γ δ T cells participate in the immune response against activated, myelin basic protein-specific, human T cells

ELSEVIER Journal of Neuroimmunology 58 (1995) 177-182

Journal of Neuroimmunology

yS T cells participate in the immune response against activated, myelin basic protein-specific, human T cells

James Burns * , Breck Bartholomew, Kim Littlefield

Veterans Administration Medical Center, Neurovirology Research 151B, Department of Neurology, University of Utah School of Medicine, 500 Foothill Drive, Salt Lake City, UT 84148, USA

Received 17 June 1994; revised 6 December 1994; accepted 30 December 1994

Abstract

y6 T cells are over-represented in some multiple sclerosis (MS) parenchyrnal lesions and in the cerebrospinal fluid (CSF) of individuals with early MS. In this investigation we studied the T cell-T cell interactions between human, myelin basic protein-reactive T cells and peripheral blood mononuclear cells (PBMC) isolated from MS and control subjects. We detected brisk proliferation by PBMC in response to activated, but not resting, MBP-specific T cells. The magnitude of proliferation approached that induced by superantigens and was distinctly greater than the response to standard recall antigens. Examination of the responding T cells revealed predominant expansion of T cells using yg rather than a/? T cell receptors. This finding suggests that the accumulation of y8 T cells noted in some MS parenchymal lesions may represent recruitment by activation markers expressed by other T cells in these lesions. The response to activated but not resting MBP-specific T cells may parallel observations that protective T cell vaccination in experimental encephalomyelitis is more effective using activated rather than resting, myelin-specific T cells.

Keywords: Multiple sclerosis; Gamma delta T cells; Anti-ergotypic response; Myelin basic protein

1. Introduction

Multiple sclerosis (MS) is a chronic central nervous system (CNS) demyelinating disease with white matter lesions characterized by mononuclear cell infiltrates including large numbers of T lymphocytes. While many of the T cells in these lesions utilize a/? T cell receptors (TCR), some investigators note an increased proportion of T cells using y6 TCR in certain MS lesions (Selmaj et al., 1991; Wucherpfennig et al., 1992; Hvas et al., 1993). The proportion of yS T cells in the CSF may also be increased in recent onset, active MS with yS T cells comprising up to 40% of the CSF lymphocytes in this patient population (Shimonkevitz et al., 1993). The biological function and the factors responsible for the accumulation of the yS T cells in MS lesions remain to be established. Peripheral blood yS T cells often recognize mycobacterial antigens or heat shock proteins (Kabelitz et al., 1990; O’Brien et al., 1991). While heat shock proteins are not normally

* Corresponding author. Phone (801) 581 6367; Fax (801) 584 1251

016%5728/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0165-5728(95)00012-7

found in the CNS, these antigens have been reported to be associated with oligodendrocytes in some MS lesions (Selmaj et al., 1991). Because yS T cells may express cytolytic activity, there has been speculation these T cells may contribute to myelin damage (Freed- man et al., 1991; Selmaj et al., 1991; Birnbaum et al., 1993).

In the current series of experiments we examined T cell-T cell interactions in an in vitro model of T cell vaccination by assessing the immune response directed against MBP-reactive T cells by freshly isolated, autologous, peripheral blood mononuclear cells (PBMC). One type of T cell-T cell response, the anti-idiotypic response, is present in some MS subjects vaccinated with irradiated, activated, MBP-specific T cells (Zhang et al., 1993). In the experimental model of MS, experimental allergic encephalomyelitis (EAE), vaccination protocols using activated, but not resting, MBP-reactive T cells frequently induce effective resistance to EAE (Naparstek et al., 1983; Lider et al., 1988; Lohse et al., 1993). In this study we found an unexpected dichotomy in the immune response of PBMC against activated T cells compared to resting T cells. The

178 J. Bums et al. /Journal of Neuroimmunology 58 (1995) 177-182

qerxphc~r;tl Blood

Mononuclear Cells

Plus.

