key: cord-0811935-gq8pusi3
authors: Shubin, Richard A.; Sussman, Mark A.; Fleming, John O.; Stohlman, Stephen A.
title: Relapsing encephalomyelitis following transfer of partial immunity to JHM virus
date: 1990-05-31
journal: Microbial Pathogenesis
DOI: 10.1016/0882-4010(90)90089-9
sha: 0c47053cbb34cd69de7c9b852a52183d7e5cffd5
doc_id: 811935
cord_uid: gq8pusi3

Abstract Mice infected with the JHM strain mouse hepatitis virus (JHMV) develop a fatal encephalomyelitis with evidence of demyelination. It has previously been shown that the adoptive transfer of 5 × 107 nylon wool adherent (NWA) spleen cells from immunized donors to lethally infected recipients clears virus from the central nervous system (CNS) and prevents demyelination. Adoptive transfer of a smaller number (1 × 107) of NWA spleen cells from immunized donors also protects from death but does not significantly alter virus replication in the CNS during the acute phase of the infection. Moreover, these mice develop a transient non-fatal encephalomyelitis which occurs approximately 3 weeks post-infection. This delayed encephalomyelitis is associated with a mononuclear cell infiltration into the CNS but little or no evidence of virus replication or increased viral antigen. A virus-specific delayed-type hypersensitivity (DTH) response precedes this delayed onset of disease by 24 to 48 h. Resolution of disease correlates with a selective and permanent suppression of the JHMV-specific DTH reactivity. In addition, no virus-specific DTH is detected following adoptive transfer of viral-specific DTH effectors derived from immunized donors. In contrast, these mice respond to a heterologous antigen, KLH, suggesting that the resolution of the encephalitis is accompanied by a profound suppression in viral-specific DTH response.

Relapsing encephalomyelitis following transfer of partial immunity to JHM virus* 8: 305-314. Mice infected with the JHM strain mouse hepatitis virus (JHMV) develop a fatal encephalomyelitis with evidence of demyelination. It has previously been shown that the adoptive transfer of 5x IO' nylon wool adherent (NWA) spleen cells from immunized donors to lethally infected recipients clears virus from the central nervous system (CNS) and prevents demyelination.

Adoptive transfer of a smaller number (1 x 107) of NWA spleen cells from immunized donors also protects from death but does not significantly alter virus replication in the CNS during the acute phase of the infection.

Moreover, these mice develop a transient non-fatal encephalomyelitis which occurs approximately 3 weeks post-infection. This delayed encephalomyelitis is associated with a mononuclear cell infiltration into the CNS but little or no evidence of virus replication or increased viral antigen. A virus-specific delayed-type hypersensitivity (DTH) response precedes this delayed onset of disease by 24 to 48 h. Resolution of disease correlates with a selective and permanent suppression of the JHMV-specific DTH reactivity. In addition, no virus-specific DTH is detected following adoptive transfer of viralspecific DTH effecters derived from immunized donors. In contrast, these mice respond to a heterologous antigen, KLH, suggesting that the resolution of the encephalitis is accompanied by a profound suppression in viral-specific DTH response.

The JHM strain of mouse hepatitis virus (JHMV) is a murine coronavirus which causes acute encephalitis and both acute and chronic demyelination in mice and rats. ' immune system in the pathogenesis of JHMV-induced disease and the ability of immune therapy to modify the course of disease. Mice and rats infected with JHMV have an acute neutralizing encephalomyelitis with neuronophagia.' To understand the pathogenesis of encephalitis and demyelination following JHMV infection of the CNS, we and others have examined the roles of the humoral and cellular immune response to JHMV and related mouse hepatitis viruses (MHV). Rodents can be protected from death by administration of monoclonal antibodies specific for the three virus structural proteins, El ,3 E24,5 and the nucleocapsid protein.6.7 Wege et a/.,5 demonstrated in rats that only anti-E2 monoclonal antibodies which neutralized virus and prevented cell fusion were protective. Protection by virus-specific monoclonal antibodies in mice is associated with sparing of neurons during JHMV infection and the subsequent development of subacute demyelination4

