Major Histocompatibility Class Class Research Articles

A phase 0/surgical window-of-opportunity study in progress, evaluating evolocumab in patients with high-grade glioma or glioblastoma.

TPS2076 Background: High-grade gliomas (HGGs) are immunologically ‘cold’ tumors. This phenomenon is partly due to reduced expression of major histocompatibility class (MHC) I on the surface of tumor cells, which prevents CD8+ cytotoxic T lymphocyte activity (CTLs). Blockade of proprotein convertase subtilisin/kexin type 9 (PCSK9) increases MHC class I expression, enhances CTL tumoral infiltration, and potentiates checkpoint inhibition in vivo. Evolocumab is an FDA-approved fully human IgG2 monoclonal antibody PCSK9 inhibitor which is clinically indicated for hyperlipidemia. This study seeks to determine whether evolocumab can cross the blood-brain barrier (BBB) and enhance MHC I expression on resected tumor cells, serving as a potential future adjunct for immunotherapy. Methods: This study will enroll ten patients over 18 years who have newly diagnosed or recurrent HGG. These patients will also need to be undergoing resection of their tumor as part of their planned treatment pathway. Following informed consent, patients will receive evolocumab (420mg, subcutaneously) 7-14 days before surgical debulking of the tumor. We will collect tissue which is not required for histological tumor analysis and compare it with a contemporaneous matched control cohort. This will consist of resected tumor specimens from patients not treated with evolocumab. Quantification of the drug will be performed using mass spectroscopy, flow cytometry, and single-cell sequencing. The primary objective of this study is to evaluate whether evolocumab can cross the BBB and be measured in resected tumor specimens taken from patients with HGG. Secondary objectives include an analysis of lipid metabolism and MHC-I expression on the tumor via flow cytometry and CITEseq. Wilcoxon rank-sum test or a two-sample t-test, will compare groups for these endpoints. Exploratory analyses will determine if evolocumab leads to changes in tumorigenic pathways and the immune profile of tumor infiltrating lymphocytes (TILs). Bioinformatic analyses will be performed using protein set enrichment, gene ontology (GO) annotations, and search tools from the retrieval of interactive genes/proteins (STRING). Progress: The study was activated on 10/04/2021 (NCT04937413) and at the time of submission has enrolled 5 participants (4 to control arm, 1 to intervention arm). Clinical trial information: NCT04937413.

MHC Class I Deficiency in Solid Tumors and Therapeutic Strategies to Overcome It.

It is now well accepted that the immune system can control cancer growth. However, tumors escape immune-mediated control through multiple mechanisms and the downregulation or loss of major histocompatibility class (MHC)-I molecules is a common immune escape mechanism in many cancers. MHC-I molecules present antigenic peptides to cytotoxic T cells, and MHC-I loss can render tumor cells invisible to the immune system. In this review, we examine the dysregulation of MHC-I expression in cancer, explore the nature of MHC-I-bound antigenic peptides recognized by immune cells, and discuss therapeutic strategies that can be used to overcome MHC-I deficiency in solid tumors, with a focus on the role of natural killer (NK) cells and CD4 T cells.

Serum β2-microglobulin levels in Coronavirus disease 2019 (Covid-19): Another prognosticator of disease severity?