MBP Reactive

Antigen T Cells (lrrad)

1) none

2) MBP

3) none

none

none

3 x lo4

4) tetanus

5) MBP

6) TSST

none

3 x lo4

none

Peripheral Blood

Mononuclear Cells

Plus:

MBP Reactive

Antigen T Cells (irrad)

1) none

2) none

3) MBP peptide

(61 - 100)

4) MBP peptide (81 - 100)

5) MBP

none

none

3 x lo4

3x lo4

CPhl S 10m3 (Thymldine IncorporatIon)

1,,,,,,,,,,,,1 III II,, ,,I I I II

5 10 15 20

CPM x 1o-3 (Thymidine Incorporation)

-3 CPM X 10 (Thymidme IncorporatIon)

CPM X 1O-3 (Thymidine Incorporation)

Fig. 1. Activated, but not resting, MBP-specific T cells induce proliferation of freshly isolated peripheral blood mononuclear cells. CD4+, MBP-reactive T cells were isolated from two individuals, a person with MS (A) and a control subject (B). The MBP-specific T cells had beerrlast stimulated with MBP and APC 9 days before these studies were performed. Freshly isolated, autologous PBMC were incubated alone or with combinations of; (1) irradiated 14000 rad) MBP-specific T cells: (2) MBP; (3) synthetic MBP peptide; (4) tetanus toxoid and; (5) the superantigen, TSST. Proliferation was measured by incorporation of [3H]thymidine for the last 18 h of a 4-day incubation. The irradiated, MBP-reactive T cells contributed no more than 400 cpm to the results shown.

J. Bums et al. /Journal of Neuroimmunology 58 (1995) 177-182 179

response of freshly isolated PBMC to resting, MBP- specific T cells was negligible. However, the response to activated, MBP-specific T cells was very brisk and, unexpectedly, yS T cells participated prominently in this response. These observations suggest that some of the yS T cells noted in MS lesions may accumulate in response to activation markers associated with other activated T cells in these lesions.

2. Materials and methods

2.1. Antigens

Human MBP was prepared by the method of Deibler and associates (Deibler et al., 1972). Tetanus toxoid was generously provided by Connaught Labs, Swiftwa- ter, PA, and was used at a concentration of 3 LF units/ml. The bacterial toxin superantigen, toxic shock syndrome toxin (TSST), was purchased from Toxin Technologies (Sarasota, FL) and used at a concentration of 0.05 ,ug/ml. Synthetic MBP peptides were generously provided by Drs. Kumar and Gomez and used as previously reported (Burns et al., 1991).

2.2. T cell lines and clones

Peripheral blood mononuclear cells were isolated from two control subjects and two individuals with definite MS. T cell lines specific for human MBP were isolated from peripheral blood mononuclear cells by the in vitro sensitization method previously described (Burns et al., 1991). T cell lines and clones were maintained by use of interleukin (IL)-2 and periodic stimulation at l-2 week intervals with irradiated antigen-presenting cells (APC) and MBP. T cell clones were isolated as previously described through limiting dilution techniques (Burns et al., 1991).

2.3. Proliferation assays

Proliferation of freshly isolated PBMC was determined by culture of 1.5 X lo5 PRMC per microwell in 0.2 ml medium (RPM1 1640 supplemented with glu- tamine, antibiotics, nonessential amino acids, and 10% autologous serum) with antigen, mitogen, or irradiated, autologous T cell populations at the concentrations indicated. Following a 4 or 5 day incubation, proliferation was determined by uptake of [3H]thymidine for the final 16 h of culture. Antigen-induced proliferation of T cell lines and clones maintained in long-term culture was measured by incubation of T cells with the appropriate antigen and irradiated (4000 rad) autologous PBMC, as a source of APC, for 72 h. Proliferation was measured as described above.

To assess the response of freshly isolated PBMC to autologous, MBP-reactive T cells, PBMC (1.5 X

lO’/microwell) were incubated with 3.0 x lo4 irradiated (4000 rad), MBP-reactive T cells. Non-activated, resting T cells were those that had last been restimu- lated with MBP and APC at least 8-10 days before the initiation of these assays. To assess the response of freshly isolated PBMC to activation markers, identical cultures were performed in parallel except that human MBP (30 pg/ml) was added to the microwells contain- ing PBMC and irradiated MBP-specific T cells. In data that are not shown, irradiated, MBP-reactive T cells generate IL-2 and up-regulate IL-2-receptor expression in response to activation. The MBP-reactive T cells were irradiated to prevent their long-term proliferation or survival in culture. This permitted assessment of the proliferative response of the PBMC at day 4 or 5, and determination of the phenotype of the responding T cells at day 10. At 4 and 5 days the irradiated MBP- specific T cells contributed less than 400 cpm to the [ 3H]thymidine incorporation.

