Ex Vivo Generation Of CD4⁺ T 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.