alpha1beta1 Integrin+ and regulatory Foxp3+ T cells constitute two functionally distinct human CD4+ T cell subsets oppositely modulated by TNFalpha blockade

The expression of the collagen receptor alpha(1)beta(1) integrin (VLA-1) on CD4(+) T cells is largely restricted to CCR7(-)CD45RO(+) cells that localize to inflamed tissues. Moreover, neutralizing alpha(1) integrin, in vivo, has been shown to compromise cell-mediated immunity. Our current study shows that the expression of VLA-1 on human CD4(+) T cells is restricted to conventional effectors. In contrast, Foxp3(+) T regulatory cells (Tregs) do not express this receptor. Moreover, Foxp3 or VLA-1 expression remained a mutually exclusive event in CD4(+) T cells even upon polyclonal anti-CD3-induced activation. Because TNFalpha blockade ameliorates certain T cell-dependent autoimmune disorders in humans, we investigated, in vitro, whether neutralizing TNFalpha affected the balance between the proinflammatory VLA-1(+) effectors and the counteracting Tregs. We found that anti-CD3 stimulation of freshly isolated PBL from healthy individuals, coupled with continuous TNFalpha blockade, inhibited the typical activation-dependent generation of CD4(+)VLA-1(+) Th1 cells. In contrast, it augmented the outgrowth of VLA-1(neg/dim)CD25(high) and Foxp3(+)CD4(+) T cells. Indeed, repeated anti-CD3 stimulation coupled with TNFalpha blockade generated CD4(+) T cell lines enriched for VLA-1(-)Foxp3(+) Tregs. Importantly, these CD4(+) T cells displayed potent suppressive functions toward autologous CD4(+) PBL, including the suppression of the activation-dependent induction of VLA-1(+) effectors. Thus, we propose a novel mechanism by which anti-TNFalpha therapy may restore self-tolerance, by shifting the balance between VLA-1(+) effectors and Foxp3(+) Tregs, during immune activation, in favor of the latter suppressor cell population.     

Large-scale expansion of CD3<Superscript>+</Superscript>CD56<Superscript>+</Superscript> lymphocytes capable of lysing autologous tumor cells with cytokine-rich supernatants

We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.     

Characterization of a novel nonclassical T cell clone with broad reactivity against human renal cell carcinomas

A CD4(+) T cell clone (HC/2G-1) was established by stimulating peripheral blood T cells from a patient with renal cell carcinoma (RCC) with dendritic cells preincubated with the autologous apoptotic renal tumor line in the presence of IFN-alpha. It recognizes the autologous RCC and most allogeneic RCC lines by IFN-gamma release (10 of 11 lines) and lysis (9 of 10 lines), but does not recognize multiple EBV B cells or fibroblasts. It shows little or no recognition of a panel of melanomas, breast cancers and non-small-cell lung cancers. Phenotypically, HC/2G-1 is CD3(+)CD4(+) TCR alphabeta(+), but CD161(-)CD16(-)NKG2D(-). Tumor recognition by clone HC/2G-1 was not blocked by Abs to HLA class I or class II, but was significantly reduced by anti-TCR alphabeta Ab. Furthermore, tumor recognition was beta(2)-microglobulin-independent. HC/2G-1 does not use a Valpha or Vbeta described for classical NKT cells, but rather Valpha14 and Vbeta2.1. Allogeneic T cells cotransfected with mRNAs encoding the alpha and beta chains of the HC/2G-1 TCR recognized renal tumor lines, demonstrating that tumor recognition is TCR-mediated. Interestingly, TRAIL appears to play a role in tumor recognition by HC/2G-1 in that reactivity was blocked by anti-TRAIL Ab, and soluble TRAIL could enhance IFN-gamma secretion by HC/2G-1 in response to renal tumors. Our findings suggest that clone HC/2G-1 represents a novel type of CD4(+) cell that has broad TCR-mediated recognition of a determinant widely expressed by RCC.     

