Absence Of CD28 Costimulation Research Articles

Cytomegalovirus Infection Facilitates the Costimulation of CD57+CD28- CD8 T Cells in HIV Infection and Atherosclerosis via the CD2-LFA-3 Axis.

CD8 T cells are emerging as important mediators in atherosclerosis and cardiovascular disease (CVD). Immune activation may play a particular role in people with HIV (PWH) who are at an increased risk of CVD, even after controlling for known CVD risk factors. Latent CMV infection is associated with increased CVD risk for both PWH and people without HIV, and human CMV-specific CD4 and CD8 T cells are enriched for an immunosenescent phenotype. We previously showed that CMV coinfection in PWH promotes vascular homing and activation of inflammatory CD4 T cells through the CD2-LFA-3 axis. However, the role of CD2/LFA3 costimulation of CD8 T cells in PWH with CMV has yet to be described. In the present study, we demonstrate that CD2 expression on CX3CR1+CD57+CD28- inflammescent CD8 T cells is increased on cells from CMV-seropositive PWH. In vitro CD2/LFA-3 costimulation enhances TCR-mediated activation of these inflammatory CD8 memory T cells. Finally, we show that LFA-3 is highly expressed in aortas of SIV-infected rhesus macaques and in atherosclerotic plaques of people without HIV. Our findings are consistent with a model in which CMV infection enhances CD2 expression on highly proinflammatory CD8 T cells that can then be stimulated by LFA-3 expressed in the vasculature, even in the absence of CD28 costimulation. This model, in which CMV infection exacerbates toxic cytokine and granzyme production by CD8 T cells within the vasculature, highlights a potential therapeutic target in atherosclerosis development and progression, especially for PWH.

Abstract 573: Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor models

Abstract Adoptive cell transfer (ACT) involving autologous tumor-specific lymphocytes (TIL) has shown promising results in the treatment of advanced cancer. Poor in vitro cell expansion, inefficient T-cell migration and T cell exhaustion in the tumor microenvironment greatly limits the broader application of this approach. The E3 ubiquitin ligase Casitas B-lineage lymphoma b (CBL-B) is highly expressed in T cells, where it limits cell activation following TCR engagement, therefore functioning as an important intracellular checkpoint that limits T cell mediated anti-tumor responses. We have developed NX-0255 a highly potent small molecule inhibitor of CBL-B that increases TCR-mediated activation of T cells in the presence or absence of CD28 co-stimulation. Here we used the OT-1 ACT/E.G7-OVA lymphoma and the Pmel-1 ACT/B16 melanoma tumor models to investigate whether adding NX-0255, during the in vitro expansion of tumor-specific T cells would have favorable effects on their ability to reject tumors following adoptive transfer in vivo. OT-1 CD8+ T cells were expanded in vitro using anti-CD3 stimulation and either NX-0255 alone, IL-2 alone, or NX-0255 combined with IL-2 and subsequently transferred in vivo to mice bearing established E.G7-OVA. ACT of OT-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 demonstrated superior anti-tumor activity against E.G7-OVA lymphoma tumors, resulting in significant improvement in survival when compared to mice receiving OT-1 CD8+ T cells cultured with NX-0255 alone or IL-2 alone. Importantly, OT-1 CD8+ T cells cultured in the presence of NX-0255 or the combination of NX-0255 plus IL-2 were found at an increased frequency in the blood and in the tumor and persisted longer in circulation when compared to cells cultured with IL-2 alone. A lower percentage of the tumor-infiltrating OT-1 cells were triple-positive for exhaustion markers PD1+TIM3+LAG3+ expression when cultured with NX-0255 alone or the combination of NX-0255 and IL-2 compared to culture with IL-2 alone. Similarly, ACT of Pmel-1 CD8+ T cells cultured with the combination of NX-0255 plus IL-2 provides a robust and durable anti-tumor response compared to mice receiving Pmel-1 CD8+ T cells cultured with IL-2 alone in the aggressive B16-OVA model. Flow cytometry analysis showed that Pmel-1 CD8+ T cells cultured in the presence of NX-0255 plus IL-2 were found at an increased frequency in the blood and showed an increased central-memory phenotype (CD44+CD62L+) after adoptive transfer when compared to cells cultured with IL-2 alone. Collectively, these data suggest that adding the CBL-B inhibitor, NX-0255, during the in vitro expansion of tumor-specific T cells increases the frequency and absolute numbers of less exhausted CD8+ memory T-cells, increasing their in vivo persistence and ability to infiltrate the tumor. Citation Format: Marilena Gallotta, Jose Gomez Romo, Alexandra Borodovsky, Austin Tenn-McClellan, Jennifer Stokes, Jennifa Gosling, Gwenn M. Hansen, Arthur Sands, Ryan Rountree, Cristiana Guiducci. Ex-vivo inhibition of CBL-B with a novel small molecule inhibitor, NX-0255, enhances persistence and anti-tumor activity of adoptively transferred CD8+ T cells in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 573.

