Presence Of Cytotoxic T Lymphocytes Research Articles

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Mathematical analysis of a within-host dengue virus dynamics model with adaptive immunity using Caputo fractional-order derivatives

AbstractDengue fever poses a significant global health threat, with over 50 million annual infections spanning more than 100 countries. Given the absence of a specific treatment, medical intervention primarily targets symptom alleviation. The present study utilizes a Caputo-type fractional-order derivative operator to investigate and analyze the dynamics of dengue virus spread within a host with adaptive immune responses. The developed model describes and analyzes the dynamics of immune cells, free dengue particles, infected monocytes, and susceptible monocytes in the presence of cytotoxic T-Lymphocytes. A range of analytical methods is employed to probe the fractional-order within-host model. The application of the generalized mean value theorem aids in investigating the model’s solutions, employing positivity and boundedness theory. Furthermore, the Banach fixed-point approach is utilized to establish the existence and uniqueness of solutions. Employing the normalized forward sensitivity approach, the fractional-order system’s response to various model parameters is scrutinized. The study reveals that the dynamics of the viral model are significantly influenced by the transmission rate and parameters representing adaptive immune responses. Numerical simulations underscore the critical role of transmission rates and adaptive immune responses in the model. Additionally, the study examines the impact of memory on the density of susceptible monocytes, infected monocytes, free dengue particles, and immune cells to optimize immune responses. Through simulations, the study illustrates the influence of memory on immune dynamics.

Prognostic Impact of the Immune-Cell Infiltrate in N1-Positive Non–Small-Cell Lung Cancer

IntroductionThe tumoral immune milieu plays a crucial role for the development of non–small-cell lung cancer (NSCLC) and may influence individual prognosis. We analyzed the predictive role of immune cell infiltrates after curative lung cancer surgery. Materials and MethodsThe tumoral immune-cell infiltrate from 174 patients with pN1 NSCLC and adjuvant chemotherapy was characterized using immunofluorescence staining. The density and distribution of specific immune cells in tumor center (TU), invasive front (IF) and normal tissue (NORM) were correlated with clinical parameters and survival data. ResultsTumor specific survival (TSS) of all patients was 69.9% at 5 years. The density of tumor infiltrating lymphocytes (TIL) was higher in TU and IF than in NORM. High TIL density in TU (low vs. high: 62.0% vs. 86.7%; p = .011) and the presence of cytotoxic T-Lymphocytes (CTLs) in TU and IF were associated with improved TSS (positive vs. negative: 90.6% vs. 64.7% p = .024). High TIL-density correlated with programmed death-ligand 1 expression levels ≥50% (p < .001). Multivariate analysis identified accumulation of TIL (p = .016) and low Treg density (p = .003) in TU as negative prognostic predictors in squamous cell carcinoma (p = .025), whereas M1-like tumor- associated macrophages (p = .019) and high programmed death-ligand 1 status (p = .038) were associated with better survival in adenocarcinoma. ConclusionThe assessment of specific intratumoral immune cells may serve as a prognostic predictor in pN1 NSCLC. However differences were observed related to adenocarcinoma or squamous cell carcinoma histology. Prospective assessment of the immune-cell infiltrate and further clarification of its prognostic relevance could assist patient selection for upcoming perioperative immunotherapies.

A fractional-order nonlinear model for a within-host chikungunya virus dynamics with adaptive immunity using Caputo derivative operator

The study of within-host dynamics of viral infection is a vital process of understanding how healthy cells in the body are affected by viral foreign bodies and how body immunity responds to such changes. This paper presents and analyses the dynamics of a within-host Chikungunya virus transmission with adaptive immune responses using a fractional-order derivative operator of the Caputo type. The formulated model describes the interaction of uninfected cells, infected cells and viral particles in the presence of Cytotoxic T-Lymphocytes and antibody immune responses. Several analytical methods are employed to analyse the fractional-order within-host model. Positivity and boundedness theory are used to investigate the properties of solutions of the model via the generalized mean value theorem approach. The existence and uniqueness of solutions are examined using the Banach fixed point method. The normalized forward sensitivity method determines how different model parameters affect the fractional-order system. It was shown that transmission rate and parameters representing adaptive immune responses played a significant role in the Chikungunya virus model’s dynamics. Further, the time-dependent optimal control fractional-order model is analysed to optimize the performance of adaptive immune responses using the optimal control theory approach made popular by Pontryagin’s maximum principle. Simulations are performed to visualize the theoretical results and show the influence of memory on the trajectories of concentrations of uninfected cells, infected cells, viral particles, Cytotoxic T-Lymphocytes and antibodies.

