Over the past few years our lab has followed various lines of research to understand T cell biology both independently or by collaborating with other investigators within MUSC or elsewhere. Our research endeavors and future plans are listed below:
Activation Induced Cell Death (AICD) of a T cell:One of the major focuses of our laboratory is to understand the pathways underlying AICD of Cytolytic T Cells (CTL) so as to develop a robust and long-term CTL response. We have earlier shown that a large fraction of melanoma epitope MART-127-35 specific primary CTL generated in an in vitro peptide-loaded DC-based CTL generation protocol undergo AICD after the very first secondary encounter of the cognate antigen (Mehrotra et al., Cancer Res. 2003; 63: 5607-14). Further, we made a novel observation that AICD in these MART-127-35 specific CTL is not triggered by the usual external death receptor (FAS, TNFR, etc.) -mediated signaling, is Caspase in-dependent and c-jun N terminal kinase (JNK) dependent (Mehrotra et al., J Immunol 2004; 173: 6017-24). More recently, we have identified that the superoxide dismutase mimetic compound, MnTBAP, and several other antioxidants also protect MART-127-35 specific CTL from AICD without interfering with the IFN- response and inhibiting JNK phosphorylation (Norell et al., Cancer Res. 2009; 69: 6282-9). Our data that formed a major part of our recently funded RO1 (funded by NIH/NCI R01CA138930; 04/01/2010 - 03/31/2015) application addresses the “Role of Oxidative Stress and Activation Induced cell Death in memory T cell persistence”. Briefly, CTL activated by cancer vaccination acquire an antigen-experienced/memory phenotype and their functional analysis confirms that although these T cells are virtually competent to attack and destroy neoplastic cells, the reason for their inefficacy in controlling tumor regression has been puzzling. Our data suggests that it is both oxidative stress and antigen mediated preferential death of antigen-experienced/memory phenotype bearing CTL that might result in inadequate function. This RO1 grant will elucidate if: a). Differential redox state of the T cell subsets (TCM vs. TEM) regulates sensitivity to oxidative stress induced apoptosis and AICD by effecting distinct intrinsic death pathways. b). Antioxidant treated or antioxidant enzyme transduced tumor epitope specific T cells would improve tumor regression, persistence and memory response in an in vivo pre-clinical model.
T cell development in immunity and tolerance:Since the TCR is a single specificity-determining molecule in T cell function, T cell adoptive immunotherapy or transfer of TCR genes into patient T cells is rapidly becoming a promising approach to treat cancer. Understanding T cell biology with respect to tumor response is thus important for modulating any T cell mediated therapy successfully. In collaboration with Dr. Michael Nishimura at MUSC we developed a novel TCR transgenic mice using HLA-A2 restricted high affinity TCR reactive to human tyrosinase-derived peptide YMDGTMSQV isolated from a class-I restricted CD4+ T cells of tumor infiltrating lymphocytes (TILs) of a patient with metastatic melanoma. Transfer of this TCR to normal human PBL-derived T cells led to HLA-A2 restricted CD4+ and CD8+ T cells capable of recognizing human melanoma cells. This led us to hypothesize that T cells engineered to express this high affinity TCR could effectively treat patients with HLA-A2+, tyrosinase+ melanomas. Thus, using funds from one of our NIH R21 grant we developed a clinically relevant mouse model for testing this hypothesis. 25% CD4+ and 75% CD8+ T cells in our transgenic mouse (named h3T on C57BL/6 background) express transgenic TCR (manuscript submitted). Surprisingly, this HLA-A2 restricted TCR was positively selected on both CD4+ and CD8+ T cells. When these mice were developed on class I deficient background TCR bearing CD8+ T cells were absent from the periphery. Similarly, absence of TCR bearing CD4+ T cells was observed in class II deficient TCR transgenic mouse. However, when this TCR transgenic mouse was developed on HLA-A2+ background then > 90 % transgenic TCR bearing T cells exhibited CD3+CD4-CD8- double negative phenotype and developed develop spontaneous autoimmune vitiligo. Interestingly, the transgenic TCR bearing CD4+, CD8+ and CD4-CD8- T cells obtained from h3T and h3TA2 mice respectively were functional and retained the capability to control growth of human melanoma in a xenograft in vivo model. Thus, our data shows that both class I and class II restricted ligands were responsible for positive selection of the same TCR on CD8+ and CD4+ T cells respectively and the presence of CD4 and CD8 co-receptor or even complete absence of co-receptors had little effect on its functional capacity. Thus, this novel TCR transgenic mouse has provided us with unique opportunity to collaborate with various investigators within MUSC or elsewhere and address issues of thymic selection, autoimmunity and tumor control.
A testimony to our successful collaboration is a recently funded RO1 grant (NIH/NCI R01AR057643; 10/01/2010 - 03/31/2015) developed with Dr. Caroline Le Poole at Loyola University, Chicago using preliminary data generated from our novel TCR transgenic mice h3TA2 that displays progressive depigmentation of the pelage in a model akin to human vitiligo we are evaluating if “Modulating tolerance in a spontaneous mouse model of autoimmune vitiligo” will help control the disease progression.
Additional lines of investigation:
a).Identifying high affinity TCR for prostate cancer:This is one other NIH R21 funded research project that we have been working on since last two years. Although the funding for this research ended last year we are continuing to work in this area in order to expand this area of research to identify a suitable TCR receptor.
b). To elucidate role of cytokines in T cell persistence:Since, cytokines play important roles in T cell priming, maintenance and memory generation our group is also evaluating the effect of key cytokines from Th1, Th2, Th9, Th17 family on long term T cell persistence and AICD. A possible modulation at the level of redox proteins or endogenous/exogenous thiol levels as the result of the presence of different cytokines has not been comprehensively evaluated. We have shown that human T cells cultured in the presence of IL-15 exhibit increased expression of anti-oxidant molecules Glutathione reductase (GSR), Thioredoxin reductase 1 (TXNDR1), Peroxiredoxin (PRDX), Superoxide dismutase (SOD) (Kaur et al., 2011; Cytokine). Overall, this study suggests that T cells cultured in IL-15 show increase persistence not only due to increased anti-apoptotic proteins but also due to increased anti-oxidant levels, which is further complimented by increased cytolytic effector functions.