Other studies have focused on the protective role that cellular immunity may play in JHMV pathogenesis. Using the adoptive transfer of JHMV-immune spleen cells into JHMV-challenged recipients, two distinct protective T lymphocyte populations have been identified. First, nylon wool non-adherent (NWNA) cells (Thy I+, CD4', Lyt-2-) will induce a delayed-type hypersensitivity (DTH) response in recipients. These cells are restricted by major histocompatibility complex (MHC) Class II (I-A) identify between donor and recipient. Protected mice show vigorous perivascular mononuclear cell responses in the CNS but viral replication is not suppressed, and surviving mice show marked demyelination in central white matter. Second, nylon wool adherent (NWA) cells (Thy l+, CD4-, Lyt-2-) will protect mice, suppress viral replication, and prevent demyelination. The effect of these cells is dependent on MHC Class I (H-2 D) identify between donor and recipient and is blocked by administration of anti-CD8' antibody to the recipient. Protection mediated by the administration of either donor NWNA CD4' or NWA CD4' populations is prevented by immunosuppression of the recipients, implying that both donor T cell populations function primarily as helpers for host cells. Thus, NWNA CD4' cells induce a DTH response, prevent a lethal encephalitis, but are unable to block viral replication and subsequent demyelination; NWA CD4' cells induce CD8' effecters cells which protect mice, reduce viral replication, and prevent demyelination.

In the present report we have focused on the influence of the second T helper population, CD4' NWA cells, on JHMV-induced demyelination. Interestingly, when a reduced number of these cells are administered, sufficient to protect only partially from an ordinarily lethal JHMV challenge, many recipient surviving mice show a biphasic, relapsing disease. This finding is consistent with the observation that chronic CNS pathology often occurs when there is an equivalence or balance between the ability of a virus to replicate and the ability of immune system to clear the virus. We have also found that the onset and resolution of delayed disease is correlated with the level of anti-JHMV cellular immunity in these mice. This system provides a useful model for dissecting the regulation of immunological responses to a viral infection of the CNS with regard to clinically relevant phenomena, such as the onset and resolution of an episode of demyelination.

The adoptive transfer of NWA, but not NWNA cells, from immunized donors to lethally infected mice mediates the clearance of JHMV." Previous results from our laboratory have indicated that the active cells in this population are CD4' T cells that function by providing help for CD8' cells, because treatment of the recipients with anti-Lyt-2 (CD8") monoclonal antibody prevents the reduction of JHMV in the CNS." Table 1 shows that adoptive transfer of 5x IO' NWA cells results in the reduction of JHMV in the brain of infected mice as previously described." To determine the number of cells required for protection, 1 x 10' and 5 x 1 O6 NWA cells were transferred. The transfer of 1 xl 0' NWA cells also protects against lethal JHMV infection; however, this cell number does not mediate reduction of the viral titer in the CNS (Table 1) . Adoptive transfer of 5 x 1 O6 or fewer cells neither protected mice nor cleared virus from the CNS (Table 1) . Complement-mediated antibody depletions prior to transfer of 1 x IO' NWA cells per recipient indicated that the phenotype of the cell responsible for protection is a CD4' T cell (data not shown), similar to our previously published report."

The quantity of NWA cells transferred during infection not only determines survival and the clearance of virus from the CNS but also the clinical course of the disease (Table 1) . Control mice which received JHMV and no NWA cells showed clinical evidence of acute encephalomyelitis (ruffled fur, hyperexcitibility, and myoclonus) on day 11 post-infection and died by day 14." Mice which received 5x10' NWA cells did not manifest any clinical signs of encephalomyelitis following infection." In contrast, most mice which received the intermediate number of NW cells (1 x IO') developed a non-fatal encephalitis. Figure 1 outlines the incidence of clinical disease in this group. All mice appeared normal until day 11. At that time, a subgroup (5 out of 24 or 22%) developed encephalomyelitis of variable severity from which a minority (1 out of 5 or 25%) died. The remainder of this subgroup recovered clinically by day 14. On approximately day 21 post-infection (17 out of 20 or 85%) develop a second episode of encephalomyelitis of variable severity. This second phase was lethal for one out of the 17 or 5% of the mice; however, the majority (15 of 16) exhibit signs of shows a representative section of cerebrum exhibiting abundant viral antigen in both neurons and glia. The degree of cellular infiltration in the CNS reaches a plateau at 7 days post-infection and remains constant until death at day 14 post-infection.