β2-microglobulin (β2-m), a 11.8 kDa protein, pairs non-covalently with the α3 domain of the major histocompatibility class (MHC) I α-chain and is essential for the conformation of the MHC class I protein complex. Shed β2-m is measurable in circulation, and various disorders are accompanied by increases in β2-m levels, including several viral infections. Therefore, we explored whether β2-m levels could also be elevated in Coronavirus disease 2019 (Covid-19) and whether they predict disease severity. Serum β2-m levels were measured in a cohort of 34 patients infected with SARS-CoV-2 on admission to a tertiary care hospital in Riyadh, Saudi Arabia, as well as in an approximately age-sex matched group of 34 uninfected controls. Mean β2-m level was 3.25±1.68 mg/l (reference range 0.8-2.2 mg/l) in patients (mean age 48.2±21.6) and 1.98±0.61 mg/l in controls (mean age 48.2±21.6). 17 patients (mean age 36.9± 18.0) with mean β2-m levels of 2.27±0.64 mg/l had mild disease by WHO severity categorization, 12 patients (mean age 53.3±18.1) with mean β2-m levels of 3.57±1.39 mg/l had moderate disease, and five patients (of whom 2 died; mean age 74.4±13.8) with mean β2-m levels of 5.85±1.85 mg/l had severe disease (P < = 0.001, by ANOVA test for linear trend). In multivariate ordinal regression β2-m levels were the only significant predictor of disease severity. Our findings suggest that higher β2-m levels could be an early indicator of severity of disease and predict outcome of Covid-19. As the main limitations of the study are a single-center study, sample size and ethnicity, these results need confirmation in larger cohorts outside the Arabian Peninsula in order to delineate the value of β2-m measurements. The role of β2-m in the etiology and pathogenesis of severe Covid-19 remains to be elucidated.

Cytomegalovirus Promotes Aberrant Memory CD4 T Cell Differentiation and Immune Function after Allogeneic Stem Cell Transplantation

Background: Cytomegalovirus (CMV) serostatus is associated with transplant related mortality, relapse and survival following allogeneic stem cell transplant (SCT). Prior studies have demonstrated that CMV seronegative recipients (R-) receiving a CMV seropositive graft (D+) experience inferior outcomes compared to the R-/D- combination, an observation that is independent of viral reactivation and due to increased bacterial and fungal sepsis (Nichols WG et al, J. Infect. Dis. 2002; 185:273-82). We therefore investigated the hypothesis that CMV exposure, including that by the donor only prior to transplant, promotes endogenous and long-term immunodeficiency after SCT. Methods: A total of 108 patient peripheral blood samples were obtained from the Chronic Graft-vs.-Disease (GVHD) Consortium, which includes both patients with and without chronic GVHD. Seventy-three baseline samples were taken at an average of 458 days (range 63 to 1,965) after SCT, and 35 paired blood samples were taken at an average of 94 days follow-up. Transplant parameters, CMV serostatus, and CMV viral titers after SCT were collected. We performed multiparameter flow cytometry to assess innate and adaptive immunity after SCT in the context of CMV, including transcription factor and cytokine expression, together with TCR/BCR sequencing to assess immune diversity. Results: We demonstrate that CMV exposure (including solely in the donor prior to transplant or in the recipient after transplant) is strongly associated with long-lasting expansion of a terminally differentiated MHC class II-restricted donor memory T cell subset identifiable by the surface markers CD4+/CD57+/CD27-(Figure 1). These cells are CCR7neg/CD45RO+/CD45RA+/-, express T-bet, eomesodermin (EOMES), granzyme B, and secret high amounts of Th1 cytokines IFNγ and TNF (Figure 1). This T cell subset represents an average of 2.8% for D-/R- transplants and &amp;gt;10% (and up to 70%) of total CD4+ T cells in all other serostatus combinations irrespective of the presence or absence of measurable CMV reactivation after SCT (p&amp;lt;0.05 across all pairwise comparisons with D-/R- subset, Figure 2). Furthermore, this phenotype persists in an expanded fashion for years following SCT (samples were taken up to 5 years post-SCT). Follow-up samples from the same patient taken up to 218 days after the initial sample confirm phenotypic stability over time (r=0.90, p&amp;lt;0.0001, Figure 3). Detectable CMV replication after SCT further augmented this memory CD4+ T cell subset, and antiviral treatment for CMV did not limit its expansion. We demonstrate that these differentiated memory CD4+ T cells are associated with a significant loss of T cell receptor diversity (r=-0.60, p=0.004) and reduced proportions of major histocompatibility class (MHC) II expressing classical monocytes (r=-0.43, p=0.0002) and plasmacytoid dendritic cells (r=-0.31, p=0.0085) in peripheral blood. Conclusion: These data describe a temporally stable, expanded MHC class II-restricted memory T cell population with high cytotoxic potential and putative mechanisms by which CMV exposure modifies immune function in the peri-transplant period, namely the limitation of TCR diversity and antigen presentation. This cell population is expanded even in the absence of detectable CMV reactivation post SCT and can be observed for several years following transplant. We thus provide a potential mechanism for the immunodeficiency and broad increase in infection mortality invoked by CMV exposure. In particular, these findings may provide a rationale for supporting the selection of CMV- donors for R- SCT recipients. Disclosures Jagasia: Ocugen: Other; Mallinckrodt: Research Funding; Janssen: Research Funding. Dahlman:Kadmon Holdings, Inc: Other. Lee:Syndax: Research Funding; Pfizer: Consultancy, Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Kadmon: Research Funding; Takeda: Research Funding; Novartis: Research Funding; Incyte: Consultancy, Research Funding. Hill:Generon: Consultancy; Roche: Research Funding; Compass Pharmaceuticals: Research Funding; CSL: Research Funding; Implicit Bioscience: Research Funding; Pharmacyclics: Research Funding.