Abstract 1595: Small molecule inhibition of the ubiquitin ligase CBL-B results in potent T and NK cell mediated anti-tumor response

Abstract The E3 ubiquitin ligase Casitas B-lineage lymphoma b (CBL-B) is expressed in T cells where it functions as an important negative regulator of immune activation. CBL-B d attenuates T-cell activation initiated by TCR engagement in part by mediating the requirement for CD28 co-stimulation, thus setting the threshold for T cell activation. CD4+ and CD8+ T cells from mice deficient in cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN-γ when stimulated ex vivo with anti-CD3. Cbl-b deficient mice also demonstrate enhanced NK cell function. Here we describe NX-1607, an investigational, orally bioavailable, small molecule inhibitor of CBL-B. NX-1607 demonstrates potent biochemical inhibition of CBL-B leading to stimulatory effects on human immune cells at low nanomolar concentrations. NX-1607 induction of IL-2 and IFN-γ secretion occurs in primary human T cells stimulated with anti-CD3 antibodies, in both the presence and absence of CD28 co-stimulation, although to a lesser degree in the absence of co-stimulation. In vivo, oral administration of NX-1607 in mice demonstrated significant tumor growth inhibition in two colon carcinoma tumor models, CT26 and MC38, as well as a triple negative breast tumor model, 4T1. The change in tumor microenvironment caused by NX-1607 treatment leads to rapid NK cell infiltration followed by infiltration of activated CD8+ T cells. Depletion of CD8+ T cells or NK cells completely abrogated NX-1607 antitumor activity. Importantly, the combination of NX-1607 and anti-PD-1 can substantially increase the median overall survival and the frequency of complete tumor rejections in all three tumor models. These studies provide significant insights into the antitumor activity of this novel, small molecule E3 ligase inhibitor and deliver experimental support for clinical development of NX-1607 given as monotherapy or in combination with PD-1 blockade. Citation Format: Ryan Rountree, Frederick Cohen, Austin Tenn-McClellan, Alexandra Borodovsky, Marilena Gallotta, Jennifer Stokes, Jose Gomez Romo, Chris Karim, Gwenn M. Hansen, Cristiana Guiducci, Arthur Sands, Jennifa Gosling. Small molecule inhibition of the ubiquitin ligase CBL-B results in potent T and NK cell mediated anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1595.

CD28 engagement inhibits CD73-mediated regulatory activity of CD8+ T cells

CD28 is required for T cell activation as well as the generation of CD4+Foxp3+ Treg. It is unclear, however, how CD28 costimulation affects the development of CD8+ T cell suppressive function. Here, by use of Hepa1.6.gp33 in vitro killing assay and B16.gp33 tumor mouse model we demonstrate that CD28 engagement during TCR ligation prevents CD8+ T cells from becoming suppressive. Interestingly, our results showed that ectonucleotidase CD73 expression on CD8+ T cells is upregulated in the absence of CD28 costimulation. In both murine and human tumor-bearing hosts, CD73 is upregulated on CD28−CD8+ T cells that infiltrate the solid tumor. UPLC-MS/MS analysis revealed that CD8+ T cells activation without CD28 costimulation produces elevated levels of adenosine and that CD73 mediates its production. Adenosine receptor antagonists block CD73-mediated suppression. Our data support the notion that CD28 costimulation inhibits CD73 upregulation and thereby prevents CD8+ T cells from becoming suppressive. This study uncovers a previously unidentified role for CD28 costimulation in CD8+ T cell activation and suggests that the CD28 costimulatory pathway can be a potential target for cancer immunotherapy.

CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo.

