Faculty Bibliography

Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators. Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9-mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) CD95 deletion inhibited T cell apoptosis upon reactivation; 2) deletion of ORAI1, a Ca²⁺ release-activated channel, abolished Ca²⁺ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.

Calcium signaling is fundamental to many cellular processes. An important pathway for increasing intracellular Ca2+ levels is store-operated Ca2+ entry (SOCE) regulated by stromal interaction molecule (STIM1-2), and Ca2+ channels formed by ORAI1-3 proteins. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. We present patients with Anhidrotic Ectodermal Dysplasia with Immunodeficiency (EDA-ID) caused by novel homozygous p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. A unifying feature of patients with null mutations in ORAI1 is EDA. Anhidrosis was present in patients P1-P4 and confirmed by pilocarpin iontophoresis. Patients had dry and exfoliate skin. They showed signs of heat intolerance and thermoregulatory instability characterized by several attacks of facial flushing accompanied by tachycardia, tachypnea, and hypertension. A skin biopsy showed the presence of eccrine sweat glands in the dermis demonstrating that anhidrosis is not due to a defect in sweat gland development. Recently, we reported that sweat glands require SOCE for opening of the Ca2+-activated chloride channel TMEM16A and thus chloride secretion and sweat production, pointing that anhidrosis in ORAI1-deficient patients could be functional. ORAI1-deficient patients had severe enamel defects diagnosed as hypocalcified amelogenesis imperfecta type III. In contrast, patients with EDA-ID caused by NF-kB signaling defects also have a tooth defect, which is characterized by hypodontia and conical teeth and thus is morphologically easily distinguishable from the enamel defects in ORAI1-deficient patients. ORAI1-deficient patients showed thin and brittle hair. To date, the diagnosis of EDA-ID is limited to patients with defects in NF-kB signaling who are prone to infections with mycobacteria, P.jirovecii, Candida albicans, and, most frequently, pyogenic bacteria caused by hypogammaglobulinemia and failure to mount a specific antibody response to polysaccharide antigens. In contrast, ORAI1-deficient patients are susceptible to an overlapping spectrum of pathogens, but they are also prone to viral infections, including CMV, EBV, RSV, and rotavirus. In addition, AIHA and autoimmune thrombocytopenia are also common in SOCE deficient patients but not NF-kB; instead, patients with NF-kB defects can have inflammatory bowel disease (NF-kB essential modulator colitis). Here we propose that mutations in ORAI1 that abolish SOCE constitute a new form of EDA-ID and are an important differential diagnosis of EDA-ID caused by defects in NF-kB signaling

BACKGROUND AND PURPOSE: Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant challenge. Prior studies have shown that low-dose oral cyclophosphamide can transiently deplete regulatory T cells (Tregs) and improve anti-tumor immunity. We investigated the combination of exemestane with cyclophosphamide in patients with advanced HR-positive breast cancer and assessed changes in circulating immune cell subsets. METHODS: This was a single-arm phase II trial of exemestane with cyclophosphamide in patients with metastatic HR-positive/HER2-negative breast cancer who had progressed on prior endocrine therapy (ClinicalTrials.gov: NCT01963481). Primary endpoint was progression-free survival (PFS) at 3 months (RECIST 1.1). Secondary objectives included median PFS, objective response rate, duration of response, and safety. Circulating Tregs (FOXP3+Helios+) and other immune cell subsets were monitored during treatment and compared with healthy controls. RESULTS: Twenty-three patients were enrolled. Treatment was well tolerated, without grade 4/5 toxicities. Objective responses were seen in 6/23 patients (26.1%; 95% CI 10.2-48.4%) and were durable (median 11.6 months). Three-month PFS rate was 50.1% (95% CI 33.0-76.0%); median PFS was 4.23 months (95% CI 2.8-11.7). No treatment-related decrease in Tregs was observed. However, elevated baseline levels of Naive Tregs [greater than 2.5 (the median of the naive Tregs)] were associated with relative risk of disease progression or death [hazard ratio 11.46 (95% CI 2.32-56.5)]. In addition, the baseline levels of Naive Tregs (adj-p = 0.04), Memory Tregs (adj-p = 0.003), CD4 + Central Memory T cells (adj-p = 0.0004), PD-1 + CD4 + Central Memory T cells (adj-p = 0.008), and PD-1 + CD4 + Effector Memory T cells (adj-p = 0.009) were significantly greater in the patients than in the healthy controls; the baseline levels of %CD4 + Naive T cells (adj-p = 0.0004) were significantly lower in patients compared with healthy controls (n = 40). CONCLUSION: Treg depletion was not observed with low-dose cyclophosphamide when assessed by the specific marker FOXP3 + Helios +; however, baseline naive Tregs were associated with 3-month PFS. Exemestane/cyclophosphamide combination had favorable safety profile with evidence of clinical activity in heavily pretreated patients.