Murine Bone Marrow-Derived Megakaryocytes Are Capable of Antigen Cross-Presentation on Major Histocompatibility Class (MHC) I Molecules

Major Histocompatibility complex (MHC) class I is a complex composed of two proteins, the MHC-encoded alpha heavy chain and the beta2-microglobulin light chain. In the endoplasmic reticulum, it receives an antigen peptide of usually eight to eleven residues in its groove and is exported to the plasma membrane. In classical MHC class I presentation, endogenous antigens are presented to CD8+ T cells. On the other hand, cross-presentation consists in the presentation of exogenously-derived proteins in the context of MHC class I molecules to CD8+ T lymphocytes, inducing a cytotoxic response against the antigen presenting cell (APC). Platelets have been previously shown to be able to cross-present immunogenic ovalbumin (OVA) peptides, as well as bacterial peptides and to trigger CD8+ T lymphocyte activation. It is thought that the antigen cross presenting function of platelets is transmitted from megakaryocytes (MK) during thrombopoiesis, but whether megakaryocytes can cross-present is still unknown. To address this question, we developed an in vitro endocytosis model of murine bone marrow-derived megakaryocytes. C57BL/6 mouse megakaryocytes are enriched by gradient sedimentation and pulsed with fluorescent ovalbumin (OVA) for various times. In addition, we used DQ-OVA, a BODIPY FL-conjugated OVA, which becomes fluorescent upon proteolysis. Cells were then analyzed by flow cytometry or confocal microscopy to assess the OVA megakaryocyte internalization, its proteolytic degradation and its loading onto MHC class I molecules. We can show the endocytosis of exogenous OVA by megakaryocytes, in a pathway involving lysosomes. The endocytosed OVA were subsequently proteolytically cleaved into the immunogenic OVA peptide SIINFEKL and the peptide is inserted into the antigen binding groove of MHC class I molecules. The MHC class I-OVA peptide complexes are stored in the MK alpha-granules and can be translocated to the plasma membrane. Thus, we have shown that MKs can effectively process and present exogenous antigens on MHC class I molecules. This opens a new avenue toward a functional characterization of this pathway and its modulation, leading to new therapeutic approaches in autoimmune diseases targeting megakaryocytes, such as immune thrombocytopenia (ITP). DisclosuresNo relevant conflicts of interest to declare.

Genetic variability of MHC class II DQB exon 2 alleles in yak (Bos grunniens)

The major histocompatibility class (MHC) DQ molecules are dimeric glycoproteins revealing antigen presentation to CD(4+) T cells. In the present study, the exon 2 of the MHC class II DQB gene from 32 yaks (Bos grunniens) was cloned, sequenced and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 25 DQB exon 2 alleles among 32 yaks, all alleles are found to belong to DQB1 loci. These alleles exhibited a high degree of nucleotide and amino acid polymorphisms with most amino acid variations occurring at positions forming the peptide-binding sites. The DQB loci were analyzed for patterns of synonymous (d S) and non-synonymous (d N) substitution. The yak was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (d N = 0.15, P < 0.001). It appears that this variability among yaks confers the ability to mount immune responses to a wide variety of peptides or pathogens.