Abstract CT145: Systemic rintatolimod and interferon-α2b selectively reprogram local tumor microenvironment in patients with metastatic triple negative breast cancer for enhanced influx of cytotoxic T-lymphocytes but not regulatory T-cells

Abstract Background: Effective immune therapies depend on the presence of cytotoxic T-lymphocytes (CTLs) in the tumor microenvironment (TME). Our preclinical data showed synergy between TLR3 ligands and interferon-α (IFN-α) in reprogramming the TME, but not healthy tissues, to selectively enhance CTL attraction, providing rationale for their systemic application to enhance local CTL densities in “cold” tumors. The pilot study NCT03599453 evaluated the safety of systemic chemokine modulating regimen (CKM) composed of i.v. rintatolimod (Ampligen; selective TLR3 ligand) and IFN-α, and its ability to promote local CTL influx to mTNBC lesions. Methods: Six evaluable patients (33-75 years) with mTNBC received 6 doses of rintatolimod (200 mg i.v.), IFN-α (INTRON-A; 20MU/m2 i.v.) and COX-2 inhibitor (celecoxib; 2 x 200 mg, p.o.) over 2 weeks, with tumor biopsies obtained before (within 6 days) and after (within 5 days) CKM. All patients received follow-up pembrolizumab (200 mg, i.v, Q3 weeks). The primary endpoint was the change in the CTL marker CD8α in the TME with a planned interim analysis after 3 patients (α=0.03) and final analysis after 6 patients (α=0.084). Correlative studies analyzed additional markers of CTLs, regulatory T-cells (Tregs), and CTL- and Treg-attracting chemokines in the TME and blood. Results: Treatment was well tolerated with mostly grade 1/2 adverse events and one grade 3 clinically significant pneumonitis and immune thrombocytopenic purpura observed during follow up pembrolizumab treatment. We observed uniform increases of intratumoral type-1 immune markers upon treatment: CD8α mRNA (6.1-fold; p=0.034), GZMB (3.5-fold; p=0.058), ratios of CD8α/FOXP3 and GZMB/FOXP3 (5.7-fold; p=0.036, and 7.6-fold; p=0.024 respectively), and CTL attractants CXCL10 (2.6-fold; p=0.104) and CCL5 (3.3-fold; p=0.019), successfully meeting the primary endpoint. In contrast, neither Treg marker Foxp3 nor Treg attractants CCL22 or CXCL12 were enhanced. These TME changes were accompanied by transient decreases in circulating CD3+CD8+ CTLs and CD3-CD56+ NK cells (but not Tregs), selectively affecting the cells expressing CXCR3 (receptor for CXCL10), but not CCR4 or CXCR4 (receptors for CCL22 and CXCL12). Three patients had stable disease lasting 2.4, 2.5 and 3.8 months, as of September 1, 2021 cut-off. An additional patient had a partial response (breast auto-amputation) with massive tumor necrosis observed in the post-CKM biopsy. Conclusion: This proof-of-concept study shows that short-term systemic CKM followed by pembrolizumab is safe and selectively enhances local CTL infiltration in the TME, providing rationale for concurrent CKM and PD1 blockade in prospective phase II studies. Citation Format: Shipra Gandhi, Mateusz Opyrchal, Melissa Grimm, Ronald Slomba, Kathleen Kokolus, Sebastiano Battaglia, Kristopher Attwood, Adrienne Groman, Lauren Williams, Mary Lynne Tarquini, Paul Wallace, Kah Teong Soh, Tracey O'Connor, Amy Early, Ellis Levine, Igor Puzanov, Marc Ernstoff, Pawel Kalinski. Systemic rintatolimod and interferon-α2b selectively reprogram local tumor microenvironment in patients with metastatic triple negative breast cancer for enhanced influx of cytotoxic T-lymphocytes but not regulatory T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT145.

Functional genomic landscape of cancer-intrinsic evasion of killing by T cells.

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic Tlymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ andTNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, thesedata expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.

