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Evidence from nonhuman animal models demonstrates an important role for immune cells in hypertension, but immune cell changes in human hypertension are less clear. Using mass cytometry, we demonstrate novel and selective reductions in CCR10(+) regulatory T cells (Tregs) and PD-1(+)CD57(−)CD8(+) memory T cells. RNA sequencing reveals that CCR10(+) Tregs exhibit gene expression changes consistent with enhanced immunosuppressive function. In addition, CITE-Seq demonstrates that PD-1(+)CD57(−)CD8(+) memory T cells exhibit features of T-cell exhaustion. Taken together, these results provide novel evidence for decreases in anti-inflammatory and/or hypofunctional T-cell populations that may contribute to enhanced inflammation in human hypertension.

SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism inWe used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of thisTrp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Background: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan (Trp) for arginine (Arg) at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. Methods: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells. Results: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8 + T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8 + T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8 + T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans.. Conclusions: Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Aims Elevated serum immunoglobulins have been associated with experimental and human hypertension for decades but whether immunoglobulins and B cells play a causal role in hypertension pathology is unclear. In this study, we sought to determine the role of B cells and high-affinity class-switched immunoglobulins on hypertension and hypertensive end-organ damage to determine if they might represent viable therapeutic targets for this disease. Methods and results We purified serum IgG from mice exposed to vehicle or angiotensin (Ang) II to induce hypertension and adoptively transferred these to wild type (WT) recipient mice receiving a subpressor dose of Ang II. We found that transfer of IgG from hypertensive animals leads does not affect blood pressure, endothelial function, renal inflammation, albuminuria, or T cell derived cytokine production compared with transfer of IgG from vehicle infused animals. As an alternative approach to investigate the role of high affinity, class-switched immunoglobulins, we studied mice with genetic deletion of activation-induced deaminase (Aicda-/-). These mice have elevated levels of IgM but virtual absence of class-switched immunoglobulins such as IgG subclasses and IgA. Neither male nor female Aicda-/- mice were protected from Ang II-induced hypertension and renal/vascular damage. To determine if IgM or non-immunoglobulin dependent innate functions of B cells play a role in hypertension, we studied mice with severe global B cell deficiency due to deletion of the membrane exon of the IgM heavy chain (μMT-/-). μMT-/- mice were also not protected from hypertension or end-organ damage induced by Ang II infusion or deoxycorticosterone acetate (DOCA)-salt treatment. Conclusions These results suggest that B cells and serum immunoglobulins do not play a causal role in hypertension pathology. Translational perspective Our results suggest that in most cases of essential hypertension, B cells are not a causal factor in the pathophysiology of disease. Thus, elevated serum immunoglobulins seen in hypertensive animals and humans may reflect a biomarker of aberrant immune activation in hypertension and not a therapeutic target. However, autoantibodies may cause hypertension in special cases, and more work is needed to determine whether specific B cell subsets might play an important role that is masked by global B cell deficiency.

The NOD, LRR, and pyrin domain-containing 3 (NLRP3) protein has been established as a central component of the inflammasome and regulates the inflammatory response to a myriad of environmental, microbial, and endogenous danger stimuli. Assembly of the NLRP3 inflammasome results in the cleavage and activation of caspase-1, in turn causing release of the pro-inflammatory interleukins 1-beta and 18. This activation response, while crucial to coordinated innate immune defense, can be aberrantly activated by the likes of cell-free DNA, and cause significant autoimmune pathology. Complications of autoimmunity induced by aberrant NLRP3 inflammasome activation have a great degree of mechanistic crossover with alloimmune injury in solid organ transplant, and stratagems to neutralize NLRP3 inflammasome activation may prove beneficial in solid organ transplant management. This article reviews NLRP3 inflammasome biology and the pathology associated with its hyperactivation, as well as the connections between NLRP3 inflammasome activation and allograft homeostasis.

Emerging evidence from animal models has demonstrated the importance of multiple innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets are altered in human hypertension. To test this, we performed high dimensional single cell profiling of human peripheral blood mononuclear cells using mass cytometry. Unsupervised computational analysis revealed a 40% decrease in CD8 + memory T cells in hypertensive individuals. Using Phenograph to identify subsets of these cells revealed a selective 60% decrease in PD-1 + CD8 + memory T cells in hypertension. This observation was confirmed in a validation cohort using flow cytometry in which PD-1 + CD8 + memory T cells were significantly decreased 44% in hypertensive compared to control individuals. To determine the phenotype of these PD-1 + CD8 + memory T cells, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) on four control and four hypertensive individuals. Using antibodies to identify PD-1 + and PD-1 - CD8 + memory T cells, gene set enrichment analysis of the coordinate single cell transcriptomic data revealed that PD-1 + cells exhibit over-representation of features of both immunologically active effector T cells and hypofunctional exhausted T cells. Thus, clustering analysis of PD-1 + CD8 + memory T cells was performed which demonstrated 4 distinct subclusters. One of these subclusters was decreased in hypertension and exhibited selective expression of multiple inhibitory receptors characteristic of exhausted T cells. At the protein level, this subcluster was marked by expression of the inhibitory receptor LAG3 and low levels of CD57. Combining these markers to identify PD-1 + LAG3 + CD57 - CD8 + memory T cells permitted identification of exhausted cells which demonstrated a significant 35% decrease in hypertensive compared to control individuals using flow cytometry. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1 + CD8 + memory T cells with features of exhaustion in human hypertension. These findings provide new insights into the pathogenesis of human hypertension including loss and/or re-invigoration of exhausted T cells.