Purpose/UNASSIGNED:The promise of precision oncology is that identification of genomic alterations will direct the rational use of molecularly targeted therapy. This approach is particularly applicable to neoplasms that are resistant to standard cytotoxic chemotherapy, like T-cell leukemias and lymphomas. In this study, we tested the feasibility of targeted next-generation sequencing in profiles of diverse T-cell neoplasms and focused on the therapeutic utility of targeting activated JAK1 and JAK3 in an index case. Patients and Methods/UNASSIGNED:Using Foundation One and Foundation One Heme assays, we performed genomic profiling on 91 consecutive T-cell neoplasms for alterations in 405 genes. The samples were sequenced to high uniform coverage with an Illumina HiSeq and averaged a coverage depth of greater than 500× for DNA and more than 8M total pairs for RNA. An index case of T-cell prolymphocytic leukemia (T-PLL), which was analyzed by targeted next-generation sequencing, is presented. T-PLL cells were analyzed by RNA-seq, in vitro drug testing, mass cytometry, and phospho-flow. Results/UNASSIGNED:missense mutations were activating, caused pathway hyperactivation, and conferred cytokine hypersensitivity. Conclusion/UNASSIGNED:These results underscore the utility of profiling occurrences of resistance to standard regimens and support JAK enzymes as rational therapeutic targets for T-cell leukemias and lymphomas.

Background/UNASSIGNED:T follicular helper (Tfh) cells are crucial for B cell differentiation and antigen-specific antibody production. Dysregulation of Tfh-mediated B cell help weakens B cell responses in HIV infection. Moreover, Tfh cells in the lymph node and peripheral blood comprise a significant portion of the latent HIV reservoir. There is limited data on the effects of perinatal HIV infection on Tfh cells in children. We examined peripheral Tfh (pTfh) cell frequencies and phenotype in HIV-infected children and their associations with disease progression, immune activation, and B cell differentiation. Methods/UNASSIGNED:In a Kenyan cohort of 76 perinatally HIV-infected children, comprised of 43 treatment-naïve (ART-) and 33 on antiretroviral therapy (ART+), and 42 healthy controls (HIV-), we identified memory pTfh cells, T cell activation markers, and B cell differentiation states using multi-parameter flow cytometry. Soluble CD163 and intestinal fatty acid-binding protein plasma levels were quantified by ELISA. Results/UNASSIGNED:ART- children had reduced levels of pTfh cells compared with HIV- children that increased with antiretroviral therapy. HIV+ children had higher programmed cell death protein 1 (PD-1) expression on pTfh cells, regardless of treatment status. Low memory pTfh cells with elevated PD-1 levels correlated with advancing HIV disease status, indicated by increasing HIV viral loads and T cell and monocyte activation, and decreasing %CD4 and CD4:CD8 ratios. Antiretroviral treatment, particularly when started at younger ages, restored pTfh cell frequency and eliminated correlations with disease progression, but failed to lower PD-1 levels on pTfh cells and their associations with CD4 T cell percentages and activation. Altered B cell subsets, with decreased naïve and resting memory B cells and increased activated and tissue-like memory B cells in HIV+ children, correlated with low memory pTfh cell frequencies. Last, HIV+ children had decreased proportions of CXCR5+ CD8 T cells that associated with low %CD4 and CD4:CD8 ratios. Conclusion/UNASSIGNED:Low memory pTfh cell frequencies with high PD-1 expression in HIV+ children correlate with worsening disease status and an activated and differentiated B cell profile. This perturbed memory pTfh cell population may contribute to weak vaccine and HIV-specific antibody responses in HIV+ children. Restoring Tfh cell capacity may be important for novel pediatric HIV cure and vaccine strategies.