CD4<Superscript>+</Superscript> T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25<Superscript>+</Superscript> T cells

Background The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8⁺ T cells recognizing h5T4 can be generated in the absence of CD4⁺ T cells from peripheral blood lymphocytes of human healthy individuals. Results We report the existence and expansion of human CD4⁺ T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25⁺ cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4⁺ T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4⁺ T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis. Conclusion Our data show that CD4⁺ T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4⁺ T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses. This work was supported by Cancer Research UK.     

Functional CD8<Superscript>+</Superscript> T cells infiltrate into nonsmall cell lung carcinoma

Infiltration of CD3(+)CD8(+) cytotoxic T cells was analyzed by multiparameter confocal laser microscopy in a panel of 16 randomly selected stage I nonsmall cell lung carcinomas. T-cell infiltration was observed in the stroma (range 57-2,093 T cells/mm(2)) but also in the tumor epithelium (range 21-892 T cells/mm(2)) and showed wide variation between individual tumors. Interestingly, a significantly higher percentage of CD3(+)CD8(+) T cells was detected in the tumor epithelium compared to the stroma illustrating that cytotoxic T cells may preferentially migrate into tumor epithelium. Aberrant HLA class I antigen expression was observed in 69% of the nonsmall-cell lung carcinoma (NSCLC) tumors. One tumor of a squamous cell lung carcinoma patient with the highest number of tumor infiltrating CD3(+) and CD3(+)CD8(+) cells was studied in detail and the majority (90%) of these cells were shown to be functionally activated granzyme B-positive cytotoxic T cells. DNA oligotyping of a lung carcinoma cell line established from this tumor revealed loss of one HLA haplotype corresponding with a translocation involving chromosome 6, as observed by COBRA-FISH. HLA class I-restricted tumor specific T cells could be isolated from PBMC. One further characterized cytotoxic CD8(+) T cell clone, that released TNF-alpha, IFN-gamma, and granzyme B upon co-incubation with the autologous tumor cells, was shown to be restricted by the remaining HLA-A11 allele, which was also shown to be expressed in the tumor tissue. Our data indicate that, despite HLA-haplotype loss a vigorous antitumor immune response mediated by CD8(+ )T-cells can be present in NSCLC offering possibilities for specific immunotherapy.     

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.     

CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30(+) T cells elicited with allogeneic APC are a major source of IFN-gamma and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30(+) T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30(+) T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30(+) cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.     

Tumor-infiltrating CD4+ T lymphocytes express APO2 ligand (APO2L)/TRAIL upon specific stimulation with autologous lung carcinoma cells: role of IFN-alpha on APO2L/TRAIL expression and -mediated cytotoxicity

In the present report, we have investigated TRAIL/APO2 ligand (APO2L) expression, regulation, and function in human lung carcinoma tumor-infiltrating lymphocytes. Using a panel of non-small cell lung carcinoma cell lines, we first showed that most of them expressed TRAIL-R1/DR4, TRAIL-R2/DR5, but not TRAIL-R3/DcR1 and TRAIL-R4/DcR2, and were susceptible to APO2L/TRAIL-induced cell death. Two APO2L/TRAIL-sensitive tumor cell lines (MHC class I(+)/II(+) or I(+)/II(-)) were selected and specific CD4(+) HLA-DR- or CD8(+) HLA-A2-restricted CTL clones were respectively isolated from autologous tumor-infiltrating lymphocytes. Interestingly, although the established T cell clones did not constitutively express detectable levels of APO2L/TRAIL, engagement of their TCR via activation with specific tumor cells selectively induced profound APO2L/TRAIL expression on the CD4(+), but not on the CD8(+), CTL clones. Furthermore, as opposed to the CD8(+) CTL clone which mainly used granule exocytosis pathway, the CD4(+) CTL clone lysed the specific target via both perforin/granzymes and APO2L/TRAIL-mediated mechanisms. The latter cytotoxicity correlated with APO2L/TRAIL expression and was significantly enhanced in the presence of IFN-alpha. More interestingly, in vivo studies performed in SCID/nonobese diabetic mice transplanted with autologous tumor and transferred with the specific CD4(+) CTL clone in combination with IFN-alpha resulted in an important APO2L/TRAIL-mediated tumor growth inhibition, which was prohibited by soluble TRAIL-R2. Our findings suggest that APO2L/TRAIL, specifically induced by autologous tumor and up-regulated by IFN-alpha, may be a key mediator of tumor-specific CD4(+) CTL-mediated cell death and point to a potent role of this T cell subset in tumor growth control.     

