Premed · Premed · Immunology
Lecture 31: Emerging Topics: Checkpoint Inhibitors, CAR-T Therapy, Microbiome-Immune Interactions
Immunology
Learning Objectives
By the end of this lecture, students will be able to:
- Explain the biology of immune checkpoints (PD-1/PD-L1, CTLA-4) and how their blockade enhances antitumor immunity
- Describe the design, clinical applications, and challenges of CAR-T cell therapy in depth
- Explain the bidirectional interactions between the gut microbiome and the immune system
- Discuss emerging immunological concepts including bispecific antibodies, next-generation checkpoint targets, and immune metabolism
- Describe the clinical significance of immune-related adverse events and cytokine release syndrome
Lecture Content
I. Immune Checkpoint Biology in Depth
Immune checkpoints are inhibitory receptor-ligand pairs that maintain self-tolerance and modulate the duration and amplitude of immune responses. In cancer, these checkpoints are co-opted by tumors to evade immune destruction.
CTLA-4 (CD152) is expressed on activated T cells, where it is upregulated after TCR engagement, and constitutively on Tregs. It competes with CD28 for binding B7-1 (CD80) and B7-2 (CD86) on APCs, binding with approximately 20 times higher affinity than CD28. CTLA-4 inhibits T cell activation through several mechanisms: it outcompetes CD28 to block the co-stimulatory signal; it performs trans-endocytosis, physically stripping B7 molecules from the APC surface and thereby reducing co-stimulatory capacity for all nearby T cells; it recruits SHP-2 and PP2A phosphatases that dampen TCR and CD28 signaling; and it enhances Treg-mediated suppression. CTLA-4 primarily regulates T cell activation in lymph nodes during the priming phase. Ipilimumab (anti-CTLA-4) was the first checkpoint inhibitor approved in 2011 for melanoma, and works by enhancing T cell priming and depleting intratumoral Tregs via ADCC in the tumor microenvironment.
PD-1 (CD279) is expressed on activated T cells, B cells, and NK cells, and becomes particularly upregulated during chronic activation and exhaustion. Its ligands are PD-L1 (B7-H1), which has broad tissue expression, is upregulated by IFN-gamma, and is expressed on many tumors, and PD-L2 (B7-DC), which is found mainly on APCs. When PD-1 is engaged, it recruits SHP-2, which dephosphorylates CD3-zeta and CD28 signaling intermediates, suppressing TCR signaling, PI3K/Akt, and Ras/MAPK pathways. PD-1 primarily regulates T cell function in peripheral tissues and tumors during the effector phase. Anti-PD-1 antibodies (nivolumab, pembrolizumab) and anti-PD-L1 antibodies (atezolizumab, durvalumab, avelumab) reinvigorate exhausted tumor-specific T cells in the tumor microenvironment.
Next-generation checkpoint targets are currently under investigation. LAG-3 (CD223) binds MHC class II and inhibits CD4+ T cells; relatlimab (anti-LAG-3) has been approved in combination with nivolumab for melanoma. TIM-3 binds galectin-9, CEACAM-1, and phosphatidylserine to inhibit T cells. TIGIT binds CD155 (PVR) and competes with the activating receptor DNAM-1, with expression on T cells and NK cells. Additional inhibitory receptors including VISTA, B7-H3, and BTLA are in clinical trials. Combinatorial approaches using dual checkpoint blockade, such as anti-PD-1 plus anti-LAG-3 or anti-PD-1 plus anti-CTLA-4, aim to overcome resistance mechanisms.
II. Immune-Related Adverse Events (irAEs)
Checkpoint inhibitors remove immune restraints and can consequently cause autoimmune-like toxicities. The underlying mechanisms include unleashed self-reactive T cells attacking normal tissues, breaking of peripheral tolerance, enhanced Th17 responses, reduced Treg function, and cross-reactivity between tumor and normal tissue antigens.
