Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following reexposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony-forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for off-on state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase I DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and antitumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.

Suppression of Heart Failure With PAR1 Pepducin Technology in a Pressure Overload Model in Mice.

BACKGROUND: PAR1 (protease-activated receptor-1) contributes to acute thrombosis, but it is not clear whether the receptor is involved in deleterious inflammatory and profibrotic processes in heart failure. Here, we employ the pepducin technology to determine the effects of targeting PAR1 in a mouse heart failure with reduced ejection fraction model. METHODS: After undergoing transverse aortic constriction pressure overload or sham surgery, C57BL/6J mice were randomized to daily sc PZ-128 pepducin or vehicle, and cardiac function, inflammation, fibrosis, and molecular analyses conducted at 7 weeks RESULTS: After 7 weeks of transverse aortic constriction, vehicle mice had marked increases in macrophage/monocyte infiltration and fibrosis of the left ventricle as compared with Sham mice. PZ-128 treatment significantly suppressed the inflammatory cell infiltration and cardiac fibrosis. Despite no effect on myocyte cell hypertrophy, PZ-128 afforded a significant reduction in overall left ventricle weight and completely protected against the transverse aortic constriction-induced impairments in left ventricle ejection fraction. PZ-128 significantly suppressed transverse aortic constriction-induced increases in an array of genes involved in myocardial stress, fibrosis, and inflammation. CONCLUSIONS: The PZ-128 pepducin is highly effective in protecting against cardiac inflammation, fibrosis, and loss of left ventricle function in a mouse model.

T-Cell MyD88 Is a Novel Regulator of Cardiac Fibrosis Through Modulation of T-Cell Activation.

BACKGROUND: Cardiac inflammation in heart failure is characterized by the presence of damage-associated molecular patterns, myeloid cells, and T cells. Cardiac damage-associated molecular patterns provide continuous proinflammatory signals to myeloid cells through TLRs (toll-like receptors) that converge onto the adaptor protein MyD88 (myeloid differentiation response 88). These induce activation into efficient antigen-presenting cells that activate T cells through their TCR (T-cell receptor). T-cell activation results in cardiotropism, cardiac fibroblast transformation, and maladaptive cardiac remodeling. T cells rely on TCR signaling for effector function and survival, and while they express MyD88 and damage-associated molecular pattern receptors, their role in T-cell activation and cardiac inflammation is unknown. METHODS: We performed transverse aortic constriction in mice lacking MyD88 in T cells and analyzed remodeling, systolic function, survival, and T-cell activation. We profiled wild type versus Myd88-/- mouse T cells at the transcript and protein level and performed several functional assays. RESULTS: Analysis of single-cell RNA-sequencing data sets revealed that MyD88 is expressed in mouse and human cardiac T cells. MyD88 deletion in T cells resulted in increased levels of cardiac T-cell infiltration and fibrosis in response to transverse aortic constriction. We discovered that TCR-activated Myd88-/- T cells had increased proinflammatory signaling at the transcript and protein level compared with wild type, resulting in increased T-cell effector functions such as adhesion, migration across endothelial cells, and activation of cardiac fibroblast. Mechanistically, we found that MyD88 modulates T-cell activation and survival through TCR-dependent rather than TLR-dependent signaling. CONCLUSIONS: Our results outline a novel intrinsic role for MyD88 in limiting T-cell activation that is central to tune down cardiac inflammation during cardiac adaptation to stress.

Antigen presentation by cardiac fibroblasts promotes cardiac dysfunction.

Heart failure (HF) is a leading cause of morbidity and mortality. Studies in animal models and patients with HF revealed a prominent role for CD4+ T cell immune responses in the pathogenesis of HF and highlighted an active crosstalk between cardiac fibroblasts and IFNγ producing CD4+ T cells that results in profibrotic myofibroblast transformation. Whether cardiac fibroblasts concomitantly modulate pathogenic cardiac CD4+ T cell immune responses is unknown. Here we show report that murine cardiac fibroblasts express major histocompatibility complex type II (MHCII) in two different experimental models of cardiac inflammation. We demonstrate that cardiac fibroblasts take up and process antigens for presentation to CD4+ T cells via MHCII induced by IFNγ. Conditional deletion of MhcII in cardiac fibroblasts ameliorates cardiac remodelling and dysfunction induced by cardiac pressure overload. Collectively, we demonstrate that cardiac fibroblasts function as antigen presenting cells (APCs) and contribute to cardiac fibrosis and dysfunction through IFNγ induced MHCII.