Tumour-expressed CD43 (sialophorin) mediates tumourmesothelial cell adhesion

Mesothelial cell intercellular adhesion molecule-1 (ICAM-1) has recently been shown to play a role in tumour cell adherence to the peritoneum. However, solid tumours poorly express its most ubiquitous ligand, beta2 integrin. The aim of this study was to investigate the role of the beta2 integrin subunit and CD43, a known ligand for ICAM-1, in the development of peritoneal metastases. beta2 Integrin subunit and CD43 expression was assessed on a number of tumour cell lines. Adhesion of SW1222 and PSN-1 cells to human peritoneal mesothelial cells was investigated using a fluorometric assay incorporating an inhibitory antibody to beta2 integrin and CD43. beta2 Integrin expression was not inducible on these tumour cell lines, but Western blotting demonstrated CD43 expression in all the cancer cell lines examined and cell surface expression was confirmed by flow cytometry. The anti-CD43 antibody significantly reduced adhesion of PSN-1 and SW1222 cells to HPMC, however beta2 integrin inhibition did not reduce tumour cell adhesion. CD43 is expressed by a variety of carcinoma cell lines, and plays a role in tumour cell-peritoneal adhesion probably via interactions with its putative ligand ICAM-1.     

Modulation of CD103 expression on human colon carcinoma-specific CTL

Recent results have shown a correlation between survival and frequency of tumor-infiltrating T cells in colorectal cancer patients. However, the mechanisms controlling the ability of human T lymphocytes to infiltrate colon carcinoma remain unclear. Although, it is known that expression of the integrin CD103alpha(E)/beta(7) by intraepithelial lymphocytes controls the retention of lymphocytes in epithelial layers, very little is known about the expression of intestinal homing receptors in human T lymphocytes. In particular, it remains unknown whether expression of CD103/beta(7) by human colon cancer-specific T lymphocytes is controlled by recognition of tumor Ags and is imprinted during T cell priming, facilitating its expression during memory T cell activation. In this study, we demonstrate that expression of CD103/beta(7) in human colon carcinoma-specific CTL is synergistically enhanced by the simultaneous TGF-beta1 stimulation and Ag recognition. These results were confirmed by using a panel of human CTL clones. Finally, we show that priming of naive CD8(+) T cells in the presence of TGF-beta1 ensures up-regulation of CD103/beta(7) in recall responses, at concentrations of TGF-beta1 significantly lower than those required by memory T cells primed in the absence of TGF-beta1. These results indicate a role of TGF-beta1 during T cell priming in modulating expression of CD103/beta(7) and controlling retention of human memory CD8(+) T cells into tumor epithelium.     

Development of a transient CD4(+)CD8(+) T cell subset in the cervical lymph nodes following intratracheal instillation with an adenovirus vector

Past studies have described the serendipitous appearance of peripheral CD4(+)CD8(+) double-positive (DP) T cells in both humans and nonhuman primates usually following a viral infection or resulting from a malignancy. However, understanding the role of DP T cells has been hampered by the lack of their reproducible generation. Herein, we describe DP T cells produced after a single intratracheal or intranasal dose of recombinant adenovirus 2 or 5 vector into mice. In a time-dependent fashion, DP T cells localized only in the deep cervical lymph nodes but not in the lungs or in any of the respiratory lymph nodes. These DP T cells were TCR(alpha)beta(+) and CD8(alpha)beta(+), but not TCR(gamma)delta(+) nor CD8(alpha)alpha(+), suggesting that these cells are unrelated to intestinally derived DP T cells. Upon co-stimulation with anti-CD3 and anti-CD28, DP T cells showed increased expression of VLA-1, VLA-2, and CD69, and were more effective than CD4(+) T cells in T helper cell activity, as evidenced by increased IgA, IgG, and IgM production. Such co-stimulation also favored the production of IFN-gamma and IL-10 where CD4(+) T cells were more inclined to produce IFN-gamma and IL-2.     

