Host-Pathogen Interaction Laboratory
© 2015 - 2016 Uzonna (Created by F. Khadem)

Identification of conserved and immunodominant cross-species protective antigen of

Leishmania and its responding clonal CD4+ T cells.

We   used   reverse   immunology   and   proteomics   approaches   to   identify   naturally   processed   L.   major    peptides presented   by   MHC   II   molecules   on   infected   mouse   dendritic   cells.   One   of   the   peptides   derived   from   highly conserved   glycosomal   phosphoenolpyruvate   carboxykinase   (PEPCK),   induced   strong   proliferation   and   IFN- g     production   by   CD4 +    T   cells   from   infected   mice.   PEPCK   is   expressed   in   glycosomes   of   L.   major    promastigotes and    amastigotes    and    also    induced    proliferation,    IFN- g     and    granzyme    production    in    peripheral    blood mononuclear   cells   (PBMC)   from   infected   human   patients   that   healed   cutaneous   leishmaniasis.   Vaccination with   PEPCK   peptide,   DNA   or   recombinant   protein   induced   strong   protective   immunity   against   both   L.   major and   L.   donovani ,   (the   causative   agents   of   cutaneous   and   visceral   leishmaniasis,   respectively)   challenge   in   both the   resistant   and   susceptible   mice.   Importantly,   we   generated   PEPCK   peptide-MHC   II   tetramer   and   reliably demonstrate    the    activation,    expansion,    effector    activity,    contraction    and    stable    maintenance    of    PEPCK- specific CD4 +  T cells in L. major -infected mice. Mou et al, 2015. Science Translational Medicine, In press

Regulation of liver immunity in visceral leishmaniasis by hepatic stellate cells (HSCs).

We   showed   for   the   first   time,   that   HSCs   are   infected   with   L.   donovani    in   vivo    and   in   vitro    and   this   infection leads   to   the   production   of   immunoregulatory   (IL-2,   IL-6   and   TGF-     cytokines   known   to   induce   Tregs.   We further   demonstrated   that   L.   donovani    infection   leads   to   expansion   of   HSCs   in   a   p110 d -dependent   manner, and   this   correlated   with   proliferation   of   hepatic   Tregs   in   vivo.   In   vitro    studies   clearly   show   that   L.   donovani - infected   HSCs   induce   CD4 +    T   cells   to   become   Tregs   and   also   expand   Tregs   in   a   p110 -dependent   manner. Targeted    depletion    of    HSCs    during    infection    caused    a    dramatic    reduction    in    liver    Treg    numbers    and proliferation,   which   was   associated   with   a   decrease   in   IL-10   production   by   hepatic   T   cells   and   a   more   efficient parasite   control.   These   studies   demonstrate   for   the   first   time,   the   critical   role   of   HSCs   in   the   pathogenesis   of VL,   and   suggest   that   the   enhanced   resistance   of   p110δ D910A    mice   to   L.   donovani    infection   is   due   in   part   to impaired expansion and inability of their HSCs to induce and expand Tregs in their liver. Khadem   et   al,   2014,   Future   Microbiol.   9(7):901-15;   PLoS   Negl   Trop   Dis.19;8(6):e2951;   Hepatology.   2015   [Epub ahead of print]

Requirement for live parasites for maintenance of resistance to Leishmania major.

In    this    study,    I    showed    that    L.    major -infected    mice    can    be    manipulated    to    mount    an    exclusive    type    1 immunity    that    completely    eliminate    parasites    (contrary    to    the    existing    dogma    that    L.    major    persists indefinitely   at   the   infection   site   and   its   draining   lymph   nodes   in   resistant   individuals   or   mice).   However, complete   parasite   elimination   leads   to   loss   of   Leishmania-specific   effector   cells   and   immunity   to   reinfection. This   seminal   observation,   which   has   been   cited   as   one   of   the   milestone   discoveries   in   immunobiology   of cutaneous leishmaniasis (Noben Trauth & Sacks, Nat Rev Immunol, 2002 11:845) , has enormous implications for vaccine    designs    against    leishmaniasis.    They    suggest    that    persistence    of    live    parasites    is    required    for maintenance   of   anti-Leishmania   immunity,   and   could   explain   why   heat-killed   Leishmania   vaccine   confers weak and short-term anti-Leishmania immunity. Uzonna, J. et al., 2001. J. Immunol. 167:6967; Uzonna, J. &. Bretscher. 2001 Eur. J. Immunol. 31:3175.

Discovery of effector memory and central memory Leishmania-specific CD4+ T cells.

In   this   seminal   manuscript,   we   showed   that   live   parasites   are   important   for   maintaining   Leishmania-specific effector   memory   cells   that   mediate   delayed   type   hypersensitivity   (DTH)   reaction,      rapid   IFN- g    secretion   and parasite   destruction.   However,   our   study   also   revealed   the   induction   of      central   memory   CD4 +    T   cells   that   are capable   of   mediating   delayed   anti-Leishmania   immunity   in   the      absence   of   live   parasites.   This   is   the   first report    of    the    induction    and    function    of    CD4 +     central    memory    T    cells    during    a    protozoan    infection. Significance:   Our   study   underscores   the   need   to   target   both   effector   and   central   memory   arms   of   T      cells   in vaccination strategies against leishmaniasis. Uzonna, J.E., C. Zaph*, S. Beverley, and P. Scott. Nat Med 2004: 1104-1110.

