Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR α-chain CDR3 loop

doi:10.1002/eji.200737389

Eur J Immunol. Author manuscript; available in PMC 2010 May 5.

Published in final edited form as:

Eur J Immunol. 2008 June; 38(6): 1756–1766.

doi: 10.1002/eji.200737389

PMCID: PMC2864538

NIHMSID: NIHMS168417

Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR α-chain CDR3 loop

Mark A. Exley,^1,⁸ Runhua Hou,² Angela Shaulov,¹ Paolo Dellabona,³ Giulia Casorati,³ Omid Akbari,^4,⁸ H. Orhan Akman,¹ Edward A. Greenfield,⁵ Jenny E. Gumperz,⁶ Jonathan E. Boyson,⁷ Steven P. Balk,^1,⁸ and S. Brian Wilson^2,⁸

¹Hematology/Oncology, Beth Israel-Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215.

²Diabetes Unit, Massachusetts General Hospital, 65 Landsdowne Street, Cambridge, MA 02139.

³Cancer Immunotherapy & Gene Therapy, DIBIT San Raffaele Scientific Institute, 20132 Milan, Italy.

⁴Children’s Hospital, Boston, MA 02115.

⁵Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115.

⁶Medical Microbiology & Immunology, University of Wisconsin, 1300 University Ave, Madison, WI 53706.

⁷University of Vermont, Burlington, VT 05405.

⁸Harvard Medical School, Boston MA 02115.

Addresses for Correspondence: Dr. M. Exley, Cancer Biology, Hematology/Oncology, NRB 1030L, Beth Israel-Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, Fax: 617-667-0610, Phone 617-667-0982, Email: mexley/at/bidmc.harvard.edu, Dr. S. B. Wilson, Diabetes Unit, Department of Medicine, Massachusetts General Hospital, 65 Landsdowne Street, Cambridge, MA 02139, Phone 617 768-8883, Fax: 617-768-8888, Email: bwilson/at/rics.bwh.harvard.edu

The publisher's final edited version of this article is available at Eur J Immunol

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Summary

A significant fraction of CD1d-restricted T cells express an invariant T cell receptor (TCR) α-chain. These highly conserved ‘iNKT’ populations are important regulators of a wide spectrum of immune responses. The ability to directly identify and manipulate iNKT is essential to understanding their function and to exploit their therapeutic potential. To that end, we sought monoclonal and polyclonal antibodies specific for iNKT by immunizing CD1d KO mice, which lack iNKT, with a cyclic peptide modeled after the TCR-α CDR3 loop. One monoclonal antibody (mAb; 6B11) was specific for cloned and primary human but not rodent iNKT and the human invariant TCR-α, as shown by transfection and reactivity with human invariant TCR-α transgenic T cells ex vivo and in situ. 6B11 was utilized to identify, purify, and expand iNKT from an otherwise minor component of human peripheral blood lymphocytes and to specifically identify human iNKT in tissue. Thus, we report a novel and general strategy for the generation of monoclonal antibodies specific for the CDR3 loop encoded by the TCR of interest. Specifically, an anti-Vα24Jα18 CDR3 loop clonotypic TCR mAb is available for the enumeration and therapeutic manipulation of human and non-human primate iNKT populations.

Keywords: anti-TCR, CD161, clonotypic, cyclic peptide, IL-4, invariant, NKT

Introduction

“Natural killer T cells” expressing surface markers found on NK cells (e.g. CD161, CD56, CD94 and / or KIR) comprise up to 20% of human peripheral blood lymphocytes (PBL), [1] whilst only ~0.05 % of PBL are CD1d-restricted. [2] [3] [4] A major proportion of such PBL CD1d-reactive T cells are “invariant NKT” cells (iNKT), in that they express an invariant TCR-α chain, Vα24Jα18 in humans and Vα24Jα18 in mice. [3] [5] [6] Both human and murine iNKT can be activated in a CD1d-dependent fashion by the synthetic glycolipid α-galactosylceramide (α-GalCer) and self or pathogen-derived glycolipid molecules. [7] [8] [9] Activation of iNKT leads to rapid production of large amounts of numerous cytokines and consequent stimulation of multiple immune components, [3] [10] [11] [12] [13] [14] perhaps most significantly, by regulating dendritic cell (DC) function. [10] [15]

The frequency and functional activity of iNKT have been shown to be important determinants for the progression of autoimmune disorders, tumors, and viral infections. [10] [16] [17] [18] In humans, a reduction in the number of circulating iNKT is accompanied by a striking Th1 polarization in certain autoimmune diseases such as type 1 diabetes, systemic sclerosis and rheumatoid arthritis. [18] [19] [20] [21] Cancer patients have decreased numbers of iNKT in their blood and impaired in vitro proliferative potential, reversible with IL-2. [22] [23] [24] As well as the reduced numbers of these cells found in many cancer patients, there is a striking reversal of cytokine polarization relative to that found in diabetes, which may reflect a reduction in iNKT anti-tumor activity during progression. [22] [25] Stimulation of healthy donor iNKT triggers both secretion of multiple cytokines including IFN-γ and CD1d-specific cytotoxic activity, which in the human involves perforin / granzyme granule secretion. [2] [4] Thus, iNKT are clearly important regulators of a widely disparate set immune responses, making them attractive targets for therapeutic intervention.