Interleukin-10 increases Th1 cytokine production and cytotoxic potential in human papillomavirus-specific CD8(+) cytotoxic T lymphocytes

Interleukin-10 (IL-10) is widely known as an immunosuppressive cytokine by virtue of its ability to inhibit macrophage-dependent antigen presentation, T-cell proliferation, and Th1 cytokine secretion. However, several studies have challenged the perception of IL-10 solely as an immunosuppressive cytokine. As part of an investigation on potentiation of the cytotoxic activity of human papillomavirus E7-specific CD8(+) cytotoxic T lymphocytes (CTL) for adoptive transfusions to cervical cancer patients, we found that IL-10 in combination with IL-2, unlike several other combinations, including IL-2 with IL-12, gamma interferon (IFN-gamma), tumor necrosis factor alpha, and transforming growth factor beta, was able to consistently increase cytotoxicity. This augmentation in cytotoxic activity correlated with a significant increase in the cytoplasmic accumulation of perforin as detected by fluorescence-activated cell sorter. Surface expression of both the alpha and beta chains of the CD8 heterodimeric coreceptor and CD56 molecules was increased by exposure of CTL to IL-10. More importantly, we found that administration of IL-10 in combination with IL-2 after antigen stimulation consistently increased the intracellular expression of Th1 cytokines (i.e., IFN-gamma and IL-2) compared to results for control CD8(+) T cells cultured in IL-2 alone. In kinetic studies, proliferation, intracellular perforin levels, cytotoxic activity, and IFN-gamma expression were consistently elevated in CTL cultures containing IL-10 compared to control cultures, both at early and late time points following stimulation. In contrast, intracellular IL-2 expression was consistently increased only at early time points following stimulation with autologous tumor cells or solid-phase anti-CD3 antibody. Taken together, these data support the use of IL-10 in combination with IL-2 for the in vitro expansion and potentiation of tumor-specific CTL for clinical use in the therapy of cancer.     

CD4(+) Epstein-Barr virus-specific cytotoxic T-lymphocytes from human umbilical cord blood.

Umbilical cord blood (CB) is increasingly used for allogeneic hematopoietic stem cell transplantation. To determine whether viral antigen-specific cytotoxic T-lymphocytes (CTL) could be generated from the predominantly naive T-cell populations in CB, CB-derived mononuclear cells were stimulated with autologous Epstein-Barr virus (EBV) transformed B-lymphoblastoid cell lines over several weeks in the presence of recombinant human interleukin-2 (IL-2). By 28 days of culture, T-lymphocytes from all six CB that had been treated with IL-2 displayed EBV-specific cytotoxicity. These cells were largely CD4(+), with complete inhibition of cytotoxicity by anti-CD3 and variable inhibition by anti-HLA DR monoclonal antibodies. The EBV-specific effectors were cloned by limiting dilution, and most of the CTL clones were CD4(+). The cytotoxicity of the CB-derived CD4(+) CTL clones was inhibited by EGTA but not by anti-Fas ligand mAb, suggesting that this cytotoxicity was mediated by perforin/granzyme B. These data indicate that virus-specific CTL can be cultivated and cloned from CB, a human T-cell source that may not have prior in vivo antigenic exposure or reactivity. This finding may have applications in adoptive immunotherapy to recipients of CB transplants.     

CD4+ T cell-induced differentiation of EBV-transformed lymphoblastoid cells is associated with diminished recognition by EBV-specific CD8+ cytotoxic T cells

EBV transformation of human B cells in vitro results in establishment of immortalized cell lines (lymphoblastoid cell lines (LCL)) that express viral transformation-associated latent genes and exhibit a fixed, lymphoblastoid phenotype. In this report, we show that CD4(+) T cells can modify the differentiation state of EBV-transformed LCL. Coculture of LCL with EBV-specific CD4(+) T cells resulted in an altered phenotype, characterized by elevated CD38 expression and decreased proliferation rate. Relative to control LCL, the cocultured LCL were markedly less susceptible to lysis by EBV-specific CD8(+) CTL. In contrast, CD4(+) T cell-induced differentiation of LCL did not diminish sensitivity of LCL to lysis by CD8(+) CTL specific for an exogenously loaded peptide Ag or lysis by alloreactive CD8(+) CTL, suggesting that differentiation is not associated with intrinsic resistance to CD8(+) T cell cytotoxicity and that evasion of lysis is confined to EBV-specific CTL responses. CD4(+) T cell-induced differentiation of LCL and concomitant resistance of LCL to lysis by EBV-specific CD8(+) CTL were associated with reduced expression of viral latent genes. Finally, transwell cocultures, in which direct LCL-CD4(+) T cell contact was prevented, indicated a major role for CD4(+) T cell cytokines in the differentiation of LCL.     

