Cancer Immunotherapy: Whence and Whither.

The current concepts and practice of cancer immunotherapy evolved from classical experiments that distinguished "self" from "non-self" and the finding that humoral immunity is complemented by cellular immunity. Elucidation of the biology underlying immune checkpoints and interactions between ligands and ligand receptors that govern the immune system's ability to recognize tumor cells as foreign has led to the emergence of new strategies that mobilize the immune system to reverse this apparent tolerance. Some of these approaches have led to new therapies such as the use of mAbs to interfere with the immune checkpoint. Others have exploited molecular technologies to reengineer a subset of T cells to directly engage and kill tumor cells, particularly those of B-cell malignancies. However, before immunotherapy can become a more effective method of cancer care, there are many challenges that remain to be addressed and hurdles to overcome. Included are manipulation of tumor microenvironment (TME) to enhance T effector cell infiltration and access to the tumor, augmentation of tumor MHC expression for adequate presentation of tumor associated antigens, regulation of cytokines and their potential adverse effects, and reduced risk of secondary malignancies as a consequence of mutations generated by the various forms of genetic engineering of immune cells. Despite these challenges, the future of immunotherapy as a standard anticancer therapy is encouraging. Mol Cancer Res; 15(6); 635-50. ©2017 AACR.

New Approaches to Investigate Drug-Induced Hypersensitivity.

The workshop on "New Approaches to Investigate Drug-Induced Hypersensitivity" was held on June 5, 2014 at the Foresight Center, University of Liverpool. The aims of the workshop were to (1) discuss our current understanding of the genetic, clinical, and chemical basis of small molecule drug hypersensitivity, (2) highlight the current status of assays that might be developed to predict potential drug immunogenicity, and (3) identify the limitations, knowledge gaps, and challenges that limit the use of these assays and utilize the knowledge gained from the workshop to develop a pathway to establish new and improved assays that better predict drug-induced hypersensitivity reactions during the early stages of drug development. This perspective reviews the clinical and immunological bases of drug hypersensitivity and summarizes various experts' views on the different topics covered during the meeting.

Assessment of Newcastle disease-specific T cell proliferation in different inbred MHC chicken lines.

In this study, we have described the establishment of an antigen-specific T cell proliferation assay based on recall stimulation with Newcastle disease (ND) antigen; further, we have described the results obtained after recall stimulation of animals containing different major histocompatibility complex (MHC) haplotypes, vaccinated against ND. First optimization of the assay was performed to lower unspecific proliferation and to enhance antigen-specific T cell proliferation. These two issues were achieved using ethylene diamine tetra acetic acid as stabilizing agent in blood samples and autologous immune serum in culture medium. The optimized assay was used to screen chickens with different MHC haplotypes for their ability to perform T cell proliferation. Results showed that the antigen-specific response of CD4(+) and CD8(+) T cells from B12 chickens was generally low, whereas B13, B130 and B201 chickens were medium in CD4(+) or CD8(+) T cell responses. High responses were seen only in few animals of each haplotype and not in general. A polymorphism in the chicken CD8α gene was found in our experimental chicken lines, resulting in incapability to detect CD8α(+) T cells using antibodies from the CT8 clone. Screening chickens with alternative antibodies showed that antibodies from the 2-398 clone were able to discriminate all CD8α(+) cells from CD8α(-) cells, and consequently this antibody was used in a second vaccination experiment performed with chickens of the haplotypes B13 and B130. This experiment showed a significant difference in antigen-specific proliferation of CD4(+) T cells between the two lines, but not in CD8α(+) T cell proliferation.

CTLs' repertoire shaping in the thymus: a Monte Carlo simulation.

