THE CRITICAL ROLE OF CD4+ TH CELLS IN CD8+ CTL RESPONSES AND ANTI-TUMOR IMMUNITY

The goal of this body of research was to elucidate the mechanism by which CD4+ T cells provide help for CD8+ cytotoxic T lymphocyte (CTL) responses in different immunization types. The establishment of diseases, such as chronic infections and cancers, is attributed to severe loss of or dysfunctions of CD4+ T cells. Even in acute infections, CD4+ T cell deficiency leads to poor memory responses. While the role of CD4+ T cells is being increasingly appreciated in these diseases, the timing and nature of CD4+ T help and associated molecular mechanisms are not completely understood. Growing evidence suggests that, depending on the type of infections or immunizations, the requirements of CD4+ T cells can vary for optimal CD8+ CTL responses. In order to understand the modulatory effects of CD4+ T cells for optimal CD8+ CTL responses, two distinct immunization types were chosen. These include: 1) non-inflammatory dendritic cell (DC) immunization, which fails to provide inflammatory/danger signals; and 2) inflammatory adenovirus (AdV) immunization, which provides profound inflammatory/danger signals. This allowed us to study CD4+ T cell’s participation under different inflammatory conditions. The studies described in Chapters 2 and 3 of this thesis were performed to further understand the concept of how CD4+ T cells mediate optimal CD8+ CTL responses. This has been called the “new dynamic model of CD4+ T helper – antigen (Ag)-presenting cells (Th-APCs),” proposed in 2005 by our laboratory. The study described in Chapter 2 shows that Th-APCs participate not only in augmenting CTL-mediated immune responses, perhaps during early phase, but also in regulating cellular immunity, perhaps during a later phase. Through enhanced IL-2, CD80 and CD40L singnaling, and weaker peptideMHC I (pMHC) signaling, Th-APCs stimulated naïve CD8+ T cells to differentiate into effector CTLs, capable of developing into, central memory CTLs. Th-APC-stimulated CD4+ T cells behaved like Th cells in function, augmenting the overall magnitude of CTL responses. In contrast, Th-APCs were able to kill DCs and other Th-APCs, predominantly through perforin-mediated pathway. The experiments described in Chapter 3 revealed a novel co-operative role of cognate Th-CTL interactions, contrary to previously known immune-regulatory mechanisms among Th-Th or CTL-CTL interactions. In our experiments, Th cells, via CD40L, IL-2, and acquired pMHC-I signaling, enhanced CTL survival and transition into functional memory CTLs. Moreover, RT-PCR, flow cytometry and western blot analysis demonstrate that increased survival of Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of FasL and TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (NFATc1, Bcl-10, Casp-3, Casp-4, Casp-7) genes/ molecules. Finally, helped CTLs were also able to induce protection against highly metastasizing tumor challenge, explaining why memory CTLs generated under cognate Th1’s help show survival and recall advantages. The studies in Chapter 4 showed how the precursor frequency (PF) of CD8+ T cells impacts CD4+ T helper requirements for functional CTL responses. At endogenous PF, CD4+ T helper signals were necessary for both primary and memory CTL responses. At increased PF, CD4+ T help, and its CD40L but not IL-2 signal became dispensable for primary CTL responses. In contrast, memory CTL responses required CD4+ T cell signals, largely in the form of IL-2 and CD40L. Thus, these results could impact the development of novel immunotherapy against cancers, since their efficacy would be determined in part by CD4+ T help and CD8+ T cell PF. Finally, the study showed the importance of CD4+ T cells for multiple phases of AdV transgene product-specific CTL responses. These include: a) cognate CD4+ T cells enhanced CTL responses via IL-2 and CD40L signaling during primary, maintenance and memory phases; b) polyclonal CD4+ T environment enhanced the survival of AdV-specific CTL survival, partially explaining protracted CTL contraction phase; and c) during the recall phase, the CD4+ T environment, particularly memory CD4+ T cells, considerably enhanced not only helped, but also unhelped, memory CTL expansion. Thus, these results suggest the participation of both cognate and polyclonal CD4+ T cells for multiple phases of AdV-specific CTLs. Taken together, the current work delineated the critical roles of CD4+ T cells in different stages of CTL responses and in the development of anti-tumor immunity. The results presented here will significantly advance our current understanding of immunity to cancers, autoimmunity and chronic infections, since pathogenesis of these diseases is largely determined by CD4+ T helper functions. As most immunization procedures use the principle that is based on functions of memory cells, the knowledge gained from this work will also have a major impact on designing vaccines against intractable diseases, including cancers and chronic infections. Moreover, in advanced tumors, vaccines developed using this knowledge may act synergistically with other cancer treatments such as irradiation, chemotherapy and microsurgery, minimizing their side effects and prolonging the lives of patients

Suppression of a broad spectrum of liver autoimmune pathologies by single peptide-MHC-based nanomedicines

Peptide-major histocompatibility complex class II (pMHCII)-based nanomedicines displaying tissue-specific autoantigenic epitopes can blunt specific autoimmune conditions by re-programming cognate antigen-experienced CD4+ T-cells into disease-suppressing T-regulatory type 1 (TR1) cells. Here, we show that single pMHCII-based nanomedicines displaying epitopes from mitochondrial, endoplasmic reticulum or cytoplasmic antigens associated with primary biliary cholangitis (PBC) or autoimmune hepatitis (AIH) can broadly blunt PBC, AIH and Primary Sclerosing Cholangitis in various murine models in an organ- rather than disease-specific manner, without suppressing general or local immunity against infection or metastatic tumors. Therapeutic activity is associated with cognate TR1 cell formation and expansion, TR1 cell recruitment to the liver and draining lymph nodes, local B-regulatory cell formation and profound suppression of the pro-inflammatory capacity of liver and liver-proximal myeloid dendritic cells and Kupffer cells. Thus, autoreactivity against liver-enriched autoantigens in liver autoimmunity is not disease-specific and can be harnessed to treat various liver autoimmune diseases broadly

Th cells promote CTL survival and memory via acquired pMHC-I and endogenous IL-2 and CD40L signaling and by modulating apoptosis-controlling pathways.

