Wettstein, Peter J.

Abstract Systemic lupus erythematosus (SLE) inflicts damage via autoantibody complexes that result in multiorgan damage and renal failure. The BXSB.Yaa mouse is a well-established model of SLE. We show that genetic disruption of multiple components of the MHC I axis greatly accelerates autoimmune disease in this model. Mice lacking both CD8 and IL15 succumb to SLE-like disease earliest (mean age 14 wks), implying a protective role for CD8 T cells and potentially natural killer cells dependent on IL15. BXSB.Yaa mice develop an expanded population of CD8 T cells with increased expression of both CD122 and IL15Rα, which we propose is due to increased expression of IL15R on these cells. Neither Foxp3 nor CD25 are elevated, and IL10 deficient BXSB.Yaa mice fail to show accelerated disease. However, lytic function appears significant as BXSB.Yaa mice deficient in perforin show decreased survival. TCR analysis failed to suggest a biased CD8 T cell repertoire, suggesting that the cellular targets of suppression are not antigenically discrete. Overall, the results suggest the existence of potent MHC I-dependent suppressor cell population(s) that normally protect BXSB.Yaa mice from an even more aggressive form of SLE-like disease. Moreover, the results support a model in which suppression is unlikely to be mediated by cytokine regulation but instead by lytic mechanisms. Harnessing this inherent regulatory cell population could provide a novel strategy to treat SLE and related syndromes.

Abstract In susceptible mouse strains, the wild-type Daniel’s (wt-DA) strain of Theiler’s murine encephalomyelitis virus induces a persistent central nervous system (CNS) infection with chronic demyelination. The virus is cleared from resistant mice with no resulting demyelination. We characterized the role of the DA L* protein in late demyelination and persistent infection. The DA genome has two alternative reading frames, encoding the virus polyprotein and L*, respectively. The mutant virus DAL*-1 fails to synthesize L* and does not persist in the CNS of wt-DA-susceptible SJL/J or B10.S mice. Since class I-restricted cytotoxicity has been shown to determine resistance to virus persistence and demyelination in this model, virus-specific cytotoxicity in the CNS of DA-resistant (B6 or B10) and -susceptible (SJL/J and B10.S) mice during the acute stage of DA and DAL*-1 infection was characterized. Following intracerebral inoculation with DAL*-1, virus-specific Db- and Kb-restricted CTLs were demonstrated in the CNS of resistant B10 mice, whereas only Db-restricted CTL were found in wt-DA-inoculated mice. CTLs specific to wt-DA or DAL*-1 recognized class I-presented peptides from either of the viruses. Of particular interest, Ks-restricted virus-specific cytotoxicity-restricted CTLs were identified in the CNS of susceptible SJL/J (H-2s) and B10.S (H-2s) mice inoculated with DAL*-1. In contrast, no virus-specific CTLs were identified in the CNS of SJL/J and B10.S mice inoculated with wt-DA. We propose that L* inhibits the generation of H-2K-restricted virus-specific cytotoxicity in the CNS, permitting a persistent infection in susceptible strains, with subsequent inflammatory demyelination in the CNS similar to that in human multiple sclerosis.

Abstract We addressed the contributions of persistent virus infection and immune cells to the pathogenesis of Theiler's virus-induced demyelination, a model for human multiple sclerosis. We developed a model involving the transfer of spleen cells into immunodeficient C.B-17-scid (SCID) mice, which normally die of overwhelming virus encephalitis without demyelination when infected with Theiler's virus. Adoptive transfer of nonimmune spleen cells from BALB/c mice into SCID mice resulted in the survival of all mice. However, these mice developed extensive demyelination and virus Ag/RNA persistence in the spinal cord white matter. The most demyelination was observed when mice received an intermediate number of spleen cells (1.8-7.5 x 10(6)), whereas too few cells (0.5 x 10(6)) did not ameliorate the SCID phenotype, and too many cells (30 x 10(6)) resulted in almost complete viral clearance with minimal demyelination. Adoptive transfer of spleen cells depleted of either CD4+ or CD8+ T cells produced vacuolar demyelination associated with virus persistence. In contrast, reconstitution with both CD4+ and CD8+ T cells produced less severe demyelination and partial clearance of virus. These experiments support the hypothesis that demyelination is the result of a balance between persistent virus infection and immune injury mediated by either CD4+ or CD8+ T cells.

