Quantifying the Generation of T Cell Immunity using a Systems Biology Approach.

The immune system is our defense against pathogens. Quantitatively predicting its response to foreign stimulation is key toward developing tools to interfere with or prevent infection (e.g. vaccines and immunotherapies). I use a systems biology approach and develop computational models describing dynamics occurring within lymph nodes, sites where activated immune cells are generated. These effector cells circulate out into blood and to sites of infection participating in immunity. I both quantitatively and qualitatively study dynamics of immune cells during a generalized infection as well as during infection with Mycobacterium tuberculosis (Mtb). The models predict that their 3-dimensional configuration enables the lymph nodes to support rare antigen-specific T cells to efficiently search for antigen-bearing dendritic cells, and this efficiency is not reduced when the precursor frequency increases in a wide range. The models also predict strategies to manipulate the differentiation of immune cells to maximize specific subtypes of T cells populations, depending on different immunomodulation goals. When coupled with Mtb infection models, our models are able to assist vaccine design by finding correlations between immune cell subsets and protection against Mtb, and also help identify mechanisms controlling different disease outcomes at host level.PhDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113563/1/changgon_1.pd

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Early and Prolonged Antiretroviral Therapy Is Associated with an HIV-1-Specific T-Cell Profile Comparable to That of Long-Term Non-Progressors

Background: Intervention with antiretroviral treatment (ART) and control of viral replication at the time of HIV-1 seroconversion may curtail cumulative immunological damage. We have therefore hypothesized that ART maintenance over a very prolonged period in HIV-1 seroconverters could induce an immuno-virological status similar to that of HIV-1 long-term non-progressors (LTNPs).Methodology/Principal Findings: We have investigated a cohort of 20 HIV-1 seroconverters on long-term ART (LTTS) and compared it to one of 15 LTNPs. Residual viral replication and reservoirs in peripheral blood, as measured by cell-associated HIV-1 RNA and DNA, respectively, were demonstrated to be similarly low in both cohorts. These two virologically matched cohorts were then comprehensively analysed by polychromatic flow cytometry for HIV-1-specific CD4(+) and CD8(+) T-cell functional profile in terms of cytokine production and cytotoxic capacity using IFN-gamma, IL-2, TNF-alpha production and perforin expression, respectively. Comparable levels of highly polyfunctional HIV-1-specific CD4(+) and CD8(+) T-cells were found in LTTS and LTNPs, with low perforin expression on HIV-1-specific CD8+ T-cells, consistent with a polyfunctional/non-cytotoxic profile in a context of low viral burden.Conclusions: Our results indicate that prolonged ART initiated at the time of HIV-1 seroconversion is associated with immuno-virological features which resemble those of LTNPs, strengthening the recent emphasis on the positive impact of early treatment initiation and paving the way for further interventions to promote virological control after treatment interruption

Understanding the critical role of microRNAs in HIV-1 pathogenesis

MicroRNAs (miRNAs) are ~22 nucleotide small RNAs which are critical regulators of mRNA translation and have key roles in HIV-1 infection. We characterized miRNA changes in CD4+ T cells from 7 patients with chronic HIV-1 infection (CHI), 7 long term non progressors (LTNPs) and 8 healthy controls (HC). There were 111 up- and 52 down-regulated miRNAs in CHI relative to HC, and 70 up- and 27 down-regulated miRNAs in LTNPs relative to HC. Let-7 miRNAs were found to be highly expressed in CD4+ T cells from HC whilst significantly and differentially decreased in CHI and LTNPs. We modulated let-7 levels in an in-vitro model and found no direct effect on HIV-1 replication kinetics. We therefore investigated whether miRNAs played a role in the altered cytokine milieu associated with chronic infection. As several in silico algorithms consistently suggested that let-7 miRNAs target the 3’ untranslated (3’UTR) region of IL10, we focused on this as a target. IL-10 was significantly increased in plasma from CHI compared to HC and also in the supernatant of HIV-1infected HUT78 cells compared to uninfected cultures. Using HUT78 cells infected with HIV-1, we noted let-7b was down-regulated within 72 hours and let-7c within 24 hours. We over-expressed let-7 miRNAs and showed decreases in IL-10 for let-7b, let-7f and let-7c. Using anti-miRNAs we showed increased IL-10 levels for let-7c, let-7f and let-7b. We confirmed that let-7 was binding to the IL10 3’UTR using a reporter construct. We performed a microarray in CD4+ T cell subsets revealing that Blimp-1 was preferentially expressed in activated regulatory T cells. Blimp-1 is a zinc finger transcriptional repressor known to play a role in IL-2 expression. We found that miR-9 played a pivotal role in regulating Blimp-1, that Blimp-1 levels were markedly elevated in CHI compared to LTNPs and HC and its expression in CHI may explain the dysfunctional endogenous IL-2 secretion noted in CHI. We investigated the expression of miRNAs in monocytes and found only two miRNAs which were differentially expressed between patient groups. By understanding the interaction between miRNAs and HIV-1, this work has resulted in unique insights into HIV-1 pathogenesis

