Selective depletion of myelin-reactive T cells with the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis.

The OX-40 protein was selectively upregulated on encephalitogenic myelin basic protein (MBP)-specific T cells at the site of inflammation during the onset of experimental autoimmune encephalomyelitis (EAE). An OX-40 immunotoxin was used to target and eliminate MBP-specific T cells within the central nervous system without affecting peripheral T cells. When injected in vivo, the OX-40 immunotoxin bound exclusively to myelin-reactive T cells isolated from the CNS, which resulted in amelioration of EAE. Expression of the human OX-40 antigen was also found in peripheral blood of patients with acute graft-versus-host disease and the synovia of patients with rheumatoid arthritis during active disease. The unique expression of the OX-40 molecule may provide a novel therapeutic strategy for eliminating autoreactive CD4+T cells that does not require prior knowledge of the pathogenic autoantigen.

Treatment of multiple sclerosis with T-cell receptor peptides: results of a double-blind pilot trial.

A T-cell receptor (TCR) peptide vaccine from the V beta 5.2 sequence expressed in multiple sclerosis (MS) plaques and on myelin basic protein (MBP)-specific T cells boosted peptide-reactive T cells in patients with progressive MS. Vaccine responders had a reduced MBP response and remained clinically stable without side effects during one year of therapy, whereas nonresponders had an increased MBP response and progressed clinically. Peptide-specific T helper 2 cells directly inhibited MBP-specific T helper 1 cells in vitro through the release of interleukin-10, implicating a bystander suppression mechanism that holds promise for treatment of MS and other autoimmune diseases.

Analysis of T cell receptor beta chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3.

This study explores the usage of T cell antigen receptor (TCR) beta chain elements in Lewis rats with experimentally induced allergic encephalomyelitis (EAE). TCRs from 15 different T cell clones and hybridomas derived from animals immunized with myelin basic protein (MBP), and all having specificity for the 21-mer encephalitogenic fragment MBP 68-88, utilized V beta 8.2. In addition, there was a marked conservation of the first two amino acid residues of the junctional complementarity determining region 3 (CDR3) associated with the V beta 8.2 receptors. 12 of 15 contained an aspartic acid followed by serine regardless of the associated J beta element. At the nucleotide level, this conservation of AspSer residues was accomplished with few or no nongermline-encoded nucleotide (N) additions. A similar pattern of AspSer usage and N region nucleotide additions was observed in a number of V beta 8.2 isolates derived from MBP-immunized lymph nodes. In contrast, V beta 8.2 polymerase chain reaction amplified isolates from Lewis T cells activated with concanavalin A or from lymph nodes of complete Freund's adjuvant-immunized animals showed no AspSer utilization (0/31) in the CDR3, and four to nine N region nucleotide additions. We conclude from this finding that AspSer residues in the CDR3, limited N region nucleotide additions, along with V beta 8.2 sequences, contribute to TCR specificity for MBP 68-88. This raises the possibility that encephalitogenic, disease-causing T cells either represent a population that derives from late fetal life or alternatively, that they are rare cells with this particular TCR phenotype contributed to the T cell pool throughout adulthood and are selected by antigen. In either case, the CDR3 AspSer sequences as well as V beta 8.2 sequences are candidates for the receptor target structures recognized by regulator T cells in recovery from and resistance to active EAE. In this respect, a preliminary analysis of TCR utilization in three T cell clones specific for MBP 68-88 isolated from animals recovered from active EAE indicates that while all three use V beta 8.2, only one contains AspSer in the CDR3.

Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V alpha and V beta chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different.

Prospects for specific immune intervention in T cell-mediated autoimmune disease via anti-idiotypic regulation depend on the degree of diversity of the responder cell antigen receptor repertoire. A highly heterogenous response against self epitopes offers little chance for such regulation. We report here that the Lewis rat autoimmune disease experimental allergic encephalomyelitis, generally considered to be a model of human multiple sclerosis, is caused by T cells that use a limited set of TCR V genes. We have cloned the rat TCR alpha and beta chain cDNAs from the Lewis rat x mouse T cell hybridoma 510, which retains the rat specificity for the encephalitogenic determinant of myelin basic protein (MBP). Using Northern blot analysis of T cell RNA with the cloned V region probes, we have found a specific, and near perfect, correlation between expression of TCR message hybridizing to the V alpha 510 and VB510 probes and specificity for the encephalitogenic determinant of MBP in both T cell hybridomas and encephalitogenic T cell clones. This restricted V gene usage provides a basis for observed idiotypic regulation of auto-reactive T cells, and possible therapy for autoimmune disease. A curious and unexplained observation is that the Lewis rat V alpha/V beta combination that dominates the encephalitogenic response to the 68-88 peptide of MBP is precisely the same V alpha/V beta combination used by the B10.PL mouse response to the encephalitogenic response to the 1-9 peptide of MBP.

