Standardized monitoring of cytomegalovirus-specific immunity can improve risk stratification of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation.

Recurrence of cytomegalovirus reactivation remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Monitoring cytomegalovirus-specific cellular immunity using a standardized assay might improve the risk stratification of patients. A prospective multicenter study was conducted in 175 intermediate- and high-risk allogeneic hematopoietic stem cell transplant recipients under preemptive antiviral therapy. Cytomegalovirus-specific cellular immunity was measured using a standardized IFN-γ ELISpot assay (T-Track® CMV). Primary aim was to evaluate the suitability of measuring cytomegalovirus-specific immunity after end of treatment for a first cytomegalovirus reactivation to predict recurrent reactivation. 40/101 (39.6%) patients with a first cytomegalovirus reactivation experienced recurrent reactivations, mainly in the high-risk group (cytomegalovirus-seronegative donor/cytomegalovirus-seropositive recipient). The positive predictive value of T-Track® CMV (patients with a negative test after the first reactivation experienced at least one recurrent reactivation) was 84.2% in high-risk patients. Kaplan-Meier analysis revealed a higher probability of recurrent cytomegalovirus reactivation in high-risk patients with a negative test after the first reactivation (hazard ratio 2.73; p=0.007). Interestingly, a post-hoc analysis considering T-Track® CMV measurements at day 100 post-transplantation, a time point highly relevant for outpatient care, showed a positive predictive value of 90.0% in high-risk patients. Our results indicate that standardized cytomegalovirus-specific cellular immunity monitoring may allow improved risk stratification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation. This study was registered at www.clinicaltrials.gov as #NCT02156479.

TNFRSF receptor-specific antibody fusion proteins with targeting controlled FcγR-independent agonistic activity.

Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.

Protection of Mice from Acute Graft-versus-Host Disease Requires CD28 Co-stimulation on Donor CD4+ Foxp3+ Regulatory T Cells.

Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell plus T cell transplantation (allo-HSCT). In this study, we investigated the requirement for CD28 co-stimulation of donor CD4+ conventional (CD4+CD25-Foxp3-, Tconv) and regulatory (CD4+CD25+Foxp3+, Treg) T cells in aGvHD using tamoxifen-inducible CD28 knockout (iCD28KO) or wild-type (wt) littermates as donors of CD4+ Tconv and Treg. In the highly inflammatory C57BL/6 into BALB/c allo-HSCT transplantation model, CD28 depletion on donor CD4+ Tconv reduced clinical signs of aGvHD, but did not significantly prolong survival of the recipient mice. Selective depletion of CD28 on donor Treg did not abrogate protection of recipient mice from aGvHD until about day 20 after allo-HSCT. Later, however, the pool of CD28-depleted Treg drastically declined as compared to wt Treg. Consequently, only wt, but not CD28-deficient, Treg were able to continuously suppress aGvHD and induce long-term survival of the recipient mice. To our knowledge, this is the first study that specifically evaluates the impact of CD28 expression on donor Treg in aGvHD. Moreover, the delayed kinetics of aGvHD lethality after transplantation of iCD28KO Treg provides a novel animal model for similar disease courses found in patients after allo-HSCT.

Targeting of the WT191-138 fragment to human dendritic cells improves leukemia-specific T-cell responses providing an alternative approach to WT1-based vaccination.