High levels of Fas ligand and MHC class II in the absence of CD80 or CD86 expression and a decreased CD4<Superscript>+</Superscript> T cell Infiltration,enables murine skin tumours to progress

It is still not clear why some tumours will be recognized and destroyed by the immune system, and others will persist, grow, and eventually kill the host. It has been hypothesized that tumour cells might evade immunological destruction by expressing Fas ligand (FasL), a molecule which induces apoptosis in Fas(+) target cells. However, the role of FasL in creating an immune privileged status within a tumour remains controversial. To determine whether FasL is associated with skin tumour progression, we developed a tumour model enabling us to compare two squamous cell carcinomas (SCC). One is a regressor SCC which spontaneously regresses after injection into syngeneic mice. The other is a progressor SCC which evades immunological destruction. Detailed flow cytometric analysis was used to study tumour cell expression of FasL, Fas, CD80, CD86 and MHC class II. We also analysed the percentage of apoptotic tumour cells in vivo using annexin V and correlated skin tumour progression with CD4 and CD8 T cell infiltration. Progressor tumours expressed high levels of FasL in vivo, which was virtually absent from regressor tumours. The percentage of progressor tumours expressing MHC II was significantly greater than regressor tumours, while neither tumour expressed CD80 or CD86 costimulatory molecules. Consistent with a regressor phenotype, the percentage of viable tumour cells was significantly lower for regressor compared to progressor tumours which coincided with a significantly larger CD4(+) T cell infiltrate into the tumour mass. The results suggest that progression of skin tumours occurs if tumour cells express high levels of MHC II but not costimulatory molecules such as CD80 or CD86. This implies that tumours may induce anergy in CD4(+) T cells via MHC II antigen presentation in the absence of costimulation. To ensure escape from the immune system, tumours may then kill these T cells via a FasL-dependent mechanism.     

Evidence for an association between CD8 molecules and the T cell receptor complex on cytotoxic T cells

The T cell differentiation molecule CD8 is thought to play an important role in class I major histocompatibility complex-restricted T cell activities but the precise function of this molecule is unknown. To explore this question, we have studied several CD3+, CD8+ class I alloantigen-specific cytotoxic T lymphocyte (CTL) lines and clones. The ability of these CTL to proliferate as well as to lyse specific targets was inhibited by either anti-CD3 or anti-CD8 monoclonal antibodies. Exposure of CTL to relevant but not irrelevant target cells induced the rapid (less than 1 hr) disappearance of approximately 20 to 30% of CD3 and CD8 molecules from the cell surface. The modulation of these molecules became maximal at 6 to 12 hr and recovered thereafter in parallel. Treatment of CTL with anti-CD8 prevented alloantigen-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD8. Incubation of CTL with the combination of anti-CD3 and goat anti-mouse Ig also resulted in modulation of CD8. In contrast, the expression of other CTL surface antigens, such as CD2 (Leu-5, T11) and HLA-DR, was not reduced by any of these manipulations. These results suggest that CD8 molecules are associated with the CD3/antigen receptor complex on the surface of CTL, and may play a direct role in antigen-induced modulation and cross-linking of the T cell receptor.     

