Additional booster doses in patients with chronic lymphocytic leukemia induce humoral and cellular immune responses to SARS-CoV-2 similar to natural infection regardless ongoing treatments: A study by ERIC, the European Research Initiative on CLL.

Profound immune dysregulation and impaired response to the SARS-CoV-2 vaccine put patients with chronic lymphocytic leukemia (CLL) at risk of severe COVID-19. We compared humoral memory and T-cell responses after booster dose vaccination or breakthrough infection. (Green) Quantitative determination of anti-Spike specific antibodies. Booster doses increased seroconversion rate and antibody titers in all patient categories, ultimately generating humoral responses similar to those observed in the postinfection cohort. In detail, humoral response with overscale median antibody titers arose in >80% of patients in watch and wait, off-therapy in remission, or under treatment with venetoclax single-agent. Anti-CD20 antibodies and active treatment with BTK inhibitors (BTKi) represent limiting factors of humoral response, still memory mounted in ~40% of cases following booster doses or infection. (Blue) Evaluation of SARS-CoV-2-specific T-cell responses. Number of T-cell functional activation markers documented in each patient. The vast majority of patients, including those seronegative, developed T-cell responses, qualitatively similar between treatment groups or between vaccination alone and infection cases. These data highlight the efficacy of booster doses in eliciting T-cell immunity independently of treatment status and support the use of additional vaccination boosters to stimulate humoral immunity in patients on active CLL-directed treatments.

Killing of laminin receptor-positive human lung cancers by tumor infiltrating lymphocytes bearing gammadelta(+) t-cell receptors.

BACKGROUND: The monomeric laminin receptor, a 67-kd high-affinity laminin-binding protein, is expressed by a variety of normal cell types. Overexpression and abnormal surface distribution of this receptor have been demonstrated in tumor cells where it appears to promote tumor invasion and metastasis. Previously, we reported the existence of an association between laminin receptor overexpression by lung cancer cells and the presence of tumor-infiltrating lymphocytes (TILs) bearing gammadelta T-cell receptors. Gammadelta(+) lymphocytes represents a sizable fraction of the TILs in approximately one fourth of lung cancers analyzed thus far. PURPOSE: The aim of this study was to determine whether gammadelta(+) TILs might participate in the immune response against lung cancer through recognition of monomeric laminin receptors expressed by tumor cells. METHODS: Tumor cells from 11 lung cancer specimens exhibiting sizable gammadelta(+) T-cell infiltrates and from 11 other specimens infiltrated predominantly by lymphocytes bearing alphabeta(+) T-cell receptors were analyzed for expression of the monomeric laminin receptor by use of the monoclonal antibody (MAb) MLuC5. Gammadalta TILs and chibeta+ TILs derived from four tumors were each examined for cytotoxic activity toward lung cancer target cells by use of a standard 51Cr-release assay and lung tumor cell lines expressing different levels of surface monomeric laminin receptor. The ability of MAbs directed against the laminin receptor (i.e., MLuC5) or against gammadelta T-cell receptors (i.e., TigammaA and A13) as well as laminin peptides known to bind to the laminin receptor to inhibit TIL-mediated target cells lysis was also determined. RESULTS: We confirmed that the association between overexpression of the monomeric laminin receptor by lung tumor cells and the presence of gammadeltadelta+ TILs is statistically significant (two sided P = .003; Fisher's exact test). We also observed a relationship between the levels of laminin receptor expression on cultured lung cancer cells and their susceptibility to specific lysis by gammadelta(+), but not alphabeta+, TILs. This specific cell killing was not T-cell receptor mediated, but it was inhibited by addition of the anti-monomeric laminin receptor MAb MLuC5 and by a synthetic peptide corresponding to amino acids 2091-2108 of the laminin A chain. CONCLUSION AND IMPLICATIONS: Our results indicate gammadelta(+) TILs localized at human lung cancer sites can kill tumor cells in a process that involves interaction with the monomeric laminin receptor. The infiltration of gammadelta(+) TILs at lung tumor sites may represent a first line of defense against cells undergoing malignant transformation.

