Low-dose carboplatin modifies the tumor microenvironment to augment CAR T cell efficacy in human prostate cancer models.

Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (LeY)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, LeY CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although LeY CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.

Early-phenotype CAR-T cells for the treatment of pediatric cancers.

Chimeric antigen receptor (CAR)-T-cell therapy is a promising approach for the treatment of childhood cancers, particularly high-risk tumors that fail to respond to standard therapies. CAR-T cells have been highly successful in treating some types of hematological malignancies. However, CAR-T cells targeting solid cancers have had limited success so far for multiple reasons, including their poor long-term persistence and proliferation. Evidence is emerging to show that maintaining CAR-T cells in an early, less-differentiated state in vitro results in superior persistence, proliferation, and antitumor effects in vivo. Children are ideal candidates for receiving less-differentiated CAR-T cells, because their peripheral T-cell pool primarily comprises naïve cells that could readily be harvested in large numbers to generate early-phenotype CAR-T cells. Although several studies have reported different approaches to successfully generate early CAR-T cells, there are only a few clinical trials testing these in adult patients. No trials are currently testing early CAR-T cells in children. Here, we summarize the different strategies used to maintain CAR-T cells in an early phenotypic stage and present evidence suggesting that this approach may be particularly relevant to treating childhood cancers.

Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Ex vivo culture of chimeric antigen receptor T cells generates functional CD8+ T cells with effector and central memory-like phenotype.

The anti-tumor efficacy of adoptively transferred T cells requires their in vivo persistence and memory polarization. It is unknown if human chimeric antigen receptor (CAR)-expressing T cells can also undergo memory polarization. We examined the functional status of CAR CD8(+) T cells, re-directed to Lewis Y antigen (LeY-T), throughout a period of ex vivo expansion. Immediately before culture CD8(+) T cells comprised a mixture of phenotypes including naive (CD45RA(+)/CCR7(+)/CD27(+)/CD28(+)/perforin-), central memory (CM, CD45RA(-)/CCR7(lo)/CD27(+)/CD28(+)/perforin(lo)), effector memory (EM, CD45RA(-)/CCR7(-)/CD27(+)/CD28(+)/perforin(mod)) and effector (Eff, CD45RA(+)/CCR7(-)/CD27(-)/CD28(-)/perforin(hi)) cells. After transduction and expansion culture of peripheral blood mononuclear cells from normal donors or multiple myeloma patients, CD8(+) LeY-T cells polarized to EM- and CM-like phenotype. CD8(+) LeY-T cells differed from starting CD8(+) CM and EM T cells in that CD27, but not CD28, was downregulated. In addition, CD8(+) LeY-T cells expressed high levels of perforin, similar to starting CD8(+) Eff. CD8(+) LeY-T cells also showed hallmarks of both memory and Eff function, underwent homeostatic proliferation in response to interleukin (IL)-15, and showed interferon (IFN)-γ production and cytotoxicity in response to Le-Y antigen on OVCAR-3 (human ovarian adenocarcinoma) cells. This study confirms CD8(+) LeY-T cells have a CM- and EM-like phenotype and heterogeneous function consistent with potential to persist in vivo after adoptive transfer.

Gene-modified T cells as immunotherapy for multiple myeloma and acute myeloid leukemia expressing the Lewis Y antigen.

We have evaluated the carbohydrate antigen Lewis(Y) (Le(Y)) as a potential target for T-cell immunotherapy of hematological neoplasias. Analysis of 81 primary bone marrow samples revealed moderate Le(Y) expression on plasma cells of myeloma patients and myeloblasts of patients with acute myeloid leukemia (AML) (52 and 46% of cases, respectively). We developed a retroviral vector construct encoding a chimeric T-cell receptor that recognizes the Le(Y) antigen in a major histocompatibility complex-independent manner and delivers co-stimulatory signals to achieve T-cell activation. We have shown efficient transduction of peripheral blood-derived T cells with this construct, resulting in antigen-restricted interferon-gamma secretion and cell lysis of Le(Y)-expressing tumor cells. In vivo activity of gene-modified T cells was demonstrated in the delayed growth of myeloma xenografts in NOD/SCID mice, which prolonged survival. Therefore, targeting Le(Y)-positive malignant cells with T cells expressing a chimeric receptor recognizing Le(Y) was effective both in vitro and in a myeloma mouse model. Consequently, we plan to use T cells manufactured under Good Manufacturing Practice conditions in a phase I immunotherapy study for patients with Le(Y)-positive myeloma or AML.