Abstract P2-04-08: BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy in mammary adenocarcinoma

Abstract The AXL receptor tyrosine kinase is associated with poor overall survival in breast cancer. Axl signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response. AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer cells, dendritic cells and tumor-associated macrophages. AXL is required for tumor immune evasion in mammary adenocarcinoma models and EMT-mediated resistance to cytotoxic T cell and natural killer (NK)-cell mediated cell killing. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms in breast cancer. We evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in the aggressive mammary adenocarcinoma (4T1) syngeneic (Balb/C) mouse modelthat display limited immunogenicity. Immune therapy with anti-CTLA-4/anti-PD-1 increased AXL and EMT-marker expression in 4T1 tumors, and correlated with lack of response to immune therapy. Combination treatment with BGB324 (50 mg/kg bid) significantly enhanced responsiveness to anti-CTLA-4/anti-PD-1 treatment (10 mg/kg of each, 4 doses) in Balb/C mice bearing established 4T1 tumors. The combination of BGB324 + anti-CTLA-4/anti-PD-1 resulted in durable primary tumor clearance in 23 % of treated mice versus 5.6% obtained with anti-CTLA-4/anti-PD-1 alone (p=0.0157). In a separate study, BGB324 + anti-CTLA-4 treated resulted in 22% long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of this model were concomitantly abrogated in the animals responding to the combination treatment. In addition, BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes (CTLs). Enhanced presence of CTLs was also detected in spleens from animals responding to treatment. BGB324 + anti-CTLA-4/anti-PD-1 treatment increased the number of NK cells, macrophages and polymorphonuclear neutrophils, but decreased the number of mMDSC. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. Together with recent results in other tumor types that support a prominent role for AXL in resistance to immune therapy and encouraging results from ongoing clinical trials with BGB324, support combining BGB324 with immune checkpoint inhibitors to improve treatment of breast cancer. Citation Format: Lorens JB, Lipinska KW, Davidsen K, Blø M, Hodneland L, Engelsen A, Kang J, Lie MK, Bougnaud S, Aguilera K, Ahmed L, Rybicka A, Nævdal EM, Deyna P, Boniecka A, Straume O, Chouaib S, Brekken RA, Gausdal G. BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy in mammary adenocarcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-08.

Cord Blood Allograft Rejection Mediated By Coordinated Donor-Specific Cellular and Humoral Immune Processes

Recent statistical analyses of the clinical outcome have revealed that the presence of donor-specific anti-HLA antibodies (DSAs) in pre-transplant recipient is correlated with increased risk for graft rejection after allogeneic hematopoietic stem cell (HSC) transplantation. However, while cytotoxic T lymphocytes (CTLs) recognizing mismatched HLA molecule of the donor have been shown to be involved in graft rejection, there has been no biological evidence in humans that graft rejection is mediated by DSAs. In the present study, we demonstrate a case of cord blood allograft rejection in which DSAs acted to mediate graft rejection. Interestingly, CTL clones specific for donor HLA molecule were also isolated, suggesting that humoral and cellular immune responses were together responsible for allograft rejection. A patient with graft rejection after HLA-mismatched cord blood transplantation was studied. The white blood cell count increased transiently, subsequently decreased to undetectable level, and finally graft rejection was diagnosed on day 34. Screening for pre-transplant anti-HLA antibodies was not routinely performed at that time. We initially presumed that DSA would be responsible for graft rejection. The patient serum on day 36 were screened for HLA antibodies and further evaluated to determine HLA specificities using a LABScreen Single Antigen Kit. An antibody against HLA-B* 54:01, which was expressed in donor cells but not in patient cells, was detected. To test the effect of the DSA against HLA-B* 54:01 on HSC engraftment, antibody-dependent cellular cytotoxicity (ADCC) activity was assessed using unrelated HLA-B* 54:01-positive bone marrow mononuclear cells (BMMNCs) pre-cultured with patient serum and pre-transplant NK cells. The patient serum clearly showed an inhibitory effect on colony formation. Complement-dependent cytotoxicity activity of the DSA could not be detected. These data suggest that the DSA impaired the cord blood engraftment through ADCC activities. We next determined if cellular immunity was also responsible for allograft rejection. Two independent CTL clones, CD4-CD8+ and CD4+CD8-, were isolated from the patient blood at the time of graft rejection by limiting dilution. Both CTL clones were of patient origin by using short tandem repeat analysis and showed cytotoxicity against donor cells but not patient cells in Cr release assay. A CTL stimulation assay using COS cells transfected with various mismatched donor HLA cDNA constructs demonstrated that CD8+ and CD4+CTL clones recognized HLA-B* 54:01 and -DRB1* 15:02 molecules, respectively, both of which were expressed in donor cells but not in patient cells. To test the effect of the CTL clones on engraftment, a colony forming assay using either HLA-B* 54:01 or -DRB1* 15:02-positive unrelated BMMNCs pre-cultured with the corresponding CTL clones was performed. Each of these CTL clones inhibited colony formation from unrelated BMMNCs sharing target HLA in a dose-dependent manner. These data suggest that the CTLs also impaired the cord blood engraftment. Furthermore, anti-HLA-B* 54:01 antibody was detected in cryopreserved pre-transplant patient serum, and in nested PCR assays specific for the HLA-B* 54:01-specific CTL clone’s uniquely rearranged T cell receptor V beta chain, PCR product was detected by amplification of cDNA from pre-transplant patient peripheral blood mononuclear cells. Thus, both HLA-B* 54:01-specific antibody and CTL clone developed in the patient prior to transplantation. The present data provide the first direct evidence showing that humoral and cellular immune responses were involved in the graft rejection. Of note is the recognition of the same mismatched HLA-B* 54:01 molecule by the DSA and CTL, suggesting that humoral and cellular immune processes acted together to mediate allograft rejection. Given the difficulty in detecting HLA-specific CTLs in pre-transplant patient blood in contrast to the easiness in screening for DSAs, the presence of DSA not only means a direct deleterious effect on donor cells but it may also reflect the presence of CTLs that causes allograft rejection. Further studies are warranted to clarify whether the present observation can be duplicated in other patients with DSA. We are conducting the study to clarify whether patients with anti-HLA antibodies have CTLs that recognize the same HLA molecules before transplantation. Disclosures: No relevant conflicts of interest to declare.