Particulate naturally processed peptides prime a cytotoxic response against human melanoma in vitro.

For an efficient antitumor cytotoxic response, tumor antigenic peptides need to be presented by professional antigen-presenting cells in association with MHC class I molecules. We established in vitro short-term human CTL lines from healthy and melanoma-bearing subjects, using as antigen-presenting cells autologous adherent cells after phagocytosis of latex beads coated with melanoma peptides. Melanoma peptides were obtained by acid extraction of melanoma cells that matched with donor peripheral blood mononuclear cells, at least for one HLA-A allele. The cytotoxic activity of the lines was specific for the melanoma from which peptides were obtained and for melanoma sharing HLA alleles. These results demonstrate that a complex mixture of naturally processed melanoma peptides conjugated to a phagocytic substrate that targets them into the MHC class I pathway of adherent cells can prime a CTL response in healthy subjects in vitro, and that peptides from allogeneic tumors may be used to propagate CTL in melanoma patients. Our data support the feasibility of active and passive vaccination procedures with nonliving vaccines in cancer patients.

In vivo administration of GM-CSF promotes the clearance of apoptotic cells: effects on monocytes and polymorphonuclear leukocytes.

The clearance of apoptotic cells is crucial to avoid chronic inflammation and autoimmunity. Little is known about the factors that regulate it in vivo. We show that granulocyte-macrophage colony-stimulating factor (GM-CSF) administration to carcinoma patients confers to their leukocytes a significantly higher ability to phagocytose apoptotic cells than before (P < 0.005). GM-CSF increased the concentration of monocytes and polymorphonuclear leukocytes in the peripheral blood and activated circulating polymorphonuclear leukocytes. Both effects abated early after treatment, whereas phagocytosis of apoptotic cells was still significantly higher after 18 days compared with basal values (P < 0.005 and P < 0.025 for monocytes and polymorphonuclear leukocytes, respectively). On in vitro phagocytosis of apoptotic cells monocytes, but not polymorphonuclear leukocytes, up-regulated MHC class II membrane expression. These findings are consistent with the possibility that GM-CSF endows both scavenger and antigen-presenting leukocytes with the ability to internalize apoptotic tumor cells.

Human melanoma cells transfected with the B7-2 co-stimulatory molecule induce tumor-specific CD8+ cytotoxic T lymphocytes in vitro.

Neoplastic cells express tumor-associated antigens, but tumor rejection seldom occurs in vivo. The absence of an effective immune response may be explained by the inability of tumor cells to deliver co-stimulatory signals. Indeed, transfection of either B7-1 or B7-2 co-stimulatory molecules into mouse tumor cells enhances antitumor immune responses. In this study, we stably transfected human melanoma cells with the cDNA encoding the B7-2 molecule to evaluate in vitro: (i) the induction of anti-melanoma cytotoxic T lymphocytes (CTL) by stimulation of CD8+ T cells, purified from healthy donors and a melanoma patient, with B7-2 transfected allogeneic HLA-matched melanoma cells; (ii) the tumor specificity and the HLA restriction of the induced CTL; and (iii) the feasibility to propagate long-term antimelanoma CTL lines. We found that B7-2 transfected, but not untransfected or mock-transfected, melanoma cells activated MHC-class I-restricted, melanoma-specific CD8+ CTL from healthy donors. More importantly, CD8+ tumor-associated lymphocytes, purified from a tumor-invaded lymph node of a melanoma patient and stimulated with B7-2-transfected melanoma cells, acquired a strong reactivity toward the autologous tumor. CTL lines with specific cytolytic activity could be propagated in long-term culture. These results indicate that: (i) the expression of the B7-2 molecule into human melanoma cells makes them immunogenic and able to act as antigen-presenting cells and (ii) purified CD8+ cells, stimulated with B7-2+ allogeneic HLA-matched melanoma cells, preferentially recognize melanoma-specific rather than allogeneic antigens. This study may have clinical implications for passive and/or active immunotherapy in melanoma patients.

