Ex Vivo Generation of CD4+ Th17 Cells to Prevent and Treat Infection from Antibiotic-Resistant Klebsiella Pneumoniae in Immunocompromised Patients
Authors: Muranski Pawel , Franco-Colon Manuel , Chinnasamy Dhanalakshmi , Chen Kong , Stegemann Scott , Sui Jinwen , Ito Sawa , Sabatino Marianna , Stroncek David F. , Palmore Tara , Kolls Jay , Barrett A John 
Published: November 2019

Abstract
Klebsiella (K.)pneumoniae is an important cause of Gram-negative nosocomial infections. Recent worldwide emergence of K. pneumoniae producing carbapenemase (KPC) poses a significant problem for stem cell transplantation (SCT) recipients and patients whose immunity is impaired. Such patients have few treatment options and face mortality rates over 50%. Novel strategies are needed. Emerging evidence suggests that, in addition to granulocytes, 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, promote hematopoietic stem cell function, myelopoiesis, and recruitment of myeloid cells. In an animal model, antigen-specific MHC class II-restricted Th17 cells recognizing K. pneumoniae outer membrane protein (Omp). 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 of normal healthy donors against Klebsiella antigens. PBL were exposedtorecombinant 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 virus and tumor antigen control peptide libraries indicating pre-established antigen-specific immunity against K. pneumoniae in normal healthy donors.
Next the feasibility of expanding the Klebsiella-specific CD4+ T cells in vitro under Th17-polarizing conditions was tested. Magnetic bead purified CD4+ memory T cells from normal donors were co-cultured for 10-14 days with autologous monocytes or irradiated PBMCs pulsed with KP lysate, recombinant OmpX or control peptide libraries derived from BK virus. Cultures were maintained in media containing Th17 polarizing cytokines (TGF-1, IL-1β, IL-6, and IL-23) and supplemented with IL-7, IL-15, and IL-2. Two rounds of stimulation were performed and resulting T cells were tested by FACS for their ability to recognize target antigens. Both KP-lysate and OmpX-stimulated T cells from majority of the donors demonstrated robust antigen-specific production of IL-17A, TNF-α, and IFN-γ upon exposure to respective cognate antigens with some cross-reactivity between them. Control autologous BK-virus specific T cells showed minimal reactivity against KP lysate or OmpX, underscoring antigen-specific nature of observed antibacterial responses. Comparable induction of anti-KP responses was achieved using CD4+ T cells from pre-transplant patients with leukemia, but there was marked impairment when T cells isolated from recipients of allogeneic SCT were used. Importantly, T cells generated using KP serotype 2 lysate demonstrated robust recognition of KP lysate from KP396 (serotype 1), while significantly lower cross-reactivity was observed against lysate from E. coli and no responses were seen upon stimulation with antigens from Gram-positive bacteria (S. aureus and S. pneumoniae),
In summary, for the first time we demonstrate that human Th17 cells from healthy donors specifically recognize K. pneumoniae OMP, analogous to the animal model of Th17-mediated serotype-independent immunity against K. pneumoniae. Notably, we efficiently expanded the KP-reactive CD4+ Th17 cells in vitro and these cells recognized KP antigens independently of serotype, indicating the feasibility of targeting simultaneously many strains of KP including the multi-drug-resistant isolates. These results raise the possibility of testing adoptively-transferred antibacterial Th17 cells as a novel strategy to augment host defenses of patients colonized with antibiotic-resistant bacteria at risk for Gram-negative sepsis.

Disclosures
No relevant conflicts of interest to declare.