Identification of genes and pathways associated with cytotoxic T lymphocyte infiltration of serous ovarian cancer

Background:Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer.Methods:For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated.Results:The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment.Conclusion:Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.

Mode-of-Action, Efficacy, and Safety of a Homologous Multi-Epitope Vaccine in a Murine Model for Adjuvant Treatment of Renal Cell Carcinoma

Background and objectiveIn a phase-III trial it was recently shown that an adjuvant renal cell carcinoma (RCC) vaccine (Reniale) reduces the risk of tumour progression following nephrectomy. This clinical trial focused on efficacy and did not investigate end-points relating to mode-of-action of the vaccine. In a murine model we investigated mode-of-action, efficacy, and safety of a homologous RENCA cell-based vaccine. Design, setting, and participantsSix groups with 12 BALB/c mice per group received five vaccinations (lysate of 1×106–1×107 RENCA cells, manufactured with or without prior IFN-γ incubation) at 3-wk intervals before tumour transplantation and one vaccination 14 d afterwards. Controls (12 mice) received only solvent. All mice were sacrificed 21 d after tumour transplantation. MeasurementsAnimal welfare, tumour growth, number of metastases, and the presence of cytotoxic T-lymphocytes as determined by a 51chromium-release assay. Adoptive immune transfer experiments (vaccination of nine mice with the RENCA vaccine or saline and transfer of serum, spleen cells, and CD4 and/or CD8 depleted spleen cells into five recipient mice each) were carried out to demonstrate involvement of different immune mechanisms. ResultsAll controls developed a renal tumour, compared to 7/72 animals (9.7%) in the vaccine groups. The mean number of lung metastases was 100 (range 3–750) in controls and 4 (range 0–196) in the vaccine groups, respectively. Tumour uptake and number of metastases were not related to the vaccine dose. The 51chromium-release assay confirmed a significant tumour-specific cytolytic activity and marginally increased NK activity of splenocytes from vaccinated mice against RENCA cells compared to controls. Adoptive immune transfer experiments showed that the antitumoural effective immune mechanisms are cell-based. ConclusionsWe could demonstrate the mode-of-action, efficacy, and safety of a homologous tumour vaccine in a RENCA model. These findings support the positive results from a phase-III trial with Reniale.

Structural and Functional Constraints Limit Options for Cytotoxic T-Lymphocyte Escape in the Immunodominant HLA-B27-Restricted Epitope in Human Immunodeficiency Virus Type 1 Capsid