Sialomucin CD43 Plays a Deleterious Role in the Development of Experimental Heart Failure Induced by Pressure Overload by Modulating Cardiac Inflammation and Fibrosis.

Sialomucin CD43 is a transmembrane protein differentially expressed in leukocytes that include innate and adaptive immune cells. Among a variety of cellular processes, CD43 participates in T cell adhesion to vascular endothelial cells and contributes to the progression of experimental autoimmunity. Sequential infiltration of myeloid cells and T cells in the heart is a hallmark of cardiac inflammation and heart failure (HF). Here, we report that CD43-/- mice have improved survival to HF induced by transverse aortic constriction (TAC). This enhanced survival is associated with improved systolic function, decreased cardiac fibrosis, and significantly reduced T cell cardiac infiltration in response to TAC compared to control wild-type (WT) mice. Lack of CD43 did not alter the number of myeloid cells in the heart, but resulted in decreased cardiac CXCL10 expression, a chemoattractant for T cells, and in a monocyte shift to anti-inflammatory macrophages in vitro. Collectively, these findings unveil a novel role for CD43 in adverse cardiac remodeling in pressure overload induced HF through modulation of cardiac T cell inflammation.

Myeloid Mineralocorticoid Receptor Transcriptionally Regulates P-Selectin Glycoprotein Ligand-1 and Promotes Monocyte Trafficking and Atherosclerosis.

Objective: MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. Approach and Results: We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. Conclusions: These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.

Isolevuglandin-Modified Cardiac Proteins Drive CD4+ T-Cell Activation in the Heart and Promote Cardiac Dysfunction.

BACKGROUND: Despite the well-established association between T-cell-mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)-modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure. METHODS: We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation-deficient MhcII-/- mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins. RESULTS: We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII-/- mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction-induced cardiac dysfunction despite the presence of left ventricle-infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species-dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation. CONCLUSIONS: Our study demonstrates an important role of reactive oxygen species-induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.

Gut dysbiosis induced by cardiac pressure overload enhances adverse cardiac remodeling in a T cell-dependent manner.

Despite the existing association of gut dysbiosis and T cell inflammation in heart failure (HF), whether and how gut microbes contribute to T cell immune responses, cardiac fibrosis and dysfunction in HF remains largely unexplored. Our objective was to investigate whether gut dysbiosis is induced by cardiac pressure overload, and its effect in T cell activation, adverse cardiac remodeling, and cardiac dysfunction. We used 16S rRNA sequencing of fecal samples and discovered that cardiac pressure overload-induced by transverse aortic constriction (TAC) results in gut dysbiosis, characterized by a reduction of tryptophan and short-chain fatty acids producing bacteria in WT mice, but not in T cell-deficient mice (Tcra-/- ) mice. These changes did not result in T cell activation in the gut or gut barrier disruption. Strikingly, microbiota depletion in WT mice resulted in decreased heart T cell infiltration, decreased cardiac fibrosis, and protection from systolic dysfunction in response to TAC. Spontaneous reconstitution of the microbiota partially reversed these effects. We observed decreased cardiac expression of the Aryl hydrocarbon receptor (AhR) and enzymes associated with tryptophan metabolism in WT mice, but not in Tcra-/- mice, or in mice depleted of the microbiota. These findings demonstrate that cardiac pressure overload induced gut dysbiosis and T cell immune responses contribute to adverse cardiac remodeling, and identify the potential contribution of tryptophan metabolites and the AhR to protection from adverse cardiac remodeling and systolic dysfunction in HF.

Heart Inflammation: Immune Cell Roles and Roads to the Heart.