Adoptive immunotherapy for cancer: the next generation of gene-engineered immune cells.

Adoptive cellular immunotherapy involving transfer of tumor-reactive T cells has shown some notable antitumor responses in a minority of cancer patients. In particular, transfer of tumor-infiltrating lymphocytes has resulted in long-term objective responses in patients with advanced melanoma. However, the inability to isolate sufficient numbers of tumor-specific T cells from most malignancies has restricted the broad utility of this approach. An emerging approach to circumvent this limitation involves the genetic modification of effector cells with T cell receptor (TCR) transgenes or chimeric single-chain variable fragment (scFv) receptors that can specifically redirect T cells to tumor. There has been much progress in the design of TCR and scFv receptors to enhance the antigen-specific activation of effector cells and their trafficking and persistence in vivo. Considerable effort has been directed toward improving the safety of this approach and reducing the immunogenicity of the receptor. This review discusses the latest developments in the field of adoptive immunotherapy using genetically modified immune cells that have been transduced with either TCR or scFv receptor transgenes and used in preclinical and clinical settings as anticancer agents.

Absence of retroviral vector-mediated transformation of gene-modified T cells after long-term engraftment in mice.

There is considerable concern regarding the transforming potential of retroviral vectors currently used for gene therapy, with evidence that retroviral integration can lead to leukemia in recipients of gene-modified stem cells. However, it is not clear whether retroviral-mediated transduction of T cells can lead to malignancy. We transduced mouse T cells with a Moloney murine retroviral gene construct and transferred them into congenic mice, which were preconditioned to enhance the engraftment of transferred T cells. Recipients were then observed long-term for evidence of cancer. Transferred T cells persisted in mice throughout life at levels up to 17% with gene copy numbers up to 5.89 x 10(5) per million splenocytes. Mice receiving gene-modified T cells developed tumors at a similar rate as control mice that did not receive T cells, and tumors in both groups of mice were of a similar range of histologies. Hematological malignancies comprised approximately 60% of cancers, and the remaining cancers consisted largely of carcinomas. Importantly, the incidence of lymphomas was similar in both groups of mice, and no lymphomas were found to be of donor T-cell origin. This study indicates that the use of retroviral vectors to transduce T cells does not lead to malignant transformation.

Tumor-specific dendritic cells generated by genetic redirection of Toll-like receptor signaling against the tumor-associated antigen, erbB2.

Dendritic cells (DC) perform an important role in the initiation of the immune response through the local secretion of inflammatory mediators within diseased tissue in response to Toll-like receptor (TLR) ligation. However, DC vaccine strategies fail to make use of this capability against cancer. To harness the TLR response capability of DC against cancer, we tested a series of recombinant genes for their ability to redirect DC function specifically against a tumor-associated antigen. Each gene encoded a cell surface chimeric protein made up of extracellular single-chain immunoglobulin anti-erbB2 linked to an intracellular TLR-signaling component composed of either myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1 (IRAK-1) or the cytoplasmic domain of TLR4. Each gene was expressed in the DC line, JAWS II, to a similar degree following retroviral transduction. However, only the chimera containing IRAK-1 was able to mediate interleukin-12 and tumor necrosis factor-alpha secretion. Since TLR engagement can also activate DC and enhance their ability to stimulate T cells, we ligated the chimeric anti-erbB2-IRAK-1 receptor and determined the effect on the stimulation of T cells. We found that JAWS II cells triggered through chimeric anti-erbB2-IRAK-1 displayed an enhanced ability to stimulate ovalbumin-specific OT-II CD4(+) T cells. This first description of the generation of tumor-reactive DC may lead to the development of new cell-based vaccines that can act at both the tumor site to induce danger and at the lymph node to stimulate a specific T-cell response.