Constitutive expression of the heat shock protein 72 kDa in human melanoma cells.

Heat shock proteins (hsp) are a family of proteins characteristically produced under stress conditions in normal cells. Overexpression of hsp 70 kDa protects tumoral cells from tumor necrosis factor cytotoxicity and is related to drug resistance. In this study we investigated whether hsp are abnormally expressed in melanoma cells in vivo. Antibodies directed against hsp 72 and hsp 90 were used to identify hsp on non-stressed neoplastic cells derived from surgical specimens of two primary and four metastatic melanomas. Hsp 72 and hsp 90 were always present at high level in the cytoplasm of melanoma cells. It is possible that the activation of hsp genes during neoplastic transformation confers a selective advantage to tumoral cells in vivo, allowing them to escape the immunological surveillance.

Autoimmune thyroiditis following interleukin-2 and LAK cell therapy for metastatic renal cell carcinoma: correlation with tumor regression.

A 63-year-old woman receiving recombinant interleukin-2 (rIL-2) + lymphokine activated killer cells for metastatic renal cell carcinoma developed autoimmune thyroiditis with clinical hypothyroidism and high titer anti-thyroglobulin and anti-microsomal antibodies. The onset of thyroid dysfunction was associated with tumor regression and resulted in complete response at the end of the treatment. Cytologic and cytofluorimetric studies on thyroid tissue showed two distinct populations, mainly consisting of small lymphocytes and large thyrocytes, and the latter expressed MHC class II antigens. After completion of rIL-2 treatment, hypothyroidism gradually decreased until resolution; complete tumor remission lasted 18 months. Mechanisms underlying the association between autoimmune thyroiditis and cancer regression are discussed.

V delta 1+ gamma/delta T lymphocytes infiltrating human lung cancer express the CD8 alpha/alpha homodimer.

Murine gamma/delta T lymphocytes localize to different epithelial tissues and are phenotypically distinct from peripheral gamma/delta T cell-populations in that they show limited TCR diversity, express the CD8 alpha/alpha homodimer and lack the CD8 beta chain. In humans, a compartmentalization of gamma/delta cells sharing similar phenotypic features has been documented to date only in the case of intestinal epithelium. In the present study we show that about half of V delta 1+ (as well as V delta 1-V delta 2-) gamma/delta lymphocytes, which can be selectively expanded from human lung cancers, coexpress the CD8 alpha/alpha homodimer. The accumulation of intraepithelial CD8+ gamma/delta+ lymphocytes might then be a more general phenomenon, possibly as a result of common mechanisms operating at those sites.

Dual-parameter flow cytometric analysis of an early lymphocyte activation antigen (CK226) and DNA content.

This study provides a direct correlation, via dual-parameter flow cytometric analysis (simultaneous assessment of surface immunofluorescence and DNA content), between activated T-cell entry into the S/G2/M phases of the cell cycle and the kinetics of expression of a novel T-cell activation antigen, termed CK226. This molecule was identified by the specific monoclonal antibody on the leukaemic T-cell line CEM/K, and it was expressed by 8-30% of resting peripheral blood lymphocytes and the majority of monocytes and granulocytes. A large fraction of activated lymphocytes acquired the CK226 antigen before DNA synthesis. Moreover, this molecule was expressed on virtually all G0/G1 and S/G2/M phase cells by day 2 after phytohaemagglutinin (PHA) activation and at day 6 after stimulation in a mixed lymphocyte culture. The time course of expression of other known activation antigens, such as Tac and transferrin receptor, was comparable to that of CK226. Based on the relationships between CK226 expression on cycling cells and the stimulatory effects of the specific monoclonal antibody, we conclude that CK226 should be considered an early activation antigen, which defines a new pathway of T-cell activation.

Unusual expression and localization of heat-shock proteins in human tumor cells.