Heart failure (HF) has been traditionally viewed as a disease of the cardiac muscle associated with systemic inflammation. Burgeoning evidence implicates immune effector mechanisms that include immune cell activation and trafficking to the heart. Immune cell infiltration in the myocardium can have adverse effects in the heart and contribute to the pathogenesis of HF. Both innate and adaptive immunity operate sequentially, and the specificity of these responses depends on the initial trigger sensed by the heart. Although the role of the immune system in the initial inflammatory response to infection and injury is well studied, what sets the trajectory to HF from different etiologies and the role of immunity once HF has been established is less understood. Herein, we review experimental and clinical knowledge of cardiac inflammation induced by different triggers that often result in HF from different etiologies. We focus on the mechanisms of immune cell activation systemically and on the pathways immune cells use to traffic to the heart.

CXCR3 regulates CD4+ T cell cardiotropism in pressure overload-induced cardiac dysfunction.

Heart failure (HF) is associated in humans and mice with increased circulating levels of CXCL9 and CXCL10, chemokine ligands of the CXCR3 receptor, predominantly expressed on CD4+ Th1 cells. Chemokine engagement of receptors is required for T cell integrin activation and recruitment to sites of inflammation. Th1 cells drive adverse cardiac remodeling in pressure overload-induced cardiac dysfunction, and mice lacking the integrin ligand ICAM-1 show defective T cell recruitment to the heart. Here, we show that CXCR3+ T cells infiltrate the heart in humans and mice with pressure overload-induced cardiac dysfunction. Genetic deletion of CXCR3 disrupts CD4+ T cell heart infiltration and prevents adverse cardiac remodeling. We demonstrate that cardiac fibroblasts and cardiac myeloid cells that include resident and infiltrated macrophages are the source of CXCL9 and CXCL10, which mechanistically promote Th1 cell adhesion to ICAM-1 under shear conditions in a CXCR3-dependent manner. To our knowledge, our findings identify a previously unrecognized role for CXCR3 in Th1 cell recruitment into the heart in pressure overload-induced cardiac dysfunction.

Sialomucin CD43 regulates T helper type 17 cell intercellular adhesion molecule 1 dependent adhesion, apical migration and transendothelial migration.

T helper type 17 lymphocytes (Th17 cells) infiltrate the central nervous system (CNS), induce inflammation and demyelination and play a pivotal role in the pathogenesis of multiple sclerosis. Sialomucin CD43 is highly expressed in Th17 cells and mediates adhesion to endothelial selectin (E-selectin), an initiating step in Th17 cell recruitment to sites of inflammation. CD43-/- mice have impaired Th17 cell recruitment to the CNS and are protected from experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. However, E-selectin is dispensable for the development of EAE, in contrast to intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). We report that CD43-/- mice have decreased demyelination and T-cell infiltration, but similar up-regulation of ICAM-1 and VCAM-1 in the spinal cord, compared with wild-type (WT) mice, at the initiation of EAE. CD43-/- Th17 cells have impaired adhesion to ICAM-1 under flow conditions in vitro, despite having similar expression of LFA-1, the main T-cell ligand for ICAM-1, as WT Th17 cells. Regardless of the route of integrin activation, CD43-/- Th17 cell firm arrest on ICAM-1 was comparable to that of WT Th17 cells, but CD43-/- Th17 cells failed to optimally apically migrate on immobilized ICAM-1-coated coverslips and endothelial cells, and to transmigrate under shear flow conditions in an ICAM-1-dependent manner. Collectively, these findings unveil novel roles for CD43, facilitating adhesion of Th17 cells to ICAM-1 and modulating apical and transendothelial migration, as mechanisms potentially responsible for Th17 cell recruitment to sites of inflammation such as the CNS.

Trypanosoma cruzi Neurotrophic Factor Facilitates Cardiac Repair in a Mouse Model of Chronic Chagas Disease.