Genetic Alterations In Immune Cell Crosstalk Genes In Diffuse Large B-Cell Lymphoma Predict Survival

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype accounting for ∼40% of all non-Hodgkin lymphomas. Although R-CHOP has significantly improved outcome in DLBCL, 35% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Immune cell signatures have been shown to impact survival in DLBCL (Lenz et al, NEJM 2008). Recurrent mutations and/or copy number alterations (CNAs) in genes that impact the tumor's immune-cell crosstalk have recently been described, but their clinical impact is unknown. Moreover, mutations and copy-number loss involving genes that foster immune escape has been implicated as a dominant oncogenic mechanism in DLBCL (Challa-Malladi et al, Can Cell 2011). We sought to determine the relationship between genetic alterations involving immune-cell crosstalk pathways and survival in a cohort of DLBCL patients treated with R-CHOP. MethodsFollowing our initial analysis of 91 whole transcriptome sequencing (RNA-seq) including 40 with whole genome sequencing, we validated SNVs in the genes of interest by Sanger sequencing in the tumor and matched germline DNAs if available. RNA-seq-derived gene expression data were also used to determine the cell of origin (COO) distinction. Affymetrix SNP 6.0 microarrays were used to ascertain the CNAs in all 91 DLBCL samples. Clinical outcome correlations were analyzed by Cox regression and the log-rank test in the 82 patients treated with R-CHOP (median follow-up; 82 months). ResultsUsing next-generation sequencing, we identified 97 SNVs in 16 genes (B2M, CD274, CD276, CD58, CD83, CIITA, NLRC4, NLRC5, RFXAP, RFXANK, TNFRSF14, TNFRSF25, TNFSF9, TAP1, TAP2 and TAPBP) that were selected based on an extensive literature review implicating these genes in immune-cell crosstalk pathways in DLBCL. Among them, eight genes were mutated in more than one case including B2M (7%), CD58 (7%), TNFRSF14 (7%), CIITA (4%), TNFSF9 (2%), NLRC5 (3%), CD83 (11%), and RFXAP (2%). These eight loci were further analyzed for CNAs, gene expression and outcome correlations. SNP 6.0 microarrays revealed either focal heterozygous or homozygous deletions in B2M (22%), CD58 (14%), TNFSF9 (10%), and heterozygous deletions in TNFRSF14 (12%), CIITA (3%), NLRC5 (7%), CD83 (2%), and RFXAP (7%). Amongst the 91 cases, 35 cases (38%) had no genetic aberration. 56 cases (62%) had at least one aberration, while 29 (32%) had multiple genetic aberrations indicating that genetic aberrations associated with immune-cell crosstalk are not mutually exclusive in DLBCL. Mutations and CNAs were not COO specific, with the exception of TNFRSF14, which was significantly restricted to the GCB-subtype (p=0.001).We evaluated outcome correlations for each genetic aberrations in the 82 cases with uniform therapy, including two recognized major histocompatibility class (MHC) groups; MHC class I (B2M and NLRC5) and MHC class II (CIITA, RFXAP and CD83). In univariate analysis, the presence of genetic aberrations in the MHCI group and NLRC5 individually were prognostic (5yr PFS, 59% vs 78%, p=0.025, 44% vs 77%, p=0.005, respectively), but not the MHCII group or other individual loci. In subgroup analyses according to COO, both MHCI and MHCII genetic aberrations were associated with inferior 5yr PFS in the ABC subtype (MHCI, 27% vs 79%, p<0.0001) (MHCII, 40% vs 68%, p=0.018), but not in GCB. We next assessed the prognostic impact of the total number of genetic hits and revealed that patients with at least one genetic hit had a 5yr OS and PFS of 65% and 57%, respectively, while those with no genetic hit had a 5yr OS and PFS of 100% and 97%, respectively (both comparisons, p<0.0001). In a Cox model of PFS including IPI and COO, any genetic hit in immune-cell crosstalk pathways remained strongly predictive (HR=15.1 [2.0-113.2], p= 0.008) and was independent of both IPI (HR= 2.1 [1.1-4.0], p= 0.030) and COO (HR= 0.6 [0.3-1.4], p=0.26). ConclusionsRecurrent genetic alterations involving genes related to immune-cell crosstalk in DLBCL are frequent (56%), co-occur and independently contribute to outcome in patients treated with R-CHOP. MHCI and MHCII perturbations are only prognostic in the ABC subtype. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression, purification and assembly of soluble multimeric MHC class II–invariant chain complexes

