Single cell proteomics microchip to profile immune function, with applications in stem cell biology, translational disease mechanism study and clinical therapeutics monitoring

In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

Function of microRNA-146a and NF-κB in physiologic and pathologic hematopoiesis

During inflammation and infection, hematopoietic stem and progenitor cells (HSPCs) are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of the gene encoding miR-146a—expressed in all blood cell types—produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to exhaustion of the HSCs and hematopoietic neoplasms. Six-week-old deleted mice are normal, with no effect on cell numbers, but by 4 months bone marrow hypercellularity can be seen, and by 8 months marrow exhaustion is becoming evident. The ability of HSCs to replenish the entire hematopoietic repertoire in a myelo-ablated mouse also declines precipitously as miR-146a-deficient mice age. In the absence of miR-146a, LPS-mediated serial inflammatory stimulation accelerates the effects of aging. This chronic inflammatory stress on HSCs in deleted mice involves a molecular axis consisting of upregulation of the signaling protein TRAF6 leading to excessive activity of the transcription factor NF-κB and overproduction of the cytokine IL-6. At the cellular level, transplant studies show that the defects are attributable to both an intrinsic problem in the miR-146a-deficient HSCs and extrinsic effects of miR-146a-deficient lymphocytes and non-hematopoietic cells. This study has identified a microRNA, miR-146a, to be a critical regulator of HSC homeostasis during chronic inflammatory challenge in mice and has provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms. This may have implications for human hematopoietic malignancies, such as myelodysplastic syndrome, which frequently displays downregulated miR-146a expression.

Learning and using taxonomies for visual and olfactory classification

Humans are able of distinguishing more than 5000 visual categories even in complex environments using a variety of different visual systems all working in tandem. We seem to be capable of distinguishing thousands of different odors as well. In the machine learning community, many commonly used multi-class classifiers do not scale well to such large numbers of categories. This thesis demonstrates a method of automatically creating application-specific taxonomies to aid in scaling classification algorithms to more than 100 cate- gories using both visual and olfactory data. The visual data consists of images collected online and pollen slides scanned under a microscope. The olfactory data was acquired by constructing a small portable sniffing apparatus which draws air over 10 carbon black polymer composite sensors. We investigate performance when classifying 256 visual categories, 8 or more species of pollen and 130 olfactory categories sampled from common household items and a standardized scratch-and-sniff test. Taxonomies are employed in a divide-and-conquer classification framework which improves classification time while allowing the end user to trade performance for specificity as needed. Before classification can even take place, the pollen counter and electronic nose must filter out a high volume of background “clutter” to detect the categories of interest. In the case of pollen this is done with an efficient cascade of classifiers that rule out most non-pollen before invoking slower multi-class classifiers. In the case of the electronic nose, much of the extraneous noise encountered in outdoor environments can be filtered using a sniffing strategy which preferentially samples the visensor response at frequencies that are relatively immune to background contributions from ambient water vapor. This combination of efficient background rejection with scalable classification algorithms is tested in detail for three separate projects: 1) the Caltech-256 Image Dataset, 2) the Caltech Automated Pollen Identification and Counting System (CAPICS) and 3) a portable electronic nose specially constructed for outdoor use.

Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein

More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design.