Clonal analysis of in vivo activated CD8+ cytotoxic T lymphocytes from a melanoma patient responsive to active specific immunotherapy

To study in vivo activated cytolytic T cells, CD8⁺ T cells clones were isolated from a melanoma patient (HLA A2, A11) treated with active specific immunotherapy for 5 years. CD8⁺ T lymphocytes, purified by fluorescence-activated cell sorting, were cloned directly from the peripheral blood without antigen-presenting cells in the presence of irradiated autologous melanoma cells and recombinant interleukin-2 (IL-2) and IL-4. These conditions were inhibitory to de novo in vitro immunization. Of the 28 cytolytic CD8⁺ T cell clones, 21 lysed the autologous melanoma cell line (M7) but not the autologous lymphoblastoid cell line (LCL-7) nor the two melanoma cell lines, M1 (HLA A28) and M2 (HLA A28, A31), used to immunize the patient. The remaining 7 clones were also melanoma-specific, although their reactivities were broader, lysing several melanoma cell lines but not HLA-matched lymphoblastoid cells. Eight clones from the first group, ostensibly self-MHC-restricted, were expanded for further analysis. All expressed cluster determinants characteristic of mature, activated T cells, but not those of thymocytes, naive T cells, B cells or natural killer (NK) cells. They also expressed CD13, a myeloid marker. Of the 8 clones, 3 expressed both CD4 and CD8, but dual expression was not correlated with specificity of lysis. Two CD8⁺ and 2 CD4⁺ CD8⁺ clones were specific for the autologous melanoma cells, the other 4 were also reactive against other HLA-A2-positive melanomas. Cytotoxicity for both singly and doubly positive clones was restricted by HLA class I but not class II antigens. Analysis of the RNA expression of the T cell receptor (TCR) V and V gene segments revealed heterogeneous usage by the A2-restricted clones and, perhaps, also by the broadly melanoma-specific clones. Apparent TCR-restricted usage was noted for the self-MHC-restricted clones; 2 of the 4 expressed the V17/V7 dimer. Since the T cell clones were derived from separate precursors of circulating cytotoxic T lymphocytes (CTL), the V17/V7 TCR was well represented in the peripheral blood lymphocytes of this patient. In summary, we show that melanoma cells presented their own antigens to stimulate the proliferation of melanoma-reactive CD8⁺ CTL. CTL with a range of melanoma specificities and different TCR dimers were encountered in this patient, perhaps as a result of hyperimmunization. Restricted TCR gene usage was noted only for classical self-MHC-restricted CD8⁺ T cell clones, although lysis of the autologous melanoma cells was effected by a variety of TCR structures. Molecular definition of the TCR repertoire of well-characterized T cell clones in this and other patients should provide new insight into the human antitumor immune response.Supported by National Institutes of Health research grants CA 36233 and EY 9031, the Lucy Adams Memorial Fund and a grant from the Concern Foundation     

Cross-primed CD8+ cytotoxic T cells induce severe Helicobacter-associated gastritis in the absence of CD4+ T cells

BACKGROUND: Although previous studies have reported important roles of CD4(+) type 1-helper T cells and regulatory T cells in Helicobacter-associated gastritis, the significance of CD8(+) cytotoxic T cells remains unknown. To study the roles of CD8(+) T cells, we examined the immune response in the gastric mucosa of Helicobacter felis-infected major histocompatibility complex (MHC) class II-deficient (II(-/-)) mice, which lack CD4(+) T cells. MATERIALS AND METHODS: Stomachs from H. felis-infected wild-type and infected MHC II(-/-) mice were examined histologically and immunohistochemically. Gastric acidity and serum levels of anti-H. felis antibodies were measured. The expression of pro-inflammatory and anti-inflammatory cytokine, Fas-ligand, perforin, and Foxp3 genes in the gastric mucosa was investigated. RESULTS: H. felis-infected MHC II(-/-) mice developed severe gastritis, accompanied by marked infiltration of CD8(+) cells. At 1 and 2 months after inoculation, mucosal inflammation and atrophy were more severe in MHC II(-/-) mice, although gastritis had reached similar advanced stages at 3 months after inoculation. There was little infiltration of CD4(+) cells, and no Foxp3-positive cells were detected in the gastric mucosa of the infected MHC II(-/-) mice. The expression of the interleukin-1beta and Fas-ligand genes was up regulated, but that of Foxp3 was down regulated in the infected MHC II(-/-) mice. Serum levels of anti-H. felis antibodies were lower in the infected MHC II(-/-) mice, despite severe gastritis. CONCLUSIONS: The present study suggests that cross-primed CD8(+) cytotoxic T cells can induce severe H.-associated gastritis in the absence of CD4(+) helper T cells and that Foxp3-positive cells may have an important role in the control of gastric inflammation.     