T cell determinants of myelin basic protein include a unique encephalitogenic I-E-restricted epitope for Lewis rats.

The major encephalitogenic epitope for Lewis rats is the 72-89 sequence of guinea pig basic protein (GP-BP) or rat basic protein (Rt-BP). T cells responsive to this epitope are I-A restricted and preferentially express the V alpha 2:V beta 8 gene combination in their TCR. In this work, we describe for the first time the delayed appearance of T cells specific for additional discrete determinant of BP, the nonencephalitogenic 55-68 sequence of GP-BP restricted by I-A, and the encephalitogenic 87-99 sequence of Rt-BP restricted by I-E. The TCR V alpha 2:V beta 8 gene combination was expressed by both encephalitogenic GP-BP S72-89 and Rt-BP S87-99 T cell specificities but not by GP-BP 44-68-specific T cells. This is the first demonstration of I-E-restricted encephalitogenic T cells in Lewis rats and supports the conclusion that the I-E class II locus is involved in autoimmune diseases.

Effect of experimental stroke on peripheral immunity: CNS ischemia induces profound immunosuppression.

The profound damage to the CNS caused by ischemic lesions has been well documented. Yet, relatively little is known about the contribution to and effects on the immune system during stroke. We have focused on both early and late events in the peripheral immune system during stroke in mice and have observed an early activation of splenocytes that conceivably could result in immune-mediated damage in the developing CNS lesion, followed by global immunosuppression that affects the spleen, thymus, lymph nodes and circulation. While this second immunosuppressive phase may not directly enhance infarction size, it without doubt leads to an inability to respond to antigenic challenges, thereby enhancing the risk for crippling systemic infection and septicemia in stroke survivors. These novel findings advocate the need to develop or effectively utilize agents that can block early neural splenic activation and modulate immune cells specific for brain antigens as a means to prevent mobilization of T and B cells carrying a cytokine death warrant to the brain. Equally important for the recovering stroke patient are approaches that can derail the second phase of immune dysfunction and restore the ability to mount a defense against systemic infectious insults.

T cell receptor peptide therapy triggers autoregulation of experimental encephalomyelitis.

Encephalitogenic T cells specific for myelin basic protein share common V beta 8 peptide sequences in their T cell receptor (TCR) that can induce autoregulatory T cells and antibodies that prevent clinical signs of experimental autoimmune encephalomyelitis (EAE). It is not known, however, if TCR peptides can treat established disease. To test its therapeutic value, TCR-V beta 8-39-59 peptide was injected into rats with clinical signs of EAE. This treatment reduced disease severity and speeded recovery, apparently by boosting anti-V beta 8 T cells and antibodies raised naturally in response to encephalitogenic V beta 8+ T cells. These results demonstrate that synthetic TCR peptides can be used therapeutically, and implicate the TCR-V beta 8-39-59 sequence as a natural idiotope involved in EAE recovery. Similarly, human TCR peptides may be effective in enhancing natural regulation of autoreactive T cells that share common V genes.

Estrogen potentiates treatment with T-cell receptor protein of female mice with experimental encephalomyelitis.

Transgenic mice expressing the BV8S2 chain, which is specific for the myelin basic protein determinant Ac1-11, possess a naturally induced set of regulatory T cells directed against BV8S2. Further activation of anti-BV8S2 T cells in male mice with recombinant BV8S2 protein can inhibit IFN-gamma release by Ac1-11-specific T cells through a cytokine-driven mechanism and prevent induction of experimental autoimmune encephalomyelitis (EAE). In contrast, naive female mice possess fewer anti-BV8S2-reactive T cells, and treatment with BV8S2 delayed but did not prevent EAE. We here demonstrate that combining T-cell receptor (TCR) vaccination with supplemental estrus doses of estrogen potentiated IL-10 production by anti-BV8S2-reactive T cells and induced Ac1-11-specific T cells to produce IL-10 and TGF-beta. This combined treatment resulted in full protection against EAE, which was not observed with either therapy alone. These findings imply that supplemental estrogen can enhance the efficacy of TCR-based immunotherapy for autoimmune diseases that predominate in females.