Due to its immunogenicity and overexpression concomitant with leukemia progression, Wilms tumor protein 1 (WT1) is of particular interest for immunotherapy of AML relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). So far, WT1-specific T-cell responses have mainly been induced by vaccination with peptides presented by certain HLA alleles. However, this approach is still not widely applicable in clinical practice due to common limitations of HLA restriction. Dendritic cell (DC) vaccines electroporated with mRNA encoding full-length protein have also been tested for generating WT1-derived peptides for presentation to T-cells. Alternatively, an efficient and broad WT1 peptide presentation could be elicited by triggering receptor-mediated protein endocytosis of DCs. Therefore, we developed antibody fusion proteins consisting of an antibody specific for the DEC205 endocytic receptor on human DCs and various fragments of WT1 as DC-targeting recombinant WT1 vaccines (anti-hDEC205-WT1). Of all anti-hDEC205-WT1 fusion proteins designed for overcoming insufficient expression, anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were identified in good yields. The anti-hDEC205-WT191-138 was capable of directly inducing ex vivo T-cell responses by co-incubation of the fusion protein-loaded monocyte-derived mature DCs and autologous T-cells of either healthy or HSCT individuals. Furthermore, the DC-targeted WT191-138-induced specific T-cells showed a strong cytotoxic activity by lysing WT1-overexpressing THP-1 leukemia cells in vitro while sparing WT1-negative hematopoietic cells. In conclusion, our approach identifies four WT1 peptide-antibody fusion proteins with sufficient production and introduces an alternative vaccine that could be easily translated into clinical practice to improve WT1-directed antileukemia immune responses after allo-HSCT.

Hsp90 inhibition ameliorates CD4+ T cell-mediated acute Graft versus Host disease in mice.

INTRODUCTION: For many patients with leukemia only allogeneic bone marrow transplantion provides a chance of cure. Co-transplanted mature donor T cells mediate the desired Graft versus Tumor (GvT) effect required to destroy residual leukemic cells. The donor T cells very often, however, also attack healthy tissue of the patient inducing acute Graft versus Host Disease (aGvHD)-a potentially life-threatening complication. METHODS: Therefore, we used the well established C57BL/6 into BALB/c mouse aGvHD model to evaluate whether pharmacological inhibition of heat shock protein 90 (Hsp90) would protect the mice from aGvHD. RESULTS: Treatment of the BALB/c recipient mice from day 0 to +2 after allogeneic CD4+ T cell transplantation with the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) partially protected the mice from aGvHD. DMAG treatment was, however, insufficient to prolong overall survival of leukemia-bearing mice after transplantation of allogeneic CD4+ and CD8+ T cells. Ex vivo analyses and in vitro experiments revealed that DMAG primarily inhibits conventional CD4+ T cells with a relative resistance of CD4+ regulatory and CD8+ T cells toward Hsp90 inhibition. CONCLUSIONS: Our data, thus, suggest that Hsp90 inhibition might constitute a novel approach to reduce aGvHD in patients without abrogating the desired GvT effect.

In vivo activation of Treg cells with a CD28 superagonist prevents and ameliorates chronic destructive arthritis in mice.

Although regulatory T (Treg) cells are necessary to prevent autoimmune diseases, including arthritis, whether Treg cells can ameliorate established inflammatory disease is controversial. Using the glucose-6-phosphate isomerase (G6PI)-induced arthritis model in mice, we aimed to determine the therapeutic efficacy of increasing Treg cell number and function during chronic destructive arthritis. Chronic destructive arthritis was induced by transient depletion of Treg cells prior to immunization with G6PI. At different time points after disease induction, mice were treated with a CD28 superagonistic antibody (CD28SA). CD28SA treatment during the induction phase of arthritis ameliorated the acute signs of arthritis and completely prevented the development of chronic destructive arthritis. CD28SA treatment of mice with fully developed arthritis induced a significant reduction in clinical and histological signs of arthritis. When given during the chronic destructive phase of arthritis, 56 days after disease induction, CD28SA treatment resulted in a modest reduction of clinical signs of arthritis and a reduction in histopathological signs of joint inflammation. Our data show that increasing the number and activation of Treg cells by a CD28SA is therapeutically effective in experimental arthritis.

High-density preculture of PBMCs restores defective sensitivity of circulating CD8 T cells to virus- and tumor-derived antigens.

Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T cells transiently lose antigen sensitivity when cellular contacts are lost (eg, by entering the circulation). Using the simple and robust protocol for resetting T cells to original reactivity (RESTORE; ie, preculturing PBMCs for 2 days at a high cell density before initiation of antigenic stimulation), we show that CD8 T-cell responses to viral and tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected, if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 protein-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of "restored" CD8 T cells is associated with phosphorylation of proximal T-cell receptor signaling components, and with the upregulation of genes involved in aerobic glycolysis, thereby increasing T-cell functionality. The RESTORE protocol permits a more meaningful monitoring of CD8 memory T-cell responses to viral infections and tumors and vaccination success. Furthermore, when generating T-cell lines for adoptive T-cell therapy, it avoids the loss of those clones, which strictly depend on the primed status conferred by cellular interactions in the tissue context for their initial reactivation by antigen. The data reported in this article have been deposited in the Gene Expression Omnibus database (accession number GSE63430).

The T cell-selective IL-2 mutant AIC284 mediates protection in a rat model of Multiple Sclerosis.

Targeting regulatory T cells (Treg cells) with interleukin-2 (IL-2) constitutes a novel therapeutic approach for autoimmunity. As anti-cancer therapy with IL-2 has revealed substantial toxicities a mutated human IL-2 molecule, termed AIC284 (formerly BAY 50-4798), has been developed to reduce these side effects. To assess whether AIC284 is efficacious in autoimmunity, we studied its therapeutic potential in an animal model for Multiple Sclerosis. Treatment of Lewis rats with AIC284 increased Treg cell numbers and protected the rats from Experimental Autoimmune Encephalomyelitis (EAE). AIC284 might, thus, also efficiently prevent progression of autoimmune diseases in humans.

In vitro polyclonal activation of conventional T cells with a CD28 superagonist protects mice from acute graft versus host disease.

Upon transplantation of T cells from a CD28 superagonist (CD28-SA) treated donor into an irradiated allogeneic host, the CD28-SA-induced activation and expansion of Treg cells inhibits acute graft versus host disease (aGvHD), while not abrogating the desired graft versus tumor effect. Human peripheral blood CD4(+) T cells, however, harbor only very few Treg cells. Therefore, we studied whether polyclonal in vitro prestimulation of conventional, that is Treg -cell-depleted, CD4(+) T cells of C57BL/6 mice with CD28-SA-coated paramagnetic beads is sufficient to protect recipient BALB/c mice from aGvHD. CD28-SA prestimulation of conventional CD4(+) T cells efficiently protected BALB/c recipient mice from aGvHD and CD28-SA-stimulated CD4(+) and CD8(+) T cells were capable of mediating long-term protection from the BCL1 lymphoma. The recently completed successful phase I testing of the human CD28-SA TGN1412/TAB08 should greatly facilitate further development of this straightforward method into a novel immunotherapy for patients.

Oligodendrocytes enforce immune tolerance of the uninfected brain by purging the peripheral repertoire of autoreactive CD8+ T cells.

Myelin-specific CD8(+) T cells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) T cells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) T cells failed to induce disease and were cleared from the immune system after antigen encounter in the central nervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) T cells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) T cells to cause demyelinating disease. Thus, in response to infection, CD8(+) T cells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

Cyclic AMP underpins suppression by regulatory T cells.

Elevated levels of intracellular cyclic adenosine monophosphate (cAMP) in naturally occurring T regulatory (nTreg) cells play a key role in nTreg-cell-mediated suppression. Upon contact with nTreg cells, cAMP is transferred from nTreg cells into activated target CD4(+) T cells and/or antigen-presenting cells (APCs) via gap junctions to suppress CD4(+) T-cell function. cAMP facilitates the expression and nuclear function of a potent transcriptional inhibitor, inducible cAMP early repressor (ICER), resulting in ICER-mediated suppression of interleukin-2 (IL-2). Furthermore, ICER inhibits transcription of nuclear factor of activated T cell c1/α (NFATc1/α) and forms inhibitory complexes with preexisting NFATc1/c2, thereby inhibiting NFAT-driven transcription, including that of IL-2. In addition to its suppressive effects mediated via ICER, cAMP can also modulate the levels of surface-expressed cytotoxic T lymphocyte antigen-4 (CTLA-4) and its cognate B7 ligands on conventional CD4(+) T cells and/or APCs, fine-tuning suppression. These cAMP-driven nTreg-cell suppression mechanisms are the focus of this review.