Common irAEs are organized by organ system. Skin manifestations include rash, pruritus, and vitiligo (especially in melanoma, where it may correlate with treatment response). Gastrointestinal toxicity manifests as colitis with diarrhea and abdominal pain, and is more common with anti-CTLA-4. Liver toxicity presents as hepatitis with elevated transaminases. Endocrine toxicities include thyroiditis causing hypothyroidism or hyperthyroidism, hypophysitis (especially with anti-CTLA-4), adrenal insufficiency, and rarely type 1 diabetes. Pulmonary toxicity manifests as pneumonitis. Neurological complications include peripheral neuropathy, myasthenia gravis (rare), and encephalitis. Cardiac toxicity in the form of myocarditis is rare but potentially fatal.
Management follows severity grading using CTCAE criteria. Mild cases can continue therapy with monitoring. Moderate cases require holding therapy and administering corticosteroids. Severe cases demand discontinuation of therapy plus high-dose corticosteroids with additional immunosuppression as needed, such as infliximab for colitis or mycophenolate for hepatitis.
III. CAR-T Cell Therapy in Depth
The chimeric antigen receptor (CAR) is an engineered molecule with a defined modular structure. The extracellular portion consists of a scFv (single-chain variable fragment derived from a monoclonal antibody) that provides MHC-independent antigen recognition. A hinge/spacer provides flexibility, and a transmembrane domain anchors the CAR in the T cell membrane. The intracellular portion contains CD3-zeta for activation plus co-stimulatory domains. Second-generation CARs include CD3-zeta plus one co-stimulatory domain, either CD28 or 4-1BB. CD28 co-stimulation produces rapid, intense effector function but shorter persistence, while 4-1BB co-stimulation enhances persistence and memory formation with slower initial expansion. Third-generation CARs incorporate two co-stimulatory domains. Fourth-generation "armored CARs" or TRUCKs include cytokine transgenes (such as IL-12) designed to modify the tumor microenvironment.
Approved CAR-T products targeting CD19 include tisagenlecleucel (Kymriah, using 4-1BB), axicabtagene ciloleucel (Yescarta, using CD28), lisocabtagene maraleucel (Breyanzi, using 4-1BB), and brexucabtagene autoleucel (Tecartus, using CD28). These are indicated for relapsed/refractory B-ALL, DLBCL, follicular lymphoma, and mantle cell lymphoma, achieving complete remission rates above 80% in B-ALL and approximately 40 to 60% in DLBCL. Anti-BCMA CARs including idecabtagene vicleucel (Abecma) and ciltacabtagene autoleucel (Carvykti) are indicated for relapsed/refractory multiple myeloma.
The major toxicities of CAR-T therapy include cytokine release syndrome (CRS), which results from massive release of cytokines (IL-6, IFN-gamma, TNF-alpha, IL-1) from activated CAR-T cells and bystander macrophages. Symptoms range from fever alone (grade 1) to hypotension, hypoxia, and capillary leak that can progress to multi-organ failure (grade 4). Treatment with tocilizumab (anti-IL-6R) rapidly reverses CRS, with corticosteroids reserved for refractory cases. Immune effector cell-associated neurotoxicity syndrome (ICANS) presents with confusion, aphasia, seizures, and rarely cerebral edema. The mechanism involves endothelial activation, blood-brain barrier disruption, and cytokine entry into the CNS. Corticosteroids are the primary treatment, as tocilizumab is less effective for ICANS. B cell aplasia is an on-target, off-tumor toxicity in which anti-CD19 CARs kill normal B cells, producing hypogammaglobulinemia managed with IVIG. Prolonged cytopenias from bone marrow suppression occur due to lymphodepletion conditioning and CRS.
Key challenges and future directions include antigen escape, in which the tumor loses the target antigen leading to resistance (such as CD19-negative relapse in B-ALL). Solid tumors present limited efficacy due to the hostile tumor microenvironment, antigen heterogeneity, poor T cell infiltration, and immunosuppression. Off-the-shelf (allogeneic) CARs using donor or gene-edited T cells aim to reduce manufacturing time and cost. Dual-targeting CARs target two antigens simultaneously to prevent escape (such as CD19 plus CD22). CAR-NK cells use NK cells as alternative effectors with lower CRS risk and potential for allogeneic off-the-shelf products.