2.4. Phenotype assessment

Monoclonal antibodies identifying specific T cell surface antigens were used. Monoclonals recognizing CD3 (Anti-Leu 4, pan T cell), CD4 (Anti-Leu 3, helper/inducer T cells), and CD8 (Anti-Leu 2, sup- pressor/cytotoxic T cells) were purchased from Becton Dickinson, Mountain View, CA. Monoclonal anti-c@ TCR (Identi-T Pan TCR cup), anti-y6 TCR (Identi-T TCR Sl), anti TCR Vy2(9) (Diversi-T yV2), and anti TCR V61 (Diversi-T SVl) were purchased from T Cell Diagnostics Inc., Cambridge, MA. Indirect immuno- fluorescence staining was performed as previously described (Burns et al., 1991) with phenotype determination by flow cytometry using an Ortho Cytofluorograph 11s (Westwood, MA) or by epifluorescence microscopy in initial experiments.

3. Results

Initial experiments demonstrated a striking differ- ence between the response of freshly isolated peripheral blood mononuclear cells to activated versus resting, autologous MBP-specific T cells. In these experiments, the term ‘resting T cell’ refers to T cells populations that have not been stimulated by antigen for at least 8-10 days. ‘Activated T cells’ are taken from the same T cell population; however, activation is achieved through MBP presentation by macrophage in the freshly isolated PBMC. Both resting and activated MBP-reactive T cell clones are irradiated to prevent proliferation and survival in long-term cultures.

An MBP-specific T cell clone was isolated from an individual with MS. As shown in Fig. lA, when freshly isolated, autologous PBMC were incubated with either

180 J. Burns et al. /Journal of Neuroimmunology 58 (1995) 177-182

Table 1 Changes in lymphocyte subpopulations following culture of PBMC with activated antigen-specific T cells

Cell population Percentage of cells staining

CD3 uP YS VY 90) VSI

(1) Freshly isolated PBMC (2) PBMC cultured with activated, MBP-specific T cells (3) PBMC cultured with activated, Can&a-specific T cells

61% 60% 6% 4% 3% 69% 25% 49% 43% 9%

64% 25% 41% 41% 9%

The phenotype of lymphocytes before and after culture with autologous, activated MBP-reactive T cells or activated Can&da-reactive T cells was assessed using a panel of monoclonal antibodies recognizing CD3 (pan T cell), the cup TCR, the yS TCR, and the two predominant subsets of yS T cells. PBMC (1.5 X lo5 per microwell) were incubated with 4 X lo4 irradiated (4000 rad), MBP or Candida-specific T cells plus the appropriate antigen. IL-2 was added at day 6 and the phenotype assessed at day 10 as described in Materials and methods.

the irradiated MBP-reactive T cell clone alone or with MBP alone, there was little subsequent proliferation. However, if both MBP and the irradiated MBP-specific T cells were incubated with the PBMC, a very brisk response was seen after 4 days. The proliferative response to tetanus toxoid and the superantigen, TSST, are also shown to permit a comparison with the proliferation generated by activated, MBP-specific T cells. Although it is not obvious due to the scale used in Fig. lA, the stimulation index for the response to tetanus toxoid is 2.5 and represents the proliferation of approximately 1 in 10000 PBMC. By contrast, TSST activates approximately lo-15% of all peripheral blood T cells and thus routinely generates very brisk proliferation even in short-term assays (Choi et al., 1990). Since the magnitude of the response against activated, MBP- specific T cells approaches that produced by TSST, it is likely that a substantial fraction of the PBMC participate in this response. A similar level of proliferative response was seen using PBMC and autologous, MBP- specific T cells from a second subject with MS.