Compared to naive T cells, differentiated T cells are thought to be less dependent on CD28 costimulation for full activation. To revisit the role of CD28 costimulation in mouse T cell recall responses, we adoptively transferred in vitro generated OT-II T helper (Th) 1 cells into C57BL/6 mice (Thy1.2+) and then either blocked CD28–ligand interactions with Fab fragments of the anti-CD28 monoclonal antibody (mAb) E18 or deleted CD28 expression using inducible CD28 knock-out OT-II mice as T cell donors. After injection of ovalbumin protein in adjuvant into the recipient mice we observed that systemic interferon (IFN)γ release strongly depended on CD28 costimulation of the Th1 cells, while secondary clonal expansion was not reduced in the absence of CD28 costimulation. For human memory CD4+ T cell responses we also noted that cytokine release was reduced upon inhibition of CD28 costimulation. Together, our data highlight the so far underestimated role of CD28 costimulation for the reactivation of fully differentiated CD4+ T cells.

Cereblon tunes c-Myc protein expression and regulates the metabolic function of activated T cells

Abstract Immunomodulatory drugs, such as lenalidomide, pomalidomide, and CC122 are glutarimide derivatives that alter T cell effector functions, reverse tolerance, and increase cytokine production. We hypothesized that cereblon (CRBN), the molecular target of these drugs, is a negative regulator of T cell function. Therefore, germline Crbn knockout mice were used to investigate its role in T cell regulation. Similar to human T cells treated with immunomodulatory drugs, stimulated Crbn−/− T cells exhibit increased proliferation and cytokine production and maintain this phenotype in the absence of CD28 co-stimulation. Using Gene Set Enrichment Analysis (GSEA) to identify transcriptional drivers of differentially expressed genes, we found that Myc and its family member Max may drive the observed activated phenotype in Crbn−/− T cell. Crbn−/− T cells have Myc-related phenotypic changes, such as increased cell size, CD98 expression, glucose uptake, glutamine and arginine uptake, increased intracellular polyamine biosynthesis, and oxygen consumption. Similar increases in Myc-related processes were observed in activated human T cells treated with immunomodulatory drugs. While c-myc mRNA is similar, Crbn−/− T cells and drug-treated human T cells have prolonged c-Myc protein expression. CRBN, a substrate receptor for the DDB1/Cul4A/Rbx1 E3 ubiquitin ligase complex, has only one known endogenous substrate which is glutamine synthetase (GS). GS interacts with CRBN through acetylated lysine residues yielding a protein that is succeptable to polyubiquitinlyation. Collectively, this suggests that CRBN tunes c-Myc-regulated pathways in activated T cells and that this process is blocked by immunomodulatory drugs.

IL-23 and IL-1β Drive Human Th17 Cell Differentiation and Metabolic Reprogramming in Absence of CD28 Costimulation

Th17 cells drive autoimmune disease but also control commensal microbes. A common link among antigens from self-proteins or commensal microbiota is relatively low activation of Tcell receptor (TCR) and costimulation signaling. Indeed, strong TCR/CD28 stimulation suppressed Th17 cell differentiation from human naive Tcells, but not effector/memory cells. CD28 suppressed the classical Th17 transcriptional program, while inducing known Th17 regulators, and acted through an Akt-dependent mechanism. Th17 cells differentiated without CD28 were not anergic: they showed robust proliferation and maintained Th17 cytokine production following restimulation. Interleukin (IL)-23 and IL-1β promoted glucose uptake and increased glycolysis. Although modestly increased compared to CD28 costimulation, glycolysis was necessary to support Th17 differentiation, indicating that cytokine-mediated metabolic shifts were sufficient to obviate the classical requirement for CD28 in Th17 differentiation. Together, these data propose that, in humans, strength of TCR/CD28/Akt activation serves as a rheostat tuning the magnitude of Th17 development driven by IL-23 and IL-1β.

Rapamycin ameliorates the CTLA4-Ig–mediated defect in CD8+ T Cell Immunity During Gammaherpesvirus Infection

Latent viral infections are a major concern among immunosuppressed transplant patients. During clinical trials with belatacept, a CTLA4-Ig fusion protein, patients showed an increased risk of Epstein-Barr virus-associated posttransplant lymphoproliferative disorder, thought to be due to a deficient primary CD8(+) T cell response to the virus. Using a murine model of latent viral infection, we observed that rapamycin treatment alone led to a significant increase in virus-specific CD8(+) T cells, as well as increased functionality of these cells, including the ability to make multiple cytokines, while CTLA4-Ig treatment alone significantly dampened the response and inhibited the generation of polyfunctional antigen-specific CD8(+) T cells. However, the addition of rapamycin to the CTLA4-Ig regimen was able to quantitatively and qualitatively restore the antigen-specific CD8(+) T cell response to the virus. This improvement was physiologically relevant, in that CTLA4-Ig treated animals exhibited a greater viral burden following infection that was reduced to levels observed in untreated immunocompetent animals by the addition of rapamycin. These results reveal that modulation of T cell differentiation though inhibition of mTOR signaling can restore virus-specific immune competence even in the absence of CD28 costimulation, and have implications for improving protective immunity in transplant recipients.

