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VIRAL IMMUNOLOGY Volume 17, Number 4, 2004 © Mary Ann Liebert, Inc. Pp. 604–608
Brief Report Effector CD8 T Cells Are Suppressed by Measles Virus Infection during Delayed Type Hypersensitivity Reaction SABINE STREIF,1 KARIN PUESCHEL,1 ANNETTE TIETZ,1 JORGE BLANCO,2 VOLKER TER MEULEN,1 and STEFAN NIEWIESK1
ABSTRACT Measles virus infection reduces or abolishes delayed type hypersensitivity reactions (DTH) in humans. We have previously shown that the primary 2,4-dinitrofluorobenzene (DNFB) response is temporarily suppressed by measles virus in cotton rats. Here, we demonstrate that also the secondary DNFB response (cutaneous hypersensitivity [CHS]) is suppressed in cotton rats by measles virus infection. As in mice, DNFB specific CD8 T cells are the predominant T cell response in cotton rats. After MV infection, CD8 T cells are reduced in their proliferative capacity whereas the CD4/CD8 ratio, the number and activation status of CD8 T cells is not affected. As a result of impaired proliferation of DNFB specific T cells the DTH response (measured as ear swelling) is reduced in measles virus infected cotton rats. At the same time as DNFB specific T cell responses are suppressed, spontaneous proliferation of lymphocytes as evidence for immune activation is found.
INTRODUCTION
M
EASLES VIRUS induces a severe immune suppression
in infected individuals which is signified by reduction or loss of mitogen and antigen responsiveness ex vivo, inhibition of delayed type hypersensitivity (DTH) reactions and increased susceptibility towards secondary infections. To study the underlying mechanism, cotton rats (Sigmodon hispidus) have been used. Cotton rats are a unique rodent model to study measles virus pathogenesis because MV replicates after intranasal inoculation in the respiratory tract of these animals. As in humans, MV infection severely reduces mitogen responsiveness in these animals (15). In order to evaluate the effect of MV
1Institut 2Virion
infection on antigen-specific T cell responses, the T cell response against the hapten 2,4-dinitrofluorobenzene (DNFB) was investigated. After application to the ear skin, dendritic cells carry DNFB into draining lymph nodes and stimulate proliferation of T cells. Stimulated lymphocytes from draining lymph nodes incorporate 3Hthymidine overnight in tissue culture which is used as a measure of proliferation. Using this model system we have previously shown that the primary DNFB-specific T cell proliferation was reduced during the acute phase of the infection (day 6) but not in the early and late phase (day 1 and 13) (2,16). A reduction of proliferation was preceded by a decrease in Akt kinase activity (2) which correlates with impaired progression through the cell cy-
fuer Virologie und Immunbiologie, Wuerzburg, Germany. Systems, Rockville, Maryland.
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EFFECTOR CD8 TYPE CELLS ARE SUPPRESSED BY MEASLES
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cle (17). At the time, investigation of the T cell subsets involved was not possible due to the lack of reagents. In this study we have analyzed the suppression of the primary DNFB T cell response in more detail and also studied the suppression of the secondary DNFB response (contact hypersensitivity [CHS]) by MV infection. In mice, application of DNFB to the flank induces the development of DNFB specific CD8 cytotoxic T cells (4,10) which after secondary application to the ear are recruited to the site of application leading to increase in ear thickness. The development of CD8 T cells is independent of CD4 T cells (5). However, DNFB specific CD4 T cells down regulate DNFB specific CD8 T cells and after depletion of CD4 T cells the ear swelling is increased (4). Our data demonstrate that in cotton rats CD8 T cells are the main effector cells in the DNFB response, which are suppressed by MV infection during the primary and secondary response. This is due to a block in proliferation and not due to a lack of activation.
nization with mouse fibroblast cell line L929 transfected with the CD8alpha gene of cotton rats (Sigmodon hispidus; accession number AY065643). Both antibodies were of the IgG1 isotype and their specificity was confirmed by flow cytometry and immune precipitation of lymphocytes (manuscript in preparation). Injection of 500 g of either antibody into cotton rats led to depletion of the respective T cell subset (as measured by flow cytometry). Antibody W6/32 [a pan-HLA antibody (3)] also reacts with cotton rat MHC class I (data not shown). Flow cytometry and Pappenheim’s stain. Cells were stained with 10–15 g/mL CR-CD4, CR-CD8 or W6/32 antibody and a donkey anti mouse serum labeled with FITC (Dianova, Germany). Subsequently cells were analyzed by flow cytometry (Facscan, Becton Dickenson). For Pappenheim’s stain, 2 105 lymph node cells were spun onto a glas slide and air-dried. Cells were stained with concentrated May-Grünwald solution and counterstained with GIEMSA solution (Firma Labor und Technik).