Non-fastidious,melanoma-specific CD8+ cytotoxic T lymphocytes from choroidal melanoma patients

To characterize the anti-melanoma reactivity of CD8⁺ cytotoxic T lymphocytes (CTL) from choroidal melanoma patients, CTL clones were isolated from the peripheral blood of three patients after mixed lymphocyte/tumor cell culture (MLTC). Clones were derived from lymphocytes stimulated by allogeneic (OCM-1, A24, A28) or autologous (OCM-3, Al, A30) melanoma cells. Their reactivity against a panel of HLA-typed melanoma and nonmelanoma cells was assessed, to determine whether a single CTL clone could recognize and lyse a variety of allogeneic melanoma cell lines. While proportionately more clones derived from autologous MLTC were melanoma-specific than allogeneic MLTC (42% versus 14%), melanoma-specific CTL were recovered from both. Notably, a novel melanoma specificity was identified. These CTL clones were termed non-fastidious because they were capable of lysing melanoma cells with which they had no HLA class I alleles in common. Nonetheless, lysis was mediated by the HLA class I molecule. Since lysis was specific for melanoma cells, these CTL appeared to recognize a shared melanoma peptide(s). Because of their prevalence, we propose that non-fastidious CTL are integral to human anti-melanoma T cell immunity. This reinforces clinical findings that allogeneic melanomas can substitute for autologous tumors in active specific immunotherapy. By circumventing the need for autologous melanoma, it is possible to treat patients after removal of the primary choroidal melanoma in an attempt to prevent metastasis.Supported by USPHS grants EY-09031 and EY-09427, and the Lucy Adams Choroidal Melanoma Research Fund to J. K.-M.     

Peptide immunization restimulates the memory CD4 T cell response but fails to induce cytotoxic T lymphocytes in cynomolgus monkeys

A potential strategy to induce peptide specific CTL in vivo was investigated. A synthetic vaccine consisting of an SIV-derived, HLA-A2.1-binding CTL epitope and a tetanus toxin-derived T helper epitope was evaluated for its capacity to induce peptide-specific CTL in monkeys. Thirteen animals were immunized and boosted twice with 150 μg of CTL plus 250 μg of the T helper peptide (p30). Peripheral blood mononuclear cells (PBMC) were regularly analysed for cytotoxic and proliferative responses before, between, and after the immunizations, and the serum was tested for anti-peptide antibodies. No unequivocal induction of SIV peptide-specific CTL in any of the monkeys was observed. However, a wide pattern of mild and transient side reactions were observed, ranging from local redness at the injection site to generalized exanthema, myalgias, arthralgias, and fever. The side-effects were related to the T helper epitope, as they were similar to the side-effects experienced after tetanus immunization, correlated to the magnitude of the p30-specific in vitro proliferative response, and occurred only if p30 was co-injected. No antibody against the SIV-derived peptides nor against p30 was detectable in the serum after repeated immunizations. The data suggest that the CTL peptide, at the concentration used in this study, failed to induce a cytotoxic immune response in vivo, although the T helper peptide seems to be capable of restimulating the specific memory T cells.     