<image>A diagram showing the molecular mechanisms of CTLA-4 and PD-1 checkpoint pathways and their blockade. Left panel (CTLA-4 pathway -- lymph node): An APC presenting antigen on MHC to a T cell. CD28 on the T cell binds B7 on the APC (activating signal, green arrow). CTLA-4 (upregulated after activation) competes with CD28 for B7 binding with higher affinity (inhibitory signal, red arrow). CTLA-4 also strips B7 from the APC (trans-endocytosis). A Treg cell with constitutive CTLA-4 is shown suppressing the interaction. Ipilimumab (anti-CTLA-4 antibody) is shown blocking CTLA-4, allowing CD28-B7 co-stimulation to proceed, and also depleting intratumoral Tregs via ADCC (Fc-mediated). Right panel (PD-1 pathway -- tumor microenvironment): A CD8+ T cell with upregulated PD-1 interacts with a tumor cell expressing PD-L1. PD-1-PD-L1 engagement recruits SHP-2 phosphatase, which dephosphorylates CD3-zeta and downstream signaling molecules (shown as inactive/gray signaling cascade). The T cell is exhausted (reduced IFN-gamma, granzyme, proliferation). Nivolumab/pembrolizumab (anti-PD-1) blocks PD-1, restoring the signaling cascade (green arrows) and reinvigorating the T cell to kill the tumor. An inset compares the sites of action: CTLA-4 blockade acts primarily during T cell priming (lymph node), while PD-1 blockade acts primarily at the effector site (tumor).</image>
IV. Microbiome-Immune Interactions
The human gut harbors approximately 38 trillion microorganisms, including bacteria, archaea, fungi, and viruses, collectively called the microbiome. The microbiome and the immune system engage in continuous bidirectional crosstalk that has profound implications for health and disease.
The microbiome shapes immune development in fundamental ways. Germ-free mice have underdeveloped gut-associated lymphoid tissue with small Peyer's patches, few IgA-positive plasma cells, reduced Tregs, and deficient Th17 cells. Colonization restores immune maturation, demonstrating that the microbiome is essential for normal immune development. Specific microbes play defined roles: segmented filamentous bacteria (SFB) are potent inducers of intestinal Th17 cells, while Clostridium clusters IV and XIV and Bacteroides fragilis (through its polysaccharide A) promote colonic Treg differentiation and immune homeostasis. Short-chain fatty acids (SCFAs), the products of dietary fiber fermentation by gut bacteria, are critical mediators. Butyrate promotes Treg differentiation by enhancing FOXP3 expression through HDAC inhibition, strengthens the epithelial barrier, and is anti-inflammatory. Propionate modulates dendritic cell function and reduces Th2 responses. Additional microbial metabolites include tryptophan derivatives (which serve as AhR ligands to activate ILC3s and promote IL-22 production), bile acid metabolites, and vitamins B12 and K.
Conversely, the immune system shapes the microbiome. Secretory IgA coats commensal bacteria to maintain spatial segregation and prevent invasion. Antimicrobial peptides such as defensins and RegIII-gamma from Paneth cells limit microbial contact with the epithelium. IgA deficiency and other immune deficiencies lead to dysbiosis.
Dysbiosis and disease are linked across multiple conditions. Inflammatory bowel disease (IBD) is associated with reduced microbial diversity, loss of Firmicutes (butyrate producers), and increased Proteobacteria, combined with a dysregulated immune response to commensals. Autoimmune diseases including T1D, MS, and RA are associated with specific dysbiotic signatures. Allergy and asthma are linked to reduced early-life microbial diversity that impairs Treg development and promotes Th2 skewing. In cancer, microbiome composition influences response to checkpoint immunotherapy. Patients with diverse gut microbiomes and specific taxa such as Akkermansia muciniphila, Faecalibacterium prausnitzii, and Bifidobacterium show better responses to anti-PD-1 therapy. Fecal microbiota transplantation from responders to non-responders can enhance checkpoint inhibitor efficacy in clinical trials. Clostridioides difficile infection results from antibiotic-induced dysbiosis causing loss of colonization resistance and C. difficile overgrowth, treated effectively with FMT.
