Regulation of key molecules of immunological synapse by T11TS immunotherapy abrogates Cryptococcus neoformans infection in rats

Highlights

• Preclinical immunotherapy with T11TS in cryptococcosis in the rat model.

• Enhanced clearance of fungus from the lung tissue in the T11TS treated groups.

• Improved RANTES level in the T11TS treated groups suggests recruitment of T cells.

• Immunological Synapse modulation in cryptococcosis and after T11TS treatment.

Abstract

Cryptococcus neoformans infects and disseminates in hosts with diminished T cell responses. The immunomodulator T11TS (T11 target structure) had profound potential in glioma as well as C. neoformans infected model for disease amelioration. It is been established by our group that T11TS potentiates Calcineurin-NFAT pathway in T cells of C. neoformans infected rats. We investigated the upstream Immunological Synapse (IS) molecules that are vital for the foundation of initial signals for downstream signaling, differentiation and proliferation in T cells. Improved RANTES level in the T11TS treated groups suggests potential recruitment of T cells. Down-regulation of TCRαβ, CD3ζ, CD2, CD45 and CD28 molecules by cryptococcus were boosted after T11TS therapy. Heightened expression of inhibitory molecule CTLA-4 in cryptococcosis was dampened by T11TS. The decline of MHC I, MHC II and CD80 expression on macrophages by C. neoformans were enhanced by T11TS. The dampening of positive regulators and upsurge of negative regulators of the IS during cryptococcosis was reversed with T11TS therapy resulting in enhanced clearance of fungus from the lungs as envisaged by our histological studies. This preclinical study with T11TS opens a new prospect for potential immunotherapeutic intervention against the devastating C. neoformans infection with positive aspect for the long-term solution and a safer immunotherapeutic regimen.

Introduction

The encapsulated basidiomycete Cryptococcus neoformans is an opportunistic fungus that causes life-threatening cryptococcosis in individuals with diminished cell-mediated immunity (Wozniak et al., 2011). It usually infects through inhalation of spores or desiccated yeast from environmental sources (Coelho et al., 2014). After an initial asymptomatic pulmonary infection, the organism is carried in the bloodstream and subsequently disseminated to other target organs (Yang et al., 2017). Intracellular survival of C. neoformans primarily in macrophages provides advantages by allowing escape from the immune response (Yang et al., 2017). Although lungs are considered to be a common site of infection, C. neoformans predominantly targets the brain causing meningoencephalitis, and the brain involvement may result in severe illness and mortality, even with adequate antifungal drug treatment (Coelho et al., 2014; Yang et al., 2017). C. neoformans mainly affects in an immunocompromised state, such as in immunosuppressive therapies, diabetes mellitus, systemic lupus erythematosus, and more frequently in AIDS (Li et al., 2017; Wozniak et al., 2011).

CD4+ and CD8 + T cells are important in controlling C. neoformans by limiting their survival within macrophages (Lindell et al., 2005) indicating that T cells play a pivotal role in modulating the outcome of the disease. RANTES is a pro-inflammatory chemokine present at the sites of multiple inflammatory conditions and could trigger T-cell signaling pathway to activate T-cells (Appay et al., 2000).

It is imperative to understand the operative mechanisms of the Immunological Synapse (IS) to comprehend the early steps in the signaling cascades and downstream signaling in T cell in C. neoformans disease setup. IS is a spatial interface between the contact of T cell and antigen-presenting cell (APC) and is responsible for the activation and functional outcome of T cells (Dustin, 2014; Ortega-Carrion and Vicente-Manzanares, 2016). IS consists of three concentric rings of membrane receptor-ligand molecules and their underlying cytoskeletal and signaling domain and/or proteins (Huppa and Davis, 2003). The innermost region, or the central supramolecular activation cluster (cSMAC), mostly comprises of TCRαβ (T cell receptor αβ chain), CD3, CD4, CD8, CD28, CTLA-4 on T cells and peptide-MHC, CD80/86 on APCs. The cSMAC is encircled by the peripheral SMAC (pSMAC) region that is mainly formed by adhesion molecules such as CD2, LFA-1 on T cells and CD58, CD48/CD59, ICAM-1 on APCs. Lastly, the outermost region encircling the pSMAC is known as distal SMAC (dSMAC), where proteins with large ectodomains are located, such as CD43 and CD45 on T cells (Alarcon et al., 2011; Huppa and Davis, 2003).