Major histocompatibility class (MHC) II molecules are essential for running adaptive immune response. They are produced in the ER and targeted to late endosomes with the help of invariant chain (Ii) trimers. Ii trimerization may be induced by the Ii TM domain. To enable mechanistic and structural studies of MHC class II–Ii assembly, soluble forms of the complexes were expressed. We show that Ii trimerizes in the absence of the transmembrane part, prior to binding of α/β chains. The biochemical analysis supports the suggestion that the MHC class II–Ii complexes are not necessarily trimers of trimers, but that the Ii trimer can also be occupied by one or two MHC class II complexes.

Natural Killer Cell Killing of Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia Blasts by Killer Cell Immunoglobulin-Like Receptor–Negative Natural Killer Cells after NKG2A and LIR-1 Blockade

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR(-) cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR(-) NK cells when inhibitory receptor-major histocompatibility class I interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR(-) NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR(-) NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

Pathobiology of Inflammation to Cell Death

When a cell dies in vivo the innate immune system investigates the scene of the crime. If the death of the cell is physiological, e.g. as part of normal cell turn over, then the corpse is cleared and little else is done. In fact, as part of this process phagocytes that ingest normal apoptotic cells can actively suppress tissue responses by elaborating anti-inflammatory cytokines (1). However, if the cell has died unexpectedly from some pathological process, then the innate immune system expands its investigation.

Network-based gene expression analysis of intracranial aneurysm tissue reveals role of antigen presenting cells

Little is known about the pathology and pathogenesis of the rupture of intracranial aneurysms. For a better understanding of the molecular processes involved in intracranial aneurysm (IA) formation we performed a gene expression analysis comparing ruptured and unruptured aneurysm tissue to a control artery.Tissue samples of six ruptured and four unruptured aneurysms, and four cerebral arteries serving as controls, were profiled using oligonucleotide microarrays. Gene ontology classification of the differentially expressed genes was analyzed and regulatory functional networks and canonical pathways were identified with a network-based computational pathway analysis tool.Real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed as confirmation.Analysis of aneurysmal and control tissue revealed 521 differentially expressed genes. The most significantly associated gene ontology term was antigen processing (P=1.64E-16).Further network-based analysis showed the top scoring regulatory functional network to be built around overexpressed major histocompatibility class (MHC) I and II complex related genes and confirmed the canonical pathway “Antigen Presentation” to have the highest upregulation in IA tissue (P=7.3E-10).Real time RT-PCR showed significant overexpression of MHC class II genes. Immunohistochemical staining showed strong positivity for MHC II molecule specific antibody (HLA II), for CD68 (macrophages, monocytes), for CD45RO (T-cells) and HLA I antibody.Our results offer strong evidence for MHC class II gene overexpression in human IA tissue and that antigen presenting cells (macrophages, monocytes) play a key role in IA formation.

CD4+ T cells are required during priming but not the effector phase of antibody‐mediated IFN‐γ‐dependent protective immunity against pulmonary Francisella novicida infection