MOTIVATION: The human immune system evolved a multi-layered control mechanism to eliminate self-reactive cells. Of these so-called tolerance induction mechanisms, lymphocytes T education in the thymus gland represents the very first one. This complicated process is not fully understood and quantitative models able to help in this endeavor are lacking. Here, we present a stochastic computational model of the thymus which combines data-driven prediction methods and a novel method based on protein-protein potential measurements for assessing molecular binding among cell receptors, major histocompatibility complex (MHC) molecules, and self-peptides. RESULTS: Of all possible specificities of immature T cells entering the thymus, only a small fraction is actually selected for maturation. Monte Carlo simulations of thymocytes selection in the thymus are performed varying the size of the self and a parameter determining the number of encounter with antigen-presenting cells (APCs). We score the fraction of self-reacting thymocytes leaving the thymus as mature naive T cells and show that self-reactivity is only marginally dependent on the number of self-molecules presented by APCs, while it is strongly affected by a parameter proportional to the time spent in the thymus. We study how this measure changes when we vary the number of MHC alleles and found an optimal number not too different from what we have in reality. The main result of this study is more methodological than biological as we show that immunoinformatics data and methods can be used in systemic level simulation of immune processes.

A comparison of rainbow trout cell lines for their expression of the major histocompatibility complex genes and the induction of beta-2-microglobulin by dsRNA.

Western blotting with polyclonal antisera to polypeptides of the rainbow trout major histocompatibility (MH) genes and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to compare expression of MH genes in rainbow trout cell lines. One line was the spleen monocyte/macrophage-like RTS11, which grew loosely on plastic surfaces. Adherent cell lines were fibroblast-like RTG-2 from gonads and four epithelial-like cell lines from gill, intestine, liver and hepatoma: RTgill-W1, RTgutGC, RTL-W1, and RTH-149 respectively. All cell lines expressed a 45 kDa MHC class I alpha chain. All cell lines expressed beta-2-microglobulin (beta2m), which was at 11 kDa, but detection was abrogated following trypsinization prior to cell collection. All cell lines expressed transcripts for MH class II alpha and MH class II beta genes; however, MH class II polypeptides were expressed only in RTS11, the only cell line from a lineage of antigen-presenting cells. We report here that double stranded RNA up regulates beta2m and that these cell lines and antisera can be employed for studying MH regulation.

Histocompatibility assessment in the chicken colonies of the RIR-Y8/NU, YL, WL-G, and BL-E closed for 28-48 years.

Histocompatibility was assessed in the RIR-Y8/NU, BL-E, YL, and WL-G chicken closed colonies by hemagglutination test using anti-red blood cell (RBC) antibodies (HT), skin transplantation test (STT), and formation of isohemagglutinins (FIHs) during STT. The YL individuals all showed the survival of skingrafts for more than 17 days with no FIHs in STT and no RBC antigenic variations in HT, indicating a histocompatible nature together with high homogeneity at serological loci. The BL-E as well as WL-G closed colonies were also found to be histocompatible in the STT with no FIHs, although the HT showed heterogeneities at serological locus/loci other than the B and C blood group loci which have significant effects on histocompatibility or FIHs in chicken. In the RIR-Y8/NU closed colonies, one individual in 6 reciprocal combinations of the STT showed early skingraft rejection with positive FIHs caused by different B locus alleles, and the HT suggested relatively high heterogeneities at the other serological loci too. The closed colonies of YL, BL-E, and WL-G will be useful avian materials for transplantation or related experiments, but RIR-Y8/NU needs further pedigree selection for serological homogeneity.

Response of the rainbow trout monocyte/macrophage cell line, RTS11 to the water molds Achlya and Saprolegnia.

The Saprolegniales are responsible for various fish mycoses worldwide and considered the most important fungi afflicting fresh water fish. Saprolegniosis leads to massive epidermal destruction and macrophage recruitment, yet little is known regarding the cytological response of their piscine hosts. The objective of this study was to explore the response of fish macrophage to members of the Saprolegniales using the rainbow trout monocyte/macrophage cell line, RTS11. After 48 h in co-culture, RTS11 demonstrated chemotaxis, adherence and homotypic aggregation to both live and heat-killed fungal spores and mycelia. This aggregation was enhanced when using conditioned media from co-cultured RTS11 and Achlya, suggesting the presence of synergistic effectors of aggregation. Although fungal toxins were not evident, as cells remained viable throughout fungal overgrowth, phagocytosis was inhibited due to large fungal spore size, allowing these molds to evade macrophage defenses. Although class I MH and other viral response genes showed no significant change in expression, calreticulin and interleukin-8 were moderately up-regulated implicating calcium modulation and chemotactic response, respectively. Cyclooxygenase (COX-2) and the cytokines IL-1beta and TNFalpha were strongly up-regulated in the presence of Achlya, while gene expression of the class II major histocompatibility (MH II) receptor and associated molecules appeared down-regulated, suggesting fungal interference of immune function. Previous studies have shown an increased dependence of macrophage in immune function at low temperatures; based upon data presented here, this reduction of macrophage MH II receptor expression and inability to phagocytose spores may limit host response thereby providing increased susceptibility to these opportunistic pathogens.