Control of human immunodeficiency virus type 1 (HIV-1) by HLA-B27-positive subjects has been linked to an immunodominant CD8(+) cytotoxic T-lymphocyte (CTL) response targeting the conserved KK10 epitope (KRWIILGLNK(263-272)) in p24/Gag. Viral escape in KK10 typically occurs through development of an R(264)K substitution in conjunction with the upstream compensatory mutation S(173)A, and the difficulty of the virus to escape from the immune response against the KK10 epitope until late in infection has been associated with slower clinical progression. Rare alternative escape mutations at R(264) have been observed, but factors dictating the preferential selection of R(264)K remain unclear. Here we illustrate that while all observed R(264) mutations (K, G, Q, and T) reduced peptide binding to HLA-B27 and impaired viral replication, the replicative defects of the alternative mutants were actually less pronounced than those for R(264)K. Importantly, however, none of these mutants replicated as well as an R(264)K variant containing the compensatory mutation S(173)A. In assessing the combined effects of viral replication and CTL escape using an in vitro coculture assay, we further observed that the compensated R(264)K mutant also displayed the highest replication capacity in the presence of KK10-specific CTLs. Comparisons of codon usage for the respective variants indicated that generation of the R(264)K mutation may also be favored due to a G-to-A bias in nucleotide substitutions during HIV-1 replication. Together, these data suggest that the preference for R(264)K is due primarily to the ability of the S(173)A-compensated virus to replicate better than alternative variants in the presence of CTLs, suggesting that viral fitness is a key contributor for the selection of immune escape variants.

Lack of MHC class I surface expression on neoplastic cells and poor activation of the secretory pathway of cytotoxic cells in oral squamous cell carcinomas

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the secretory pathway of perforin/granzymes to kill their target cells. In contrast to NK cells, CTL responses are MHC class I restricted. In this study we analysed the relative activation of CTL and NK cells in relation with MHC class I expression on oral squamous cell carcinomas (OSCCs). MHC class I expression was investigated in 47 OSCCs by immunohistochemistry using HCA2, HC10 and β2-m antibodies. The presence of CTLs, NK cells, and its activation, was investigated in 21 of these OSCCs using respectively, CD8, CD57 and GrB7 antibodies. The Q-Prodit measuring system was used for quantification of cytotoxic cells. All OSCCs showed weak or absent staining of β2-m on the cell surface. The absence of β2-m was significantly associated with absent expression of MHC class I heavy chain as detected by HC10 antibody (P = 0.004). In tumour infiltrates CTLs always outnumbered NK cells, as reflected by the ratio CD57/CD8 being always inferior to one (mean: 0.19; SD: 0.15). The proportion of activated cytotoxic cells as detected by granzyme B expression was generally low (mean: 8.6%; SD 8.9). A clear correlation between MHC class I expression and the relative proportion of NK cells/CTLs was not found. This study shows that the majority of OSCCs show weak or absent expression of MHC class I molecules on the cell surface, possibly due to alterations in the normal β2-m pathway. The low proportion of granzyme B-positive CTLs/NK cells indicates that the secretory pathway of cytotoxicity is poor in these patients. The lack of correlation between MHC class I expression and CTL/NK cell activation as detected by granzyme B expression suggests that, next to poor antigen presentation, also local factors seem to determine the final outcome of the cytotoxic immune response. © 1999 Cancer Research Campaign

Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: Evidence for immunoselection of antigen-loss variantsin vivo

Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen-specific tumor vaccines. In this study, we examined the expression of Melan A/MART-1, tyrosinase and gp100lPmel17 in fresh melanoma tissues of HLA-A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART-1 and tyrosinase in patients with metastatic melanoma. In 2 patients with advanced disease, CTL responses against Melan A/MART-1 and tyrosinase were induced by intradermal immunization with synthetic nona- or deca-peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen-positive tumors and select for antigen-loss variants in vivo.

Polyomavirus DNA is damaged in target cells during cytotoxic T-lymphocyte-mediated killing

Target cell DNA damage is an early event in cytotoxic T-lymphocyte (CTL)-mediated killing. It has been hypothesized that this DNA damage may serve as one mechanism of destroying viral genetic material inside infected cells. We directly examined the fate of viral DNA in target cells during CTL-mediated lysis. Polyomavirus DNA in transfected murine P815 mastocytoma targets was digested along with cellular DNA into oligonucleosome-sized fragments, although intact forms, possibly virion-associated DNA, were also present. In infected BALB/3T3 murine fibroblasts, which normally undergo single-stranded nicks when killed by CTL, polyomavirus DNA was converted to relaxed forms in the presence of CTL. These results suggest that the fate of the viral DNA depends on the stage of the viral life cycle and corresponds to the fate of the host cell DNA. Cleavage of the viral genome prior to assembly may thus be an important mechanism in specific antiviral immunity.

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice.

DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration. The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay. For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10(6) to 10(5) lymphocytes and 10(3) to 10(2) target cells respectively, no tumor neutralization was obtained. The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity. In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.