MATERIALS AND METHODS
RESULTS
Infection of cotton rats. Four to eight week old inbred cotton rats (strain cotton N/Ico from Iffa Credo, France) of both sexes were used. Animals were infected with 5 106 pfu MV Edmonston strain intranasally in ether narcosis. MV was grown and titrated on Vero cells as described previously (15). Primary sensitization and secondary cutaneous hypersensitization (CHS). For primary sensitization, the right ear of an animal was painted with 40 L of 2% 2,4dinitrofluorobenzene (Sigma) in DAE (40 v/% dimethylacetamid, 30 v/% ethanol, 30 v/% aceton) on three consecutive days. On day four the draining lymph nodes were removed and lymphocytes plated in triplicates in a 96well plate at 5 105 cells/well with 0.5 Ci (3H-thymidine, Amersham). At 18–20 h later, cells were harvested onto a Whatman filter and counted in a Wallac betaplate counter. For induction of CHS, on day 10, 40 L of a 2% DNFB solution was applied to the shaved flank of cotton rats. On day 0, 40 L of a 2% DNFB solution was applied to the right ear and ear thickness was measured on day 1 and 2 using a Peacock dial thickness gauge (Labtek). Ear swelling was measured in duplicate as the difference between the treated (right) ear and the untreated (left) ear. Monoclonal antibodies specific for cotton rat CD4 and CD8. The CD4 specific antibody CR-CD4 was obtained by immunization of BALB/c mice with Concanavalin A stimulated cotton rat spleen cells and the CD8 specific antibody CR-CD8 was obtained by immu-
In order to characterize the cellular composition of lymph node cells after stimulation with DNFB, lymph node cells were isolated after application of DNFB to ear skin. The number of cells was fivefold higher in lymph nodes draining the right (treated) ear compared to the left (untreated) ear and by Pappenheim staining lymph node cells were identified as lymphocytes. To analyze the CD4 and CD8 T cell subsets, the CR-CD4 and CR-CD8 antibodies were used. The respective hybridomas were obtained after immunization of mice, and their specificity determined by immune precipitation. Antibody CR-CD4 precipitated a cell surface molecule of 55 kDa and antibody CR-CD8 precipitated a cell surface molecule of 34 kDa from cotton rat thymocytes (data not shown). After staining with antibody CR-CD4 and CR-CD8 the ratio of CD4 to CD8 T cells was found to be 1.5:1 in cells from untreated lymph nodes by flow cytometry. In lymph node cells from activate (treated) lymph nodes, the ratio was reversed (1:1.7), indicating a strong stimulation of DNFB specific CD8 T cell growth as seen in mice. To investigate whether CD8 T cells were also responsible for ear swelling, CHS was induced in three groups of cotton rats. On day 10, 40 L of a 2% DNFB solution was applied to the shaved flank of cotton rats. On day 0, 40 L of a 2% DNFB solution was applied to the right ear and ear thickness was measured on day 1 and 2 using a Peacock dial thickness gauge (Labtek). Two groups of cotton rats received 0.4 to 0.5 mg of either CR-CD4 or CR-CD8 on day 11, 10, 9, 6, 0 and 2 whereas the control group received no antibody (Table 1). The depletion of
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STREIF ET AL. TABLE 1. AVERAGE DIFFERENCE IN EAR THICKNESS BETWEEN TREATED AND UNTREATED EAR OF COTTON RATS INJECTED WITH MONOCLONAL ANTIBODY AGAINST CD4 AND CD8 Ear swelling, m
Treatment DNFB ear paint DNFB ear paint and monoclonal antibody against CD4 DNFB ear paint and monoclonal antibody against CD8
Day 1
Day 2
Day 3
Day 4
370 71 251 21 p 0.009 123 57 p 0.003
344 66 242 90 p 0.16 133 82 p 0.02
340 81 236 37 p 0.11 158 64 p 0.04
391 90 269 15 p 0.08 144 48 p 0.01
Cotton rats (four animals per group) were painted at the flank with DNFB on day 10, on day 0 challenged by ear paint and ear swelling was measured as the difference in ear thickness between the treated and untreated ear on day 1, 2, 3 and 4 after application. The thickness of untreated ears varied up to 40 m in comparison to day 0. Two groups received antibody (0.4 to 0.5 mg/i.p. injection) specific for cotton rat CD8 or CD4 on day 11, 10, 9, 6, 0 and 2. Statistically significant differences between controls and animals injected with antibody are bold faced (two-sided t-test).
