Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model

<p>Abstract</p> <p>Background</p> <p>The anti-tumor efficacy of human immune effector cells, such as cytolytic T lymphocytes (CTLs), has been difficult to study in lung cancer patients in the clinical setting. Improved experimental models for the study of lung tumor-immune cell interaction as well as for evaluating the efficacy of adoptive transfer of immune effector cells are needed.</p> <p>Methods</p> <p>To address questions related to the <it>in vivo </it>interaction of human lung tumor cells and immune effector cells, we obtained an HLA class I ⁺lung tumor cell line from a fresh surgical specimen, and using the infiltrating immune cells, isolated and characterized tumor antigen-specific, CD8⁺CTLs. We then established a SCID mouse-human tumor xenograft model with the tumor cell line and used it to study the function of the autologous CTLs provided via adoptive transfer.</p> <p>Results</p> <p>The tumor antigen specific CTLs isolated from the tumor were found to have an activated memory phenotype and able to kill tumor cells in an antigen specific manner <it>in vitro</it>. Additionally, the tumor antigen-specific CTLs were fully capable of homing to and killing autologous tumors <it>in vivo</it>, and expressing IFN-γ, each in an antigen-dependent manner. A single injection of these CTLs was able to provide significant but temporary control of the growth of autologous tumors <it>in vivo </it>without the need for IL-2. The timing of injection of CTLs played an essential role in the outcome of tumor growth control. Moreover, immunohistochemical analysis of surviving tumor cells following CTL treatment indicated that the surviving tumor cells expressed reduced MHC class I antigens on their surface.</p> <p>Conclusion</p> <p>These studies confirm and extend previous studies and provide additional information regarding the characteristics of CTLs which can be found within a patient's tumor. Moreover, the <it>in vivo </it>model described here provides a unique window for observing events that may also occur in patients undergoing adoptive cellular immunotherapy as effector cells seek and destroy areas of tumor growth and for testing strategies to improve clinical effectiveness.</p

Tumor antigen-specific CTLs release cytokines and exert cytotoxic activity when incubated with autologous tumor cells

<p><b>Copyright information:</b></p><p>Taken from "Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model"</p><p>http://www.translational-medicine.com/content/5/1/29</p><p>Journal of Translational Medicine 2007;5():29-29.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1933531.</p><p></p> . CTLs (1 × 10) were incubated with an equal number of 81–86 (allogeneic tumor, a negative control), 81-86-1203 tumor cells (allogeneic tumor transfected with HLA-Cw1203, a positive control), or LT-391-06 tumor cells (autologous tumor). CTLs and tumor cells were also incubated alone as controls. Cells were incubated overnight at 37°C. Supernatants were analyzed by sandwich ELISA for the presence of IFN-γ and TNF-α. . The cytotoxic activity of CTLs was analyzed by a chromium release assay. Serial dilutions of CTLs (starting from 1 × 10) were incubated with either (1 × 10) LT-391-06 (●) or Daudi (▲, negative control) or K562 (■, negative control)

Tumor specific CTLs suppress the growth of autologous lung tumors in SCID mice

<p><b>Copyright information:</b></p><p>Taken from "Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model"</p><p>http://www.translational-medicine.com/content/5/1/29</p><p>Journal of Translational Medicine 2007;5():29-29.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1933531.</p><p></p> SCID mice were injected with 5 × 10tumor cells intravenously. At day 5, after tumor foci were established in the lungs, mice were received either 5 × 10tumor specific or anti-flu CTLs (i.v. injection). At day 20 and 35, 6 mice from each group (I. control; II. tumor only; III. tumor plus flu-specific CTL or IV. tumor plus tumor-specific CTL were sacrificed and lung and liver were excised for further studies. . Lung weights; . Histology of normal lung; . Histology of lung from tumor bearing mice treated with anti-flu specific CTLs and . Histology of lung from tumor bearing mice treated with tumor specific anti-LT391-06 CTLs. Differences in lung weight between groups was evaluated by the student t-test: II versus III, < 0.95; II vs. IV, < 0.001; III vs. IV, P < 0.004