Background Patients with end-stage kidney disease require complex and expensive medical management. Kidney transplantation remains the treatment of choice for end-stage kidney disease and is considered superior to all other modalities of renal replacement therapy or dialysis. However, access to kidney transplant is limited by critical supply and demand, making it extremely important to ensure longevity of transplanted kidneys. This is prevented through lifelong immunosuppression, with caution not to overly suppress the immune system, resulting in toxicity and harm. Transition of care to community nephrologists after initial kidney transplantation and monitoring at a transplant center is an important process to ensure delivery of effective and patient-centric care closer to home. Once transplanted, laborious surveillance of the immune system and monitoring for potential rejection and injury are undertaken through an armamentarium of screening modalities. Posttransplant surveillance for kidney function and injury remains key to follow-up care. While kidney function, quantified by estimated glomerular filtration rate and serum creatinine, and kidney injury, measured by proteinuria and hematuria, are standard biomarkers used to monitor injury and rejection posttransplant, they have recently been demonstrated to be inferior in performance to that of AlloSure (CareDx Inc, Brisbane, CA) circulating donor-derived, cell-free DNA (dd-cfDNA). Objective The outcomes and methods of monitoring renal transplant recipients posttransplant have remained stagnant over the past 15 years. The aim of this study is to consider intensive surveillance using AlloSure dd-cfDNA in an actively managed protocol, assessing whether it increases long-term allograft survival in kidney transplant recipients compared with current standard clinical care in community nephrology. Methods The study protocol will acquire data from a phase IV observational trial to assess a cohort of renal transplant patients managed using AlloSure dd-cfDNA and patient care managers versus 1000 propensity-matched historic controls using United Network for Organ Sharing U.S. Scientific Registry of Transplant Recipients data. Data will be managed in a centralized electronic data server. The primary outcome will be superior allograft survival, as a composite of return to dialysis, retransplant, death due to allograft failure, and death with a functional graft (infection, malignancy, and cardiovascular death). The secondary endpoints will assess improved kidney function through decline in estimated glomerular filtration rate and immune activity through development of donor-specific antibodies. Results The total sample is anticipated to be 3500 (2500 patients managed with AlloSure dd-cfDNA and 1000 propensity-matched controls). Active enrollment began in November 2020. Conclusions Based on a significant literature base, we believe implementing the surveillance of dd-cfDNA in the kidney transplant population will have a positive impact on graft survival. Through early identification of rejection and facilitating timely intervention, prolongation of allograft survival versus those not managed by dd-cfDNA surveillance protocol should be superior. International Registered Report Identifier (IRRID) PRR1-10.2196/25941

BACKGROUND Patients with end-stage kidney disease require complex and expensive medical management. Kidney transplantation remains the treatment of choice for end-stage kidney disease and is considered superior to all other modalities of renal replacement therapy or dialysis. However, access to kidney transplant is limited by critical supply and demand, making it extremely important to ensure longevity of transplanted kidneys. This is prevented through lifelong immunosuppression, with caution not to overly suppress the immune system, resulting in toxicity and harm. Transition of care to community nephrologists after initial kidney transplantation and monitoring at a transplant center is an important process to ensure delivery of effective and patient-centric care closer to home. Once transplanted, laborious surveillance of the immune system and monitoring for potential rejection and injury are undertaken through an armamentarium of screening modalities. Posttransplant surveillance for kidney function and injury remains key to follow-up care. While kidney function, quantified by estimated glomerular filtration rate and serum creatinine, and kidney injury, measured by proteinuria and hematuria, are standard biomarkers used to monitor injury and rejection posttransplant, they have recently been demonstrated to be inferior in performance to that of AlloSure (CareDx Inc, Brisbane, CA) circulating donor-derived, cell-free DNA (dd-cfDNA). OBJECTIVE The outcomes and methods of monitoring renal transplant recipients posttransplant have remained stagnant over the past 15 years. The aim of this study is to consider intensive surveillance using AlloSure dd-cfDNA in an actively managed protocol, assessing whether it increases long-term allograft survival in kidney transplant recipients compared with current standard clinical care in community nephrology. METHODS The study protocol will acquire data from a phase IV observational trial to assess a cohort of renal transplant patients managed using AlloSure dd-cfDNA and patient care managers versus 1000 propensity-matched historic controls using United Network for Organ Sharing U.S. Scientific Registry of Transplant Recipients data. Data will be managed in a centralized electronic data server. The primary outcome will be superior allograft survival, as a composite of return to dialysis, retransplant, death due to allograft failure, and death with a functional graft (infection, malignancy, and cardiovascular death). The secondary endpoints will assess improved kidney function through decline in estimated glomerular filtration rate and immune activity through development of donor-specific antibodies. RESULTS The total sample is anticipated to be 3500 (2500 patients managed with AlloSure dd-cfDNA and 1000 propensity-matched controls). Active enrollment began in November 2020. CONCLUSIONS Based on a significant literature base, we believe implementing the surveillance of dd-cfDNA in the kidney transplant population will have a positive impact on graft survival. Through early identification of rejection and facilitating timely intervention, prolongation of allograft survival versus those not managed by dd-cfDNA surveillance protocol should be superior. INTERNATIONAL REGISTERED REPORT PRR1-10.2196/25941