Preculture of PBMCs at high cell density increases sensitivity of T-cell responses, revealing cytokine release by CD28 superagonist TGN1412.

Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-γ, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs.

A superagonistic monoclonal antibody for CD28 ameliorates crescentic glomerulonephritis in wistar-kyoto rats.

Regulatory T (Treg) cells play an important role in the resolution of crescentic glomerulonephritis, where a T helper 1 (Th1)-predominant immune response promotes crescent formation. Therefore, agents that increase Treg cells appear to be ideal for suppressing T-cell-mediated renal pathology. We hypothesized that a superagonistic monoclonal antibody for CD28 (JJ316), which has been known to preferentially expand Treg cells in vivo, could prevent nephrotoxic serum-induced nephritis in Wistar-Kyoto rats, one of the experimental models of crescentic glomerulonephritis. Administration of JJ316 attenuated crescent formation, proteinuria and glomerular accumulation of macrophages and CD8(+) T cells. These changes were accompanied by increased infiltration of Treg cells. Among glomerular macrophages, the CD163(+) subset was significantly increased after treatment, suggesting that Treg cells may modulate the phenotype of macrophages leading to resolution of glomerulonephritis. In an adoptive transfer experiment, two T-cell subsets (CD4(+)CD25(+) and CD4(+)CD25(-) T cells) purified from spleens and lymph nodes of donor rats primed with JJ316 3 d before were inoculated into nephritic recipient rats, which recapitulated the beneficial effects of in vivo administration of JJ316. Furthermore, a single injection of JJ316 administered 3 d after disease induction completely protected nephritic rats from death for 2 months. In conclusion, we demonstrated that treatment with JJ316 has a dramatic therapeutic effect on an experimental crescentic glomerulonephritis, possibly due to expansion and activation of Treg cells.

Anti-protease and immunomodulatory activities of bacteria associated with Caribbean sponges.

Marine sponges and their associated bacteria have been proven to be a rich source of novel secondary metabolites with therapeutic usefulness in cancer, infection, and autoimmunity. In this study, 79 strains belonging to 20 genera of the order Actinomycetales and seven strains belonging to two genera of the order Sphingomonadales were cultivated from 18 different Caribbean sponges and identified by 16S rRNA gene sequencing. Seven of these strains are likely to represent novel species. Crude extracts from selected strains were found to exhibit protease inhibition against cathepsins B and L, rhodesain, and falcipain-2 as well as immunomodulatory activities such as induction of cytokine release by human peripheral blood mononuclear cells. These results highlight the significance of marine sponge-associated bacteria to produce bioactive secondary metabolites with therapeutic potential in the treatment of infectious diseases and disorders of the immune system.

Regulatory T cells facilitate the nuclear accumulation of inducible cAMP early repressor (ICER) and suppress nuclear factor of activated T cell c1 (NFATc1).

Inducible cAMP early repressor (ICER) is a transcriptional repressor, which, because of alternate promoter use, is generated from the 3' region of the cAMP response modulator (Crem) gene. Its expression and nuclear occurrence are elevated by high cAMP levels in naturally occurring regulatory T cells (nTregs). Using two mouse models, we demonstrate that nTregs control the cellular localization of ICER/CREM, and thereby inhibit IL-2 synthesis in conventional CD4(+) T cells. Ablation of nTregs in depletion of regulatory T-cell (DEREG) mice resulted in cytosolic localization of ICER/CREM and increased IL-2 synthesis upon stimulation. Direct contacts between nTregs and conventional CD4(+) T cells led to nuclear accumulation of ICER/CREM and suppression of IL-2 synthesis on administration of CD28 superagonistic (CD28SA) Ab. In a similar way, nTregs communicated with B cells and induced the cAMP-driven nuclear localization of ICER/CREM. High levels of ICER suppressed the induction of nuclear factor of activated T cell c1 (Nfatc1) gene in T cells whose inducible Nfatc1 P1 promoter bears two highly conserved cAMP-responsive elements to which ICER/CREM can bind. These findings suggest that nTregs suppress T-cell responses by the cAMP-dependent nuclear accumulation of ICER/CREM and inhibition of NFATc1 and IL-2 induction.