Major histocompatibility complex (MHC) tetramer technology: an evaluation.

Single-cell assays are currently favored to quantitate T-cell responses. Staining antigen-specific T-cells with fluorescently labeled tetrameric major histocompatibility complex (MHC)/peptide complexes has greatly enhanced the ability to assess the cellular dynamics of an immune response at the single-cell level. This article reviews MHC tetramer technology, defining it, discussing how MHC tetramers are made, outlining the benefits of this technology, comparing and contrasting it to other methods for evaluating immune responses, and describing current applications.

Comparative assessment of MHC antigen expression in bladder and testis tumour biopsies and established urological tumour cell lines: the relevance of cytokine and gene transfection for correction of defective MHC antigens.

The aim of this study using radio-binding (RB), peroxidase-anti-peroxidase (PAP) and immunoprecipitation (IP) techniques was to investigate the pattern of major histocompatibility complex (MHC) antigen expression in urological malignancies and to compare the results with those seen in established urological human tumour cell lines. The results showed that using PAP technique, the percent bladder cases showing complete loss or cases with greater than 90% of tumour cells negative with W6/32 (detects all class I antigens), HC10 (detects free heavy chain) and BMM.1 (detects beta2-mirogobulin) monoclonal antibodies (Mab) were 16%, 44% and 2% respectively. In a subgroup of 37 cases, the intensity of MHC class II antigen expression for strong, weak and negative cases were 9 (24%), 8 (22%) and 20 (54%) respectively. The expression for class I antigens on testis tumours was mainly negative and when positive, it was present in a small percent of tumour cells. This was also observed for class II antigens where only 8% of cases showed some degree of positivity. Using RB technique, 10 of 12 (85%) of tumour lines expressed class I antigens spontaneously and following interferon gamma (IFNgamma) stimulation, the 2 negative lines one testis (Tera I) and one bladder (Fen) remained negative and 2 lines (both testis lines Tera II and Ep2102) showed a significant class I up-regulation. None of the lines expressed class II antigens spontaneously and following IFNgamma stimulation, 8 of 12 (66%) were induced. The absence of class I and II antigens in the negative lines was confirmed using IP technique. In the case of one class I negative bladder cell line i.e. Fen, the biochemical analysis showed the absence of beta2-m gene product which could not be restored by IFNgamma stimulation. However, transfection of the cells with beta2-m gene resulted in the expression of fully assembled class I antigens, indicating that the loss of antigens was due to the absence of functional beta2-m gene. These results indicated the similarity between the pattern of expression of MHC antigens on tumour biopsies and established tumour cell lines. They also demonstrated that both cytokine stimulation and gene transfection could be used to correct defective class I antigens in tumour cell lines. These approaches might have important implications for pre-selection of bladder cancer patients for cytokine or gene therapies.

In vivo microscopic assessment of cremasteric microcirculation during hindlimb allograft rejection in rats.