Therapeutic efficacy of antitumor dendritic cell vaccinations correlates with persistent Th1 responses,high intratumor CD8+ T cell recruitment and low relative regulatory T cell infiltration

Despite the increasing number of immunotherapeutic strategies for the treatment of cancer, most approaches have failed to correlate the induction of an anti-tumor immune response with therapeutic efficacy. We therefore took advantage of a successful vaccination strategy-combining dendritic cells and irradiated GM-CSF secreting tumor cells-to compare the immune response induced against 9L gliosarcoma tumors in cured rats versus those with progressively growing tumors. At the systemic level, the tumor specific cytotoxic responses were quite heterogeneous in uncured vaccinated rats, and were surprisingly often high in animals with rapidly-growing tumors. IFN-gamma secretion by activated splenic T cells was more discriminative as the CD4+ T cell-mediated production was weak in uncured rats whereas high in cured ones. At the tumor level, regressing tumors were strongly infiltrated by CD8+ T cells, which demonstrated lytic capacities as high as their splenic counterparts. In contrast, progressing tumors were weakly infiltrated by T cells showing impaired cytotoxic activities. Proportionately to the T cell infiltrate, the expression of Foxp3 was increased in progressive tumors suggesting inhibition by regulatory T cells. In conclusion, the main difference between cured and uncured vaccinated animals does not depend directly upon the induction of systemic cytotoxic responses. Rather the persistence of higher CD4+ Th1 responses, a high intratumoral recruitment of functional CD8+ T cells, and a low proportion of regulatory T cells correlate with tumor rejection.     

Emperipolesis mediated by CD8+ T cells correlates with biliary epithelia cell injury in primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell‐in‐cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty‐six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early‐stage PBC (stages I and II, n = 39) and late‐stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin‐eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3⁺ and CD8⁺ T cells correlated with the advancement of emperipolesis (R² = 0.318, P < .001; R² = 0.060, P < .05). The cell numbers of TUNEL‐positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R² = 0.236, P < .001; R² = 0.267, P < .001). We conclude that emperipolesis mediated by CD8⁺ T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts.     

PSGL-1 attenuates early TCR signaling to suppress CD8+ T cell progenitor differentiation and elicit terminal CD8+ T cell exhaustion

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4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

The role of CD4+ T cell help and CD40 ligand in the in vitro expansion of HIV-1-specific memory cytotoxic CD8+ T cell responses

CD4(+) T cells have been shown to play a critical role in the maintenance of an effective anti-viral CD8(+) CTL response in murine models. Recent studies have demonstrated that CD4(+) T cells provide help to CTLs through ligation of the CD40 receptor on dendritic cells. The role of CD4(+) T cell help in the expansion of virus-specific CD8(+) memory T cell responses was examined in normal volunteers recently vaccinated to influenza and in HIV-1 infected individuals. In recently vaccinated normal volunteers, CD4(+) T cell help was required for optimal in vitro expansion of influenza-specific CTL responses. Also, CD40 ligand trimer (CD40LT) enhanced CTL responses and was able to completely substitute for CD4(+) T cell help in PBMCs from normal volunteers. In HIV-1 infection, CD4(+) T cell help was required for optimal expansion of HIV-1-specific memory CTL in vitro in 9 of 10 patients. CD40LT could enhance CTL in the absence of CD4(+) T cell help in the majority of patients; however, the degree of enhancement of CTL responses was variable such that, in some patients, CD40LT could not completely substitute for CD4(+) T cell help. In those HIV-1-infected patients who demonstrated poor responses to CD40LT, a dysfunction in circulating CD8(+) memory T cells was demonstrated, which was reversed by the addition of cytokines including IL-2. Finally, it was demonstrated that IL-15 produced by CD40LT-stimulated dendritic cells may be an additional mechanism by which CD40LT induces the expansion of memory CTL in CD4(+) T cell-depleted conditions, where IL-2 is lacking.     

Rapid G0/1 transition and cell cycle progression in CD8+ T cells compared to CD4+ T cells following in vitro stimulation

T‐cell population consists of two major subsets, CD4⁺ T cells and CD8⁺ T cells, which can be distinguished by the expression of CD4 or CD8 molecules, respectively. Although they play quite different roles in the immune system, many of their basic cellular processes such as proliferation following stimulation are presumably common. In this study, we have carefully analyzed time–course of G0/1 transition as well as cell cycle progression in the two subsets of quiescent T‐cell population following in vitro growth stimulation. We found that CD8⁺ T cells promote G0/1 transition more rapidly and drive their cell cycle progression faster compared to CD4⁺ T cells. In addition, expression of CD25 and effects of its blockade revealed that IL‐2 is implicated in the rapid progression, but not the earlier G0/1 transition, of CD8⁺ T cells.     