In contrast, the transfer of 5~10~ NWA cells at the time of intercerebral (i.c.) innoculation dramatically reduces viral titer in the CNS" and results in minimal histological-evidence of disease. A photomicrograph of the same region of brain as above [ Fig. 2(b) ] reveals an absence of viral antigen in both neurons and glia following the transfer of 5x 1 O7 NWA cells. By contrast, Table 1 shows that the protection mediated by adoptive transfer of 1 x 1 O7 NWA cells only reduces viral titer 0.8 logro pfu/g relative to the untreated controls at day 5, although the transfer of this number of NWA cells protects mice from death. Although it results in earlier clearance of virus (Table 2) , histological examination shows that there is abundant viral antigen in both neurons and glial cells and a marked infiltration into the brain parenchyma, primarily in the white matter [Fig. 2(c) ], similar to the controls [ Fig. 2(a) ].

The second phase of clinically apparent disease begins at day 21 post-infection and is associated with a marked cellular infiltration into both white and gray matter [Fig. 2(d)] . A corresponding increase in infectious virus or viral antigen was not observed at this time, although occasional mice had low levels of infectious virus or occasional foci of viral antigen in areas not associated with cellular infiltration or demyelination (data not shown). These findings are consistent with previous studiesg~'2 showing that viral antigen is generally not present in lesions at the time of demyelination and suggesting that disease may develop in areas in which active clearance of virus occurs.

Immune response in protected mice JHMV-specific antibody levels were examined to determine the role of humoral immunity in protection afforded by adoptive transfer of 1 ~10~ NWA cells. The anti-JHMV IgG response'* In both recipients and controls not given NWA cells was similar in both in kinetics and magnitude (data not shown). Similar antibody titers were obtained following the adoptive transfer of CD4' DTH inducer T cells, which also protected without altering virus replication in the CNS.'

The JHMV-specific DTH response was assessed as a measure of cell-mediated immunity in mice protected by adoptive transfer of 1 x 1 O7 NWA cells. Six days after intraperitoneal (i.p.) immunization with JHMV, mice exhibit a vigorous DTH response elicited by the injection of viral antigen into the footpad."

We have previously shown that NWA cells do not mediate a virus-specific DTH response;" in addition, mice infected with JHMV by intracerebral inoculation and not protected by donor splenocytes do not develop a measurable DTH response prior to death (data not shown). Table 3 (experiment 1) confirms that mice immunized i.p. exhibit an excellent virusspecific DTH response. Furthermore, the adoptive transfer of unseparated spleen ceils or NWNA ceils from immunized donors to naive recipients also results in a virusspecific DTH response. However, as shown previously," the transfer of NWA cells to naive recipients does not result in the induction of a virus-specific DTH response (Table 3 , experiment 2). Table 3 (experiment 3) shows that mice infected ic. with JHMV and subsequently given NWA cells by adoptive transfer do not exhibit a DTH response until 18 days post-infection. This is coincident with the development of the late phase of the disease (Fig. 1) . The DTH response is transient and disappears by day 24. The loss of DTH responsiveness corresponds to the resolution of the late disease by day 24. Table 4 shows the results of two additional experiments. First, naive mice and JHMV-infected NWAcell recipients were immunized and then challenged with keyhole lympet hemocyanin (KLH). In experiment 1, we demonstrated that the DTH response to KLH was similar in both groups. To determine if the loss of DTH reactivity to JHMV was due to active JHMV-specific suppression, we performed experiment 2 in which NWNA DTH effector T cells from JHMV immunized mice were adoptively transferred into naive recipients and NWA cell recipients which had recovered from the late encephalomyelitis.

All mice were challenged immediately with JHMV antigen and the DTH response measured 24 h later. The average DTH response in naive mice was 27.2~1 Om2 mm. In contrast, an average DTH response of only 5.1 x 10 -' mm was found in NWA cell protected mice. This suggests that the lack of a DTH response to JHMV in NWA cell protected mice is the result of viral-specific suppression, since both groups responded equally to KLH. soluble products participate in the induction of this response.17 The suppression of the DTH response may reflect an attempt of the host to reduce the adverse effects of increased vascular permeability and the infiltration of activated monocytes. Consistent with our previous results, protection from death was not due to the suppression of viral replication in the CNS, .a,'0 however, in contrast to our previous data, protection was not correlated with a virus-specific DTH response.'," The data presented in this report have demonstrated that the adoptive transfer of partial immunity to mice lethally infected with JHMV results in protection from death and the induction of a relapse of disease that is correlated with the virus-specific DTH response. 

The biology

We are grateful to Cindy Fabricius-Segal and Ligaya Pen for excellent technical assistance and to Carol Flores for excellent editorial assistance.