Antitumor activity of dual-specific T cells and influenza virus.

Activation and expansion of T cells are important in disease resolution, but tumors do not usually satisfy these immune requirements. Therefore, we employed a novel strategy whereby dual-specific T cells were generated that could respond to both tumor and influenza virus, reasoning that immunization with influenza virus would activate and expand tumor-specific cells, and inhibit tumor growth. Dual-specific T cells were generated by gene modification of influenza virus-specific mouse T cells with a chimeric gene-encoding reactivity against the erbB2 tumor-associated antigen. Dual-specific T cells were demonstrated to respond against both tumor and influenza in vitro, and expanded in vitro in response to influenza to a much greater degree than in response to tumor cells. Following adoptive transfer and immunization of tumor-bearing mice with influenza virus, dual-specific T cells expanded greatly in numbers in the peritoneal cavity and spleen. This resulted in a significant increase in time of survival of mice. However, tumors were not eradicated, which may have been due to the observed poor penetration of tumor by T cells. This is the first demonstration that the potent immunogenic nature of an infectious agent can be utilized to directly impact on T-cell expansion and activity against tumor in vivo.

Frizzled-7 dictates three-dimensional organization of colorectal cancer cell carcinoids.

Progression of colorectal cancer (CRC) involves spatial and temporal occurrences of epithelial-mesenchymal transition (EMT), whereby tumour cells acquire a more invasive and metastatic phenotype. Subsequently, the disseminated mesenchymal tumour cells must undergo a reverse transition (mesenchymal-epithelial transition, MET) at the site of metastases, as most metastases recapitulate the pathology of their corresponding primary tumours. Importantly, initiation of tumour growth at the secondary site is the rate-limiting step in metastasis. However, investigation of this dynamic reversible EMT and MET that underpins CRC morphogenesis has been hindered by a lack of suitable in vitro models. To this end, we have established a unique in vitro model of CRC morphogenesis, which we term LIM1863-Mph (morphogenetic). LIM1863-Mph cells spontaneously undergo cyclic transitions between two-dimensional monolayer (migratory, mesenchymal) and three-dimensional sphere (carcinoid, epithelial) states. Using RNAi, we demonstrate that FZD7 is necessary for MET of the monolayer cells as loss of FZD7 results in the persistence of a mesenchymal state (increased SNAI2/decreased E-cadherin). Moreover, FZD7 is also required for migration of the LIM1863-Mph monolayer cells. During development, FZD7 orchestrates either migratory or epithelialization events depending on the context. Our findings strongly implicate similar functional diversity for FZD7 during CRC morphogenesis.

Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux.

P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.

P-glycoprotein inhibits caspase-8 activation but not formation of the death inducing signal complex (disc) following Fas ligation.

Previous studies by our laboratory have shown that the drug transporter protein P-glycoprotein, P-gp, can specifically inhibit Fas-induced caspase-3 activation and apoptosis. Importantly, inhibition of both caspase-3 activation and cell death could be reversed by pharmacological and antibody inhibitors of P-gp function. However, the molecular mechanisms underpinning P-gp-mediated resistance to Fas-induced cell death and caspase activation remained unknown. We therefore sought to identify the point(s) within the death receptor pathway at which P-gp exerted its inhibitory effect and to determine whether the ATPase activity of P-gp was required. Structure-function analysis determined that ATP hydrolysis was necessary for P-gp to confer resistance to Fas-induced caspase activation and cell death. Importantly, although both FADD and caspase-8 were recruited to the Death Inducing Signal Complex (DISC) in wild-type P-gp expressing cells following Fas ligation, subsequent activation of caspase-8 at the DISC was inhibited. The ability of P-gp to inhibit caspase-8 activation was also ATP dependent. These studies demonstrate that P-gp inhibits Fas-induced caspase-8 activation but not formation of the DISC and that this activity of P-gp is dependent on ATP hydrolysis.