Interaction of lactoferrin, monocytes, and T lymphocyte subsets in the regulation of steady-state granulopoiesis in vitro.

Colony-stimulating activities (CSA) are potent granulopoietic stimulators in vitro. Using clonogenic assay techniques, we analyzed the degree to which mononuclear phagocytes and T lymphocytes cooperate in the positive (production/release of CSA) and feedback (inhibition of CSA production/release) regulation of granulopoiesis. We measured the effect of lactoferrin (a putative feedback regulator of CSA production) on CSA provision in three separate assay systems wherein granulocyte colony growth of marrow cells from 22 normal volunteers was stimulated by (a) endogenous CSA-producing cells in the marrow cells suspension, (b) autologous peripheral blood leukocytes in feeder layers, and (c) medium conditioned by peripheral blood leukocytes. The CSA-producing cell populations in each assay were varied by using cell separation techniques and exposure of isolated T lymphocytes to methylprednisolone or to monoclonal antibodies to surface antigens and complement. We noted that net CSA production increased more than twofold when a small number of unstimulated T lymphocytes were added to monocyte cultures. Lactoferrin's inhibitory effect was also T lymphocyte dependent. The T lymphocytes that interact with monocytes and lactoferrin to inhibit CSA production are similar to those that augment CSA production because their activities are neither genetically restricted not glucocorticoid sensitive, and both populations express HLA-DR (Ia-like) and T3 antigens but not T4 or T8 antigens. These findings are consistent with results of our studies on the mechanism of lactoferrin's inhibitory effect with indicate that mononuclear phagocytes produce both CSA and soluble factors that stimulate T lymphocytes to produce CSA, and that lactoferrin does not suppress monocyte CSA production, but does completely suppress production or release by monocytes of those factors that stimulate T lymphocytes to produce CSA. We conclude that mononuclear phagocytes and a subset of T lymphocytes exhibit important complex interactions in the regulation of granulopoiesis.

Immune response to melanoma extracts in three melanoma-prone families.

Immune reactivity to melanoma extracts was measured by the leukocyte adherence inhibition (LAI) test in 40 members of 3 melanoma-prone families. The melanoma patients had a wide range of responsiveness to the extract, the highest responder being a 10-year survivor. As a group, family members (including spouses) without disease had significantly elevated LAI responses compared to those of unrelated controls (P less than 0.01). Within the families, members with close exposure to melanoma patients for 10 years or more had a significantly higher response to melanoma antigen than did members with 0-5 years of close exposure (P less than 0.05). Responses of spouses and members at high risk of developing melanoma (B-K mole syndrome) also correlated with length of exposure to patients, which suggests that the elevated LAI response was not genetically determined. The high frequency of positive responses to melanoma antigens in these families, particularly in spouses, suggests the presence of transmissible melanoma-associated material capable of immunizing persons in contact with melanoma patients.

Immunity to tumor-associated antigens in vasectomized men.

Leukocytes from 49 vasectomized and 57 age-matched nonvasectomized men were tested in the leukocyte adherence inhibition (LAI) assay for reactivity to sperm and various 3-M KCl human tumor extracts. Forty-four percent of the vasectomized men and 15% of the control group were reactive to the sperm antigen preparation (P less than or equal to 0.03). Similarly, a significantly higher percentage of vasectomized men responded to 3 of 5 tumor extracts tested: melanoma I (34.7 vs. 15.8%, P less than or equal to 0.04), squamous cell carcinoma (48.8 vs. 26.0%, P less than or equal to 0.04), and breast carcinoma (19.5 vs. 4%, P less than or equal to 0.04). Thirty percent of vasectomized versus 4% of the control group responded to more than two tumor antigens (P less than or equal to 0.03). The degree of LAI reactivity to each tumor extract was highly correlated with degree of antisperm LAI reactivity, and the degree of LAI responsiveness to one of the melanoma extracts was significantly correlated with antisperm antibody titer as measured by the sperm-immobilization assay. Furthermore, nonresponsive leukocytes from the control population converted to tumor antigen-responsive when incubated with sera from vasectomized LaI-positive men. Data from this study indicated that a large percentage of vasectomized men with sperm immunity were responsive to tumor-associated antigens in the LAI test and that antisperm antibodies or other serum factors played a role in this response. These results and the evidence that most tumor antigen-responsive cancer patients also have serum antibodies that reacted with sperm suggested that vasectomized men and cancer patients frequently responded to immunologically cross-reactive antigenic determinants present on both sperm and tumor cells.

Assessment of the mechanism of the leukocyte adherence inhibition test.