Type D SRV-2 virus-specific CD8+ and CD4- CD8- T cells that regulate virus-induced T cell proliferation in Celebes macaques

These studies defined SRV-2 envelope peptides 96-102, 127-152, and 233-249 as T cell epitopes that induce significant T cell proliferation. Peripheral blood lymphocytes of Celebes macaques (Macaca nigra) exposed to SRV-2 and currently virus^- antibody⁺, cultured with SRV-2 virus show strongly suppressed T cell responses and have two immunoregulatory T cell populations.     

T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%–8% of T cell from healthy individuals expressed the V19 TCR. One BMT patient exhibited V19 expression on more than 60% of peripheral T cells, while additional patients expressed V19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8⁺ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V repertoire of CD4⁺ and CD8⁺ T cell populations. We found that biased V expression characterized both CD4⁺ and CD8⁺ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8⁺ T cells alone may not fully correct repertoire abnormalities following BMT.     

HIV-specific CD8+ T cell responses to HXB2 Gag and Nef peptide pools in Chinese HIV/AIDS patients

HXB2 is primarily used as a template strain in developing HIV vaccines in Europe and the US. However,it is not yet known whether the strain can induce strong HIV-specific CD8+ T cell responses in Chinese HIV/AIDS patients. In the present study,two groups of subjects were investigated:9 AIDS patients and 7 long-term nonprogressors (LTNPs). HIV-specific CD8+ T cell responses were examined in all patients through the ELISPOT assay. CD4+ T cell counts,CD8+ T cell counts,viral load and HIV subtype of each patien...     

CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells

Absence of CD4⁺ T cell help has been suggested as a mechanism for failed anti-tumor cytotoxic T lymphocytes (CTL) response. We examined the requirement for CD4⁺ T cells to eliminate an immunogenic murine fibrosarcoma (6132A) inoculated into the peritoneal cavity. Immunocompetent C3H mice eliminated both single and repeat intraperitoneal (IP) inoculums, and developed high frequency of 6132A-specific interferon-γ (IFNγ)-producing CTL in the peritoneal cavity. Adoptive transfer of peritoneal exudate cells (PEC) isolated from control mice, protected SCID mice from challenge with 6132A. In contrast, CD4 depleted mice had diminished ability to eliminate tumor and succumbed to repeat IP challenges. Mice depleted of CD4⁺ T cells lacked tumor-specific IFNγ producing CTL in the peritoneal cavity. Adoptive transfer of PEC from CD4 depleted mice failed to protect SCID mice from 6132A. However, splenocytes isolated from same CD4 depleted mice prevented tumor growth in SCID mice, suggesting that 6132A-specific CTL response was generated, but was not sustained in the peritoneum. Treating CD4 depleted mice with agonist anti-CD40 antibody, starting on days 3 or 8 after initiating tumor challenge, led to persistence of 6132A-specific IFNγ producing CTL in the peritoneum, and eliminated 6132A tumor. The findings suggest that CTL can be activated in the absence of CD4⁺ T cells, but CD4⁺ T cells are required for a persistent CTL response at the tumor site. Exogenous stimulation through CD40 can restore tumor-specific CTL activity to the peritoneum and promote tumor clearance in the absence of CD4⁺ T cells.Supported in part by grants from Children’s Hospital of Wisconsin Foundation, Society of University Surgeons Foundation, Florence and Marshall Schwid Foundation, Elsa Pardee Foundation, Kathy Duffy Fogarty Fund of the Greater Milwaukee Foundation (JS) and NIH grant RO1-CA-37156 (HS); Andrew Lodge and Ping Yu have contributed equally to this work.     