Regulation of anti-Leishmania primary and memory responses by phosphoinositide 3 

kinase (PI3K).

My   laboratory   has   continued   to   be   in   the   forefront   of   identifying   host   factors   that   regulate   anti-   Leishmania immunity.   We   focused   on   PI3K   because   they   play   important   role   in   regulating   cellular   processes   including inflammation,   immune   regulation,   proliferation,   etc.   We   found   that   mice   with   an   inactive   knock-in   mutation are   hyper-resistant   to   L.   major    (developed   minimal   or   no   lesion   and   rapidly   control   parasite).   This   enhanced resistance    was    independent    of    mouse    genetic    background.    Paradoxically,    the    enhanced    resistance    was associated   with   dramatically   impaired   T   cell   proliferation      and   IFN- g    response.   We   found   that   enhanced resistance   was   related   to   impaired   regulatory   T   cell      expansion   and   function.   Interestingly,   despite   this enhanced   primary   resistance,   deficiency   of   p110 d       signaling   significantly   impaired   secondary   (memory)   anti- Leishmania   immunity   such   that   these   mice   were   unable   to   control   secondary   virulent   L.   major    challenge.   This impaired   memory   response   was   related   to   inability   to   generate   effector   memory-like   T   cells.   Significance:   For the   first   time,   this   study   demonstrated   the   role   of   p110 d    isoform   of   PI3K   in      resistance   to   L.   major    that   in   the absence   of   concomitant   induction   of   Treg,   minimal   T   cell      proliferation   and   IFN- g    is   sufficient   for   complete resolution   of   L.   major    infection.   It   further   showed   that      this   enzyme   could   be   a   novel   therapeutic   target   for treatment   of   leishmaniasis.   Indeed,   we   have      patented   this   as   a   treatment   regimen   for   leishmaniasis.   In addition, this study suggests that the  quality of anti-Leishmania memory response is regulated by PI3K. Liu, D, et al. 2009 J. Immunol 199:1559-1566; Liu & Uzonna, 2010 J. Immunol 5;184(6):3098-105.

Inoculation of killed parasites causes loss of established anti-Leishmania immunity.

While   vaccination   with   killed   parasites   or   parasite-derived   molecules   do   not   elicit   any   significant   protective immunity,   recovery   from   primary   (natural   or   experimental)   L.   major    infection   in   both      humans   and   animals results    in    long    lasting    and    solid    immunity    against    virulent    challenge.    This    so-    called    infection-induced resistance   is   regarded   as   a   gold   standard   for   development   of   any   vaccine      against   leishmaniasis,   and   is   a   very strong   indication   that   vaccination   against   leishmaniasis   is      feasible.   For   the   first   time,   we   showed   that inoculation   of   killed   Leishmania   major   into   immune   mice      leads   to   rapid   and   sustained   loss   of   infection- induced   immunity   by   an   IL-10   and   Treg   dependent      mechanisms.      Significance:   This   study   could   help   explain why   unlike   active   infection,   vaccination   with   whole   heat-      killed   Leishmania   does   not   confer   immunity   and   in some   cases   could   predispose   individuals   in      endemic   regions   who   may   have   developed   immunity   due   to natural   exposure   to   become   more      susceptible   to   new   infections.   It   suggests   a   new   approach   that   can   lead   to a   truly   effective   anti-      Leishmania   vaccine.   This   work   has   been   cited   more   than   30   times   and   has   been   cited as a milestone  in Leishmania immunology research. Okwor et al., 2009. Proc Natl Acad Sci U S A. 106:13951-6.

Discovery of a novel pathologic T cell population in experimental African


In    the    process    of    determining    the    cellular    origin    of    IL-10    and    IFN- g     in    trypanosome-infected    mice,    I      discovered    and    characterized    a    novel    matrix-adherent    lymphocyte    population    that    play    critical    role    in      regulating   the   outcome   of   T.   congolense   infection   in   mice.   These   cells   express   the   ab   TCR   receptor,      CD3   and CD4   molecules   and   appear   to   be   restricted   by   MHC   class   II   molecules.   The   induction   and      activation   of   this unique    cell    is    trypanosome-dependent,    but    its    effect    is    trypanosome-independent,        suppressing    both antigen-   and   mitogen-activated   T   and   B   cells.   Significance:   I   take   pride   in   my   contribution   to   this   aspect   of research   because   until   then,   very   little      was   known   about   the   nature   of   immune   cells   mediating   suppression in    Trypanosomiasis.    Generalized        immunosuppression    has    been    known    to    be    a    feature    of    African trypanosomiasis,   but   hitherto,   the      cells   mediating   it   and   the   mechanisms   have   remained   elusive.   This discovery   formed   a   strong      foundation   upon   which   numerous   important   discoveries   on   the   mechanisms   of acute death  (susceptibility) and resistance in experimental African trypanosomiasis were made. Uzonna, J. et al., 1998. J. Immunol., 161:6189
Professor, Manitoba Health Research Chair Professor in Immunology, Department of Immunology, Department of Medical Microbiology, Brady Faculty of Health Sciences, College of Medicine, University of Manitoba. Address: Department of Immunology, Brady Faculty of Health Sciences, College of Medicine, University of Manitoba, 750 McDermot Avenue, 425 Apotex Centre, Winnipeg, Manitoba, R3E 0T5. Phone: 1-204-977-5659 Fax: 1-204-789-3921 Email:
Dr. Jude Uzonna Dr. Jude Uzonna R G