This study was designed to elucidate the mechanism of the leukocyte adherence inhibition (LAI) test in man. To identify the reactive cell types, enriched leukocyte populations (dextran-separated leukocytes and Hypaque-Ficoll-isolated mononuclear cells and neutrophils, as well as rosette-isolated B- and T-lymphocytes) were tested for leukocyte adherence in the absence of serum to tumor-specific antigens. LAI reactivity was not restricted to any of the enriched populations, suggesting the involvement of multiple cell types. Attempts to demonstrate soluble lymphocyte factors in the LAI mechanism have been uniformly negative. In contrast, factors in serum of immune donors were able to arm naive cells to be specifically responsive. This suggests a role for serum factors in the mechanism of LAI reactivity and partially explains the participation of multiple cell types in the responses observed. In additional studies, we could not document a correlation between the magnitude of the dermal test (delayed cutaneous hypersensitivity) and the magnitude of the LAI response in patients with squamous cell carcinoma of the head and neck. In 34 of 54 of these patients, there was agreement between the two tests (both positive, 27 of 54; both negative, 7 of 54). In the remaining 20 patients, the dermal test was greater than 5 mm while the LAI test was negative (less than 30% inhibition).

Association of melanoma tumor antigen activity with beta2-microglobulin.

The majority of melanoma tumor antigen activity present in melanoma extracts derived from fresh tumor tissue binds to a Sepharose-anti-beta2-microglobulin adsorbent. Removal of HLA antigens from the extracts of melanoma tissue by using a KBr flotation technique did not reduce either the tumor antigen activity of the extracts or the binding of melanoma tumor antigen (MTA) activity to the Sepharose-anti-beta2-microglobulin adsorbent. The complete blocking of MTA activity by pretreating the anti-beta2-microglobulin adsorbent with beta2-microglobulin and the lack of detectable MTA binding to a Sepharose anti-normal human serum adsorbent demonstrated the specificity of the binding of MTA to the anti-beta2-microglobulin adsorbent.

The role of 5'-methylthioadenosine phosphorylase in 5'-methylthioadenosine-mediated inhibition of lymphocyte transformation.

To determine if increased 5'-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5'-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5'-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5'-methylthioadenosine, 5'-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5'-Methylthiotubercidin acted as a potent inhibitor, but not a substrate, of the 5'-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5'-methylthioadenosine. However, in direct contrast to the 5'-methylthioadenosine effect, inhibition by 5'-methylthiotubercidin could not be completely reversed. These data suggest the 5'-methylthioadenosine phosphorylase plays an important role in reversing 5'-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5'-methylthiotubercidin are due to its resistance to 5'-methylthioadenosine phosphorylase degradation.

Human TCR as antigen: homologies and potentially cross-reactive HLA-DR2-restricted epitopes within the AV and BV CDR2 loops.

The major function of the T-cell receptor is to confer antigen specificity to T cells. However, nascent TCR proteins that are not assembled into functional heterodimers may be processed and displayed with self MHC molecules on the T-cell surface, and are thought to be the genesis of autoregulatory T cells that can limit inflammatory responses through T-T network interactions. In previous work, we and others have exploited this natural regulatory system using TCR peptides to amplify regulatory T cells that potentially can treat human autoimmune diseases such as multiple sclerosis (MS) and arthritis. The development of this approach is limited by the diversity of human TCR V gene sequences, and by lack of knowledge of exactly which regions of the V gene proteins are immunogenic in association with various MHC alleles. To identify similar amino acid sequences within and among human V gene families that might have immunologic cross reactivity, we aligned 74 known AV and 109 known BV protein sequences into homologous groups using the ClustalX program. Moreover, with a focus on CDR2 peptides that have previously been used to induce regulatory T cells in clinical trials, we established homologous peptide groups, and then identified the optimal amino acid motifs for binding to two alleles, HLA-DRB1*1501 and DRB5*0101, that have been associated with susceptibility to MS. From this analysis, > 75% of AV and BV CDR2 sequences were predicted to bind with at least moderate avidity to each of the DR2 alleles, thus enhancing the likelihood that they could be antigenic. Further ordering of putative TCR contact residues revealed a different set of homology groupings, including many intrafamily sequence matches and some interfamily matches that might allow immunological cross reactivity. Particularly striking were DRB1*1501-restricted IH-S and IY-S motifs shared by BV11, BV12, and BV13 and BV3, BV12, BV13, and BV17 family members, respectively, and DRB5*0101-restricted RL-H and RL-Y motifs shared by BV11, BV12, and BV13 and BV13 and BV17 family members, respectively. This analysis may be useful in designing an array of clinically useful homologous peptides with optimal MHC binding properties and highly cross-reactive TCR binding motifs.

