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

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

trypanosomiasis.

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: jude.uzonna@umanitoba.ca
Dr. Jude Uzonna Dr. Jude Uzonna R G
Host-Pathogen  Interaction Laboratory
© 2015 - 2016 Uzonna (Created by F. Khadem)
Contributions

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

trypanosomiasis.

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
R G