Group 9: Naila Nawaz ( 2021-ag – 8526 ) Namal Rauf (2021-ag-8527) Nimra Akram (2021-ag – 8528)

Noor Fatima (2021-ag-8529) Noor Fatima (2021-ag-8530)

Article: Alteration of Tumor Metabolism by CD4+Tcells leads to TNF alpha dependent intensification of
Oxidative stress and Tumor Cell Death.

Q 1 Pathway of drug: In adoptive Immunotherapy using CD4 T cells and CTX( cyclophosphamide), the
pathway typically involves several steps. First, CTX is administrated into the patient as a
lymphodepleting agent. CTX selectively removes certain immune cells, including regulatory T cells,
creating space for the infused Tcells. Then, T cells are removed from patient’s blood and activated and
expanded in the laboratory using various techniques such as genetic modification. The expanded Tcells
are engineered to express specific receptors such as CARs and TCRs, enabling them to recognize and
target cancer cells. Once the desired amount is achieved, these cells are infused back into patient’s
blood. These adoptively transferred cells navigate through patient’s bloodstream and tumor
microenvironment, seeking out cancer cells expressing the targeted antigens. Upon encountering their
target, they exert their anti-tumor effects through various mechanisms including increasing ROS level
and direct killing of cancer cells etc.

Q2 Rationale and Novelty : Adoptive T cell therapy represents a novel and innovative approach in
cancer treatment. Here are some key aspects that highlight the novelty of adoptive T cell therapy in
cancer:

Personalized therapy Targeting solid tumors Overcoming immune evasion Long-term

immunological memory Combination potential Success in hematological malignancies

The rationale behind adoptive T cell therapy in cancer is to harness the power of the immune system to
specifically target and eliminate the cancer cells. Here are some key rationales of the adoptive T cell
therapy:

Specificity Enhanced anti-tumor response Persistence and memory Personalized Medicine

Q3 Conclusion of the full article: The inhibitory effects of cancer on the T cell metabolism have studied
with detail but the metabolic procedure of immune therapy is not understood. So here, we used the
adoptive T cell therapy method using the CD4 T cells and CTX. It is based on increasing ROS level and
decreasing glutathione levels in tumors. This effect is enhanced by TNF- alpha released from T cells
which synergizes with chemotherapy to enhance oxidative stress and consequently, the tumor necrosis
starts. The effect of T cells with cytokines is seen by performing several experiments or different staining
methods. While, if the oxidative stress is reduced, then the effect is reversed. The findings in this article
imply the importance of oxidative stress in anti-cancer therapy and hence, its efficiency.

Q.4: Conclusion of every figure: Figure 1 provides a comprehensive set of experimental results
illustrating the efficacy of CTX pre-conditioning followed by CD4 AT in eradicating established colorectal
tumors, supported by data on tumor growth, immune response, apoptosis, and changes in the tumor
microenvironment. Fig 02: In this figure, the metabolomic analysis revealed extensive reprogramming of
tumor metabolism after CTX + CD4 AT treatment, characterized by defects in pathways controlling redox
balance, bioenergetics, and biosynthesis. These changes likely contribute to the observed therapeutic
effects in tumor regression. Fig 03: It demonstrates the methods used to assess oxidative stress in tumor
cells and the responses of these cells to different treatments. The experiments include flow cytometry
for ROS levels, immunofluorescence staining for DNA damage, and gene expression analysis to examine
how specific genes related to antioxidant defense are affected by the treatments. Fig 04: In this figure,
orthotopic tumor models are used to demonstrate that the combination of chemotherapy and T cell
therapy intensifies oxidative stress in tumor cells, and this effect is clinically relevant. The findings
suggest that inducing oxidative stress in tumor cells could be an effective approach in adoptive
immunotherapy. Fig 05: A series of experiments show that TNF-a, when combined with chemotherapy,
significantly enhances ROS production and tumor cell death, and GSH deficiency contributes to this
effect. This highlights the importance of TNF-a in mediating the response of tumor cells to
chemotherapy in the presence of polyfunctional CD4+ T cells. Fig 06: It evidence that p22phox (encoded
by Cyba) plays a significant role in ROS generation in tumor cells when exposed to melphalan and TNF-a.
The data suggest that p22phox is involved in the production of ROS, which is linked to increased
oxidative stress and cell death in response to this treatment. Fig 07: It provides strong evidence that
p22phox and TNF-a-induced oxidative stress is a critical component of the success of CTX + CD4 AT in
tumor rejection in mice. It highlights the importance of oxidative stress as an active contributor to the
effectiveness of adoptive immunotherapy and demonstrates the relevance in tumor treatment.

Q.5: New findings present in research article: Immunotherapy which is a combination of any type of
WBCs and glycoproteins that are attached on the surfaces of lymphocytes is an effective treatment for
cancer at any part of body especially in colorectal and lungs portions.

Q.6: Application of findings: When Immunotherapy before chemotherapy provided to cancer patients,
it will induce positive results very efficiently. Metabolism of cancer cells and increase in ROS enhances
the effects of chemotherapy and it leads to death of tumors in body.

Q.7: Future perspectives: The study proposes combining chemotherapy (CTX) with CD4 T cell adoptive
therapy (CD4 AT) for effective tumor regression. Future directions include clinical trials, targeted
therapies focusing on identified metabolic pathways, understanding and overcoming resistance
mechanisms, optimizing treatment protocols, exploring immunometabolism, investigating combination
therapies, discovering biomarkers, and assessing clinical applications across various cancer types.
Additionally, understanding immune memory development and long-term effects is crucial for
enhancing treatment durability.. The goal is to enhance the effectiveness of adoptive immunotherapy
while minimizing potential challenges and side effects.

Q8: Applications of the work done: The work done in adoptive T cell therapy using CD4 cells and CTX
has several applications and implications in the field of cancer treatment. Here are some notable
applications: Treatment of solid tumors Enhanced T cell expansion and persistence Combination
with checkpoint inhibitors Targeting neoantigens Immune modulation and Immune memory
Translational potential
Q9: Principle of ever technique used in article. Bioluminescent Imaging(BLI): It is a technique based on
the detection of light emitted by living cells expressing a luciferase gene.

Fluorescence- Activated Cell Sorting (FACS): It uses fluorescent markers to target and isolate group
cells.

Transfection: It is a method of artificially inducing nucleic acids (DNA and RNA) into cells.

NOX-dependent mechanism: In phagocytic leukocytes, NOX- dependent superoxide generation plays a

crucial in destroying phagocytized organisms and facilitating the anti-microbial function of cells.