Initiation of T cell signaling occurs when Signal 1 is generated through the ligation of TCRαβ to peptide-MHC complexes along with the binding of T cell co-receptors, such as CD4 and CD8 to MHC class II or MHC class I respectively (on APCs). A Signal 2 is primarily delivered through ligation of CD28 (on T cell) to CD80/86 (on APC), and impairment of this CD28-CD80 co-stimulation may result in T cell anergy (Bachmann et al., 1997). Co-stimulation is pivotal for self/nonself antigen discrimination. On the contrary, CTLA-4 expressed on T cell also ligates to CD80/86 (on APC), but it negatively regulates T cell activation (Frauwirth and Thompson, 2002). CD3 molecular complex associates with TCR and its ζ chain encompasses a signaling sequence known as Immunoreceptor Tyrosine-based Activation Motif (ITAM) in their cytoplasmic domain. ITAM conveys tyrosine phosphorylation in response to stimulation by Src family kinases (SFK) such as LCK (p56‑LCK) and FYN (p59‑FYN), where LCK is connected to the cytoplasmic domains of CD4 and CD8, and FYN is connected to TCR/CD3 complex. CD45 has a phosphatase activity and is important for the maintenance of the active state of SFKs that is involved in the activation of downstream signaling partners (Irles et al., 2003). CD2 on T cells binds to CD58 of APCs during T cell-APC interaction. This association is required for the development of specific T-cell lineages (Ohno et al., 1991; Ortega-Carrion and Vicente-Manzanares, 2016). These initial interactions establish a foundation, following which a signal transduction relay follows and leads to the T-cell activation by triggering multiple signaling branches, e.g. up-regulation of cell adhesion molecules, expression of transcription factors (Brownlie and Zamoyska, 2013; Ortega-Carrion and Vicente-Manzanares, 2016). Transcriptional activation that ensues in T cells following downstream intracellular signaling is crucial for T cell survival, proliferation and differentiation into diverse effector subtypes. The effector subtypes then produce pro-inflammatory cytokines like IFN-γ, TNF-α and IL-17/22. These effector functions are vital for the activation of other cells (macrophages, CTLs, and NK cells) for an effective anticryptococcal response (Huppa and Davis, 2003; Medici and Del Poeta, 2015; Ortega-Carrion and Vicente-Manzanares, 2016). If the above signaling events are not synchronized accurately, immune dysfunction will result (Brownlie and Zamoyska, 2013).

The optimum expression of IS molecules is a primary requirement for the successful onset of intracellular T cell signaling and their effector functions to counterattack the C. neoformans mediated immunosuppression and/or infection. In the context of cryptococcosis, the role of IS molecules that cause dampening of T cell functions is largely unexplored till date.

T11TS, a glycoprotein isolated sheep erythrocyte (SRBC) membrane, binds to CD2/T11/E-rosette receptor on immunocytes (Chatterjee et al., 2011; Chaudhuri et al., 2014; Sarkar et al., 2002). Previous findings with T11TS showed that it rejuvenates the immune system and activate T cells to overcome glioma induced immune suppression (Chaudhuri et al., 2015, 2014; Ghosh et al., 2010; Sarkar et al., 2002). T11TS also activates hematopoietic stem cells (Mondal et al., 2018). T11TS was nontoxic as determined by acute and sub-acute toxicity testing (Sarkar et al., 2010; Sarkar et al., 2007). The unavailability of standardized vaccines and potential side effects of the current antibiotic regimen, such as amphotericin B, flucytosine, and fluconazole for the prevention of cryptococcosis in humans thus far necessitates the need of safer immunotherapies, which T11TS qualifies in this disease context (Saag et al., 2000; Wozniak et al., 2011).

The microglial presentation of antigen to brain infiltrating lymphocyte was previously demonstrated by our group along with other microglial parameters during C. neoformans infection and post T11TS administration (Hazra et al., 2019). Just recently, our group also showed that T11TS immunotherapy increased the expressions of the downstream signaling partners of Calcineurin-NFAT pathway in T cells. As a result, IL-2 expression was increased that leads to the activation and proliferation of T cells and clearance of C. neoformans burden from lung and spleen (Sk Md et al., 2020). Encouraged by the previous findings, we tried to descry the key initial signals within the IS of splenic T cell and macrophages that may be involved in determining the T cell signaling in cryptococcal infection and their modulation post-T11TS immunotherapy in our present study.