Ndfip1 Enforces a Requirement for CD28 Costimulation by Limiting IL-2 Production

Although the pathways that permit IL-2 production and the full activation of T cells upon Ag encounter are fairly well defined, the negative regulatory circuits that limit these pathways are poorly understood. In this study, we show that the E3 ubiquitin ligase adaptor Ndfip1 directs one such negative regulatory circuit. T cells lacking Ndfip1 produce IL-2, upregulate IL-2Rα, and proliferate, in the absence of CD28 costimulation. Furthermore, T cells in mice lacking both Ndfip1 and CD28 become activated, produce IL-4, and drive inflammation at barrier surfaces. Ndfip1 constrains T cell activation by limiting the duration of IL-2 mRNA expression after TCR stimulation. Ndfip1 and IL-2 have a similar expression pattern, and, following TCR stimulation, expression of both Ndfip1 and IL-2 requires the activity of NFAT and Erk. Taken together, these data support a negative regulatory circuit in which factors that induce IL-2 expression downstream of TCR engagement also induce the expression of Ndfip1 to limit the extent of IL-2 production and, thus, dampen T cell activation.

CD160Ig fusion protein targets a novel costimulatory pathway and prolongs allograft survival.

CD160 is a cell surface molecule expressed by most NK cells and approximately 50% of CD8+ cytotoxic T lymphocytes. Engagement of CD160 by MHC class-I directly triggers a costimulatory signal to TCR-induced proliferation, cytokine production and cytotoxic effector functions. The role of CD160 in alloimmunity is unknown. Using a newly generated CD160 fusion protein (CD160Ig) we examined the role of the novel costimulatory molecule CD160 in mediating CD4+ or CD8+ T cell driven allograft rejection. CD160Ig inhibits alloreactive CD8+ T cell proliferation and IFN-γ production in vitro, in particular in the absence of CD28 costimulation. Consequently CD160Ig prolongs fully mismatched cardiac allograft survival in CD4−/−, CD28−/− knockout and CTLA4Ig treated WT recipients, but not in WT or CD8−/− knockout recipients. The prolonged cardiac allograft survival is associated with reduced alloreactive CD8+ T cell proliferation, effector/memory responses and alloreactive IFN-γ production. Thus, CD160 signaling is particularly important in CD28-independent effector/memory CD8+ alloreactive T cell activation in vivo and therefore may serve as a novel target for prevention of allograft rejection.

Dendritic Cells and CD28 Costimulation Are Required To Sustain Virus-Specific CD8+ T Cell Responses during the Effector Phase In Vivo

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.

Effects of Hydrocortisone on the Differentiation of Human T Helper 2 Cells

We investigated the effects of the neuroendocrine modulator hydrocortisone (HC) on Th2 differentiation of human naive CD4+ T cells with and without CD28 co-stimulation. Human naive CD4+ T cells were isolated and purified from umbilical cord blood mononuclear cells from full-term newborn infants. CD4+ T cells were treated with different concentrations of HC under Th0 or Th2 culture conditions. Th0 conditions included stimulation by immobilized monoclonal antibodies (mAbs) against CD3 and CD28; Th2 conditions were the same + rhIL-4. Parallel cultures excluded the CD28 mAb. Th1 (IL-2, INF-γ)- and Th2 (IL-4, IL-5)-type cytokines were quantified in culture supernatants by ELISA and within cells by flow cytometry. For both Th0 and Th2 culture conditions, HC significantly inhibited Th1 cytokines' release (IL-2 and INF-γ). For Th0 culture conditions, HC slightly increased IL-4 expression (Th2 cytokine). However, for Th2 culture conditions, HC inhibited the IL4-induced production of IL-4. Although the absolute cytokine amounts were decreased, absence of CD28 co-stimulation did not alter these 'trends'. Our findings indicate that HC can alter the Th1/Th2 balance by inhibiting the production of Th1-type cytokines. HC can also diminish the extensive Th2 differentiation induced by IL-4.