Collaborations: Our other on going collaborative efforts include:
i).P01CA154778 Michael I. Nishimura (PI) 09/21/2011 – 08/31/2016 Title: Impact of AICD on TCR transduced T cells for Adoptive Immunotherapy Role on Project: PI - Project 3, Christina Voelkel-Johnson (Co-PI)
ii).Title: Anti-Tumor Response by Class I Restricted CD4+ vs. CD8+ T Cells Grant # 1R01CA155274-01 (NIH/NCI), pending resubmission Role on Project: PI
iii). Role of Sphingosine Kinase in T cell persistence:An important collaboration that we have developed is with Dr. Besim Ogretmen in the Department of Biochemistry at MUSC. Our research effort is directed towards understand the role of Sphingosine kinase -1 in tumor immunotherapy. Using the preliminary data generated through this collaboration we had been successful in securing the SCTR grant and are hopeful that our continued effort would yield strong data that could firmly establish the role of sphingosine kinase in T cell function.
iv). Effect of Tyrosinase specific T cells on Eye:It has been convincingly demonstrated that vitiligo is a systemic autoimmune disease affecting pigmented cells throughout the body rather than a purely cutaneous problem. The development of vitiligo can occur with both cutaneous and ocular melanoma and results in a more favorable course. Furthermore, there is a strong association between vitiligo and uveitis, and this appears to be most common in vitiligo associated with melanoma (19%). This is seen in non-melanoma vitiligo patients as well. It has also been recently reported that ocular autoimmunity develops after successful Tumor-Infiltrating Lymphocyte Immunotherapy for metastatic melanoma. Thus, in collaboration with Dr. Shahid Husain in Storm Eye Institute at MUSC – an expert in ocular research we propose to investigate if there exists a correlation between development of ocular autoimmunity and spontaneous vitiligo in h3TA2 mice. We have initiated our studies to determine the parameters that influence the visual function, such as pattern electroretinograms (ERG); retinal ganglion cells counts and intra-ocular pressure. It is known that retinal ganglion cells are the first one to die under stress/ischemic conditions so a legitimate endpoint will be to determine the structural integrity of retinal ganglion cells under pathological conditions. With the data obtained thus far we are very hopeful that our continued effort could help us identify a role for T cell in pathology of eye.
v). Effect of Tyrosinase specific T cells on Ear:Accumulating evidence suggests that vitiligo is a systemic disease affecting the entire pigmentary system. Using h3TA2 mice that bear tyrosinase epitope specific T cells in h3TA2 mice that result in spontaneous depigmentation also affect the function ear – another organ that has melanocyte expression. We hypothesize that cochlear melanocytes may be affected in vitiligo and interfere with the conduction of action potentials in the auditory pathway. To investigate the abnormalities in melanin-containing cellular elements of the auditory system in mice with spontaneous vitiligo we would perform the conventional audiometric investigations and auditory brainstem response threshold determination in collaboration with Dr. Su-Hua Sha in Department of Pathology at MUSC – an expert in auditory research.
vi). We have also initiated collaborations with Drs. David Cole, Chentha Vasu, Chrystal Paulos and Mark Rubinstein to use their expertise and critical input to address various research aims of our lab.
Thus, while following different lines of investigation the overall goal of our laboratory remains to understand how a robust and long-lived Tumor Antigen Associated (TAA) Cytolytic T Lymphocytes (CTL) response can be orchestrated. While we are interested in understanding mechanisms for T cell immunity, T cell tolerance and how these can be modulated to control tumor and autoimmunity, we are also devoting our efforts to identify signaling pathways (as TCR and sphingosine) and other chemopreventive agents as T cell response modifiers.
R21AR056524 (NIH/NIAMS) Dates: 04/18/2009 - 03/31/2012 Title: Development & Characterization of Tyrosinase Epitope Specific TCR Transgenic Mice Role: Principal Investigator
R01CA138930 (NIH/NCI) Dates: 06/02/2010 - 04/30/2015 Title: Oxidative stress and AICD in memory T Cell persistence Role: Principal Investigator
R01AR057643-01 (Loyola University Chicago) Dates: 09/01/2010 - 04/31/2015 Title: Modulating Tolerance in a Spontaneous Mouse of Autoimmune Vitiligo Role: Co-Investigator
P01CA154778 (NIH/NCI) Michael I. Nishimura (PI) Dates: 09/21/2011 – 08/31/2016 Title: Impact of AICD on TCR transduced T cells for Adoptive Immunotherapy Role: Principal Investigator (dual PI)
The Charlotte Geyer Foundation Dates: 09/01/2008 - 08/30/2009 Title: Isolation and Characterization of Prostrate Antigen Specific High Affinity TCR Role: Principal Investigator
NIH R21CA137725 Dates: 06/01/2009 – 5/31/2010 Title: Isolation and Characterization of Prostrate Antigen Specific High Affinity TCR Role: Principal Investigator
South Carolina Clinical & Translational Research Institute Pilot Projects Program (Discovery Grants) Dates: 10/15/2010 - 10/14/2011 Title: Sphingolipid metabolism in tumor metastasis and immunotherapy Role: Co-Investigator, Besim Ogretmen (PI)