We have previously demonstrated the protective efficacy of intranasal vaccination with a defined Francisella tularensis subsp. novicida DeltaiglC mutant (KKF24) against pulmonary F. novicida U112 challenge. In this study, we further characterized the mechanisms of KKF24-induced immunity. Intranasally vaccinated KKF24 C57BL/6 major histocompatibility class (MHC) class II-/- mice produced minimal antigen-specific interferon (IFN)-gamma and serum antibodies and were highly susceptible (0% survival) to F. novicida challenge, compared to MHC class I-/- or wild-type mice (both 100% survival). Protective immunity could be transferred by immune serum into recipient wild type, but not IFN-gamma-/- mice. The protective effect of KKF24 vaccination against the respiratory F. novicida U112 challenge was not abrogated by anti-CD4 neutralizing antibody treatment and was not conferred by adoptive transfer of KKF24-specific CD4+ T cells. The protective effect of antibody was partially dependent upon Fc receptor-mediated clearance. Taken together, our data indicate that CD4+ T cells are required for priming, but not during the effector phase, of anti-KKF24 antibody-mediated IFN-gamma-dependent immunity against pulmonary F. novicida infection.

Autoimmunity in atherosclerosis: a protective response losing control?

Atherosclerosis is a chronic inflammatory disease characterized by accumulation of oxidized lipoproteins, increased cell death and hypertrophic degeneration of the arterial intima. The disease process is associated with local formation of modified self antigens that are targeted by both innate and adaptive immune responses. Although it remains to be firmly established it is likely that these autoimmune responses initially have a beneficial effect facilitating the removal of potentially harmful rest products from oxidized LDL and dying cells. However, studies performed on hypercholesterolaemic mice deficient in different components of the immune system uniformly suggest that the net effect of immune activation is pro-atherogenic and that atherosclerosis, at least to some extent, should be regarded as an autoimmune disease. These observations point to the possibility of developing new treatments for atherosclerosis based on modulation of immune responses against plaque antigens, an approach presently tested clinically for several other chronic inflammatory diseases with autoimmune components. Pilot studies in animals have provided promising results for both parental and oral vaccines based on oxidized LDL antigens. The time when this concept is ready for clinical testing is rapidly approaching but it will be important not to underestimate the difficulties that will be encountered in transferring the promising results from experimental animals into humans.

Lentiviral transduction of human dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play a pivotal role in stimulating antigen-specific T cells in vivo. The cardinal properties of DCs are: the ability to take up, process, and present antigens; (2) the ability to migrate through different tissues into lymphoid organs; and (3) the ability to interact with and stimulate T cells (3). Because of their unique capability of generating primary CD4+ and CD8+ T-cell responses, DCs are of particular interest for immunotherapeutic approaches to infectious disease and cancer. There are many ways to load DCs with antigen and to subsequently induce specific immune responses in vivo and in vitro. One strategy relies on genetically modified peripheral blood mononuclear cell (PBMC)-derived DCs to establish expression of a gene that encodes a specific antigen. In contrast to pulsing DCs with antigenic peptides or proteins, genetic engineering of DCs has the advantages of providing multiple epitopes for major histocompatibility class (MHC) class I-and MHC class II-restricted immune responses as well as a continuous supply of antigen for presentation alone or together with immunomodulatory proteins (cytokines, for example).

Activation of intraperitoneal T cells after stimulation with antigen specific major histocompatibility class (MHC) I tetramer

Introduction: Appropriate trafficking and activation of CD8+ T cells in the peritoneal cavity are essential in the response to a number of insults, including intra-abdominal sepsis and carcinomatosis. The dynamics of the CD8+ T cell response in the peritoneal cavity however, have not been well characterized. Antigen specific MHC I tetramers activate CD8+ T cells directly, without requiring antigen presenting cells, and have been used extensively to analyze CD8+ T cell function in vivo. In this study, we used antigen specific MHC class I tetramer to evaluate intraperitoneal T cell activation and trafficking of CD8+ T cells after adoptive transfer in vivo. Methods: We adoptively transferred (AT) CFSE labeled P14 T cells (TCR specific for LCMV peptide gp33) into the peritoneal cavity of C57BL/6 mice and stimulated them via intraperitoneal route (IP) with PE-labeled MHC I tetramer bound to gp33 or control peptide. At various time points, we harvested peritoneal washings and lymphoid organs and assessed AT lymphocytes by flow cytometry. Results: Five days following adoptive transfer, unstimulated AT cells still constituted over 50% of CD8+ T cells in the peritoneal cavity. Following intraperitoneal injection of antigen specific MHC 1 tetramer, the majority of AT T cells in the peritoneal cavity (compared to AT T cells in other lymphoid organs, such as the spleen) are bound to MHC I tetramer within 2 hours. Even 4 days after intraperitoneal MHC class I tetramer injection, dramatic increases in CD44 expression, representing activation, and vigorous CFSE dilution, representing proliferation, are observed in AT T cells in the peritoneal cavity compared to AT cells in other lymphoid organs. In addition, 50% of the AT T cells activated with MHC class I tetramer, regardless of location, are functionally active, producing interferon gamma, and cytotoxic T lymphocyte activity is detected. Conclusions: This study demonstrates the unique activation, proliferation, and effector phenotype profile of CD8+ T cells following adoptive transfer into the peritoneal cavity and stimulation with antigen specific MHC I tetramer. Further investigation of the intraperitoneal CD8+ T cell response to infection and malignancy in vivo is warranted.