Automated counting of T cell rosettes.

Human T cell rosettes were enumerated using an automated particle counter, the Bio/Physics Cytograf 6300A. An electronic oscilloscope representation of particle absorbance and scatter of a focused laser beam allows the separation and enumeration of both rosetted and non-rosetted lymphocytes. Repeated Cytograf sampling of a single rosette preparation gave highly reproducible results, and sampling from replicate tubes produced the same degree of variation as microscopic analysis. T cell rosettes prepared from 27 volunteers and compared by both methods of quantitation showed a high degree of correlation. This method can objectively measure at least 100 times as many cells for their rosette-forming capability as the tedious microscopic technique.

Detection of antigen specific rosette formation with the FACS.

Donors previously sensitized to conventional antigens PPD and KLH were evaluated for their antigen binding responses, utilizing a rosette forming technique with antigen-conjugated autologous erythrocytes. Reactivity is directly correlated with prior sensitization. Furthermore, antigen specificity is suggested by inhibition of rosette formation by prior incubation with the relevant antigen. The frequency of RFCs detected cytofluorometrically was compared with conventional fluorescent microscopy determinations. RFCs detected in this manner were identified as antibody armed monocytes by cell depletion and histochemical studies. The usefulness of the rosette forming technique for the routine evaluation of donor immunity is discussed.

Characterization of basic protein-specific T cell lines selected from Lewis rats with relapsing experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by intraperitoneal immunization with guinea pig whole central nervous system tissue. Basic protein (BP)-specific T cell lines selected from rats with relapsing EAE proliferated in response to BP, the 44-89 peptidase fragment of BP and the synthetic peptide, S72-89, as did lines selected from rats with non-relapsing EAE induced by immunization with guinea pig BP. BP-specific T cell lines selected from rats with relapsing EAE transferred acute but not relapsing EAE. BP-specific T cell lines selected from Lewis rats with relapsing EAE appear not to differ from those selected from rats with non-relapsing EAE.

Delayed type hypersensitivity to gangliosides in the Lewis rat.

Systematic study of the immunologic properties of gangliosides has been hampered by the lack of a suitable assay. In this study, significant delayed type hypersensitivity reactions to gangliosides were observed in Lewis rats immunized with whole guinea pig spinal cord (GP-SC) in complete Freund's adjuvant (CFA). The reaction was manifested by an increase in ear thickness after intradermal injection of a mixture of gangliosides and methylated bovine serum albumin (mBSA). No responses were observed to either gangliosides or mBSA alone. The reaction to gangliosides increased after immunization, persisted for 48 h, and was characterized by perivascular infiltration of mononuclear cells. Further evidence for a cellular response was demonstrated by the transfer of ganglioside-specific ear swelling by cultured spleen cells. The response to gangliosides was not due to contamination with myelin basic protein (BP) since no reaction to gangliosides was observed in GP-BP/CFA-immunized rats, and no reaction to BP was observed in ganglioside/CFA-immunized rats. In BP-immunized rats, responsiveness to BP persisted after recovery from clinical EAE for at least 60 days. However, no response to gangliosides was observed in BP-immunized animals after recovery from clinical EAE, suggesting the lack of autosensitization to gangliosides due to the disease process itself.

Lymphocyte vaccination against experimental autoimmune encephalomyelitis: evaluation of vaccination protocols.

Repeated vaccination with encephalitogenic but not other T cell lines could effect marked resistance to 'active' experimental autoimmune encephalomyelitis (EAE) induced by injection of GP-BP in adjuvant. Partial resistance to active EAE was observed in rats recovered from 'passive' line-mediated EAE and in rats vaccinated with T cells attenuated by irradiation or ganglioside treatment. However, no resistance was observed in animals given low doses of activated encephalitogenic T cells. Treatment with hydrostatic pressure alone was found to be ineffective as a means of attenuation, and vaccination with pressure-treated encephalitogenic T cells actually induced mild signs of EAE. However, vaccination with cells that were first pressure treated and then irradiated prevented both clinical and histologic signs of active EAE. In contrast, protection against passive EAE appeared to be clonotypic. Lymphocyte vaccination induced delayed type hypersensitivity (DTH) reactions against autologous T cells, mostly to shared antigens, demonstrating the immunogenicity of multiple antigens on the vaccinating cells.