Multiple factors involved in T cell depletion in AIDS patients with immune non-responder to cART

Background: The replication of HIV-1 can be effectively controlled by combination antiretroviral therapy (cART) at present. However, in approximately 20% of AIDS patients with undetectable viral load under effective cART, the amount of CD4+ T cells continues to be lower than normal, showing poor immune reconstitution, which is called immune non-responder (INR). The mechanism of immune reconstitution in AIDS patients is still unclear, and thus far there is no effective improvement strategy. Aim: To investigate what factors involved in T lymphocyte depletion in INR patients. Methods: A retrospective analysis of a cohort from Suzhou Infectious Disease Hospital was performed. From October 2015 to April 2019, 29 AIDS patients, whose CD4+ T cells were less than 100/ul before treatment, were included in this study. Laboratory results were obtained from the medical records, including basic clinical information, and the results of Flow cytometer detection for CD4 T cells and ELISA for T cell-related cytokines. Results: According to the number of CD4+ T cells after 24 months of treatment, the 29 patients were divided into two groups: 15 patients with CD4+ T cells <200/ul were INR, and 14 patients with CD4+ T cells >200/ul were immune responder (IR). When Compared with IR patients, the total number of red blood cells and white blood cells, especially T lymphocytes in INR patients, was significantly lower, while programmed cell death protein 1PD-1on CD4+T and CD8+ T lymphocyte surface were significantly higher in INR group, which implies that the probability of T cell apoptosis was greatly increased (p<0.05). ELISA results showed that the overall production of T cell-related cytokines were lower in INR group, but the amount of cytokine secreted from each T cell was higher in INR group than that in IR group due to the significant decrease of T cell in this group. Furthermore, the proportion and the killing efficiency of CD56dim subgroup of NK cells were significantly higher in INR group.

Conclusion: Multiple factors are involved in the immune reconstitution in AIDS patients, including myelosuppression, T cell destruction enhanced through PD-1 pathway, overactivation of T lymphocyte, and higher killing efficiency of CD56dim subgroup of NK cells.

Background
Approximately 20% AIDS patients are unable to achieve good immune reconstitution although virus replication is well controlled by combination antiretroviral therapy (cART).
Immune non-responder (INR) state usually occurs in patients who start treatment late, and whose CD4 + T cell numbers still stay at a lower level, i.e. [1]. The definition of INR is that the virus replication is controlled by cART for 1 year, but the CD4 + T cell count is still less than 200/ul by at least two years after the treatment [2]. On the other hand, the immune responder (IR) is defined when CD4 + T cell count is greater than 200/ul after the treatment.
It has been reported that HIV-1 infection affects bone marrow function [3],while the immune system returns to normal in 60%-80% AIDS patients after effective cART [4], indicating that HIV-1 infection not only induces myelosuppression but also increases lymphocyte destruction. T cells are produced by bone marrow hematopoietic stem cells and enter the peripheral blood after thymic maturation. In the meantime, the programmed cell death protein 1 (PD-1) is one of the inhibitory receptors associated with lymphocyte destruction [5], which can be expressed on the surface of T lymphocytes, and lymphocytes that overexpress PD-1 can trigger their own apoptosis resulting in a reduction in their number [6].
T cell activation-associated cytokines (IL-4, IL-6, IL-10, IL-17 and IFN-γ) secreted in various stages of T cell reflect the activation state of T cells, and cytokine disorders can be observed during HIV infection [7], especially IFN-γ, one of the most important T cell activation markers [8][9][10]. Studies have showed that lymphocytes with excessive cytokine secretion can also induce activation induced cell death (AICD) [11].
NK cells are mainly differentiated from hematopoietic stem cells. According to the distribution of CD56 molecules, they are divided into CD56 bright with more cytokine secretion function and CD56 dim with more cells killing function [12]. When stimulated by certain cytokines (such as IL-15), CD56 bright NK cells can differentiate into CD56 dim NK cells, and the latter contain Perforin and Granzyme B intracellular for the killing function and accounts for 80%-90% of the total number of NK cells [13,14]. Activated NK cells can effectively clear virus-infected cells, including other virus-associated lymphocytes [15].
In this study, we found that poor immune reconstitution in INR patients is due to reduced production resulted from the myelosuppression, and increased destruction of lymphocyte, which is caused by the over expression of PD-1 on cell surface, over-activation of T cell and enhanced killing activity of NK cells. Our finding suggested that multiple factors are involved in the immune reconstitution in AIDS patients.