Characterization of the T-cell repertoire in autologous graft-versus-host disease (GVHD): evidence for the involvement of antigen-driven T-cell response in the development of autologous GVHD

AbstractAdministration of cyclosporine A (CsA) after autologous stem cell transplantation elicits an autoimmune syndrome with pathology similar to graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with the appearance of autoreactive T cells directed at major histocompatibility class (MHC) class II antigens. In the rat model of autologous GVHD, clonal analysis reveals that the effector T cells are highly conserved and recognize a peptide from the invariant chain peptide presented by MHC class II. Although human autologous GVHD effector T cells share a similar phenotypic specificity, clonality of the response in humans has not been determined. To examine the human effector T-cell response, the T-cell repertoire of peripheral blood lymphocytes was assessed by complementarity-determining region 3 (CDR3) size distribution analysis and T-cell clonotype analysis in 26 patients treated with CsA after transplantation. Autologous GVHD developed in 3 of 4 patients with human leukocyte antigen (HLA)-DRB1*0701, and clonal expansions of β-chain variable region (BV)16+ T cells were shared. Clonal expansions within BV15+ and BV22+ T cells were also detected in 4 of 6 patients with HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively. Sequencing of BV16 cDNA for which the CDR3 size pattern exhibited apparent clone predominance revealed an identical CDR3 peptide sequence in 2 different patients, one with HLA-DRB1*0701 and the other with HLA-DRB1*1502. These findings indicate that the discrete antigen-driven expansion of T cells is involved in autologous GVHD.

Contribution of MHC class I-dependent immune mechanisms induced by attenuated recombinant Salmonella typhimurium secreting superoxide dismutase to protection against murine listeriosis

A recombinant (r)Salmonella typhimuriumaroA strain secreting the naturally non-secreted superoxide dismutase (SOD) of Listeria monocytogenes controls murine listeriosis dependent on ‘transporter associated with antigen processing’ (TAP)-mediated immune mechanisms. TAP1-deficient mice (devoid of most CD8 T cells) vaccinated with this rSalmonella SODs strain succumbed to lethal L. monocytogenes challenge, whereas C57BL/6 mice were protected by this vaccine. Moreover, vaccination of H-2I-Aβ-deficient mice (lacking major histocompatibility class (MHC) II molecules and thus devoid of mature CD4 TCR-αβ cells), of TAP1-deficient as well as of β2microglobulin-deficient mice (devoid of conventional CD8 T cells) with a sublethal dose of L. monocytogenes and subsequent challenge with rSalmonella control or SODs strain revealed contribution of both MHC class I- and MHC class II-dependent immune mechanisms to the control of secondary Salmonella infection. Finally, the clearance of rSalmonella SODs bacteria was achieved in TAP1-deficient animals vaccinated with L. monocytogenes. Our data suggest a role of TAP-dependent mechanisms in priming of protective immunity by rSalmonella micro-organisms secreting SOD.

Antitumor effects of the combination therapy with TNF-alpha gene-modified tumor cells and interleukin 12 in a melanoma model in mice.