PILAR is a novel modulator of human T-cell expansion

AbstractRobust T-cell responses without autoimmunity are only possible through a fine balance between activating and inhibitory signals. We have identified a novel modulator of T-cell expansion named proliferation-induced lymphocyte-associated receptor (PILAR). Surface PILAR is markedly up-regulated on CD4 and, to a lesser extent, on CD8 T cells on T-cell receptor engagement. In absence of CD28 costimulation, PILAR signaling through CD161 supports CD3 antibody-dependent and antigen-specificT-cell proliferation by increasing the expression of antiapoptotic Bcl-xL and induces secretion of T helper type 1 cytokines. These effects are abrogated by PILAR blockade with specific antibodies, which decrease surface levels of CD28. In contrast, PILAR induces apoptotic death on naive and early activated T cells if CD161 engagement is blocked. PILAR is expressed by approximately 7% to 10% of CD4 T cells in 2 samples of inflammatory synovial fluid, suggesting a potential role in the pathogenesis of joint inflammation. In addition, in the ovarian cancer microenvironment, effector T cells express PILAR, but not CD161, although expression of both can be augmented ex vivo. Our results indicate that PILAR plays a central role in modulating the extent of T-cell expansion. Manipulation of PILAR signaling may be important for treatment of autoimmune diseases and cancer.

Memory CD8+ T Cells Require CD28 Costimulation

CD8(+) T cells are a critical component of the adaptive immune response against infections and tumors. A current paradigm in immunology is that naive CD8(+) T cells require CD28 costimulation, whereas memory CD8(+) T cells do not. We show here, however, that during viral infections of mice, costimulation is required in vivo for the reactivation of memory CD8(+) T cells. In the absence of CD28 costimulation, secondary CD8(+) T cell responses are greatly reduced and this impairs viral clearance. The failure of CD8(+) T cells to expand in the absence of CD28 costimulation is CD4(+) T cell help independent and is accompanied by a failure to down-regulate Bcl-2 and by cell cycle arrest. This requirement for CD28 costimulation was shown in both influenza A and HSV infections. Thus, contrary to current dogma, memory CD8(+) T cells require CD28 costimulation to generate maximal secondary responses against pathogens. Importantly, this CD28 requirement was shown in the context of real infections were multiple other cytokines and costimulators may be up-regulated. Our findings have important implications for pathogens, such as HIV and measles virus, and tumors that evade the immune response by failing to provide CD28 costimulation. These findings also raise questions about the efficacy of CD8(+) T cell-based vaccines against such pathogens and tumors.

CD28 costimulation is required by effector and memory CD8+ T cells (43.2)

Abstract CD28 costimulation is crucial for priming of CD8+ T cell responses to pathogens, however its role in the later phase of the primary and in the secondary immune responses is not clear. To dissect the role of CD28, C57Bl/6 mice were primed intranasally with influenza virus and then treated with a non-depleting blocking anti CD28 antibody at days 6 and 8 post infection. The frequency and absolute number of virus-specific CD8+ T cells were reduced in anti CD28 treated (3.9±0.4%; 1.9±0.5x105) vs. untreated mice (11.5±1.2%; 7.4±1.7 x105). We found that anti-CD28 treatment induces apoptosis of virus-specific CD8+ cells while it did not affect the number of actively dividing cells. To study the role of CD28 during secondary CD8+ T cell responses, C57Bl/6 mice were primed with flu virus strain PR8 and day 60 memory cells were transferred into C57Bl/6 or CD80−/−CD86−/− deficient mice. Mice were then rechallenged with flu virus strain X31 and 7 days post rechallenge CD80−/−CD86−/− deficient mice exhibited a reduction in virus-specific CD8+ T cells in the lung, compared to C57Bl/6 mice (1.2±0.7x106 vs. 5.8±1.1x106, respectively). Failure of memory CD8+ T cells to expand in the absence of CD28 costimulation is due to a failure to downregulate Bcl-2 and to cell cycle arrest. Thus, effector and memory CD8+ T cells require CD28 costimulation to generate optimal immune responses against pathogens.