It has been suggested that members of HSP families represent the surface target of immune responses leading to tumor rejection in mice. Here we report that tumor cells, compared with normal cells, constitutively expressed 2- to 10-fold higher levels of intracellular HSP90. Moreover, in the absence of environmental stress, 2 lines (out of 6) expressed the "inducible" HSP72, which was also detectable in fresh tumor cells. HSP72 expression was not regulated during the cell cycle, in contrast with what has been observed with normal cells. Both HSP90 and HSP72 proteins exhibited a heterogeneous pattern of intracellular distribution in most cells, HSP72 being confined mainly to the nuclear compartment. Finally, we could detect both HSP90 and, to a lesser extent, HSP72 (that are generally believed to be located intracellularly) at the surface of some tumor cell lines. We conclude that tumor cells differ from normal cells in their pattern of HSP expression; this might imply a role of HSPs in eliciting an immune response against cancer.

Daudi lymphoma killing triggers the programmed death of cytotoxic V gamma 9/V delta 2 T lymphocytes.

A common feature of human V gamma 9/V delta 2 gamma delta T lymphocytes is their ability to kill Daudi lymphoma cells. We show here that during this killing a substantial fraction of cytotoxic gamma delta T cells dies as well. Their death has morphologic, cytometric, and biochemical features of apoptosis and depends on TCR triggering. We suggest that interaction with the target physiologically induces the programmed death of the cytotoxic gamma delta effector. The death of activated gamma delta T cells upon challenge with the relevant Ag may represent a negative feedback mechanism and contribute to explain the limited time span of gamma delta T cell expansions during infectious diseases, even when pathogens are not eliminated and persist in the host.

Autocrine nitric oxide modulates CD95-induced apoptosis in gammadelta T lymphocytes.

Gammadelta T lymphocytes play an important early role in the defense against pathogens. Their function is terminated by acquisition of susceptibility to CD95-triggered apoptosis. Here we show that the regulation of this process depends on the activity of the endothelial NO synthase expressed by gammadelta T lymphocytes, which is modulated in an activation-dependent way. The effects of nitric oxide thus generated, mediated via cGMP generation, are exerted at at least two sites along the CD95 signaling cascade: one at, or upstream, and the other downstream of ceramide generation. At either site, nitric oxide/cGMP action is sufficient for protection from apoptosis. The effect of NO is selective for apoptosis induced by CD95 cross-linking, since it does not affect apoptotic program triggered by other stimuli. The evidence here reported demonstrates a new physiological role for nitric oxide, acting as a survival factor for T lymphocytes.

Evaluation of cytotoxic cell-target cell conjugates. Comparison between flow cytometric analysis and 51Cr binding assay.

Two methods for evaluating the conjugate formation between either bulk cultured or cloned NK cells and fresh tumor target cells or tumor cell lines were compared. Flow cytometric analysis and 51Cr binding assay yielded comparable results using NK clones as effector cells; however, flow cytometry provides less accuracy using large granular lymphocytes or unfractionated lymphocytes.

Evidence for the involvement of phosphatidylinositol 3-kinase in fMLP-stimulated neutrophil adhesion to ICAM-1-transfected cells.

Phosphatidylinositol 3-kinase (PI-3K) controls important intracellular steps involved in inflammation, immunity, and cell growth. PI-3K also modulates leukocyte integrin adhesiveness. In this study we evaluated the role of PI-3K on neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-transfected cells. N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated neutrophil adhesion was inhibited by wortmannin and LY294002, two unrelated PI-3K inhibitors, whereas phorbol myristate acetate (PMA)-induced neutrophil adhesion was not inhibited by them. After fMLP stimulation, a rapid activation of AKT and ERK was observed. However, only activation of AKT was reversed by the PI-3K inhibitors. Neutrophil expression of the beta2-integrins Mac-1, lymphocyte function-associated antigen-1(LFA-1), and gp150.95 was not affected by wortmannin, nor was expression of the activation epitope recognized by MAB24. We conclude that (a) PI-3K is involved in fMLP-activated neutrophil adhesion to ICAM-1-transfected cells, (b) the mechanism involved is not mediated by the modulation of beta2-integrin expression or activation, and (c) another mechanism seems to involve the adhesion to ICAM-1 when a cellular system of adhesion is used.

