**3. Organoids in the immunology field**

The knowledge concerning the interactions of the immune system with other tissues has been gained mainly from animal models and/or cell lines co-cultures. Nevertheless, some interactions between human cells cannot be addressed with murine models or cell lines which are usually transformed or genetically modified [19]. For instance, a specific immune cell morphology is required to maintain the tissue properties and, moreover, the immune system needs of multiple cell types interactions for appropriate functioning. Similarly, there are some aspects that cannot be extrapolated in mice due to, for example, different protein pattern expressions in human and mice. Thus, immunology researchers are starting to get the benefits of using organoids, for a better comprehension of the immune cell interactions with other tissues, its development, homeostasis and in the bout of disease. The organoids approach maintains those cells in a near-native state, mimicking more accurately its original state and environment, providing researchers with a new effective tool.

The main challenge in the use of organoids in immunology resides in the fact that the organoids technology cultures only epithelial cells. However, a more complete resource for immunological research can be developed by co-culturing these organoids with other elements.

The number of publications showing multiple co-cultures has spiked up in the past decade, particularly in the last five years [20–22]. In order to develop effective interventions to preserve health and defeat diseases it is necessary to know how immune cells coordinate their activities to initiate, modulate, and terminate inflammation. Immune cells and molecules released by immune cells promote inflammation processes that are mediating the interactions between these cells [23].

These studies have revealed not only the importance of the presence (or absence) of immune cell derived factors in the epitheliums in culture, but also the need of the reciprocal communication with the immune system. A work concerning the role of macrophages and fibroblasts on myoblast proliferation and migration highlights the importance of multicellular communication [24]. Thus, co-culture of either macrophages or fibroblasts with myoblasts prompted a significant increase in myoblast proliferation. Conversely, in the triple co-culture, although macrophages continued promoting myoblast proliferation, they had a negative effect over the ability of fibroblasts to enhance myoblast migration [25]. Another study, using single-cell transcriptomics, highlighted that intestinal stem cells can function as non-classical antigen-presenting cells for CD4+ T cells. Moreover, these interactions, directly or through activated T cell-derived cytokines, seem to play a role in the intestinal epithelium differentiation [26].

The intestinal mucosal barrier function and the immune responses against invading pathogens seem to be regulated by the interaction between intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) [27]. IELs represent a heterogeneous population of activated and antigen-experienced T cells. A novel culture system of intestinal 'enteroids' has allowed the study of the complex interactions between IECs and immune peripheral T cells in long-term co-cultures. The development of these long-term co-cultures allowed the study of cell survival, proliferation, differentiation and IECs behavior. Moreover, IECs and T cells co-cultures revealed that peripheral T cells activated in the presence of enteroids acquire several features of IELs, including morphology, membrane markers and movement in the epithelial layer [27]. Similarly, mouse-derived enteroids cocultured with intestinal myofibroblasts and macrophages boosted their growth and differentiation [28].

In the same line, another study with intestinal organoids underlined the importance of the interactions between immune cells and other tissues for optimal maturation. In this work, the inclusion of the immune component (co-cultured with human T lymphocytes) into the differentiation protocol to form human pluripotent stem cell-derived intestinal organoids (hIOs) from hPSCs, enabled hIOs maturation. hIOs co-cultured with human T lymphocytes displayed expression levels of mature intestinal markers equivalent to adult intestinal epithelium, as well as increased intestine-specific functional activities, retaining their maturation status even after their *in vivo* engraftment. This study has proven the needless for animal models and *in vivo* maturation when working with organoids [29].

Holokai, L. *et al.* were among the first researchers to successfully obtain a multiple organoid-co-culture involving cytotoxic T lymphocytes (CTLs) and Helicobacter pylori-infected gastric organoids. CTLs express programmed death 1 (PD1) on the surface. When PD1 interacts with its ligand, CTLs cannot induce apoptosis. Thanks to this approach they discovered that PD-L1 signaling induces cellular proliferation and survival, leading to an increased expression of PD-1, IL-2 and IFNγ in lymphocytes [30].

Overall, epithelial organoid cultures, whether derived from iPSCs or AdSCs, constitute a promising platform for immunological research for several applications, allowing, among others, to study immune cell–epithelial cell interactions in the context of pathogenic infections or sterile tissue damage [19]. In this sense, the vast majority of organoid studies about the immune system and its effects on epithelial differentiation and function have been performed on intestine-like structures. However, it would be useful to have similar works with different organoid systems such as skin or lung, which also interact with both immune cells and commensal microorganisms [31].

Despite the amount of work already accomplished regarding the immune system, there is still a long way to go in inflammation research, due to the current lack of optimal immune cells organoids cultures.