Mucosal Delivery of Fusion Proteins with Bacillus subtilis Spores Enhances Protection against Tuberculosis by Bacillus Calmette-Guerin

Tuberculosis (TB) is the most deadly infectious disease in existence, and the only available vaccine, Bacillus Calmette-Guérin (BCG), is almost a century old and poorly protective. The immunological complexity of TB, coupled with rising resistance to antimicrobial therapies, necessitates a pipeline of diverse novel vaccines. Here, we show that Bacillus subtilis spores can be coated with a fusion protein 1 (“FP1”) consisting of Mycobacterium tuberculosis (Mtb) antigens Ag85B, ACR, and HBHA. The resultant vaccine, Spore-FP1, was tested in a murine low-dose Mtb aerosol challenge model. Mice were primed with subcutaneous BCG, followed by mucosal booster immunizations with Spore-FP1. We show that Spore-FP1 enhanced pulmonary control of Mtb, as evidenced by reduced bacterial burdens in the lungs. This was associated with elevated antigen-specific IgG and IgA titers in the serum and lung mucosal surface, respectively. Spore-FP1 immunization generated superior antigen-specific memory T-cell proliferation in both CD4+ and CD8+ compartments, alongside bolstered Th1-, Th17-, and Treg-type cytokine production, compared to BCG immunization alone. CD69+CD103+ tissue resident memory T-cells (Trm) were found within the lung parenchyma after mucosal immunization with Spore-FP1, confirming the advantages of mucosal delivery. Our data show that Spore-FP1 is a promising new TB vaccine that can successfully augment protection and immunogenicity in BCG-primed animals

Mathematical modelling of T cell homeostasis

T cell homeostasis describes the process through which the immune system regulates cell survival, proliferation, differentiation and death to maintain T cell numbers and diversity in a range of different conditions. The aim of this thesis is to better understand how this process leads to the development of the naive CD4+ T cell compartment during childhood. Mathematical modelling is used in combination with experimental observations to estimate naive T cell kinetics over the lifetime of an individual. The analysis described here shows that post-thymic proliferation contributes more than double the number of cells entering the pool each day from the thymus. This ratio is preserved from birth to age 20 years; as the thymus involutes, the average time between naive T-cell divisions in the periphery lengthens with age and the naive population is maintained by improved naive cell survival. Thymic output is quantified from birth to age 60 years by combining models to interpret naive T cell TRECs and Ki67 expression data. Three distinct phases of thymic T cell output are identified: (i) increasing production from birth to age 1 year; (ii) steep decline to age 8 years; (iii) slow decline from age 8 years onwards. Finally, the role of inter-cellular variation in T cell residency times is explored. It is able to explain the persistence of PTK7+ naive CD4+ T cells in thymectomised individuals. Importantly, the model predicts the accumulation of veteran PTK7+ T cells in older individuals and suggests that the residual population in thymectomised individuals will also consist predominantly of veteran PTK7+ T cells. The model has implications for the use of PTK7 as a marker of recent thymic emigration and also naturally explains improved T cell survival in older individuals