In further experiments using a control subject, an MBP-specific T cell clone that recognized human MBP and the synthetic MBP peptide (81-100) was isolated. As shown in Fig. lB, resting MBP-reactive T cells generate minimal responses by fresh PBMC. However, if the MBP-specific T cells are activated by whole MBP or by the appropriate MBP peptide (81-lOO), there is a brisk proliferative response by the PBMC. MBP or peptide alone, in the absence of MBP-specific T cells, generated no response by the PBMC.

To be certain that the proliferation was not an exaggerated response to MBP by the fresh PBMC, the cells obtained from primary cultures, as described above, were assayed for responsiveness to MBP using methods previously described (Burns et al., 1991). In three separate experiments, the cells recovered from the primary cultures did not respond to MBP but continued to respond to activated T cells (data not shown).

To identify the cell population in the PBMC responding to the activated T cells, the responding cell population was characterized using monoclonal antibodies recognizing common T cell surface antigens. These studies were performed 9 or 10 days after the initiation of the cultures. IL-2 was added after 6 or 7 days to maintain proliferation of T cells. Representa- tive experiments using monoclonal antibodies specific for TCR types ap and yS are shown in Table 1. In the

/’

Control #l

LL

l-9 70 t / Control #2

Peripheral

Blood Mononuclear Cells

Following Culture with Activated T Cells

Fig. 2. Expansion of yg T cells in fresh PBMC after culture with MBP-specific T cells. MBP-reactive T cells were isolated from two individuals with MS and two control subjects. Freshly isolated PBMC were incubated with the irradiated, activated MBP-reactive T cells as described in Table 1. The phenotype profile of the fresh PBMC and the responding lymphocyte cultures were assessed using a monoclonal antibody recognizing the yS T cell receptor. The values shown represent the percentage of the T cells that utilized the y8 TCR. The percentage of T cells in the final cultures at 9 days averaged 70%.

J. Burns et al. /Journal of Neuroimmunology 58 (1995) 177-182 181

example using MBP-specific T cells, approximately 69% of cells present after 9 days are T cells and y6 TCR- positive T cells comprise 71% of the T cells. The majority of the y6 T cells utilize the Vy9(2) TCR. In a series of six similar experiments using MBP-specific T cells with this subject, an average of 70 f 6% of the T cells recovered were y6 T cells. As also shown in Fig. 1, when CD4+ cells reactive with a control antigen, candida, were used instead of MBP-specific T cells, a similar, disproportionate expansion of yS T cells oc- curred. This result indicates that T cells reactive with antigens other than MBP may also provide the stimu- lus for proliferation of yS T cells. In freshly isolated PBMC from this individual, 8-10% of T cells expressed y6 T cell receptors. Similar results were noted in three additional experiments with a second control subject (not shown).

Fig. 2 demonstrates the enrichment of y6 T cells with each of the four subjects tested in this manner. The percentage of yS T cells in the freshly obtained PBMC increased from an average of 8% in these subjects to greater than 70% following exposure to the autologous, activated, MBP-specific T cells.

4. Discussion

This study has two major findings. First, activated, but not resting, autologous MBP-specific T cells generate a brisk proliferative response in freshly isolated, peripheral blood mononuclear cells of both MS and control subjects. Second, this response appears to prominently involve T cells utilizing the y6 TCR. An enrichment of yS T cells in CNS lesions or CSF of patients with MS has been noted in a number of studies (Selmaj et al., 1991; Wucherpfennig et al., 1992; Hvas et al., 1993; Shimonkevitz et al., 1993). To ac- count for this accumulation of y6 T cells, some investigators suggest that these lymphocytes may recognize heat shock proteins present on glial cells within the lesions (Freedman et al., 1991; Selmaj et al., 1991; Birnbaum et al., 1993). The results of the current study suggest an alternative explanation for the presence of y6 T cells. If yS T cells respond to T cell activation markers associated with T cells already in the lesions, this recognition may lead to expansion of the yS T cells in the parenchyma and CSF.

