Ex Vivo Generation of CD4+ Th17 Cells to Prevent and Treat Infection from Antibiotic-Resistant Klebsiella Pneumoniae in Immunocompromised Patients

Antibiotic resistance is becoming an increasingly significant challenge facing the healthcare system. Klebsiella (K.)pneumoniae is an important cause of Gram-negative nosocomial infections. Recently strains of K. pneumoniae producing carbapenemase (KPC) have emerged worldwide. KPC- Klebsiella infection is a significant problem for stem cell transplantation (SCT) recipients and patients whose immunity is impaired by leukemia, aplastic anemia, cancer or genetic abnormalities of host defense. Immunocompromised patients infected with KPC- Klebsiella (frequently pan-resistant to antibiotics) have few treatment options and face mortality rates over 50%. Novel strategies to improve the odds of survival for these patients are needed. Emerging evidence suggests that, in addition to neutrophil bacterial defense, Th17 cells and IL-17 augment immunity against many bacteria including K. pneumonia. Th17 cells bridge innate and adaptive responses preventing bacterial translocation by maintaining tight intestinal mucosal junctions. IL-17 promotes hematopoietic stem cell function, myelopoesis, and recruitment of myeloid cells. In an animal model, antigen-specific MHC class II-restricted Th17 cells recognizing K. pneumoniae outer membrane protein (OMP) have been described. These T cells provide serotype-independent protective mucosal immunity against K. pneumoniae, including the multi-drug resistant strains. Here we studied the endogenous reactivity of T cells derived peripheral blood of normal healthy donors against Klebsiella antigens. For antigenic stimulation PBL were exposed to recombinant K. pneumoniae outer membrane protein X (OmpX) and lysate of K. pneumoniae serotype 2 (KP). Flow cytometry revealed intracellular production of IL-17A by CD4+ T cells selectively in 4 out of 5 donors upon stimulation with KP lysate and in 3 out of 5 donors upon stimulation with OmpX protein. The observed Th17 reactivity was confined only to the effector memory compartment (TEM), suggesting an antigen-specific mechanism, but not to unrelated peptide libraries (cancer testis antigen SSX2 and CMV pp65) indicating the existence of pre-established antigen-specific immunity against K. pneumoniae in normal healthy donors. Next we tested the feasibility of expanding the Klebsiella-specific CD4+ T cells in vitro under Th17-polarizing conditions. We used irradiated autologous PBMCs as antigen presenting cells. Significant enrichment of OmpX- and KP-specific cells was achieved in 2 out of 4 tested donors following one week antigenic stimulation. Resulting Th effector cells retained a clinically-desirable feature of polyfunctionality in terms of ability to specifically produce not only IL-17A, but also IFN-γ and IL-2. In summary, for the first time we demonstrate that human Th17 cells derived from normal healthy donors can specifically recognize outer membrane proteins derived from K. pneumoniae . Our observation is analogous to findings from an animal model of Th17-mediated serotype-independed immunity against K. pneumoniae . Importantly, we also show that it is feasible to expand the OmpX-reactive CD4+ T cells in vitro . These results raise the possibility of testing the role of adoptively-transferred antibacterial Th17 cells as a novel strategy of augmenting the host defenses of vulnerable patients colonized with antibiotic-resistant bacteria who are at risk for Gram-negative sepsis. Disclosures: No relevant conflicts of interest to declare.