The anti-tumor effect of anti-LT391-06 CTLs against two HLA matched allogeneic tumor cells is antigen specific

<p><b>Copyright information:</b></p><p>Taken from "Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model"</p><p>http://www.translational-medicine.com/content/5/1/29</p><p>Journal of Translational Medicine 2007;5():29-29.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1933531.</p><p></p> SCID mice in control groups (●) received a s.c. injection of 5 × 10tumor cells alone and mice in the treated groups (○) received a co-injection of 1 × 10anti-LT391-06 CTLs s.c. . Mice bearing A549 tumors, an allogeneic bronchioalveolar carcinoma cell line which does not express the LT391-06 Ag recognized by the tumor specific CTLs; . Mice bearing 936T, an allogeneic tumor cell line derived from a squamous lung carcinoma which does express this antigen. (= 5 mice/group

The anti-tumor effect of CTLs on autologous tumor xenografts is dependent on the time of administration, but not the number of doses

<p><b>Copyright information:</b></p><p>Taken from "Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model"</p><p>http://www.translational-medicine.com/content/5/1/29</p><p>Journal of Translational Medicine 2007;5():29-29.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1933531.</p><p></p> . Mice (5/group) received 5 × 10tumor cells (i.v.). Once tumors were established in the lung, three of these groups (5 mice/group) were injected with CTLs (5 × 10): Group III on day 5, Group IV on day 15 and Group V on both days 5 and 15. (The control group I did not receive tumor, Group II received tumor but not CTLs). At day 30, mice were sacrificed and lungs were excised and weighed. Differences in lung weight between treatment groups were evaluated by the Student t-test: I vs. II, < 0.001; I vs. III, < 0.1; I vs. IV, < 0.001; II vs. IV, P < 0.35; III vs. IV, P < 0.001. . Mice (groups II-IV, 5 mice/group) were injected with 5 × 10tumor cells (i.v.). After lung tumors were established, 4 groups (III-VI) received CTLs (5 × 10) as follows: Group III – one dose of CTL on day 3; Group IV – days 3 and 4; Group V-days 3, 4 and 5; Group VI-days 3, 4, 5 and 6. Normal lung weight was derived from Group I, which did not receive tumor cells; Group II received PBS and no CTL. On day 30, mice were sacrificed and lungs were excised and weighed. The growth of lung tumors in mice treated with tumor specific CTLs was significantly inhibited by one dose on day 3; there was no advantage to the administration of additional CTL doses

Tumor antigen-specific CTL and expression of cytokines are detected in the lungs of SCID mice bearing autologous lung tumor

<p><b>Copyright information:</b></p><p>Taken from "Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model"</p><p>http://www.translational-medicine.com/content/5/1/29</p><p>Journal of Translational Medicine 2007;5():29-29.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1933531.</p><p></p> Mice were injected with 5 × 10tumor cells intravenously. On day 5, after tumor foci were established in the lungs, 5 × 10CTLs were injected intravenously. On day 10, mice were sacrificed and lung and liver were excised for immunohistochemical and PCR analysis. and . IHC analysis of lungs for the presence of CD8 positive CTLs. Lungs from mice injected with . tumor cells + tumor antigen-specific CTLs . tumor cells only, . tumor cells + influenza nucleoprotein antigen-specific CTL or . tumor antigen-specific CTL alone. Original magnification: ×200, brown staining shows CD8 positive T cells. and . RT-PCR analysis. Primers used are specific for human . CD8, . IFN-γ and . β-actin. , 100 bp DNA ladder; , cDNA from lung of SCID mice bearing human tumor only; , cDNA from liver of SCID mice bearing with tumor only; , cDNA from lung of SCID mice bearing human tumor and CTLs; , cDNA from liver of SCID mice bearing tumor and CTLs