In the present study, TNF-alpha gene-transduced B78 melanoma cells (B78/TNF) were used as a vaccine and combined with interleukin (IL)-12 in the treatment of B78 melanoma-bearing mice. The combined administration of genetically modified melanoma cells and IL-12 induced specific protective antitumor immunity resulting in a decreased rate of the tumor take following a rechallenge with parental B78 cells. When used therapeutically, intratumoral injections of irradiated B78/TNF melanoma cells and IL-12 exerted strong antitumor effects and led to complete regression of established tumors in 50% of mice. Injections of irradiated B78/TNF cells alone did not influence tumor development and IL-12 itself significantly delayed tumor growth but without curative effect. FACS analysis of parental B78 melanoma cells and its B78/TNF genetically modified variant showed that a proportion of cells of both cell lines expressed 87-1 (CD80) costimulatory molecule and that the expression of this molecule was increased during incubation with IFN-gamma. Moreover, IFN-gamma markedly augmented expression of major histocompatibility class (MHC) class I and II molecules on B78/TNF cells that were primarily MHC class I and II negative with no substantial effect on MHC-negative parental B78 melanoma. IFN-gamma also synergized in cytostatic/cytotoxic effects with TNF-alpha against B78 melanoma in vitro. Lymphocyte depletion studies in vivo showed reduction of the antitumor response in mice treated with anti - NK monoclonal antibodies (mAbs) as well as in mice treated with anti-CD4+ anti-CD8 mAbs. The results suggest that, when used therapeutically, IL-12 and a vaccine containing TNF-alpha gene-transduced tumor cells may reciprocally augment their overall antitumor effectiveness by facilitating development of systemic antitumor immunity and by stimulating local effector mechanisms of the tumor destruction.

Epithelial Lymphocyte and Macrophage Distribution in the Adult and Fetal Equine Lung

Leucocytes in the lung epithelium play an important role in the ability of an animal to respond appropriately to inhaled pathogens. The distribution of lymphoid and myeloid cells associated with the lung epithelium was examined immunohistochemically throughout the respiratory tract of four horses, comprising two adults from an abattoir, one pregnant mare, and her fetus (in the final stage of gestation). Cross and tangential cryosections were labelled with monoclonal antibodies against T-cell, B-cell, macrophage/dendritic myeloid cell, and major histocompatibility Class (MHC) II surface antigens. Cell numbers were determined by microscopy. In the three adult horses, epithelial CD3+ T-cell numbers decreased progressively from the upper to the lower respiratory tract, but in the fetus there were low numbers of T cells (at most, 10% of those seen in the adult airways) and little variation in different parts of the respiratory tract. MHC Class II was expressed on the airway epithelium of the two abattoir horses, but not that of the mare and her fetus. In these two animals occasional large, mostly irregularly-shaped, Class II-positive cells were seen. Very few epithelium-associated cells in any animal were labelled by anti-CD21 antibody, which identifies B cells, or anti-myeloid cell antibodies; an anti-rat macrophage antibody (ED2) was shown, for the first time, to identify mature equine alveolar macrophages. Despite the small number of animals, the results suggest that in normal adult horses the greatest numbers of epithelial T cells are found where there is greatest contact with airborne antigens, and that there is constitutive epithelial MHC Class II expression. The low level of MHC Class II expression in the fetus, together with the reduced numbers of T cells, was consistent with the suggestion that the fetal immune system requires exposure to airborne stimuli for full development. The low level of MHC Class II expression in the mare may have reflected the immunosuppression that accompanies pregnancy.

Isolation of Tumor-Specific Cytotoxic CD4+ and CD4+CD8dim+ T-Cell Clones Infiltrating a Cutaneous T-Cell Lymphoma

We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4+CD8dim+ and CD4+CD8− phenotype. Both clones mediated a specific MHC class I–restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vβ gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vβ7/Jβ2.3, Vβ13/Jβ2.5, and Vβ22/Jβ2.5 rearrangements. Phenotypic analysis using specific anti-Vβ monoclonal antibodies indicated that only Vβ13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vβ gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vβ transcript, corresponding, respectively, to Vβ5/Jβ2.3 and Vβ17/Jβ2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vβ TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.