Most patients acutely infected with Trypanosoma cruzi undergo short-term structural and functional cardiac alterations that heal without sequelae. By contrast, in patients whose disease progresses to chronic infection, irreversible degenerative chronic Chagas cardiomyopathy (CCC) may develop. To account for the contrast between cardiac regeneration in high-parasitism acute infection and progressive cardiomyopathy in low-parasitism CCC, we hypothesized that T. cruzi expresses repair factors that directly facilitate cardiac regeneration. We investigated, as one such repair factor, the T. cruzi parasite-derived neurotrophic factor (PDNF), known to trigger survival of cardiac myocytes and fibroblasts and upregulate chemokine chemokine C-C motif ligand 2, which promotes migration of regenerative cardiac progenitor cells (CPCs). Using in vivo and in vitro models of Chagas disease, we tested whether T. cruzi PDNF promotes cardiac repair. Quantitative PCR and flow cytometry of heart tissue revealed that stem-cell antigen-1 (Sca-1+) CPCs expand in acute infection in parallel to parasitism. Recombinant PDNF induced survival and expansion of ex vivo CPCs, and intravenous administration of PDNF into naïve mice upregulated mRNA of cardiac stem-cell marker Sca-1. Furthermore, in CCC mice, a 3-week intravenous administration of PDNF protocol induced CPC expansion and reversed left ventricular T-cell accumulation and cardiac remodeling including fibrosis. Compared with CCC vehicle-treated mice, which developed severe atrioventricular block, PDNF-treated mice exhibited reduced frequency and severity of conduction abnormalities. Our findings are in support of the novel concept that T. cruzi uses PDNF to promote mutually beneficial cardiac repair in Chagas disease. This could indicate a possible path to prevention or treatment of CCC.

Th1 effector T cells selectively orchestrate cardiac fibrosis in nonischemic heart failure.

Despite emerging data indicating a role for T cells in profibrotic cardiac repair and healing after ischemia, little is known about whether T cells directly impact cardiac fibroblasts (CFBs) to promote cardiac fibrosis (CF) in nonischemic heart failure (HF). Recently, we reported increased T cell infiltration in the fibrotic myocardium of nonischemic HF patients, as well as the protection from CF and HF in TCR-α-/- mice. Here, we report that T cells activated in such a context are mainly IFN-γ+, adhere to CFB, and induce their transition into myofibroblasts. Th1 effector cells selectively drive CF both in vitro and in vivo, whereas adoptive transfer of Th1 cells, opposite to activated IFN-γ-/- Th cells, partially reconstituted CF and HF in TCR-α-/- recipient mice. Mechanistically, Th1 cells use integrin α4 to adhere to and induce TGF-ß in CFB in an IFN-γ-dependent manner. Our findings identify a previously unrecognized role for Th1 cells as integrators of perivascular CF and cardiac dysfunction in nonischemic HF.

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo.

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27(-) phenotype was associated with active TB in HIV(-) (p = 0.0003) and HIV(+) (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV(-) subjects, MTB-specific CD4 T cell populations from HIV(+) TB-asymptomatic subjects were often dominated by CD27(-) cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression.

Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection.

HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common opportunistic pathogens, cytomegalovirus (CMV) and Mycobacterium tuberculosis (MTB). CMV-specific CD4 T cells persisted after HIV infection, whereas MTB-specific CD4 T cells were depleted rapidly. CMV-specific CD4 T cells expressed a mature phenotype and produced very little IL-2, but large amounts of MIP-1ß. In contrast, MTB-specific CD4 T cells were less mature, and most produced IL-2 but not MIP-1ß. Staphylococcal enterotoxin B-stimulated IL-2-producing cells were more susceptible to HIV infection in vitro than MIP-1ß-producing cells. Moreover, IL-2 production was associated with expression of CD25, and neutralization of IL-2 completely abrogated productive HIV infection in vitro. HIV DNA was found to be most abundant in IL-2-producing cells, and least abundant in MIP-1ß-producing MTB-specific CD4 T cells from HIV-infected subjects with active tuberculosis. These data support the hypothesis that differences in function affect the susceptibility of pathogen-specific CD4 T cells to HIV infection and depletion in vivo, providing a potential mechanism to explain the rapid loss of MTB-specific CD4 T cells after HIV infection.

Minor viral and host genetic polymorphisms can dramatically impact the biologic outcome of an epitope-specific CD8 T-cell response.