SH2B3, also known as LNK, is an adaptor protein that negatively regulates growth factor and cytokine signaling. The minor allele of a single nucleotide polymorphism in SH2B3 (rs3184504) encodes a tryptophan (Trp) at amino acid 262 as opposed to arginine (Arg) and is strongly associated with hypertension in genome-wide association studies. Whether this variant is causal and how it impacts hypertension development and end-organ damage is unknown. We used CRISPR-Cas9 to engineer mice homozygous for the major and minor alleles of this SH2B3 polymorphism, resulting in Arg/Arg and Trp/Trp mice, respectively. Trp/Trp mice exhibited increased systolic blood pressure (SBP) by radiotelemetry during weeks 3-4 of angiotensin II (Ang II) infusion compared to Arg/Arg mice (nighttime SBP 168 vs 158 mm Hg, respectively). Renal dysfunction was also exacerbated in Ang II-treated Trp/Trp compared to Arg/Arg mice, as evidenced by significantly increased urinary albumin/creatinine ratio (0.41 vs 0.17), renal perivascular fibrosis (fibrosis score 2.0 vs 1.0), and renal macrophages (8,341 vs 6,413 per kidney). In addition, renal CD8 + T cells from Ang II-treated Trp/Trp mice produced significantly more IFNγ compared to Arg/Arg controls (median fluorescence intensity 20,455 vs 14,134), and ex vivo stimulated splenic CD8 + T cells from Trp/Trp compared to Arg/Arg mice made 2.7-fold more IFNγ. To determine a mechanism for increased T cell IFNγ production, dendritic cells and naïve T cells from Trp/Trp and Arg/Arg mice were co-cultured in different combinations. The greatest increase in GM-CSF-induced IFNγ production occurred when both dendritic cells and T cells came from Trp/Trp mice (3.4-fold greater than both cell types from Arg/Arg mice). This effect appears to be due to loss of SH2B3-mediated suppression of GM-CSF signaling, as overexpression of Trp-encoding SH2B3 in HEK cells exhibited significantly less repression of GM-CSF-induced Stat5 activation compared to Arg-encoding SH2B3. Taken together, these findings suggest that the Trp encoding allele of rs3184504 is a causal variant promoting blood pressure elevation and renal dysfunction, at least in part through loss of SH2B3-mediated repression of T cell IFNγ production.

The role of B cells and serum immunoglobulins (Ig) in hypertension is unclear. Elevated serum IgG has been observed in some hypertensive patients and in animal models of hypertension. Mice with a severe B cell deficiency due to lack of B cell activating factor receptor (BAFF-R) and mice lacking the cytokine interleukin 21 (IL21) - which is a potent inducer of B cell Ig class switching and high affinity Ig production - are protected from Ang II-induced hypertension and exhibit reduced serum IgG levels. Thus, we sought to determine whether hypertensive IgGs play a causative role in hypertension and end-organ dysfunction. First, we purified serum IgG from normotensive and hypertensive wild type (WT) mice and adoptively transferred equal amounts of IgG by intraperitoneal injection into normotensive recipient mice followed by infusion of a sub-pressor dose of angiotensin II (Ang II) for 2 weeks. Flow cytometric analysis of aortas revealed a modest increase in F4/80+ macrophages (p=0.04) and CD8+ T cells (p=0.02) in mice that received hypertensive IgG. However, there was no difference in endothelial dependent relaxation, renal inflammation, albuminuria, and blood pressure (BP) between the two groups. As an alternative method to investigate the role of class-switched high affinity Igs, we studied mice with genetic deletion of activation-induced deaminase (AID) which is an enzyme necessary for somatic hypermutation (to generate high affinity Igs) and isotype class switching. AID -/- mice exhibit undetectable IgG and higher levels of IgM in serum compared to WT mice. Ang II infusion for 4 weeks revealed no difference in BP, aortic inflammation, renal inflammation, and albuminuria. We also studied mice with deficiency of the IgM heavy chain (uMT). These mice exhibit undetectable levels of all immunoglobulins and are severely deficient in mature B cells. uMT-/- mice are also not protected from hypertension induced by Ang II infusion or deoxycorticosterone acetate (DOCA-salt) treatment. Together, these results demonstrate that while elevated serum immunoglobulins seen in hypertensive animals and humans may represent a biomarker of aberrant immune activation, they are likely not playing a causal role in hypertension pathophysiology.

Hypertension is the leading risk factor for morbidity and mortality worldwide. Emerging evidence in animal models demonstrates the importance of a variety of innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of specific circulating immune cell subsets is altered in human hypertension reflecting disease pathophysiology. We performed unbiased high dimensional, single cell profiling of peripheral blood mononuclear cells in humans with a panel of 31 cell surface markers using mass cytometry. Unsupervised computational analysis from 11 control and 10 hypertensive individuals matched for age, gender, race, and body mass index revealed consistent increases in a novel memory helper T cell subset in hypertension. Manual two dimensional gating revealed that this CD4 + CD45RO + CD62L + CCR7 - CD161 lo memory cell population is nearly 2-fold increased in hypertensive subjects (1.0 vs 1.9%). As an alternative to starting with unsupervised analysis, manual gating for major known immune cell populations revealed a 40% decrease in memory cytotoxic T cells and a 29% decrease in regulatory T cells in hypertensive individuals (3.2 vs 1.9% and 1.3 vs 0.93%, respectively). Unsupervised analysis of the cellular subsets of these populations using Phenograph revealed a selective 63% decrease in PD-1 + memory cytotoxic T cells and 49% decrease in CCR10 + regulatory T cells in hypertension (0.67 vs 0.25% and 0.37 vs 0.19%, respectively). Taken together, these results demonstrate that in human hypertension there are increases in a novel memory helper T cell subset and decreases in PD-1 + memory cytotoxic and CCR10 + regulatory T cell populations. These findings may provide new insights into hypertension pathogenesis and potential therapeutic targets.