Cytologic and flow cytometric DNA analysis of multinucleated tumor cells and derived microcells.

Microcell production by means of Colcemid-induced micronucleation and subsequent enucleation with the density gradient technique was adjusted for use with the murine T-lymphoma line ESb-M. Modification of the standard protocol for a cell type on which no experiments had previously been performed required careful monitoring of the multiple steps in the procedure in order to optimize the final microcell yield. Traditional microscopic verification may sometimes be ambiguous, due to the lack of a clear cutoff point between small whole cells and cell fragments; in these conditions, the level of variability increases, thus impairing quantitative estimations. Flow cytometric (FCM) analysis of DNA content and size of donor cells and microcells was therefore applied in parallel to provide additional quantitative information. The FCM results supplemented the microscopic data in assessing which fraction recovered from the gradient has the lowest percentage of contaminant whole cells; however, FCM analysis may provide more statistically significant data due to the large size of the sample examined. Moreover, FCM is of prospective use in providing the basis for subsequent sorting of either pure microcells or specific subpopulations of defined DNA content and size.

Recombinant interleukin-2 and lymphokine-activated killer cells in renal cancer patients: II. Characterization of cells cultured ex vivo and their contribution to the in vivo immunomodulation.

Peripheral blood mononuclear cells, obtained from patients with renal cell cancer and cultured ex vivo, exhibit high natural killer (NK) and lymphokine-activated killer (LAK) activity (also against allogeneic fresh tumour cells), which is transcribed into the hosts' immune status after reinfusion. Phenotypic analysis shows a slight increase in the percentage of CD56+ and CD8+ lymphocytes, while CD4+ lymphocytes decrease slightly. As a sign of activation an increase of cells expressing DR and CD25 antigens is observed. At the peripheral blood level, mononuclear cells show an increase, compared to basal values, of NK and LAK activity, especially at the end of the first infusion cycle. Phenotypic analysis of the patients' PBMC shows a decrease of CD3+CD4+ T lymphocytes and an increase of NK cells (CD3-CD56+CD16+) and of cells expressing activation markers (DR and CD25), particularly evident by the end of the second infusion cycle. Finally, in addition to the changes induced by IL-2 alone, reinfusion of incubated cells results in an activation of CD56+ and LeuM3+ cells.

Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.

Age-related modifications of the human alphabeta T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets.

We have studied the effects of a life-long antigen stimulation on the clonal heterogeneity of human peripheral T cell subsets, as defined by their CD45 isoform expression. CD4+ or CD8+ T cells were obtained from healthy donors ranging in age from 20 to 100 years, and sorted into CD45RA+ and CD45RO+ populations. A modified PCR-heteroduplex analysis was then used to directly compare the TCR Vbeta clonal make up of either compartment pair. We find that the CD4+ T cell repertoire remains largely polyclonal throughout life, since CD4+ expanded clones are rare and accumulate predominantly in the CD45RO+ compartment of exceptionally old donors (100 years old). In contrast, the CD8+ T cell subset contains expanded clones which are already detectable in young adults and become very frequent in 70- to 75-year-old donors in both CD45RA+ and CD45RO+ compartments analyzed. Interestingly, some expanded clones are detectable in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments of either CD4+ or CD8+ T cells. These results indicate that the age-dependent accumulation of expanded clones starts earlier and is more pronounced in the CD8+ than in the CD4+ T cell subset, reinforcing the concept that clonal expansion in the two subsets is controlled by substantially different mechanisms. Furthermore, whereas the finding of expanded CD45RO+ T cell clones is explained by antigen-driven proliferation, the detection of expanded clones in the CD45RA+ or in both CD45RA+ and CD45RO+ compartments would support the hypothesis of reversion from the CD45RO+ to the CD45RA+ phenotype after antigen encounter.