Involvement of CD4(+) helper T (Th) cells is crucial for CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4(+) Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+) Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+) T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b)/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4(+) Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+) Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+) Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy

CD4⁺ T cell signals delivered during priming and recall phase are required for optimal secondary responses.

<p>(<b>a</b>) A schematic protocol. After 90 days following immunization, total CD8⁺ T cells containing memory CTLs were purified from WT (helped memory CTLs) mice and adoptively transferred equally to the naïve secondary recipients, WT and MHCII^−/− mice (∼15×10⁶/mouse). The MHCII^−/− mice were additionally reconstituted with different types of CD4⁺ T cells (∼15–20×10⁶/mouse) along with CD11c⁺ DCs (∼0.5–1.0×10⁶/mouse). Both groups were boosted with AdVova and assessed for memory CTLs expansion. (<b>b</b>) Naïve B6.1/OTII CD4⁺ T cells were co-cultured with irradiated BM DCova, as detailed in material and methods to generate Th1 cells. Th1 cells were then transferred to naïve congenic WT mice (∼10–15×10⁶/mouse). After 45 days, these cells were triple stained and phenotypically characterized for the expression of activation or memory markers (grey-shaded area) as shown in the figure. Irrelevant isotype-matched Abs were used as control (dotted thin lines). The value in the dot plot indicates % of OVA-specific CD4⁺ T memory cells remaining in total CD4⁺ T cell population. One representative of the two independent experiments is shown. (<b>c</b>) Naïve MHCII^−/− mice were adoptively transferred with helped memory CTLs with naïve OTII T cells (∼1.5×10⁶/mouse), polyclonal CD4⁺ T cells (∼15–20×10⁶/mouse) or polyclonal CD4⁺ T cells containing OVA-specific memory CD4⁺ T cells (∼15–20×10⁶/mouse) and CD11c⁺ DCs (∼0.5–1.0×10⁶/mouse) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047004#pone-0047004-g006" target="_blank">Fig. 6a</a>. Three days later, all the groups were boosted with AdVova and the recall potential of the memory CTLs were assessed 6.5 days later. The values represent mean %±SD of OVA-specific tetramer⁺ CTLs in total CD8⁺ T cell population and are representative of two independent experiments with four to five mice per group. *<i>P</i><0.05 or **<i>P</i><0.01, versus MHCII^−/− mice with helped memory CTLs alone.</p

CD4⁺ T cell signals provided during priming and recall phase are required for optimal secondary responses.

<p>(<b>a</b>) A schematic protocol. After 90 days of immunization, total CD8⁺ T cells containing memory CTLs were purified from WT B6 mice with helped CTLs or MHCII^−/− mice with unhelped CTLs, adoptively transferred in equal numbers into the naïve secondary recipients, WT and MHCII^−/− mice (∼15×10⁶/mouse), and assessed for recall potential after boosting. (<b>b</b>) Three days after adoptive transfer of helped or unhelped memory CTLs into naïve WT and MHCII^−/− mice, all the mice groups were boosted with AdVova and monitored for the expansion of memory CTLs 6.5 days later. The values represent mean %±SD of OVA-specific tetramer⁺ CTLs in total CD8⁺ T cell population and are representative of two independent experiments with five to six mice per group. **<i>P</i><0.01, versus MHCII^−/− mice with helped or unhelped memory CTLs.</p

Molecular mechanisms of CD4⁺ T-helper signals required for functional AdV-specific memory CTL responses^a.

a<p>One day prior to immunization, MHCII^−/− mice were adoptively transferred with CD11c⁺ DCs (∼0.5–1.0×10⁶/mouse) and naïve polyclonal (∼15–20×10⁶/mouse) or OTII CD4⁺ T cells (∼1.5×10⁶/mouse) with or without designated gene deficiency, as indicated. 120 days later, all the immunized mice were challenged with BL6-10ova tumor cells. Twenty-four days after the challenge, lung tumor colonies were counted and graded. The data are cumulative of two independent experiments, each comprising five to six mice per group.</p

CD4⁺ T cells impact the kinetics of AdV transgene-specific CTL populations.

<p>Following immunization, AdVova-specific CTLs were analyzed in the peripheral blood at different time points by tetramer (<b>a</b>) and intracellular IFN-γ (<b>b</b>) stainings. The values are presented as mean%±SD of OVA-specific tetramer⁺ CTLs (<b>a</b>) or IFN-γ⁺ CTLs (<b>b</b>) in total CD8⁺ T cell population and are representative of two to three independent experiments with three to four mice per group. **<i>P</i><0.01, versus MHCII^−/− mice. (<b>c</b>) Ten days following immunization, the proportions of CFSE^high-OVAI-pulsed target cells lysed by effector CTLs were determined in the spleens by <i>in vivo</i> cytotoxicity assay. The values represent mean %±SD of targets remaining in spleens relative to controls and are representative of two independent experiments with three to four mice per group. (<b>d</b>) On day 75, following immunization, OVA-specific memory CTLs were characterized in spleen for PD-1 expression by flow cytometry. A representative figure from immunized groups along with matching isotype control is shown on the left. The values in the bar diagram represent the mean %±SD of PD-1⁺ tetramer⁺ CTLs in total tetramer⁺ CTL population and are representative of two independent experiments with 3 to 4 mice per group. **<i>P</i><0.01, versus WT mice.</p