T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce interleukin 21 (IL21) promote germinal center (GC) B cell responses leading to immunoglobulin (Ig) production. Here we investigate the role of IL21 and Tfh cells in hypertension. In response to angiotensin (Ang) II-induced hypertension, T cell IL21 production is increased, and Il21-/- mice develop blunted hypertension, attenuated vascular end-organ damage, and decreased interleukin 17A (IL17A) and interferon gamma production. Tfh-like cells and GC B cells accumulate in the aorta and plasma IgG1 is increased in hypertensive WT but not Il21-/-mice. Furthermore, Tfh cell deficient mice develop blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL21 neutralization reduces blood pressure (BP) and reverses endothelial dysfunction and vascular inflammation. Moreover, recombinant IL21 impairs endothelium-dependent relaxation ex vivo and decreases nitric oxide production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL21 correlates with systolic BP and IL17A production. These data suggest that IL21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.

Emerging evidence from animal models demonstrates the importance of a variety of innate and adaptive immune cells in the pathogenesis of hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets is altered in human hypertension reflecting disease pathophysiology. We thus performed high dimensional, single cell profiling of peripheral blood mononuclear cells in humans with a panel of 31 cell surface markers using mass cytometry. Unsupervised computational analysis from 11 control and 10 hypertensive individuals matched for age, gender, race, and body mass index revealed an approximately two-fold increase in a novel memory helper CD62L+CCR7−CD161lo T cell subset in hypertensive subjects. In addition, manual gating for major known immune cell populations revealed a 40% decrease in memory cytotoxic T cells and a 29% decrease in regulatory T cells in hypertensive individuals. Unsupervised analysis of the cellular subsets of these populations using Phenograph demonstrated a selective 63% decrease in PD-1+ memory cytotoxic T cells and 49% decrease in CCR10+ regulatory T cells in hypertension. In a validation cohort, circulating PD-1+ memory cytotoxic and CCR10+ regulatory T cells were significantly decreased by 30% and 50%, respectively, without a difference in the memory helper T cell subset. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1+ memory cytotoxic and CCR10+ regulatory T cell populations in human hypertension. These findings may provide new insights into hypertension pathogenesis and potential therapeutic targets.

Aortic dissection (AD) is a life-threatening vascular disease with limited treatment strategies. Here, we show that loss of the GWAS-identified SH2B3 gene, encoding lymphocyte adaptor protein LNK, markedly increases susceptibility to acute AD and rupture in response to angiotensin (Ang) II infusion. As early as day 3 following Ang II infusion, prior to the development of AD, Lnk–/– aortas display altered mechanical properties, increased elastin breaks, collagen thinning, enhanced neutrophil accumulation, and increased MMP-9 activity compared with WT mice. Adoptive transfer of Lnk–/– leukocytes into Rag1–/– mice induces AD and rupture in response to Ang II, demonstrating that LNK deficiency in hematopoietic cells plays a key role in this disease. Interestingly, treatment with doxycycline prevents the early accumulation of aortic neutrophils and significantly reduces the incidence of AD and rupture. PrediXcan analysis in a biobank of more than 23,000 individuals reveals that decreased expression of SH2B3 is significantly associated with increased frequency of AD-related phenotypes (odds ratio 0.81). Thus, we identified a role for LNK in the pathology of AD in experimental animals and humans and describe a new model that can be used to inform both inherited and acquired forms of this disease.

Chronic inflammation characterized by vascular T cell and macrophage infiltration is associated with angiotensin II (Ang II)-induced hypertension and end-organ damage. However, the role of B cells in the development of hypertension is not well established. Elevated levels of serum immunoglobulins have been observed in experimental and human hypertension. Prior studies demonstrated that B-cell-activating factor receptor-deficient (BAFF-R -/- ) mice, which lack mature B cells, are protected from Ang II-induced hypertension and vascular remodeling. However, BAFF-R can be expressed on non-B cells as well. Thus, to further investigate the role of B cells in hypertension, we studied mice with targeted disruption of the membrane exon of the immunoglobulin μ heavy chain gene (μMT -/- ) that encodes the constant region of the IgM isotype. In these mice, B cells are arrested at the pre-B cell maturation stage. Serum immunoglobulins were undetectable at a 1:30,000 dilution in μMT -/- mice. Interestingly, μMT -/- mice exhibited similar blood pressure increase in response to 4 weeks of Ang II infusion compared to age-matched wild type (WT) mice (p=0.675). Flow cytometry of aortae from Ang II-treated μMT -/- mice demonstrated fewer CD45+ cells (p=0.047), similar levels of CD8+ and CD4+ T cells (p=0.631 and p=0.721 respectively), a trend for more F4/80+ monocytes/macrophages (p=0.2567), and virtually no CD19+ B cells (p=0.002) compared to WT mice. Endothelium-dependent vasodilatation to acetylcholine was comparable between μMT -/- mice and WT mice after Ang II treatment (p=0.223), however μMT -/- mice displayed enhanced endothelium-independent vasodilatation to sodium nitroprusside than WT mice (p=0.024). Urinary albumin/creatinine ratio was similar between Ang II-treated μMT -/- mice and WT mice (p=0.488), indicating similar renal damage. Taken together, μMT -/- mice are not protected from Ang II-induced hypertension, suggesting that B cells and immunoglobulin production are not critical for hypertension and the associated end-organ damage.