V. Immunometabolism
Immunometabolism is an emerging field that recognizes how the metabolic programs of immune cells are tightly linked to their function and differentiation. Effector T cells and M1 macrophages rely on aerobic glycolysis (the Warburg effect) and glutaminolysis, which support rapid biosynthesis for proliferation and effector function. In contrast, memory T cells and Tregs rely on oxidative phosphorylation (OXPHOS) and fatty acid oxidation, which supports their longevity and regulatory function.
The tumor microenvironment creates metabolic competition between tumor cells, which exhibit high glycolysis leading to glucose depletion and lactate accumulation, and T cells that are consequently starved of metabolic fuel, resulting in metabolic exhaustion. Two master regulators govern metabolic switching in immune cells: mTOR activation promotes glycolysis and effector function, while AMPK activation promotes OXPHOS and memory/regulatory function. These insights have therapeutic implications, as metabolic manipulation may enhance antitumor immunity. For instance, mTOR inhibition promotes memory T cell formation, and targeting IDO in tumors can restore tryptophan availability for T cells.
VI. Bispecific Antibodies and Novel Immunotherapeutics
Bispecific T cell engagers (BiTEs) are engineered antibodies with two different binding specificities: one arm binds a tumor antigen while the other binds CD3 on T cells. This physically bridges T cells to tumor cells, activating T cells to kill the tumor regardless of TCR specificity. Blinatumomab, an anti-CD19 by anti-CD3 BiTE, is approved for B-ALL. Advantages over CAR-T include being off-the-shelf with no patient-specific manufacturing and easier administration. Disadvantages include short half-life (requiring continuous infusion for some formats), CRS risk, and no memory formation.
Other emerging modalities are expanding the immunotherapy toolkit. Antibody-drug conjugates (ADCs) are expanding rapidly across cancer types. NK cell engagers are bispecific antibodies that engage NK cells instead of T cells. Oncolytic viruses are engineered viruses that selectively replicate in tumor cells, causing lysis and immune activation, with talimogene laherparepvec (T-VEC) approved for melanoma. Toll-like receptor agonists injected intratumorally activate innate immunity within the tumor microenvironment. STING agonists activate the cGAS-STING pathway to produce type I interferons that drive antitumor immunity.
<image>A diagram illustrating microbiome-immune interactions in the gut. The intestinal lumen is shown at the top with a diverse community of commensal bacteria (different colored shapes representing Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria). Dietary fiber is shown being fermented by bacteria into short-chain fatty acids (SCFAs -- butyrate, propionate, acetate), which diffuse through the epithelium. In the lamina propria, butyrate is shown acting on naive CD4+ T cells to promote FOXP3+ Treg differentiation (via HDAC inhibition). Segmented filamentous bacteria (SFB) are shown adhering to epithelial cells in the ileum, with arrows indicating they promote Th17 cell differentiation (IL-17 production). Bacteroides fragilis is shown producing polysaccharide A (PSA), which activates DCs via TLR2 to produce IL-10 and promote Tregs. Secretory IgA is shown coating commensal bacteria in the lumen (immune shaping of microbiome). Paneth cells at the crypt base secrete antimicrobial peptides (defensins). A "dysbiosis" inset shows reduced microbial diversity, loss of butyrate-producing bacteria, expansion of Proteobacteria, and the resulting immune imbalance: reduced Tregs, increased Th1/Th17 inflammation → arrows pointing to IBD, autoimmunity, and impaired cancer immunotherapy response.</image>