Methods
From October 2015 to April 2019, 29 AIDS patients who had less than 100/ul CD4 + T cells before cART treatment and consistently completed same cART regimen for 24 months were enrolled for the study. Informed consent was signed for all patients and testing involved the project has been certified by the relevant ethics committee. The antiviral treatment regimen used was tenofovir/zidovudine + lamivudine + efavirenz/nevirapine. After cART, their plasma viral load was below the detection limit (< 50 copies/ml).  (Table 1).  group than in IR group (Fig. 3.a). However, the amount of most cytokines, i.e. IL-4, IL-10, IL-17 and IFN-γ (except IL-2), secreted from a single T cell is significantly higher in INR patient than in IR patients ( Fig. 3.a). This is due to the significantly lower T cell number in INR than in IR group as showed in Fig. 1.e-f. The increased cytokine secretion in single cell reflects the over-activation of T lymphocytes, and might further induce the occurrence of 7 AICD in T lymphocytes.

The proportion and the killing ability of CD56 d i m NK cells significantly increased in INR patient
NK cells play a key role in innate immunity and immune surveillance, and regulate immune responses, as well as interact with T cells through a complex network of [18]. Our flow cytometry analysis showed that there is no significant difference in the percentage of NK cells in peripheral blood between two group patients ( Fig. 4.a) (p < 0.05). However. the proportion of CD56 dim subgroup of NK cells was significantly higher in INR group than that in IR group ( Fig. 4.b) (p < 0.05). We further tested the killing capability of CD56 dim NK cells by determining the expression level of CD107a, Granzyme B and Perforin in these cells. The data showed that these killing factors were surprisingly increased in the INR group when compared with the IR group (Fig. 4.c-e, p < 0.05), these results suggest that the increased number and function of CD56 dim NK cells might be a factor responsible for the depletion of T lymphocytes in the INR patients.

Discussion
During HIV-1 infection, CD4 + T cells are largely destroyed. Therefore, maintaining sufficient CD4 + T cell level may depend primarily on the ability of lymphocyte production in the host. It was reported that CD34 + hematopoietic progenitor cells in the blood of HIVinfected patients were significantly destroyed and unable to maintain sufficient lymphocyte count in AIDS patients [10], and the disease progression is associated with exhaustion of lymphopoiesis in spite of suppression of viral replication [19]. All of these suggest that the progression of AIDS is associated with sustained damage to the lymphoid system, individual genetic differences and latent infections [20]. In this study, we found that bone marrow suppression and PD-1 overexpression on the surface of T lymphocytes were more severe in INR group, which are likely to be responsible for T lymphocyte depletion.
At present, it is believed that the cause of abnormal activation of the immune system in AIDS patients is related to the persistent low-level viral replication from HIV latent reservoir, ectopic parasitization of intestinal flora, and other viral infections [21]. Cytokine production disorders are one of the manifestations of abnormal activation of the immune system. Studies have found that the level of IL-7 in peripheral blood of HIV-infected patients is elevated, and is negatively correlated with CD4 + T-lymphocyte count,but the level of IL-7 receptor is decreased [22]. Cytokines IL-2, IL-4, IL-10, IL-17 and IFN-γ are closely related to the activation, proliferation and function of T lymphocytes [23][24][25]. Our study found that those cytokine concentrations in patients with INR were much lower than that in the IR group. The overall low-level concentration of T cell-related cytokines in INR is insufficient to stimulate the bone marrow hematopoiesis. However, when we converted it to single T cell level, we were surprised to find that the INR patients had higher cytokine productionthan the IR patients. This suggests that T lymphocytes might be over-activated and cause AICD and finally lead to the depletion of T lymphocytes. Other studies have reported that IL-2 is of limited help for HIV treatment [26]. We have also found that there is no significant difference in IL-2 secretion at single T cell level in the two groups ( Fig. 3.b), further proved that there is no significant correlation between IL-2 and AIDS development.

NK cells play an important role in regulating T cells by directly lysing activated T cells. For
example, during LCMV infection, selective deletion of NK cells can restore the number of activated CD8 + T cells, thereby promote virus clearance [27]. NK cells can also directly remove CD4 + T cells through perforin pathway, which causes CD8 + T cells to fail to exert antiviral effects [28]. Our present study found that the average proportion and the killing function of CD56 dim NK cells in the INR group were higher than that in the IR group. We speculated that even through systemic therapy, virus components from HIV-1 latent infection continue to stimulate NK cells, resulting in an increased proportion of CD56 dim NK cell subsets and enhanced killing capability, which might destroy more T lymphocytes and finally lead to the depletion of CD4 + T lymphocytes.
In summary, the poor immune reconstitution in AIDS patients might be resulted from