T cell derived cytokines, interleukin 17A (IL17A) and interferon gamma (IFNg), promote angiotensin II (Ang II)-induced hypertension and end-organ damage. Interleukin 21 (IL21), produced primarily by T follicular helper (Tfh) cells, has been shown to induce IL17A and IFNγ production from T effector cells. IL21 is also a potent activator of germinal center (GC) B cells and immunoglobulin (Ig) class switching. We hypothesized that IL21 plays a fundamental role in hypertension and hypertensive end-organ damage through its effects on pro-inflammatory T cell polarization, vascular dysfunction, and enhanced Ig secretion. CD4+ T cells from Ang II infused WT mice exhibit 1.5-fold increased IL21 mRNA expression and 2-fold increased IL-21 production compared to vehicle. We found IL21 -/- mice exhibit a 30mmHg reduction in systolic blood pressure (SBP) in response to 4 weeks of Ang II infusion compared to age-matched wild type (WT) mice. Further, IFNg and IL17A production from CD8+ or CD4+ T cells, respectively, was abrogated in IL21 -/- mice vs. WT mice infused with Ang II. We also found Tfh cells and GC B cells significantly increased in the aortas and mesenteric vessels of Ang II WT mice but not IL21 -/- mice. Ang II induced an increase in percent of lymph node GC B cells and IgG/IgM ratio, consistent with Ig class switching. Ang II-induced increase in renal injury was also blunted in IL21 -/- mice vs. WT mice. Mesenteric vessels from IL21 -/- mice were protected from Ang II-induced endothelial dysfunction. IL21 -/- mice also developed blunted aortic fibrosis and reduced smooth muscle cell hypertrophy after 4 weeks of Ang II vs. WT mice. IL-21 neutralization beginning 2 weeks after Ang II resulted in a 15 mmHg reduction in SBP and reversal of aortic inflammation and vascular endothelial dysfunction compared to isotype control treated animals. Lastly, CD4+ T cell production of IL21 positively correlated with SBP in human volunteers, and in fact, IL-21 production was significantly higher in hypertensive compared to normotensive subjects. Taken together, these studies provide compelling evidence that IL21 may function as a master cytokine in the pathogenesis of hypertension and suggest that therapies targeting IL21 may reduce hypertension and the associated end-organ damage.

Hypertension is the leading risk factor for global morbidity and mortality. Emerging evidence in animal models implicates a variety of innate and adaptive immune cells in hypertension pathogenesis, and limited studies in humans have demonstrated increased circulating memory and senescent T cells in hypertensive individuals. However, the abundance and role of specific immune cells in human hypertension remains unclear. To perform unbiased high dimensional profiling of peripheral immune cells in humans, we developed and validated a novel approach using mass cytometry to detect 31 extracellular markers at the single cell level. Initial results in a normotensive individual using this panel revealed robust identification of major circulating immune cell subsets including CD4 + cells (60% of CD45 + cells), CD8 + cells (21%), γδ T cells (6%), CD25 + CD127 lo T regulatory cells (5%), CD19 + B cells (3%), and CD11c + myeloid cells (3%). Unsupervised viSNE plots using this panel of 31 markers demonstrates grouping of distinct circulating immune cell subsets based on similarity of marker levels (Figure) . We are extending this analysis to peripheral blood mononuclear cells collected from 16 additional control and 17 hypertensive individuals matched for age, gender, race, and BMI to determine differences in the abundance of known and potentially novel circulating immune cell subsets. The combination of high dimensional mapping and unsupervised computational analysis will permit rigorous immunophenotyping of human hypertension and potentially identify novel therapeutic targets.

Background In cardiac transplant recipients, the development of antibodies to the endothelial intermediate filament protein vimentin (antivimentin antibodies, AVA) has been associated with rejection and poor outcomes. However, the incidence of these antibodies prior to transplantation and their association with early rejection has not been investigated. Methods Pre-transplant serum was analyzed from 50 patients who underwent de novo cardiac transplant at Johns Hopkins Hospital from 2004 to 2012. Demographic, one-yr rejection, and survival data were obtained from the transplant database. Results The incidence of pre-transplant AVA was 34%. AVA-positive patients were younger (p = 0.03), and there was an a trend toward incidence in females (p = 0.08). Demographic data were similar among both groups. AVA positivity did not predict rejection in the first year post-transplant. There was no difference in rejection-free graft survival (53 vs. 52%, p = 0.85) at one yr. Similarly, there was no difference in graft survival at one yr (82 vs. 88%, p = 0.56) or graft survival at a median follow-up of 23 and 26 months, respectively (76 vs. 85%, p = 0.41). Conclusions AVA is common in the cardiac pre-transplant population with a higher incidence in the young. The presence of detectable AVA did not correlate with early post-transplant rejection or graft survival.

Background: Acute aortic dissection (AD) is a life-threatening vascular disease associated with an inflammatory response. A polymorphism in the gene SH2B3 that encodes LNK has been associated with several cardiovascular and autoimmune diseases in humans. LNK is an adaptor protein expressed in hematopoietic and endothelial cells that serves as a brake to cellular proliferation and cytokine production. We hypothesize that loss of LNK promotes AD through an exacerbation of the acute immune response. Methods: Angiotensin II (Ang II) was infused for 3 or 14 days into wild type (WT) and LNK -/- mice. Kaplan-Meier survival curves were generated. After 3 days, the aortic remodeling was accessed by standard histological staining methods and microscopy. The aortic inflammation was characterized by flow cytometry and immunohistochemistry. Results: Ang II infusion induces a rapid and drastic mortality in LNK -/- mice compared to WT mice (66% vs 8%, P -/- mice show significant remodeling with more elastin fragmentation than WT mice (10.5 vs 4.6 breaks, P 4 μm 2 , P =0.057). Strikingly, collagen qualitative analysis reveals thinner collagen fibers and several areas of disruption and disorganization in the aorta of LNK -/- mice prior to AD development. In parallel, the aorta of LNK -/- mice show an increase in the number of neutrophils and macrophages but not in T cells compared to WT mice. Conclusion: In our model, LNK seems to play a key role in maintaining the aortic wall integrity. Loss of LNK promotes acute inflammation and matrix degradation, leading to the development of AD. Targeting LNK could be a potential therapeutic strategy for the management of aortic dissection.