Human immunodeficiency virus-1 subtypes A and C differ in the highly conserved Gag-TL9 epitope at a single amino acid position. Similarly, the TL9 presenting human leukocyte antigen (HLA) class I molecules B42 and B81 differ only at 6 amino acid positions. Here, we addressed the influence of such minor viral and host genetic variation on the TL9-specific CD8 T-cell response. The clonotypic characteristics of CD8 T-cell populations elicited by subtype A or subtype C were distinct, and these responses differed substantially with respect to the recognition and selection of TL9 variants. Irrespective of the presenting HLA class I molecule, CD8 T-cell responses elicited by subtype C exhibited largely comparable TL9 variant cross-recognition properties, expressed T-cell receptors that used almost exclusively the TRBV 12-3 gene, and selected for predictable patterns of viral variation within TL9. In contrast, subtype A elicited TL9-specific CD8 T-cell populations with completely different, more diverse TCRBV genes and did not select for viral variants. Moreover, TL9 variant cross-recognition properties were extensive in B81(+) subjects but limited in B42(+) subjects. Thus, minor viral and host genetic polymorphisms can dramatically alter the immunologic and virologic outcome of an epitope-specific CD8 T-cell response.

Early depletion of Mycobacterium tuberculosis-specific T helper 1 cell responses after HIV-1 infection.

BACKGROUND: The acid-fast bacillus Mycobacterium tuberculosis is often the first manifestation of acquired immunodeficiency syndrome in patients infected with human immunodeficiency virus (HIV). This study was conducted to better understand the mechanism underlying M. tuberculosis-specific pathogenicity early after onset of HIV infection. METHODS: M. tuberculosis-specific T helper 1 (Th1) cells were studied in HIV negative (n=114) and chronically HIV infected (n=68) Tanzanian subjects by using early secreted antigenic target 6 (ESAT6) protein or tuberculin (purified protein derivative) with interferon-gamma ELISPOT and intracellular cytokine staining. In a longitudinal study, the effect of acute HIV infection on M. tuberculosis-specific Th1 cells was determined by polychromatic flow cytometric analysis in 5 subjects with latent M. tuberculosis infection who became infected with HIV. RESULTS: In tuberculosis (TB)-asymptomatic subjects (i.e., subjects with unknown TB status who did not show clinical signs suggestive of TB), chronic HIV infection was associated with a decreased percentage of subjects with detectable M. tuberculosis-specific Th1 cells (P< .001) a decrease which was not observed among subjects with active TB. Acute HIV infection induced a rapid depletion of M. tuberculosis-specific Th1 cells in 4 subjects remained TB asymptomatic, whereas the population of these cells remained stable in subjects who remained HIV negative (P< .01). CONCLUSIONS: Taken together, these data suggest a mechanism of rapid M. tuberculosis-specific Th1 cell depletion that may contribute to the early onset of TB in individuals with latent M. tuberculosis infection who become HIV infected.

Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis.

Stomata are specialized epidermal structures that regulate gas (CO(2) and O(2)) and water vapor exchange between plants and their environment. In Arabidopsis thaliana, stomatal development is preceded by asymmetric cell divisions, and stomatal distribution follows the one-cell spacing rule, reflecting the coordination of cell fate specification. Stomatal development and patterning are regulated by both genetic and environmental signals. Here, we report that Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6, two environmentally responsive mitogen-activated protein kinases (MAPKs), and their upstream MAPK kinases, MKK4 and MKK5, are key regulators of stomatal development and patterning. Loss of function of MKK4/MKK5 or MPK3/MPK6 disrupts the coordinated cell fate specification of stomata versus pavement cells, resulting in the formation of clustered stomata. Conversely, activation of MKK4/MKK5-MPK3/MPK6 causes the suppression of asymmetric cell divisions and stomatal cell fate specification, resulting in a lack of stomatal differentiation. We further establish that the MKK4/MKK5-MPK3/MPK6 module is downstream of YODA, a MAPKKK. The establishment of a complete MAPK signaling cascade as a key regulator of stomatal development and patterning advances our understanding of the regulatory mechanisms of intercellular signaling events that coordinate cell fate specification during stomatal development.