During normal T cell development in mouse and human, a low-frequency population of immature CD4 − CD8 − double-negative (DN) thymocytes expresses early, mature αβ T cell antigen receptor (TCR). We report that these early αβ TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αβ T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Abstract Introduction: Immunotherapy is most likely to work in a setting of low tumor burden, making vaccine strategies attractive as maintenance therapy post autologous peripheral blood stem cell transplantation (PBSCT) for multiple myeloma (MM). However, it is difficult to ascertain the effects of the vaccines from delayed responses to the transplant. A novel immunotherapeutic, APC8020 (Mylovenge), was studied as consolidation therapy after for MM post PBSCT (Mayo vaccine trial). Mylovenge is prepared from autologous antigen presenting cells, including dendritic cells, partially purified from an unmobilized leukapheresis product by gradient density isolation and then incubated for two days with autologous serum containing M protein obtained pretransplant. We report long term results of the Mayo vaccine trial compared retrospectively to a consecutive cohort of MM patients who underwent autologous stem cell transplant at Mayo Clinic during the same time period. Methods: The Mayo vaccine trial patients had transplants between July of 1998 and May of 2001. Using these cutoff dates we analyzed 151 total MM patients, 27 from the vaccine trial (9 newly diagnosed and 18 relapsed) and 124 from database (DB) (50 newly diagnosed patients and 74 relapsed). The median (range) of follow-up for alive patients in vaccine trial is 6.5 years (2.9 – 8), and in the DB is 7.1 years (6 – 8). Two-sided Fisher’s exact tests and stratified log rank tests (with newly diagnosed or relapsed as the stratum) were used to compare baseline patient characteristics and time to event distributions (overall survival – OS, progression free survival – PFS, and time to progression – TTP) between the two groups. Results: The median (range) of age in vaccine trial and DB was 56 (30–69) and 57 (36–71) years respectively. There were no significant differences in the known prognostic factors including PCLI, B2M, and CRP. The median (95% confidence interval - CI) TTP for the vaccine trial and DB patients was 1.5 years (1.3 – 2.4) and 1.6 years (1.3 – 1.8); stratified log rank p value = 0.46. The median (95% CI) PFS for the vaccine trial and DB patients was 1.5 years (1.3 – 2.4) and 1.5 years (1.1 – 1.8), stratified logrank p value = 0.30. The median (95% CI) OS for the vaccine trial and DB patients was 5.3 years (4.0 -N/A), and 3.4 years (2.7 – 4.6); stratified logrank p value = 0.02. Conclusions: Despite the fact that no difference was seen in TTP or PFS, post-transplant vaccine therapy was associated with prolonged survival. This is similar to what has been reported in other tumor systems including prostate cancer and glioblastoma multiforme. This approach warrants further investigation, including preclinical studies aimed at optimizing the immune response and randomized trials to define the role of vaccine therapy in myeloma. Figure Figure

Mice expressing the H2b haplotype are resistant to infection with Theiler's murine encephalomyelitis virus (TMEV), which causes chronic demyelination in susceptible mice. The prominent cytolytic T-lymphocyte (CTL) response to the VP2 antigen encoded by TMEV led us to the identification of a class I-binding peptide derived from the VP2 antigen. Escherichia coli transformants overexpressing a series of 11 overlapping VP2 protein fragments were subjected to lysis and alkali digestion, and the resultant peptide pools were tested for their abilities to sensitize RMA-S targets for lysis by CTLs. The source of effector CD8+ T cells for the assays was either freshly harvested central nervous system-infiltrating lymphocytes (CNS-IL) or CNS-IL-derived VP2-specific CTL clones and lines. A 10-residue peptide at VP2 positions 121 to 130 (VP2(121-130)) (FHAGSLLVFM) was identified that sensitized targets for lysis and formed stable complexes with H2Db class I molecules. The VP2(121-130) peptide sensitized target cells for lysis by freshly harvested CNS-IL CTLs at femtomolar concentrations. Despite its relative high level of biological activity, the VP2(121-130) peptide is distinguished from other Db-binding peptides by its lack of an asparagine residue at position five, which had been previously proposed to be a requirement for Db-peptide complexing.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.

ABSTRACTA newly identified intraerythrocyticBabesia-like organism, WA1, and its relatives were recently shown to be infectious for humans in the western United States. The purpose of the present study was to determine the susceptibilities of selected mouse genotypes to WA1 infection in an attempt to develop a murine model of the human disease. Several mouse strains were inoculated intraperitoneally with various passages of WA1-parasitized erythrocytes. Parasitemia was evaluated by blood smears and by PCR with blood samples collected at various intervals after inoculation. Hematologic parameters were monitored in blood samples at all intervals. C57BL/6 and C57BL/10 mice exhibited mortality rates of <10%. BALB/cJ, CBAJ, and 129/J mice had higher peak parasitemias than did C57BL mice, with mortality rates of 40, 50, and 50%, respectively. A/J, AKR/N, C3H, and DBA/1J mice also had higher peak parasitemia and mortality rates (>95%). An F1cross of C57BL/6 (resistant) and C3H.RKK (susceptible) mice had a mortality rate similar to that of the resistant parental strain. Histopathology of BALB/cJ and C3H mice at 9 and 14 days after inoculation revealed erythrophagocytosis and deposition of an iron-negative pigment in multiple organs. Morbidly ill C3H mice at 14 days had severe pulmonary edema, hemoglobinuria, and glomerulonephritis.