T-cell tolerance or function is determined by combinatorial costimulatory signals

Activated in immune responses, T lymphocytes differentiate into effector cells with potent immune function. CD28 is the most prominent costimulatory receptor for T-cell activation. However, absence of CD28 costimulation did not completely impair effector function of CD4 or CD8 T cells. Moreover, increasing number of costimulatory molecules are recently found on antigen-presenting cells to regulate T-cell activation. To understand the molecular mechanisms that determine T-cell function or tolerance, we have collectively examined the roles of positive and negative costimulatory molecules. Antigen-specific naïve CD4 and CD8 T cells, only when activated in the absence of both CD28 and ICOS pathways, were completely impaired in effector function. These tolerant T cells not only were anergic with profound defects in TcR signal transduction but also completely lacked expression of effector-specific transcription factors. T-cell tolerance induction in this system requires the action by negative costimulatory molecules; T-cell proliferation and function was partially restored by inhibiting PD-1, B7-H3 or B7S1. This work demonstrates that T-cell function or tolerance is controlled by costimulatory signals.

Homeostatic T cell proliferation as a barrier to T cell tolerance

The maintenance of T cell numbers in the periphery is mediated by distinct homeostatic mechanisms that ensure the proper representation of naïve and memory T cells. Homeostatic proliferation refers to the process by which T cells in lymphopenic hosts divide in the absence of cognate antigen to reconstitute the peripheral lymphoid compartment. During this process T cells acquire effector-memory like properties, including the ability to respond to low doses of antigen in the absence of CD28 costimulation. Furthermore, this capacity is retained long after proliferation has ceased. Accumulating data implicates homeostatic proliferation in autoimmune diseases and transplant rejection, and suggests that it may represent a barrier to tolerance in protocols that use T cell depletion. Implementing combination therapies that aim to promote the development and expansion of regulatory T cell populations while specifically targeting alloresponsive T cells may be the soundest approach to attaining allograft tolerance in the aftermath of T cell depletion and homeostatic proliferation.

Analysis of the Role of Negative T Cell Costimulatory Pathways in CD4 and CD8 T Cell-Mediated Alloimmune Responses In Vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.

Signals from CD28 Induce Stable Epigenetic Modification of the IL-2 Promoter

CD28 costimulation controls multiple aspects of T cell function, including the expression of proinflammatory cytokine genes. One of these genes encodes IL-2, a growth factor that influences T cell proliferation, survival, and differentiation. Antigenic signaling in the absence of CD28 costimulation leads to anergy, a mechanism of tolerance that renders CD4+ T cells unable to produce IL-2. The molecular mechanisms by which CD28 costimulatory signals induce gene expression are not fully understood. In eukaryotic cells, the expression of many genes is influenced by their physical structure at the level of DNA methylation and local chromatin remodeling. To address whether these epigenetic mechanisms are operative during CD28-dependent gene expression in CD4+ T cells, we compared cytosine methylation and chromatin structure at the IL-2 locus in fully activated CD4+ effector T cells and CD4+ T cells rendered anergic by TCR ligation in the absence of CD28 costimulation. Costimulation through CD28 led to marked, stable histone acetylation and loss of cytosine methylation at the IL-2 promoter/enhancer. This was accompanied by extensive remodeling of the chromatin in this region to a structure highly accessible to DNA binding proteins. Conversely, TCR activation in the absence of CD28 costimulation was not sufficient to promote histone acetylation or cytosine demethylation, and the IL-2 promoter/enhancer in anergic cells remained completely inaccessible. These data suggest that CD28 may function through epigenetic mechanisms to promote CD4+ T cell responses.

CD28 Costimulation Controls Histone Hyperacetylation of the Interleukin 5 Gene Locus in Developing Th2 Cells

Interleukin 5 (IL-5) plays a unique role in allergic inflammatory responses, and the understanding of molecular mechanisms underlying the generation of IL-5-producing cells is crucial for the regulation of allergic disorders. Differentiation of naive CD4 T cells into type-2 helper (Th2) cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13, and IL-5 genes. Histone hyperacetylation of the IL-5 gene displayed a delayed kinetics compared with that of the IL-4 and IL-13 genes, suggesting a distinct remodeling mechanism for the IL-5-gene locus. Here we studied the role of CD28 costimulation in the generation of IL-5-producing cells and the histone hyperacetylation of the IL-5 gene locus. CD28-costimulation selectively enhanced histone hyperacetylation of the IL-5 gene locus that appeared to be mediated through NF-kappaB activation and subsequent up-regulation of GATA3. The CD28 costimulation-sensitive histone hyperacetylation spanned almost the entire intergenic region between the IL-5 and RAD50 accompanied with intergenic transcript. Thus, this is the first demonstration that CD28 costimulation controls a chromatin-remodeling process during Th2 cell differentiation.