the respective T cell subset was 98% effective for both antibodies for at least 3 days (data not shown). Depletion of CD8 T cells with CR-CD8 lead to a significant reduction of ear swelling on day 1, 2, 3 and 4 after challenge (between p 0.04 and p 0.003, two-sided ttest). Depletion with CR-CD4 resulted not in an increase of ear thickness (as has been shown for the mouse model). In contrast, ear swelling was reduced but only significantly so on day 1. These results demonstrate that as in the mouse CD8 T cells are the main effector T cells in CHS but the role of CD4 T cells seems to deviate in that they do not down regulate the effector function of CD8 T cells and probably contribute to CHS. To test the effect of MV infection on CHS, cotton rats were painted at the flank with 40 L of a 2% DNFB solution on day 10 and were infected with 5 106 pfu MV on day 0. They were challenged on day 3 and ear measurements were taken on day 5 (2 days after challenge). The difference in ear thickness for MV infected animals was
TABLE 2. AVERAGE DIFFERENCE IN EAR THICKNESS BETWEEN TREATED AND UNTREATED EARS OF COTTON RATS INFECTED WITH MV OR UNINFECTED CONTROLS Treatment DNFB ear paint DNFB ear paint and MV infection
Ear swelling, m 148 50 93 48 p 0.04
Cotton rats (five animals per group) were painted at the flank with DNFB on day 10, on day infected with MV and on day 3 challenged by ear paint and ear swelling was measured on day 5 (2 days after challenge) as the difference of ear thickness between treated and untreated ears. Statistically significant difference between infected and not infected animals is bold faced (two-sided t-test).
93 48m, the difference for control animals 148 50m (p 0.04, two sided t-test; Table 2). To test proliferation of DNFB specific T cells, proliferation of lymph node cells from the left and right lymph nodes were compared. No difference in proliferation in MV infected animals was found between lymph node cells from the treated to untreated side (3645 1019 cpm and 2273 990 cpm) whereas in control animals a stronger proliferation was seen in cells from the treated to the untreated side (3073 405 cpm to 1363 660 cpm; p 0.05). The low T cell response in draining ear lymph nodes is due to the fact that during CHS the already primed DNFB specific T cells migrate mainly to the ear. To investigate the T cell response in more detail the primary reaction towards DNFB was used during which all DNFB specific T cells accumulate in the ear lymph nodes. Three days after infection with 5 106 pfu MV (Edmonston B strain) cotton rats (six animals per group) were ear painted for 3 days and lymph nodes were taken on day 6 after infection. As reported earlier (16), we again observed that proliferation of DNFB specific T cells was reduced in MV infected animals (Table 3), although the number of lymph node cells was comparable between infected animals (2.1 1.1 107 cells) and from control animals (2.0 1 107 cells). To further analyze the T cell response, lymph node cells were stained with antibodies against MHC I, CD4 and CD8 expression. In both infected and non infected animals MHC I was strongly up regulated (from an average of 15 mean fluorescence units (MFU) to an average of 550 MFU), but no difference was seen between the two groups. In infected animals, the ratio of CD4:CD8 T cells was 1.6:1 in the untreated ear and 1:1.6 in the treated ear. This reflects the induction of DNFB specific CD8 T cells and was similar to the ratios seen in non-infected animals (1.5:1 and 1:1.7). In mandibular lymph nodes draining the untreated
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EFFECTOR CD8 TYPE CELLS ARE SUPPRESSED BY MEASLES TABLE 3.
SUPPRESSION
OF
DNFB-SPECIFIC PROLIFERATION
607 BY
MV INFECTION 5 106 pfu MV
Not infected
Organ LN right (treated) LN left (untreated) LN from animals treated with carrier LN from naive animals
Cell number (106) SD
Proliferation in counts per minute SD
20 10 p 0.9 3.6 20 p 0.02 3.9 50
14,904 4810 p 0.02 434 316 p 0.003 620 324
3.3 40
510 223
Cell number (106) SD
Proliferation in counts per minute SD
21 11
6341 3939
13.5 8.5
3884 2256
n.d.
n.d.
n.d.
n.d.