T cell lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from transformed thymocytes. Most human T-ALL involves hyperactive NOTCH signaling that is often caused by activating NOTCH mutations. However, the identification of specific molecular signals that might induce or select for mutation and transformation are incompletely understood. We report that an understudied low-frequency, natural thymocyte subset expresses αβ T cell antigen receptor (TCR) earlier than most cells in mice and humans; engagement of the early αβTCR by major histocompatibility complexes (MHC) can cause outgrowth of NOTCH1 mutant clones and T-ALL leukemogenesis in a mouse model of T-ALL. Assessment of 5 recent human T-ALL cases found one to present this unique CD4-CD8- double- negative (DN) stage as the earliest identifiable developmental stage. These studies present a model of T-ALL leukemogenesis that identifies (i) a natural cell stage of origin susceptible to transformation, (ii) a matching mouse model showing that a signaling receptor (αβTCR) and its ligand (MHC) drive leukemogeneis and outgrowth of tumors bearing activating NOTCH1 mutations, and (iii) a human case that presents with a tumor consistent with this model and mechanism. In past work, the pre-TCR has been shown to impact T-ALL development in mice (Campese et al, Blood 2006), but an oncogenic role for the mature αβTCR is less well characterized and somewhat surprising. This is because, although T-ALL tumor cells may express variable levels of surface αβTCR/CD3, the earliest cell stages that are thought to transform are also thought to precede stages with αβTCR expression. Most conventional αβ thymocytes rearrange TCRβ and TCRα loci in separate, ordered developmental stages. However, some thymocytes in the conventional pathway rearrange both at DN stage thus exhibiting 'precocious' αβTCR (PAT) expression. Importantly, these PAT cells are indeed part of the conventional αβ lineage, being a 'subset' only due to early αβTCR expression but without known distinction in ultimate immune function (Aifantis et al, JEM 2006). We found that ~0.01% of mouse and human thymocytes are such PAT cells at steady state. To interrogate the PAT thymocyte surface phenotype, we performed multi-parametric flow cytometry with Spanning-tree Progression Analysis of Density-normalized Events (SPADE). This revealed that PAT thymocytes constitute a DN subset that is not associated with other well-described unconventional DN thymocytes known to express αβTCR, consistent with as the expectation that PATs are part of the conventional developmental pathway. We observed that the OT1 TCR transgene is expressed in mice with parallel timing and level to the natural PAT subset, allowing use of this model to study antigen-dependent signaling and oncogenesis. In a cohort study, no T-ALL was observed in wild-type C57BL/6 or OT-1.β2M-/- mice (deficient in endogenous antigen presentation), but MHC-sufficient OT-1 mice developed PAT-stage-specific T-ALL with activating NOTCH1 mutations. Transplant experiments corroborated a requirement for antigen presentation and TCR signaling for tumor maintenance as transplanted tumors grew in MHC+ but not MHC-deficient mice. This predicted that PAT thymocytes might have an unusual ability to signal through αβTCR even without coreceptor expression. When cultured in the presence of either exogenously added β2M or antigen presenting cells, both untransformed and neoplastic PAT cells upregulated CD69 in response to the OT-1 antigenic peptide, OVA. Furthermore, ex vivo analysis of PAT cells from polyclonal C57BL/6 versus MHC-deficient mice showed intrinsic upregulation of TCR-signaling-dependent Nur77 in an MHC-dependent manner. These data revealed a unique ability of PAT cells to engage in co-receptor independent but antigen-dependent signaling. Microarray analysis showed that the gene expression profile of neoplastic PAT cells from OT-1 T-ALL most closely resembled that of conventional post β-selection DN thymocytes, in agreement with the natural PAT stage during normal T cell development. These data support a model in which transformation occurred in the naturally occurring αβ PAT thymocyte subset as cell-of-origin. Collectively, our data suggest that precocious αβTCR expression and coreceptor-independent antigen engagement can cause activating NOTCH mutation and T-ALL development. Disclosures No relevant conflicts of interest to declare.

Quantitative activity through the TCR-proximal protein network specifies thymic positive versus negative selection.