T cell derived cytokines such as interferon gamma (IFNγ) and interleukin 17A (IL17A) are upregulated by and can promote angiotensin II-induced hypertension and end-organ damage. Interleukin 21 (IL21) is produced primarily by T follicular helper (Tfh) cells and has the potential to promote IL17A and IFNγ production from T effector cells while inhibiting T regulatory cells. IL21 is also a potent activator of germinal center (GC) B cells. Thus, we hypothesize that IL21 promotes hypertension and hypertensive end-organ damage through its effect on T cell polarization and GC B cell activation. The data indicate that indeed IL21 -/- mice exhibit a 30 mmHg reduction in blood pressure in response to 4 weeks of angiotensin II (Ang II) infusion compared to age-matched wild type (WT) mice (p=0.0275). IL21 mRNA expression increases 1.5 fold in splenic T cells in response to Ang II infusion (p=0.015). Moreover, Tfh cells and GC B cells are increased in the aortas of WT mice after Ang II infusion (p=0.0136 and p=0.0067, respectively). Further, IL17A production from splenic CD4+ T cells and IFNγ production from splenic CD8+ T cells was reduced in IL21 -/- mice compared to WT mice (p=0.0025 and p=0.0237, respectively) following Ang II infusion. Renal function was assessed by measuring albuminuria. WT mice developed 2-fold more albuminuria compared to IL21 -/- mice in response to Ang II infusion (p=0.0349). Lastly, Ang II infusion impaired endothelium-dependent relaxation to acetylcholine in mesenteric vessels from WT mice (52.9% vs 17.5%; p=-/- mice demonstrated moderately impaired endothelium-dependent relaxation at baseline (34.9%) but interestingly, there was no further impairment in these vessels following Ang II infusion. Taken together, these studies suggest that IL21 and the Tfh cell–GC B cell axis may play a key role in hypertension and hypertensive end-organ damage.

We previously showed that angiotensin II (Ang II) increases T cell production of IL-17A, and that mice deficient in IL-17A have blunted hypertension and attenuated renal and vascular dysfunction. It was recently shown that salt enhances IL-17A production from CD4+ T cells via a serum- and glucocorticoid-regulated kinase 1–dependent (SGK1-dependent) pathway. Thus, we tested the hypothesis that SGK1 signaling in T cells promotes hypertension and contributes to end-organ damage. We show that loss of T cell SGK1 results in a blunted hypertensive response to Ang II infusion by 25 mmHg. Importantly, renal and vascular inflammation is abrogated in these mice compared with control mice. Furthermore, mice lacking T cell SGK1 are protected from Ang II–induced endothelial dysfunction and renal injury. Loss of T cell SGK1 also blunts blood pressure and vascular inflammation in response to deoxycorticosterone acetate–salt (DOCA-salt) hypertension. Finally, we demonstrate that the Na+-K+-2Cl– cotransporter 1 (NKCC1) is upregulated in Th17 cells and is necessary for the salt-induced increase in SGK1 and the IL-23 receptor. These studies demonstrate that T cell SGK1 and NKCC1 may be novel therapeutic targets for the treatment of hypertension and identify a potentially new mechanism by which salt contributes to hypertension.

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

We have previously shown that the T cell-derived pro-inflammatory cytokine, interleukin 17A (IL17A), is upregulated by and promotes angiotensin II (Ang II)-induced hypertension and contributes to renal and vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride enhances IL17A production from CD4+ T cells in an SGK1 dependent manner. Since dietary salt intake is associated with hypertension, we hypothesized that T cell SGK1 promotes hypertension and contributes to end-organ dysfunction. To test this hypothesis, we crossed SGK1 fl/fl mice with Tg CD4cre mice to delete SGK1 in T lymphocytes. Loss of T cell SGK1 resulted in a blunted blood pressure response following 2 weeks of Ang II infusion (24.8 mmHg reduction, p=0.01). Moreover, renal and vascular inflammation in response to Ang II infusion was abrogated in these mice compared to SGK1 fl/fl control mice. Ang II infusion increased total (CD45+) leukocytes in the kidney from 55.4 to 120.4 x10 3 (pfl/fl mice while there was no increase in mice with T cell deletion of SGK1 (48.1 to 47.5x10 3 , p=ns). Similarly, Ang II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4x10 3 (pfl/fl mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1x10 3 , p=ns). Furthermore, relaxation of mesenteric arterioles isolated from Ang II infused SGK1 fl/fl mice in response to acetylcholine was impaired by 22.37% (pfl/fl mice infused with Ang II compared to 3.23-fold (p=ns) in mice without T cell SGK1, demonstrating that these mice are protected from Ang II-induced renal injury. Finally, we found that total numbers of splenic CD4+IL17A+ cells increase from 0.44% to 1.15% (pfl/fl mice infused with Ang II compared to no change (0.45% to 0.47%, p=ns) in mice without T cell SGK1. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and the associated end-organ dysfunction.