CD8 alpha beta T cells are not essential to the pathogenesis of arthritis or colitis in HLA-B27 transgenic rats

The class I MHC allele HLA-B27 is highly associated with the human spondyloarthropathies, but the basis for this association remains poorly understood. Transgenic rats with high expression of HLA-B27 develop a multisystem inflammatory disease that includes arthritis and colitis. To investigate whether CD8alphabeta T cells are needed in this disease, we depleted these cells in B27 transgenic rats before the onset of disease by adult thymectomy plus short-term anti-CD8alpha mAb treatment. This treatment induced profound, sustained depletion of CD8alphabeta T cells, but failed to suppress either colitis or arthritis. To address the role of CD8alpha(+)beta(-) cells, we studied four additional groups of B27 transgenic rats treated with: 1) continuous anti-CD8alpha mAb, 2) continuous isotype-matched control mAb, 3) the thymectomy/pulse anti-CD8alpha regimen, or 4) no treatment. Arthritis occurred in approximately 40% of each group, but was most significantly reduced in severity in the anti-CD8alpha-treated group. In addition to CD8alphabeta T cells, two sizeable CD8alpha(+)beta(-) non-T cell populations were also reduced by the anti-CD8alpha treatment: 1) NK cells, and 2) a CD4(+)CD8(+)CD11b/c(+)CD161a(+)CD172a(+) monocyte population that became expanded in diseased B27 transgenic rats. These data indicate that HLA-B27-retricted CD8(+) T cells are unlikely to serve as effector cells in the transgenic rat model of HLA-B27-associated disease, in opposition to a commonly invoked hypothesis concerning the role of B27 in the spondyloarthropathies. The data also suggest that one or more populations of CD8alpha(+)beta(-) non-T cells may play a role in the arthritis that occurs in these rats.     

Induction and large-scale expansion of CD8+ tumor specific cytotoxic T lymphocytes from peripheral blood lymphocytes by in vitro stimulation with CD80-transfected autologous melanoma cells.

Human melanoma cell lines may induce a specific T cell response against tumor cells in vitro. However, after repeated restimulation with autologous tumor cells, expansion of CTL is limited and often apoptosis of the T cells occurs. In order to improve conditions inducing primary T cell responses and thus allowing further expansion of tumor specific T cells for an adoptive transfer, we transfected human melanoma cells with the B7.1 gene (CD80), known to be a potent costimulatory molecule for T cell activation. CD80 expression on melanoma cells resulted in improved primary T cell activation, especially of CD8+ T cells. Furthermore, restimulation with CD80+ tumor cells gave rise to long term proliferating CD8+ T cell lines demonstrating an 100-fold expansion of T cells compared to the 20-30-fold increased numbers obtained with the controls (parental tumor cells +/- anti-CD28). T cells stimulated with CD80+ melanoma cells were found to display a MHC class I-restricted cytotoxic activity against the autologous tumor cells. In conclusion, these studies demonstrate the requirement of costimulation in generating large numbers of tumor specific T cells in vitro that may be used for an adoptive transfer in tumor immunotherapy.     

Constitutive expression of functional CD40 on mouse renal cancer cells: induction of Fas and Fas-mediated killing by CD40L

CD40, a member of the TNF receptor superfamily, is expressed on B cells, dendritic cells, and some tumor cells, including melanoma and bladder carcinoma. In this study, we report that both mouse and human renal carcinoma cells (RCC) also constitutively express functional CD40. Treatment of mouse RCC with CD40L induced strong expression of genes and proteins for ICAM-1 and Fas, and this expression was further enhanced by combining CD40L with IFN-gamma. Similar effects were demonstrated using an agonist anti-CD40 antibody. The increased levels of Fas expression on RCC after treatment with CD40L plus IFN-gamma resulted in potent killing by either FasL-positive effector cells or agonistic anti-Fas antibody. The combination of CD40L plus IFN-gamma also significantly enhanced killing of RCC by tumor-specific CTL lines. Our results demonstrate that constitutively expressed CD40 is functionally active and may provide a molecular target for the development of new approaches to the treatment of RCC.     

Intratumoral interleukin-2/agonist CD40 antibody drives CD4<Superscript>+</Superscript>-independent resolution of treated-tumors and CD4<Superscript>+</Superscript>-dependent systemic and memory responses

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4⁺ and CD8⁺ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8⁺ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4⁺ T cells were not required for eradicating treated-site tumors, they, plus CD8⁺ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4⁺ and CD8⁺ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4⁺ and CD8⁺ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4⁺ and CD8⁺ T cells for a complete long-term protective cure.