Experimental and clinical studies of vascular allogenic extremity transplantation have yielded disappointing results and have not been clinically useful. With recent advances in transplantation immunology, considerable interest has focused on the understanding of leukocyte-endothelial interaction at the microcirculatory level. The objective of this study was to characterize the alterations in leukocyte-endothelial interaction in the early stages of rat hindlimb allograft rejection. To study the changes at the microcirculatory level, a new microsurgical model was developed; the cremaster muscle was incorporated into the transplanted hindlimb. The purpose of this study was to report on the microcirculatory changes during rat hindlimb allograft rejection. A total of 24 transplantations were performed among the four experimental groups. In a control group, 12 rat hindlimb-cremaster grafts were transplanted between genetically identical animals, Lewis to Lewis. Microcirculatory measurements of graft survival were taken at 24 hours (group 1A, n = 6) and at 72 hours (group 1B, n = 6). In the rejection control group, 12 transplantations were performed across a major histocompatibility barrier between Lewis-Brown Norway and Lewis rats. Microcirculatory measurements were taken at 24 (group 2A, n = 6) and 72 hours (group 2A, n = 6) as above. The following parameters were evaluated to discover the leukocyte-endothelial interaction: endothelial edema index and the number of rolling, adherent, and transmigrating leukocytes and lymphocytes in the postcapillary venule. Physical signs of limb rejection, such as edema, erythema, scaling, plaque formation on the skin, hair loss, and skin surface temperature, were monitored. Microcirculatory signs of rejection included the following. There was a significant increase in the number of adherent leukocytes in allograft transplants at both 24 hours (205 percent; 2.05 +/- 0.38) and 72 hours (431 percent; 9.11 +/- 3.41) when compared with isograft controls (1.00 +/- 0.89 at 24 hours; 2.11 +/- 0.34 at 72 hours) (p < 0.05). The activation of leukocyte transmigration increased more than 7-fold in muscle allografts at 24 hours (0.55 +/- 0.25 versus 4.16 +/- 1.89) and more than 6-fold at 72 hours (0.72 +/- 0.38 versus 4.38 +/- 1.28) after transplantation (p < 0.05). Endothelial edema index, a measure of endothelial swelling and cellular deposit accumulation, increased more than 119 percent in the allograft group 72 hours after transplantation (1.23 +/- 0.07 versus 1.46 +/- 0.09) (p < 0.05). The first clinical signs of limb rejection were scaling of the skin or hair loss; they were observed between the seventh and ninth postoperative days. The composite rat hindlimb-cremaster model presented in this study introduces a new in vivo approach to monitor acute graft rejection using the intravital microscopy system. This is a valuable model for defining the timing, sequence, and correlation between immunologic events and clinical signs during the acute phase of allograft rejection.

Dissociation of mouse cardiac transplant rejection and donor alloantigen-specific T cell responsiveness.

Mouse hearts transplanted into MHC disparate donors are usually rejected 1 week after placement. It is widely accepted that alloantigen-reactive helper T lymphocytes (HTL) and cytotoxic T lymphocytes (CTL) are the key mediators of acute allograft rejection. This report demonstrates that the presence or absence of 'traditional' graft-reactive HTL and CTL is not necessarily related to allograft survival. In these studies, donor/recipient combinations disparate only at MHC or only at minor histocompatibility (mH) loci were employed. Allograft survival was monitored, donor-reactive IL-2 (interleukin-2) producing HTL and CTL were quantified by modified limiting dilution analysis, and serum levels of cytolytic alloantibody were determined. C57BL/10 hearts (H-2b) transplanted into B10.BR (H-2k) recipients (full MHC disparity) enjoyed prolonged survival despite massive infiltration of the allograft by donor-reactive HTL and CTL. IgM, but not IgG, donor-reactive alloantibody was present in the sera of these mice. Hence, traditional IL-2 producing HTL and CTL were not capable of mediating acute graft rejection, nor of providing help for alloantibody isotype switching in this MHC disparate combination. In contrast, C3H/HeN (H-2k) hearts transplanted into B10.BR (H-2k) recipients (mH disparity only) were acutely rejected. Donor-reactive HTL and CTL were rare or not detectable in these recipients, and cytolytic alloantibody was not detectable. Similar observations were made when B10.BR hearts were transplanted into C3H/HeN recipients. Interestingly, treatment of recipients with anti-CD4 monoclonal antibody prevented rejection of mH disparate allografts. Therefore, CD4+ T cells were required for rejection of mH disparate allografts, but this process was independent of detectable IL-2 production or CTL function. Hence, the significance of monitoring 'traditional' T cell functions should be questioned in certain donor/recipient combinations.