Angiotensin II–induced hypertension is associated with an increase in T-cell production of interleukin-17A (IL-17A). Recently, we reported that IL-17A −/− mice exhibit blunted hypertension, preserved natriuresis in response to a saline challenge, and decreased renal sodium hydrogen exchanger 3 expression after 2 weeks of angiotensin II infusion compared with wild-type mice. In the current study, we performed renal transporter profiling in mice deficient in IL-17A or the related isoform, IL-17F, after 4 weeks of Ang II infusion, the time when the blood pressure reduction in IL-17A −/− mice is most prominent. Deficiency of IL-17A abolished the activation of distal tubule transporters, specifically the sodium–chloride cotransporter and the epithelial sodium channel and protected mice from glomerular and tubular injury. In human proximal tubule (HK-2) cells, IL-17A increased sodium hydrogen exchanger 3 expression through a serum and glucocorticoid-regulated kinase 1–dependent pathway. In mouse distal convoluted tubule cells, IL-17A increased sodium–chloride cotransporter activity in a serum and glucocorticoid-regulated kinase 1/Nedd4-2–dependent pathway. In both cell types, acute treatment with IL-17A induced phosphorylation of serum and glucocorticoid-regulated kinase 1 at serine 78, and treatment with a serum and glucocorticoid-regulated kinase 1 inhibitor blocked the effects of IL-17A on sodium hydrogen exchanger 3 and sodium–chloride cotransporter. Interestingly, both HK-2 and mouse distal convoluted tubule 15 cells produce endogenous IL-17A. IL17F had little or no effect on blood pressure or renal sodium transporter abundance. These studies provide a mechanistic link by which IL-17A modulates renal sodium transport and suggest that IL-17A inhibition may improve renal function in hypertension and other autoimmune disorders.

We have previously shown that the T cell-derived pro-inflammatory cytokine, interleukin 17A (IL-17A), is upregulated by and promotes angiotensin II-induced hypertension and contributes to vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride in culture enhances IL-17A production from CD4+ T cells in a Serum and Glucocorticoid-Regulated Kinase 1 (SGK1) dependent manner. We confirmed the effect of salt on CD4+ T cell differentiation and extended this finding to CD8+ T cells in which 40mM of excess salt increased the expression of IL-17A (4.7 fold, p=0.0003) and the salt-sensing kinase SGK1 (2.2 fold, p=.001) in naive CD8+ T cells cultured under Th17 polarizing conditions. Since dietary salt intake is associated with hypertension, we hypothesized that T cell SGK1 promotes hypertension and contributes to vascular dysfunction. To test this hypothesis, we crossed SGK1 fl/fl mice with CD4cre mice to delete SGK1 in most T lymphocytes. Loss of T cell SGK1 resulted in a blunted blood pressure response to angiotensin II infusion (24.8 mmHg reduction, p=0.01) and DOCA-salt treatment (15.51 mmHg reduction, pfl/fl control mice. Angiotensin II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4x10 3 (pfl/fl mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1x10 3 , p=ns). DOCA-salt induction increased total (CD45+) leukocytes in the aorta in SGK1 fl/fl mice from 7.4 to 20.6x10 3 (p3 , p=ns). Furthermore, preliminary data show that angiotensin II infusion impairs vascular relaxation in mesenteric arteries isolated from SGK1 fl/fl control mice, while this effect is blunted in angiotensin II treated mice with T cell deletion of SGK1. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and its associated vascular dysfunction.

Hypertension is a leading cause of cardiovascular and renal morbidity, and mortality. Genome-wide association studies identified a single-nucleotide polymorphism in the gene SH2B3 encoding the lymphocyte adaptor protein, LNK, but, until recently, little was known about how LNK contributes to hypertension. This review summarizes recent work highlighting a central role for LNK in inflammation and hypertension.Using a systems biology approach that integrates genomic data with whole blood transcriptomic data and network modeling, LNK/SH2B3 was identified as a key driver gene for hypertension in humans. LNK is an intracellular adaptor protein expressed predominantly in hematopoietic and endothelial cells that negatively regulates cell proliferation and cytokine signaling. Genetic animal models with deletion or mutation of LNK revealed an important role for LNK in renal and vascular inflammation, glomerular injury, oxidative stress, interferon-γ production, and hypertension. Bone marrow transplantation experiments revealed that LNK in hematopoietic cells is primarily responsible for blood pressure regulation.LNK/SH2B3 is a key driver gene for human hypertension, and alteration of LNK in animal models has a profound effect on inflammation and hypertension. Thus, LNK is a potential therapeutic target for this disease and its devastating consequences.

Transplantation can significantly extend the life of patients with end-stage renal disease and provide a lifesaving treatment for patients with failure of other major organs. Extending the long-term survival of these transplanted organs requires a better understanding of the mechanisms of immune injury, including humoral immunity. Alloantibodies were determined early on to be a contraindication to transplantation, but until recently the primary focus has been on HLA-specific antibodies (1). Historically, the lymphocyte crossmatch was the primary tool used to detect HLA antibodies in the sera of transplant recipients and to assess the risk of antibody-mediated rejection posttransplantation. Now, with the advent of solid-phase immunoassays, we can detect HLA antibodies at very low levels and provide a more accurate characterization of their antigen specificities (2).

Rationale: Accumulating evidence supports a role of adaptive immunity and particularly T cells in the pathogenesis of hypertension. Formation of memory T cells, which requires the costimulatory molecule CD70 on antigen-presenting cells, is a cardinal feature of adaptive immunity. Objective: To test the hypothesis that CD70 and immunologic memory contribute to the blood pressure elevation and renal dysfunction mediated by repeated hypertensive challenges. Methods and Results: We imposed repeated hypertensive challenges using either N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME)/high salt or repeated angiotensin II stimulation in mice. During these challenges effector memory T cells (T EM ) accumulated in the kidney and bone marrow. In the L-NAME/high-salt model, memory T cells of the kidney were predominant sources of interferon-γ and interleukin-17A, known to contribute to hypertension. L-NAME/high salt increased macrophage and dendritic cell surface expression of CD70 by 3- to 5-fold. Mice lacking CD70 did not accumulate T EM cells and did not develop hypertension to either high salt or the second angiotensin II challenge and were protected against renal damage. Bone marrow–residing T EM cells proliferated and redistributed to the kidney in response to repeated salt feeding. Adoptively transferred T EM cells from hypertensive mice homed to the bone marrow and spleen and expanded on salt feeding of the recipient mice. Conclusions: Our findings illustrate a previously undefined role of CD70 and long-lived T EM cells in the development of blood pressure elevation and end-organ damage that occur on delayed exposure to mild hypertensive stimuli. Interventions to prevent repeated hypertensive surges could attenuate formation of hypertension-specific T EM cells.

