**5.1 Memory CD8 T cells**

The memory T cell population formation process of CD8 T cells during *C. trachomatis* infection differs from the responses detected against acute infection agents. Some expansion of CD8 T cells is observed during primary infection in mouse models. However, the fact that *C. trachomatis* CrpA antigen-specific CD8 T cells does not proliferate in the expected number and rate during secondary infection with *C. trachomatis* indicates insufficient formation of memory CD8 T cells [105]. While the elimination of the infected cell will deprive the organism of its intracellular niche, with the deterioration of the adaptive immune response, both the infections cannot be cleared and permanent immunity is not formed.

Differential programming of memory CD8 T cells when stimulated by agents such as *C. trachomatis* that cause persistent infection is attributed to the environment at the onset of infection. Namely, for the activation of Chlamydia-specific naïve T cell clones, both T cell receptors must recognize Chlamydia-derived peptides presented by dendritic cells, and co-stimulatory molecules on the dendritic cell must interact with those on the T cell. Interactions of some of these co-stimulatory molecules cause upregulation of the T cell response, while others cause downregulation of the T cell response [81]. One of the inhibitory interactions is the binding of programmed death ligand 1 (PD-L1) on the dendritic cell with PD-1 on the T cell [106]. A study of murine infection with *C. trachomatis* found PD-L1 upregulation in the uterus and PD-L1 upregulated in *in vitro* infected cells. As a result, CD8 T cell expansion is impaired and the development of CD8 memory responses is inhibited. This upregulation leads CrpA-specific CD8 T cells to the Tcm phenotype, which is found in secondary lymphoid organs and lymphatic vessels but has limited effect in peripheral tissues, instead of the Tem phenotype, which contributes to clearance of pathogens in peripheral tissues. When antibodies that block the interaction of PD-1 with PD-L1 were used during primary infection, or when knockout animals were used in both molecules, there was a marked increase in the number of T cells responding to secondary C. trachomatis infection, with more IFN-γ producing CD8 T cells. The memory CD8 T cell population shifted toward the Tem phenotype, resulting in faster clearance of infection [105].

On the other hand, there are studies suggesting that *C. muridarum* CD8 T cell response contributes to the pathology [101, 107, 108]. For this reason, it has been suggested that PD-L1-mediated inhibition may be a mechanism that prevents cellmediated uterine pathology by CD8 T cells [101]. In the study by Peng et al., immunoinhibitory molecules TIM3 and PD-L1 were blocked in C. muridarum-infected mice. As a result, it was observed that uterus and oviduct pathology increased [109]. This finding reveals that immunoinhibitory molecules regulate inflammation by preventing T cell activation and cytokine production.

#### **5.2 Interferon-gamma**

IFN-γ, which is released from both innate cells such as macrophages and NK cells and CD4 and CD8 T cells in response to chlamydial infection, is a critical cytokine for inhibiting chlamydial growth [104]. Gamma interferon is responsible for the upregulation of some interferon-induced genes that may help control intracellular bacterial replication in infected epithelial cells [110]. As a result, some protective mechanisms emerge in infected cells. Iron metabolism, a critical mineral for Chlamydia, is blocked [111, 112]. Expression of the tryptophan-decyclizing enzyme indoleamine-2,3-dioxygenase (IDO) is induced, which breaks down tryptophan necessary for the survival of most Chlamydia species. Also, IFN-γ enhances the phagocytic abilities of macrophages and also ingestion and destruction of *C. trachomatis* [71, 113].

The effect of IFN-γ on tryptophan metabolism was reviewed by Vasilevsky et al. during *C. trachomatis* infection in humans and the IFN-γ signaling cascade leads to upregulation of the IDO enzyme in genital tract epithelial cells. The enzyme catalyzes the breakdown of tryptophan to N-formylkynurenine and kynurenine, thereby disrupting intracellular tryptophan stores [58]. *C. trachomatis* deprived of this essential amino acid has been shown to die due to tryptophan starvation. There are also chlamydia species that have adapted to tryptophan starvation by transforming into the non-replicating persistent form. After IFN-γ removal and subsequent tryptophan production, these persistent forms rapidly become replicative [76, 114, 115]. Since some genital tract strains also express tryptophan synthase, they can overcome tryptophan depletion by producing their tryptophan using exogenous indole [116]. In addition, kynurenine, a by-product of tryptophan catabolism, inhibits host CD4 T cells, thus reducing IFN-γ production and ultimately limiting its overall production. It has been reported that it can lead to the re-activation of *C. trachomatis* [110].