Three days after MV infection (5 106 pfu), cotton rats (six animals per group) were ear painted for 3 consecutive days. One day later, numbers of lymphocytes from mandibular lymph nodes were counted and plated out in 96-well plates to incorporate3H-thymidine overnight. Although there is no reduction in cell numbers, proliferation is strongly reduced. Statistically significant differences between infected and not infected animals are bold faced (two-sided t-test). Lymph nodes from not infected animals which were either treated with carrier solution or not treated at all did not differ in cell numbers and proliferation from the lymph node cells from left ears of uninfected and treated animals.
ear of infected animals, the number of lymphocytes was threefold increased compared to lymphocytes from control animals (p 0.02, two sided t-test). In addition, spontaneous proliferation was higher in lymph nodes draining the untreated ear than in lymph nodes from not infected animals (p 0.003, two sided t-test; Table 3).
DISCUSSION Cotton rats (Sigmodon hispidus) are in contrast to rats and mice susceptible to MV replication in their respiratory tract after intranasal inoculation. Our studies demonstrate that MV infection suppresses both the primary and the secondary CD8 DNFB specific T cell response. In humans it has been observed that the ratio of CD4 to CD8 T cells in peripheral blood lymphocytes is not changed during MV infection (1). Similarly, in lymph node cells from infected cotton rats no change in the CD4/CD8 ratio in comparison to lymph node cells from not infected animals was observed in either non stimulated lymph node cells or after antigenic stimulation with DNFB. In infected animals the same number of lymph node cells was found in lymph nodes draining the DNFB treated ear as in not infected animals. Lymphocytes also expressed high levels of MHC I. In the absence of more specific reagents for the cotton rat model, this increase is an indication of immune activation. However, proliferation of these DNFB specific T cells was reduced. These data indicate that the antigen specific T cell response was initiated but expansion of T cells during both the primary and
secondary T cell responses were inhibited after MV infection. Direct contact of lymphocytes with MV glycoproteins leads to reduced proliferation in tissue culture (18) and in cotton rats (15) due to down-regulation of the cell cycle (13,14,17,19). However, as no infectious virus was found in mandibular lymph node cells by co-cultivation (data not shown) a direct contact between MV glycoproteins and lymphocytes cannot explain proliferation inhibition. Using previously published primers for nucleocapsid (N) mRNA (6), PBL were found to be positive for MV-N mRNA by PCR (1 in 10000 lymphocytes to 1 in 20,000). An alternate explanation of proliferation inhibition would be the incomplete replication of MV with expression of the N protein which has been shown to reduce replication of T cells (12). Alternatively, rather than direct contact of MV (proteins) with mandibular lymph node cells, infection of lung and lung draining lymph nodes might lead to indirect regulatory mechanisms of T cell proliferation as has been proposed previously (8). This would be supported by the fact that in the mouse model of CHS, i.p. injection of UV-inactivated MV leads to reduction in ear swelling although the site of MV (protein) deposition is distant from the site of T cell action (12). In tissue culture, infection of dendritic cells with MV leads to reduction in IL12 secretion after stimulation (7,9,11), which in vivo might lead to the development of a TH2 response. In the mouse, the number of dendritic cells expressing interleukin 12 was reduced after injection of UV-inactivated MV which would be consistent with development of a TH2 response (12). In cotton rats, the development of monoclonal antibodies
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against CD4 and CD8 was part of the effort to establish reagents necessary to analyze the role and regulation of T cells in MV infection. These reagents will also be required to analyze immune activation by MV infection as seen in lymph nodes draining the not treated ear of cotton rats. In patients, spontaneously proliferating PBL are found which secrete cytokines. In addition, a higher activity of natural killer cells has been observed. How immune activation is achieved and why it takes place simultaneously with suppressed immune reaction remains to be investigated.
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Address reprint requests to: Dr. Stefan Niewiesk Department of Veterinary Biosciences Ohio State University 1925 Coffey Rd. Columbus, OH 43210 E-mail:
[email protected] Received April 15, 2004; accepted August 18, 2004.