Background: During human immunodeficiency virus (HIV) disease, chronic immune activation leads to T-cell exhaustion. PD-1 identifies "exhausted" CD8 T cells with impaired HIV-specific effector functions, but its role on CD4 T cells and in HIV-infected children is poorly understood. Methods: In a Kenyan cohort of vertically HIV-infected children, we measured PD-1+ CD4 T-cell frequencies and phenotype by flow cytometry and their correlation with HIV disease progression and immune activation. Second, in vitro CD4 T-cell proliferative and cytokine responses to HIV-specific and -nonspecific stimuli were assessed with and without PD-1 blockade. Results: HIV-infected children have increased frequencies of PD-1+ memory CD4 T cells that fail to normalize with antiretroviral treatment. These cells are comprised of central and effector memory subsets and correlate with HIV disease progression, measured by viral load, CD4 percentage, CD4:CD8 T-cell ratio, and immune activation. Last, PD-1+ CD4 T cells predict impaired proliferative potential yet preferentially secrete the Th1 and Th17 cytokines interferon-gamma and interleukin 17A, and are unresponsive to in vitro PD-1 blockade. Conclusions: This study highlights differences in PD-1+ CD4 T-cell memory phenotype and response to blockade between HIV-infected children and adults, with implications for potential immune checkpoint therapies.

BACKGROUND: Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. OBJECTIVES: To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. METHODS: Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. RESULTS: Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin alphaIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. CONCLUSIONS: Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

Improving the ability to predict the efficacy and toxicity of drug candidates earlier in the drug discovery process will speed up the introduction of new drugs into clinics. 3D in vitro systems have significantly advanced the drug screening process as 3D tissue models can closely mimic native tissues and, in some cases, the physiological response to drugs. Among various in vitro systems, bioprinting is a highly promising technology possessing several advantages such as tailored microarchitecture, high-throughput capability, coculture ability, and low risk of cross-contamination. In this opinion article, we discuss the currently available tissue models in pharmaceutics along with their limitations and highlight the possibilities of bioprinting physiologically relevant tissue models, which hold great potential in drug testing, high-throughput screening, and disease modeling.

Regulatory T cells (Tregs) are functionally suppressive CD4 T cells, critical for establishing peripheral tolerance and controlling inflammatory responses. Previous reports of Tregs during chronic HIV disease have conflicting results with higher or lower levels compared to controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently up-regulate expression as a result of immune activation. Helios is an Ikaros family transcription factor that marks natural Tregs with suppressive activity and does not up-regulate expression after activation. Coexpression of FOXP3 and Helios has been suggested as a highly specific marker of "bona fide" Tregs. We evaluated Treg subsets by FOXP3 co-expressed with either CD25 or Helios and their association with HIV disease progression in perinatally-infected HIV positive children. Identifying Tregs by FOXP3 coexpression with Helios rather than CD25 revealed markedly higher Treg frequencies, particularly in HIV+ children. Regardless of ART, HIV infected children had a selective expansion of memory FOXP3+Helios+ Tregs. The rise in memory Tregs correlated with declining HIV clinical status, indicated by falling CD4 percentages and CD4:CD8 ratios and increasing HIV plasma viremia and immune activation. In addition, untreated HIV+ children exhibited an imbalance between the levels of Tregs and activated T cells. Finally, memory Tregs expressed immune activation markers CD38 and Ki67 and exhaustion marker, PD-1, that tightly correlated with a similar phenotype in memory CD4 T cells. Overall, HIV infected children had significant disruptions of memory Tregs that associated with advancing HIV disease.