Immunological memory provides protection to repeated antigen challenges and is a cardinal feature of adaptive immunity. We have previously shown that adaptive immunity contributes to hypertension and have observed memory T cells in several models. We hypothesized that memory T cells contribute to long-term renal damage in response to repeated hypertensive challenges. To impose repeated episodes of hypertension, we treated C57BL/6 mice with L-NAME (0.5mg/ml) in drinking water for two weeks, allowed a two-week normotensive interval and then fed high salt (4% NaCl) for three weeks. L-NAME followed by high salt increased SBP to 151 ± 14 mmHg and caused a two-fold increase in CD4 + and CD8 + memory T cells in the kidney and bone marrow, as identified by the surface marker CD44hi. Intracellular staining showed that memory T cells were predominant sources of the inflammatory cytokines IL-17A and IFN-γ. Development and reactivation of memory T cells require the interaction of CD27 on T cells with CD70 on antigen presenting cells. Flow cytometry revealed that L-NAME/High salt increased expression of CD70 on splenic macrophages by 5-fold and dendritic cells by 3-fold. Because memory T cells are a major source of IFN-γ, we examined the hypertensive response to the L-NAME/high salt protocol in IFN-γ -/- mice. The hypertension caused by L-NAME was identical between WT, CD70 -/- and IFN-γ -/- mice. In contrast, the hypertension induced by subsequent salt administration was markedly attenuated in CD70 -/- mice (123 ± 1.3 mmHg, p-/- and IFN-γ -/- mice failed to develop memory T cell formation in the kidney. The L-NAME/high salt caused striking albuminuria and increased urinary N-gal in WT mice, and these were absent in CD70 -/- and IFN-γ -/- mice. In contrast, L-NAME/high salt had no effect on renal angiotensinogen levels. Thus, repeated hypertensive stimuli lead to accumulation of long-lived effector memory T cells that are major sources of inflammatory cytokines, which in turn promote renal dysfunction, salt sensitivity and hypertension. These studies provide further insight into how the adaptive immune system promotes hypertension.

Aim To investigate the unexplained HLA antibody specificity observed in a post-transplant serum specimen from a lung transplant recipient. Methods Antibody (Ab) evaluation was performed using phenotype and single antigen panels tested on the Luminex ® platform (Lifecodes, One Lambda). Results Patient JD is a 47 year old female with a history of cystic fibrosis. The patient received a bilateral lung transplant on October 3, 2013 from a deceased donor at Johns Hopkins Hospital. A serum specimen drawn immediately prior to transplant was negative for IgG, HLA class I-specific antibody and demonstrated very weak reactivity for IgG, class II-specific antibody. At day T + 4, a post-transplant serum specimen exhibited a substantial antibody increase. Although the timing of antibody appearance indicated an anamnestic response, the specificity was inconsistent with the patient’s historical HLA-specific antibody and appeared to be specific for non-donor antigens. Repeated testing and testing with additional assays confirmed the initial results. Transplanted lungs can be accompanied by substantial donor lymphoid tissue. We therefore tested donor serum for HLA specific antibody. Tests of the deceased donor serum revealed strong HLA-specific antibody nearly identical to the pattern observed with the recipient’s Day T + 4 post-transplant serum. Upon further investigation, we found that, in fact, the donor has been the recipient of a kidney transplant. The deceased donor’s previous transplant information was obtained. The antibody detected in the donor serum was specific for multiple mismatched antigens present in the HLA phenotype of the deceased donor’s kidney transplant. Tests of sera obtained monthly from the lung recipient have shown a steady reduction in antibody strength and breadth. The pattern and the course of the antibody over time suggest that the antibody was of donor origin. Conclusion In this case, having access to the deceased donor’s serum allowed us to investigate the anomalous HLA-specific antibody testing results. Retrieving the deceased donor’s past medical history listed in UNET provided evidence of the transfer of donor antibody-producing cell to the patient JD.

Aim The intermediate filament vimentin is not normally exposed to the immune system, however when exposed, anti-vimentin antibodies (AVA) can be provoked. AVA have been implicated in the development of cardiac allograft vasculopathy (CAV), limiting the long-term benefits of cardiac transplantation. Our aim was to develop a more specific and sensitive assay for the detection of AVA than currently available. Methods A Luminex® multiplexed assay for AVA was designed and validated by parallel testing with a commercially available ELISA for AVA. Sera titration and competitive inhibition with soluble vimentin were used to assess sensitivity and specificity. Pre- and post-transplant sera from forty-six patients were tested under IRB approval. Post-transplant sera were obtained within 12 months after transplantation. Results In parallel titration studies, the bead-based assay was found to be twice as sensitive as the commercially available ELISA. Competitive inhibition studies of five sera resulted in a mean of 60% ± 28% reduction of antibody binding, confirming the Luminex® assay specificity. The incidence of AVA in per-transplant sera was 47.8%.[figure1] Conclusions A Luminex® bead based assay for AVA was developed that is both specific and twice as sensitive as a commercially available ELISA that may have future clinical application. Although a high incidence of AVA among cardiac transplant candidates was observed pre-transplant, longer term studies will be needed to confirm any association with CAV post-transplant.