Immunology of the genital tract - a review

The objective of this work was to systematically review and discuss recent studies and articles dealing with the subject of the immunology of female genital tract mucosal tissue. The emphasis hereby lies on the evaluation of studies concerning the basics of female reproductive immunology, research on immunology of the most important genital infections and vaccination strategies, immunologic principles at the fetomaternal interface during normal pregnancy and its complications as well as on immunologic data on infertility and immunocontraception. It is now established that the mucosal immune system is a distinct and separate component of the host`s immune apparatus and differs from the lymphoid tissues in peripheral sites. Furthermore, despite some common features, the female genital tract mucosal system displays some distinct characteristics which outlines its special role. Analysis of the female genital tract indicates that the key cells of the innate and adaptive immune systems are present and functionally responsive to antigens; however, there is a certain degree of compartmentalization within the tract. The identification of TLRs in the fallopian tubes, uterus, cervix, and vagina and the presence of ECs, macrophages, DCs, NK cells, and neutrophils throughout the reproductive tract along with their responsiveness to selected PAMPs indicate that the female reproductive tract has evolved to meet the challenges of STDs, while at the same time supporting an immunologically distinct fetal placental unit. To meet these diverse challenges, innate and adaptive immune system in the female genital tract are precisely regulated not only by a network of cytokines and chemokines, but also by the sex hormones estrogen and progesterone. Understanding the specialty of the genital tract immune system is of critical importance, because STDs are and will be a major worldwide health problem. Despite extensive efforts, only limited success has been achieved in dealing with a growing list of STDs. The role of immune factors in the control of genital viral and bacterial infections appears complex and needs further studying, also with respect to creating vaccines. Despite the recognition that innate immunity as the first line of defense and adaptive immunity, especially Th1 immune responses, play a critical role in preventing infection and in limiting viral replication, factors such as antimicrobials and TLRs that contribute to the mucosal response in the female genital tract have only recently begun to receive attention. Further studies are also needed to elucidate the relationship between mucosal immunity, the hormonal environment, and response to pathogen challenge. More data must be collected on the mechanisms of immune evasion by several pathogens such as HSV, N. gonorrheae or Chlamydia. While considerable information can be obtained from animal experiments, important differences in the physiology of reproduction and the immune sytem result in the need for studies in humans. Further knowledge on female tract immunology will also impact on immunological approaches to contraception, immunological infertility and the immunological aspects of pregnancy. This does not only involve new options for diagnostics but also for treatment of pregnancy complications such as preeclampsia, preterm birth and early pregnancy loss as well as for infertility. Pregnancy involves maternal tolerance of the semiallogenic histoincompatible fetus and is characterized by the enhancement of the innate immune system and suppression of the adaptive immune response, probably with progesterone as the important regulator. In opposite to normal pregnancy, improper immune responses and an unbalanced cytokine network may characterize implantation failures, pregnancy loss and obstetric complications. These are the presence of elevated Th1/Th2 cell ratios, high concentrations of Th1 cytokines, elevated NK cell cytotoxicity and levels, and emergence of various autoantibodies. These immunological approaches needs to be investigated and evaluated further with respect to widening of treatment options by modification of immune responses

Indoleamine 2,3-Dioxygenase in Human Hematopoietic Stem Cell Transplantation

In recent years tryptophan metabolism and its rate limiting enzyme indoleamine 2,3-dioxygenase (IDO) have attracted increasing attention for their potential to modulate immune responses including the regulation of transplantation tolerance. The focus of this review is to discuss some features of IDO activity which particularly relate to hematopoietic stem cell transplantation (HSCT). HSCT invariably involves the establishment of some degree of a donor-derived immune system in the recipient. Thus, the outstanding feature of tolerance in HSCT is that in this type of transplantation it is not rejection, which causes the most severe problems to HSCT recipients, but the reverse, graft-versus-host (GvH) directed immune responses. We will discuss the peculiar role of IDO activity and accelerated tryptophan metabolism at the interface between immune activation and immune suppression and delineate from theoretical and experimental evidence the potential significance of IDO in mediating tolerance in HSCT. Finally, we will examine therapeutic options for exploitation of IDO activity in the generation of allo-antigen-specific tolerance, i.e. avoiding allo-reactivity while maintaining immunocompetence, in HSCT