Prevention of graft-versus-host diseases by in vivo supCD28mAb-expanded antigen-specific nTreg cells.

Naturally occurring CD4(+)CD25(+) Treg cells (nTregs) can be exploited to establish an immunologic tolerance to non-self-antigens. The in vivo administration of a single superagonistic CD28-specific monoclonal antibody (supCD28mAb) to naive rat preferentially expanded the nTregs, which induced a potent inhibition of lethality of the graft-versus-host (GvH) diseases. The appearance of increased Foxp3 molecules was accompanied with a polarization towards a Th2 cytokine profile with a decreased production of IFN-γ and increased production of IL-4 and IL-10 in the serum of the antibody-treated rat. The peripheral Foxp3 nTregs are decreased in acute GvHD, while supCD28mAb administration showed that nTregs were preferentially proliferating in vivo, thus resulting in the significant prevention of the GvH disease. Furthermore, antigen-specific nTregs could suppress conventional T-cell proliferation stimulated with alloantigen in vitro. Taken together, our findings demonstrate that the potent regulatory functions of the Tregs for the treatment of GvHD are antigen specific. These data also provide evidence that GvHD is associated with decrease of Tregs in the periphery of the host. The determination of the Foxp3 Tregs can be a helpful tool to discriminate GvHD severity and lethality after allogeneic stem cell transplantation.

Regulatory T cells protect from local and systemic bone destruction in arthritis.

We previously demonstrated the suppressive effects of regulatory T cells (Treg cells) on osteoclast differentiation in vitro. In this article, we show that blood markers of bone resorption inversely correlate with the amount of circulating Treg cells in healthy controls and rheumatoid arthritis patients, further suggesting that Treg cells may control bone destruction in vivo. Indeed, bone marrow from Foxp3-transgenic (Foxp3tg) mice fully protected human TNF transgenic (hTNFtg) mice from TNF-alpha-induced bone destruction, whereas Foxp3-deficient bone marrow enhanced local and systemic bone loss. The same protective effect was also obtained by treating hTNFtg mice with the CD28 superagonist mAb (CD28 SA), which increased Treg cell numbers. In both models, bone protection by Treg cells was associated with reduced osteoclast numbers, resulting in less bone-resorbing activity. Reduced osteoclast numbers were not caused by an intrinsic defect in osteoclast differentiation because osteoclast precursors from hTNFtg/Foxp3tg chimeras responded normally to M-CSF and receptor activator of NF-kappaB ligand. Although a decrease in the clinical signs of arthritis was observed in Foxp3tg bone marrow-transferred and CD28 SA-treated hTNFtg mice, the bone-protective effect of Treg cells was independent of the suppression of inflammation, as demonstrated by the increased systemic bone density observed in wild-type mice treated with CD28 SA. This work demonstrated that increasing Treg cell numbers improved clinical signs of arthritis and suppressed local and systemic bone destruction. Thus, enhancing the activity of Treg cells would be beneficial for the treatment of inflammation-induced bone loss observed in rheumatoid arthritis.

CD28 and IL-4: two heavyweights controlling the balance between immunity and inflammation.