It has been demonstrated repeatedly that myelin antigen-reactive T cells can induce inflammatory CNS disease in experimental animals. While the antigen specificity of the T cell is critical, a state of functional activation is also a prerequisite for encephalitogenicity. As many as 7 X lo6 resting, MBP-specific T cells may not induce disease while as few as 1 X lo5 activated T cells of the same cell line can induce EAE (Vanden- bark et al., 1985). This suggests that, when autoimmu-

nity is concerned, the immune system may have a particular interest in monitoring the state of activation of autoreactive T cells. Control of activated, autoreactive T cells might reduce overt autoimmune disease even if non-activated T cells are not deleted from the lymphocyte repertoire. A state of functional activation is also necessary in certain T cell vaccination protocols (Naparstek et al., 1983; Lider et al., 1988; Lohse et al., 1993). Some property associated with activation of T cells provokes more effective protection than the use of non-activated T cells. Thus the presence of idiotype alone seems to not be sufficient (Naparstek et al., 1983; Lider et al., 1988; Lohse et al., 1993). Whether the anti-ergotypic response itself augments protection or a synergistic, adjuvant-like effect occurs has not been established. The conspicuously brisk nature of the anti-ergotypic response seen in this study, with non- vaccinated human subjects, suggests there may be a physiological role for this type of response.

T cell recognition of activation markers on other T cells has been termed anti-ergotypic responsiveness by investigators studying this response in experimental autoimmune encephalomyelitis (EAE) (Lohse et al., 1989; Lohse et al., 1993). In these experiments, Lohse and colleagues noted anti-ergotypic responses could reduce the severity and incidence of EAE. This was not as effective as anti-idiotypic responses in generat- ing protection in EAE, but appeared to act synergisti- cally with anti-idiotypic responses (Lohse et al., 1989). The activation marker recognized by the anti-ergotypic cells in these experiments appeared to be a structural component of the activated T cells and not a secreted T cell product or lymphokine (Lohse et al., 1989; Lohse et al., 1993). Additional studies are underway to identify the human T cell activation antigen or lymphokine responsible for the yS T cell expansion noted in the current study. In agreement with other investigators, we find that the addition of IL-2 alone to PBMC does not result in the level of proliferation or yS T cell expansion noted in these experiments (Pechhold et al., 1994) (data not shown).

The immunological function of yS T cells remains uncertain (Modlin et al., 1989; Kabelitz et al., 1990; O’Brien et al., 1991; Schild et al., 1994). In humans these form a subset of T cells with about S-10% of T cells in the peripheral blood utilizing y6 TCR. Some yS T cells appear to recognize a class of antigens known as heat shock or stress proteins (Born et al., 1990; Kabelitz et al., 1990; O’Brien et al., 1991). Other investigators suggest a possible role for y6 T cells in the control of immune responses (Kaufmann et al., 1993; Fu et al., 1994). For example, recent work has demonstrated that y6 T cells limit potentially destruc- tive inflammatory responses to experimental listeriosis in mice (Fu et al., 1994). In humans, regulatory interac- tion between yS and a/3 T cells may occur in Plusmod-

182 .I. Burns et al. /Journal of Neuroimmunology 58 (1995) 177-182

ium falciparum infections (Roussilhon et al., 1994). MS is not the only possibly autoimmune disorder in which yS T cells have been noted to accumulate in disease lesions. Similar findings have been noted in rheumatoid arthritis with oligoclonality in yS T cells noted in the synovial tissue (Olive et al., 1992).

The current study illustrates that y8 T cell expansion in MS lesions may occur in response to markers expressed by activated T cells within these lesions. This effect is not specific for MBP-reactive T cells and other CD4+, activated T cells will induce the same response. T cells recognizing activation markers on autoreactive T cells have been shown to function in immune regulation in animal models of demyelinating disease (Lohse et al., 1989; Lohse et al., 1993). Whether the human, anti-ergotypic T cell response described in this report contributes to immune regulation remains to be established.

Acknowledgements

The authors wish to thank Mr. Steve Lobo, Ms. G. Morrison, and Ms. K. Clark for invaluable assistance, and Dr. Demming Sun for discussions leading to pre- liminary investigations. This work was supported by Veterans Administration Research Funds and a grant from the NIH (NS27556) and CA 42104 supporting the Flow Cytometry Facility of the Utah Cancer Center.

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γ δ T cells participate in the immune response against activated, myelin basic protein-specific, human T cells