R. T., Parks, D. R. and Herzenberg, L. A. 1995. Heterogeneous calcium flux in peripheral T cell subsets revealed by five-color flow cytometry using log-ratio circuitry. Cytometry 21:187.[ISI][Medline]
-
Picker, L. J., Singh, M. K., Zdraveski, Z., Treer, J. R., Waldrop, S. L., Bergstresser, P. R. and Maino, V. C. 1995. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 86:1408.[Abstract/Free Full Text]
-
Roederer, M., Raju, P. A., Mitra, D. K., Herzenberg, L. A. and Herzenberg, L. A. 1997. HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28. J. Clin. Invest. 9:1555.
-
Azuma, M., Cayabyab, M., Buck, D., Phillips, J. H. and Lanier, L. L. 1993. CD28 interaction with B7 co-stimulates primary allogeneic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes. J. Exp. Med. 175:353.[Abstract]
-
Picker, L., Treer, J., Ferguson, D., Collins, P., Buck, D. and Terstappen, L. 1993. Control of lymphocyte recirculation in man. I. Differential regulation of the peripheral lymph node homing receptor L-selectin on T cells during the virgin to memory cell transition. J. Immunol. 150:1105.[Abstract/Free Full Text]
-
Okumura, M., Fujii, Y, Takeuchi, K., Inada, K., Nakahara, K. and Matsuda, H. 1993. Age-related accumulation of LFA-1high cells in a CD8+CD45RAhigh T cell population. Eur. J. Immunol. 23:1057.[ISI][Medline]
-
Roederer, M., Dubs, J. G., Anderson, M. T., Raju, P. A. Herzenberg, L. A. and Herzenberg, L. A. 1995. CD8 naive T cell counts decrease progressively in HIV-infected adults. J. Clin. Invest. 95:2061.[ISI][Medline]
-
Seder, R. A. and Le Gros, G. G. 1995. The functional role of CD8+ T helper type 2 cells. J. Exp. Med. 181:5.[ISI][Medline]
-
Field, E. H., Noelle, R. J., Rouse, T., Goeken, J. and Waldschmidt, T. 1993. Evidence for excessive Th2 CD4+ subset activity in vivo. J. Immunol. 151:48.[Abstract/Free Full Text]
-
Modlin, R. L. 1994. Th1-Th2 paradigm: insights from leprosy. J. Invest. Dermatol. 102:828.[Abstract]
-
Sieling, P. A. and Modlin, R. L. 1994. Cytokine patterns at the site of mycobacterial infection. Immunobiology 191:378.[ISI][Medline]
-
Mocci, S. and Coffman, R. L. 1997. The mechanism of in vitro T helper cell type 1 to T helper cell type 2 switching in highly polarized Leishmania major-specific T cell populations. J. Immunol. 158:1559.[Abstract]
-
Mathewson, J. J. and DuPont, H. L. 1996. Impaired TH1-like immune response in Schistosoma mansoni infection [letter]. J. Infect Dis. 174:677.[ISI][Medline]
-
Szabo, S. J., Dighe, A. S., Gubler, U. and Murphy, K. M. 1997. Regulation of the interleukin (IL)-12R beta 2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817.[Abstract/Free Full Text]
-
Rogge, L., Barberis-Maino, L., Biffi, M., Passini, N., Presky, D. H., Gubler, U. and Sinigaglia, F. 1997. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J. Exp. Med. 185:825.[Abstract/Free Full Text]
-
Austrup, F., Vestweber, D., Borges, E., Lohning, M., Brauer, R., Herz, U., Renz, H., Hallmann, R., Scheffold, A., Radbruch, A. and Hamann, A. 1997. P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues. Nature 385:81.[ISI][Medline]
-
Hosken, N. A., Shibuya, K., Heath, A. W., Murphy, K. M. and O'Garra, A. 1995. The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model. J. Exp. Med. 182:1579.[Abstract]
-
Paul, W. E. and Seder, R. A. 1994. Lymphocyte responses and cytokines. Cell 76:241.[ISI][Medline]
-
Abbas, A. K., Murphy, K. M. and Sher, A. 1996. Functional diversity of helper T lymphocytes. Nature 383:787.[ISI][Medline]
-
O'Garra, A. and Murphy, K. 1994. Role of cytokines in determining T-lymphocyte function. Curr. Opin. Immunol. 6:458.[ISI][Medline]
-
Scott, B., Liblau, R., Degermann, S., Marconi, L. A., Ogata, L., Caton, A. J., McDevitt, H. O. and Lo, D. 1994. A role for non-MHC genetic polymorphism in susceptibility to spontaneous autoimmunity. Immunity 1:73.[ISI][Medline]
-
Oro, A. S., Guarino, T. J., Driver, R., Steinman, L. and Umetsu, D. T. 1996. Regulation of disease susceptibility: decreased prevalence of IgE-mediated allergic disease in patients with multiple sclerosis. J. Allergy Clin. Immunol. 97:1402.[ISI][Medline]
-
Shirakawa, T., Enomoto, T., Shimazu, S. and Hopkin, J. M. 1997. The inverse association between tuberculin responses and atopic disorder. Science 275:77.[Abstract/Free Full Text]