The costimulatory receptor CD28 and IL-4Ralpha containing cytokine receptors play key roles in controlling the size and quality of pathogen-specific immune responses. Thus, CD28-mediated costimulation is needed for effective primary T-cell expansion and for the generation and activation of regulatory T-cells (Treg cells), which protect from immunopathology. Similarly, IL-4Ralpha signals are required for alternative activation of macrophages, which counteract inflammation by type 1 responses. Furthermore,immune modulation by CD28 and IL-4 is interconnected through the promotion of IL-4 producing T-helper 2 cells by CD28 signals. Using conditionally IL-4Ralpha and CD28 deleting mice, as well as monoclonal antibodies, which block or stimulate CD28, or mAb that deplete Treg cells, we have studied the roles of CD28 and IL-4Ralpha in experimental mouse models of virus (influenza), intracellular bacteria (L. monocytogenes, M. tuberculosis), and parasite infections (T. congolense, L. major). We observed that in some, but not all settings, Treg cells and type 2 immune deviation, including activation of alternative macrophages can be manipulated to protect the host either from infection or from immunopathology with an overall beneficial outcome. Furthermore, we provide direct evidence that secondary CD8 T-cell responses to i.c. bacteria are dependent on CD28-mediated costimulation.

Modulating T cell functions does not alleviate chronic inflammatory skin lesions in K5.TGF beta 1 transgenic mice.

To use mice with chronic hyperproliferative skin inflammation as psoriasis models, their thorough phenotypic and functional characterization is indispensable. Mice with keratin 5 promoter-controlled overexpression of latent human Transforming Growth Factor (TGF)beta1 within the basal epidermis (K5.TGF beta 1 mice) show a psoriasiform phenotype, but the underlying pathogenic mechanisms are not entirely clear. To elucidate the contribution of T lymphocytes to the pathogenesis in K5.TGF beta 1 mice, we used three complementary approaches: first, peripheral T cells were eradicated via systemic treatment with CD3- or CD4-depleting antibodies. However, this elimination did not alleviate the chronic inflammatory disorder. Second, bone marrow transplantation from transgenic mice into wildtype recipients and vice versa resulted in the expected reconstitution of both adaptive and innate immune system but had little effect on the cutaneous phenotype both in wildtype and transgenic chimeras. Third, based on the hypothesis that the disease course could be modulated by regulatory T cells (Tregs), we expanded Tregs in vivo using a superagonistic anti-CD28 antibody. While this treatment achieved a threefold increase in Foxp3-expressing Tregs, there was little, if any, effect on the chronic skin inflammation. We conclude from our findings that T cells play little, if any, role in the skin lesions of K5.TGF beta 1 mice.

Collateral neuronal apoptosis in CNS gray matter during an oligodendrocyte-directed CD8(+) T cell attack.

Demyelination and death of oligodendrocytes accompanied by transection of neurites and neuronal apoptosis are pathological hallmarks of cortical and subcortical gray matter lesions in demyelinating viral and autoimmune inflammatory CNS disorders. In these disorders, leukocortical lesions, containing the perikarya of most efferent neurons, display pronounced infiltration by CD8(+) T cells of putative specificity for oligodendrocyte- and myelin-related antigens. Hence, neuronal apoptosis in gray matter lesions may be a collateral effect of an oligodendrocyte-directed attack by CD8(+) T cells. To challenge this hypothesis, we transferred activated antigen-specific CD8(+) T cells (OT-I T cells) into acute coronal brain slices from mice selectively expressing ovalbumin as a cytosolic neo-self-antigen in oligodendrocytes (ODC-OVA mice). We studied mechanisms and kinetics of oligodendroglial and neuronal apoptosis in the neocortex and hippocampus, using multicolor staining for different cell types and activated caspase-3. Within the gray matter, a single OT-I T cell caused simultaneous caspase-3 activation in about 30 ODCs and 10 neurons within 6 h in a strictly antigen-dependent manner. Experiments with OT-I T cells genetically deficient for perforin or the granzyme B-cluster and with blocking anti-FasL antibodies as well as proinflammatory cytokines revealed, that collateral apoptosis of neurons was likely due to a spillover of perforin and granzyme(s) from the OT-I T cell itself or the immunological synapse that it selectively formed with antigen-presenting oligodendrocytes. Collateral neuronal apoptosis could contribute to substantial neuronal loss in gray matter lesions and cause persistent neurological impairment in both acute and chronic gray matter lesions in various inflammatory CNS disorders.