Identification and characterization of a novel family of mammalian ependymin-related proteins (MERPs) in hematopoietic, nonhematopoietic, and malignant tissues.

Evidence is presented for a family of mammalian homologs of ependymin, which we have termed the mammalian ependymin-related proteins (MERPs). Ependymins are secreted glycoproteins that form the major component of the cerebrospinal fluid in many teleost fish. We have cloned the entire coding region of human MERP-1 and mapped the gene to chromosome 7p14.1 by fluorescence in situ hybridization. In addition, three human MERP pseudogenes were identified on chromosomes 8, 16, and X. We have also cloned the mouse MERP-1 homolog and an additional family member, mouse MERP-2. Then, using bioinformatics, the mouse MERP-2 gene was localized to chromosome 13, and we identified the monkey MERP-1 homolog and frog ependymin-related protein (ERP). Despite relatively low amino acid sequence conservation between piscine ependymins, toad ERP, and MERPs, several amino acids (including four key cysteine residues) are strictly conserved, and the hydropathy profiles are remarkably alike, suggesting the possibilities of similar protein conformation and function. As with fish ependymins, frog ERP and MERPs contain a signal peptide typical of secreted proteins. The MERPs were found to be expressed at high levels in several hematopoietic cell lines and in nonhematopoietic tissues such as brain, heart, and skeletal muscle, as well as several malignant tissues and malignant cell lines. These findings suggest that MERPs have several potential roles in a range of cells and tissues.

Redirecting mouse CTL against colon carcinoma: superior signaling efficacy of single-chain variable domain chimeras containing TCR-zeta vs Fc epsilon RI-gamma.

The structurally related TCR-zeta and Fc receptor for IgE (Fc epsilon RI)-gamma are critical signaling components of the TCR and Fc epsilon RI, respectively. Although chimeric Ab receptors containing zeta and gamma signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the zeta and gamma subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-zeta and scFv-gamma receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-zeta chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-gamma receptors. T cells expressing scFv-zeta chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the zeta signaling pathway.

Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL.

The redirection of autologous lymphocytes to predefined tumor target Ags has considerable potential for the immunotherapeutic treatment of cancer; however, robust experimental systems for comparing various approaches have not been developed. Herein, we have generated a single chain variable domain anti-carcinoembryonic Ag (CEA) Fcepsilon receptor I gamma-chain fusion (scFv anti-CEA) receptor and demonstrated high-level expression of this chimeric receptor in naive mouse T lymphocytes by retroviral gene transduction. These gene-modified CTL were able to lyse CEA+ targets and secrete high levels of IFN-gamma following Ag stimulation. Depletion studies demonstrated that specific tumor cell cytotoxicity was mediated by gene-modified CD8+ T cells. Importantly, in increasingly stringent tests of efficacy in vivo, transduced CTL were sequentially shown to reject CEA+ colon carcinoma cells in a Winn assay and then reject established s.c. colon carcinoma in scid or syngeneic mice. Furthermore, using gene-targeted and scFv anti-CEA receptor-transduced donor CTL, perforin and IFN-gamma were demonstrated to be absolutely critical for the eradication of colon carcinoma in mice. In summary, we have developed a highly efficient gene transfer system for evaluating chimeric receptor expression in cytotoxic lymphocytes. This series of experiments has revealed the utility of scFv anti-CEA chimeras in providing mouse T cells the capacity to reject colon carcinoma in an Ag- and perforin-specific manner.

Fas-ligand-mediated lysis of erbB-2-expressing tumour cells by redirected cytotoxic T lymphocytes.

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor gamma chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-gamma clones, and lysis of erbB-2+ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-gamma, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL.

Adoptive transfer: the role of perforin in mouse cytotoxic T lymphocyte rejection of human tumor xenografts in vivo.