Tuberculosis patients display a high proportion of CD8+ T cells with a high cytotoxic potential

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) and remains a major cause of morbidity and mortality worldwide. In the host's immune response system, T cells play a critical role in mediating protection against Mtb infection, but the role of CD8⁺ T cells is still controversial. We evaluated the phenotypical characterization and cytotoxic ability of CD8⁺ T cells by flow cytometry‐based assay. Cytokine levels in serum were measured by multiplex cytokine assay. Our data show that cells from TB patients have an increased percentage of peripheral blood CD8⁺αβ⁺ T (p = 0.02) and CD56⁺CD8⁺ T (p = 0.02) and a decreased frequency of NKG2D⁺CD8⁺ T (p = 0.02) compared with healthy donors. Unlike CD8⁺ T cells from healthy donors, CD8⁺ T cells from TB patients exhibit greater cytotoxicity, mediated by HLA class I molecules, on autologous monocytes in the presence of mycobacterial antigens (p = 0.005). Finally, TB patients have a proinflammatory profile characterized by serum high level of TNF‐α (p = 0.02) and IL‐8 (p = 0.0001), but, interestingly, IL‐4 (p = 0.002) was also increased compared with healthy donors. Our data show evidence regarding the highly cytotoxic status of CD8⁺ T cells in Mtb infection. These cytotoxic cells restricted to HLA‐A, B, and C could be used to optimize strategies for designing new TB vaccines or for identifying markers of disease progression.     

Global activation of CD8+ cytotoxic T lymphocytes correlates with an impairment in regulatory T cells in patients with generalized vitiligo

Melanocyte-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a pivotal role in vitiligo-induced depigmentation. Yet, the mechanisms underlying the high frequency of generalized autoimmune disorders associated with generalized vitiligo (GV) are unknown. We hypothesized that an imbalance between activated CD8(+) CTLs and regulatory T cells (Tregs) exists in patients with GV . Assessment of the circulating CD8(+) CTLs and Tregs by flow cytometric analysis revealed an obvious expansion of CD8(+) CTLs and a concomitant decrease in Treg cells in GV patients. The percentages of skin infiltrating CD8(+) CTLs and Tregs were evaluated by immunohistochemistry and revealed dramatically increased numbers of both CD8(+) CTLs and Tregs in the perilesional skin of GV patients. However, peripheral Tregs were impaired in their ability to suppress the proliferation and cytolytic capacity of autologous CD8(+) T cells, suggesting that a functional failure of Tregs and the hyper-activation of CD8(+) CTLs may contribute to progressive GV. Our data indicate that reduced numbers and impaired function of natural Tregs fail to control the widespread activation of CD8(+) CTLs, which leads to the destruction of melanocytes and contributes to the elevated frequency of various associated autoimmune diseases. This knowledge furthers our understanding of the mechanisms of immune tolerance that are impaired in GV patients and may aid in the future development of effective immunotherapy for GV patients.     

T Cell receptor clonotype influences epitope hierarchy in the CD8+ T cell response to respiratory syncytial virus infection

CD8+ T cell responses are important for recognizing and resolving viral infections. To better understand the selection and hierarchy of virus-specific T cell responses, we compared the T cell receptor (TCR) clonotype in parent and hybrid strains of respiratory syncytial virus-infected mice. K(d)M2(82-90) (SYIGSINNI) in BALB/c and D(b)M(187-195) (NAITNAKII) in C57Bl/6 are both dominant epitopes in parent strains but assume a distinct hierarchy, with K(d)M2(82-90) dominant to D(b)M(187-195) in hybrid CB6F1/J mice. The dominant K(d)M2(82-90) response is relatively public and is restricted primarily to the highly prevalent Vβ13.2 in BALB/c and hybrid mice, whereas D(b)M(187-195) responses in C57BL/6 mice are relatively private and involve multiple Vβ subtypes, some of which are lost in hybrids. A significant frequency of TCR CDR3 sequences in the D(b)M(187-195) response have a distinct "(D/E)WG" motif formed by a limited number of recombination strategies. Modeling of the dominant epitope suggested a flat, featureless structure, but D(b)M(187-195) showed a distinctive structure formed by Lys(7). The data suggest that common recombination events in prevalent Vβ genes may provide a numerical advantage in the T cell response and that distinct epitope structures may impose more limited options for successful TCR selection. Defining how epitope structure is interpreted to inform T cell function will improve the design of future gene-based vaccines.