Induction And Evaluation Of Melanoma- Specific CTL Responses In Mice

The purpose of immunotherapy in cancer is to induce an effective anti-tumorspecific cytotoxic T lymphocytes (CTL) response, however, major histocompati-bility complex (MHC) class I is usually expressed at low levels by tumors andcant elicit effective immune responses. Self-antigens are the most common anti-gens that are recognized in human cancer. Immune responses to self-antigens aredifficult to induce because of self-tolerance. Even if the immune system recogni-zes and makes a response to self antigens, it is often too weak to reject thetumor.FasL is a member of TNF protein family; expression of FasL can confer im-mune privilege to some organs, allowing them to kill infiltrating lymphocytes andinflammatory cells. However, the expression of FasL in a number of instancesprovokes an intense neutrophil infiltrating. Neutrophils expressing Fas can be at-tracted and activated by FasL to become cytotoxic. It is believed that this neu-trophil activation impairs tumor growth, then induces an adaptive immune re-sponse possibly mediated by CTLs. In this study we tested whether FasL ex-pressed by tumors is able to induce a CTL response. TheB16F10 melanomamodel was used because it is a poorly immunogenic tumor and thus a good modelto test strategies to enhance tumor immunity. The best-characterized melanomaantigens are the non-mutated differentiation antigens expressed in both melanomaand melanocytes: gp100, Tyrosinase, Tyrosinase-Related-Protein (TRP) 1 and2 and Melan-A. Three epitopes were chosen from these antigens known to be in-volved in the rejection of the tumor: murine gp100 (mgp100) and its more im-munogenic counterpart in human (hgp100) and TRP2. Furthermore twoepitopes from endogenous virus described to be expressed by B16F10 were alsochosen, peptides were synthesized and tetramers generated. The MHC class I tetramer technology was recently developed by Oxford andStanford universities. It can quantify and analyze antigen specific CTL at a sin-gle cell level by FACS. Mice were immunized with irradiated B16F10 expressingFasL and then challenged with wild type B16F10. Protected mice were chosenfor tetramer analysis. To improve the response, protected mice were boostedwith recombinant vaccinia virus expressing the melanocyte differentiation anti-gens. It is known that vaccinia virus can strongly induce T cell-mediated immu-nity as well as humoral immune response. Furthermore, antigen specific killingwas measured in vivo using a novel technique. We hope our observations mayhelp for the definition of new cell surface tumor markers and may provide an ad-ditional route to stimulate a broader anti-tumor response.Material and MethodsWe used the B16F10 melanoma C57BL/6 model. Two techniques wereused to detect antigen-specific CTL activity in immunized mice: 1) MHC classI-peptide tetramers. The protein sequence of five relevant epitopes are as fol-lows: mdm(K~b):YAMIYRNL,p15E (K~b) : KSPWFTTL, trp-2 (v) (K~b): VYD-FFVWL,trp-2(s) (K~b) :SVYDFFVWL hgp100 (D~b) : KVPKNQDWL. Makingcorresponding tetramers :Briefly, the cDNA for the MHC class-I and beta 2 mi-croglobulin are cloned into expression plasmids, and protein expressed as inclu-sion bodies within E. coli. After solubilisation, the class I molecule,theβ2mand synthetic peptide are added in relatively large quantities(10-30mg) to abuffer solution containing protease inhibitors at 4℃, in which the formation of astable trimolecular complex can occur, after concentration, the complexes areenzymatically biotinylated and purified by size. Monomeric complexes may thenbe frozen for future use or immediately formed into tetramers around streptavidin-phycoerythrocin (SA-PE) conjugates. 2) In vivo killing assay: CFSE is a mo-lecular probe that emits green fluorescence at 488nm, thus can be detected byFACS. Combine with DC, it can be found in draining lymphatic nodes. It canbe a target for CTL if DC integrates with MHC class I Label syngeneic targetcells (Spleen)with CFSE and pulse with relevant peptides, then inject into pro- tected mice, bleed mice 5～18 hours later and perform FACS analysis of bloodsamples. Target cells labeled with the relevant peptide should disappear, as theywill be specifically killed by CTLs recognizing the peptide. Mice were dividedinto 4 groups for priming and boosting 1) B16F10-FasL followed by rVV(mgp100, NP), B16F10-FasL, rVV ,naive mice. Sometimes mice were alsoboosted with spleen cells loaded with peptides one week later, Tetramer stainingand vivo killing were performed. Spleen cells of the different groups are culturedto set up CTL lines. All cells were divided into two groups, one group was stim-ulated with hgp100 and tested by two kinds of tetramers: hgp100, mgp100 aweek later after final in vitro stimulation. The other six CTL lines were stimula-ted by six peptides and crisscross staining was performed after finial stimulation.ResultsWe established a technique whereby tetramer staining was combined withPropidium iodide (PI). Excluding PI positive cells helped to reduce the unspe-cific staining of dead cells. No antigen specific CTLs were detected ex vivo, tes-ting blood, lymph nodes and spleens except a mouse that received vaccinia-NPand was stained with NP tetramer used as a positive control. We performed 5 vi-vo killings, which are difficult to interpret. Both the irrelevant control peak andthe relevant peak from the CFSE labeled and peptide-pulsed targets are verylow, as we did not retrieve enough cells from the mice. Blood testing yielded twopeaks, while spleen only one. Compare with other groups, the peaks of FasL+rVVgp100 disposing group are remarkably lower, but it is obvious that they arenot antigen specific killings as not only the peak with relevant peptide, but alsothe one with irrelevant peptide low. Tetramer positive CTLs were observed incell lines made from mice that received rVVgp100 and FasL+rVVgp100, testedby tetramer hgp100 and mgp100. All other groups are negative. The cell linemade from FasL+rVVgp100 mice also stained positive with irrelevant tetramer,while the other CTL lines negative, indicating that there is non-specific back-ground occurring in some of the samples. Conclusions1. Tetramer can detect antigen specific CTL accurately.2. Combination of FasL and rVVgp100 can break self-tolerance to B16F10melanoma.3. Combination of FasL and rVVgp100 induces possibly an innate immuneresponse, leading to unspecific killing.4. Combination of FasL and rVVgp100 augments the overall immune reac-tion, leading to non-specific binding of irrelevant tetramer.5. Success of immune response in mice varies among individual mice andtherefore bigger groups of mice need to be included in the future.6. In combination with PI, the results of tetramer staining of CTL lines byFACS axe remarkably improved.7. Best result was obtained after two weeks after final stimulation for theCTL lines.8. Our observation and results will provide a basis to develop new anti-tumor vaccine and immunotherapy.