The popliteal lymph node cells of immunocompetent mice generated a strong in vitro cytotoxic response to footpad injection of several human tumor cell lines and the resulting mouse effector cells predominantly used a perforin-mediated cytotoxic mechanism. A relatively minor FasL-dependent cytotoxic response to CEM-CCRF and Jurkat leukemias, but not colon carcinoma COLO 205 cells, was also detected in immunized perforin-deficient mice. In vitro depletion of CD3+ CD8+ T cells, but not CD4+ T or NK1.1+ cells, completely inhibited lysis of human tumor cells, suggesting that CD3+ CD8+ T cells were effectors of perforin-mediated xenospecific cytotoxicity. Xenospecific cytotoxic T cells from wild-type mice were extremely efficient at rejecting tumor when adoptively transferred into scid mice bearing established COLO 205, CEM-CCRF, or Jurkat tumor xenografts. By contrast, cytotoxic T lymphocytes of perforin-deficient mice had no effect on the growth of established tumor xenografts. These data indicate that perforin, and hence direct cytotoxicity, plays a key role in the ability of adoptively transferred CD8+ cytotoxic T lymphocytes to eradicate established xenografts.

Tumor-specific IgE-mediated inhibition of human colorectal carcinoma xenograft growth.

Antibodies have found application in the diagnosis and therapy of cancer. These antitumor antibodies are confined to isotypes of IgG and little is known of the potential usefulness of other classes of immunoglobulin. In order to determine a possible antitumor effect of IgE antibody a tumor-specific mouse monoclonal IgE antibody was constructed. This antibody was derived from the mouse monoclonal antibody 30.6 that detects an antigenic determinant expressed on the surface of colorectal adenocarcinoma cells, including COLO 205. Mouse IgE 30.6 inhibited the growth of established COLO 205 tumor growing subcutaneously in scid mice. This effect was transient with tumor growth returning to pretreatment levels after 48 h. By contrast, a mouse IgG 30.6 and a chimeric human/mouse IgE 30.6 were without antitumor effect. This isotype-specific antitumor effect was not attributable to differences in antibody affinity, tumor localization, or serum half-life as these were essentially the same for all three isotypes of antibody. In addition, none of the 30.6 monoclonal antibodies inhibited the growth of COLO 205 cells in vitro. As little as 1 microgram per mouse of the tumor-specific mouse IgE antibody was sufficient to inhibit COLO 205 tumor growth, which is in contrast to previous results in which the comparatively weak antitumor effect of a chimeric human/mouse IgG1 required an optimum dose of 4 x 250 micrograms per mouse. This antitumor effect of mouse IgE 30.6 was specifically abrogated by prior administration of a nonspecific mouse IgE. Given this potency, and the fact that mouse Fc epsilon RI binds mouse IgE, but not human IgE, a role for Fc epsilon receptor bearing effector cells in the observed antitumor effect is discussed.

Xenospecific CD8+ cytotoxic T lymphocyte generation: accessory function for CD4+ T cells and natural killer 1.1+ cells.

BACKGROUND: Controversy exists as to whether natural killer (NK)1.1+ cells additionally support cytotoxic T lymphocyte (CTL) generation. We have previously demonstrated that mice generate a strong in vitro xenospecific CTL response in local popliteal lymph nodes (LN) to footpad immunizations with large numbers of human tumor cells. METHODS: In vivo depletion of various LN subsets using cytotoxic monoclonal antibodies was used to determine their relative importance in stimulating xenospecific CD8+ CTL responses to human Jurkat tumor cells. Depletion of functional NK cells in vivo was evidenced by the relative lack of NK1.1+ cells and NK activity in the spleens and LN of anti-NK1.1 monoclonal antibody-treated mice. CONCLUSION: Depletion of LN subsets indicated that CD4+ T cells were critical in generating an effective xenospecific CD8+ CTL response, but also suggested that NK1.1+ cells play a significant additional accessory role in the development of mouse anti-human xenospecific CTL.