Human iNKT were originally identified with Vα24 and Vβ11 mAb, [26] but even the combination of these two relatively selective reactivities does not formally define iNKT. [5] A number of groups have reported selective identification of CD1d-restricted T cells ex vivo with CD1d multimers specifically loaded with α-GalCer. [4] [27] Whilst this approach has been powerful for enumeration of α-Galcer-reactive T cells, limitations include the possibility of identifying CD1d-reactive cells that are non-invariant and whose functionality is unclear, as well as missing iNKT with sufficiently divergent TCR that do not react with α-GalCer. [28] [29] [30] Furthermore, functional application of tetramer reagents is complex and they are generally not useful in histology. [4] Here we report a novel and general strategy for the isolation and characterization of polyclonal and monoclonal antibodies (mAbs) specific for TCR CDR3. We have used this approach to generate mAbs reactive with human CD1d-reactive invariant T cells that can be used to identify in situ and detect iNKT and to selectively stimulate and expand this rare population ex vivo. Since iNKT appear to contribute to regulation of or directly to anti-viral, anti-tumor, autoimmune and other immune responses, these new reagents should be helpful in understanding the role of these cells and in development of improved diagnostics and immunotherapies for such diseases.

Results

Isolation of invariant TCR CDR3-loop specific antibodies

The generation of the invariant Vα24Jα18 TCR chain results from joining of the Vα24 segment to the Jα18 element in germ line configuration. The cysteine encoded by the end of the Vα24 segment (-CVVS) is the beginning of the CDR3 loop and is conserved in all known mammalian TCR α-chain sequences. This residue forms one of the two-intrachain disulfide bonds that form the Ig-fold domains present in the TCR α-chain structure. To generate antibodies cyclic peptides matching the human invariant TCR α-chain CDR3 sequence and modeled after the available crystal structures were synthesized. In order to maximize exposure of the CDR3 loop and facilitate screening of antibodies, the peptides were synthesized with a reduced sulfhydryl group on the conserved cysteine. The free sulfhydryl residue was then used to couple the cyclic peptide hapten to activated carrier proteins KLH, BSA, or Ova.

CD1d KO animals, selected because they do not have iNKT cells and would not be expected to be tolerant to the highly homologous murine iNKT TCR, were first immunized with cyclic peptides covalently coupled to KLH as carrier in adjuvant. This was followed by two boosts with the same peptide coupled to antigenically unrelated carrier BSA and iNKT clone D2.N6 cells on the second BSA immunization (Figure 1A). Sera and hybridomas were screened for specific binding to cyclic peptide by ELISA on cyclic peptide coupled to a further unrelated carrier, ovalbumin (Ova) and to Ova alone. More than 10% of wells contained antibodies reactive with peptide-Ova, approximately half of which also reacted with Ova alone. Since prior to cloning some of the latter wells could also contain invariant TCR-specific hybridomas, all 119 positives were screened for reactivity with iNKT clones by FACS analysis. A total of 19 hybridomas secreting mAbs that stained human iNKT clones were identified. A panel of 8 hybridomas whose staining of iNKT clones could be blocked by the addition of cyclic peptide, but did not stain a series of control CD8⁺ or CD4⁺ T cell lines and clones, were identified (data not shown). Six of these hybridomas were lost on subcloning and not evaluated further. However, two hybridomas, 3A6 (IgM) and IgG1 6B11 (IgG1,κ1), that reacted specifically with the iNKT clones (Figure 1B) were stably subcloned. Of these two, only 6B11 retained staining specificity after conjugation. 6B11 was functionally active in pure form or after conjugation with FITC, Alexa488, biotin, or PE. To confirm the specificity of the mAb, 6B11 was used to stain TCR-negative Jurkat cells transiently transfected to express the full-length TCR transcripts for the invariant TCR Vα24Jα18 and a Vβ11 mRNA cloned from an iNKT clone (Figure 2A). The antibody did not react with singly transfected cells or Jurkat transfected to express TCR pairings specific for other CD1 molecules (data not shown). [31] Staining by 6B11 paralleled that of anti-Vβ11; the higher degree of staining by the anti-Vα24 mAb may be related to over expression of Vα24 protein and the fact that this antibody recognizes a pan Vα24 linear epitope encoded by the CDR2 region of this protein. To further validate the specificity of 6B11 for the Vα24Jα18 CDR3 loop, splenocytes from C57BL/6 mice that express the human invariant Vα24Jα18 transgene were stained with 6B11. Staining of iNKT by 6B11 was only noted in those mice that expressed the human transgene. (Figure 2B) Interestingly, although Vα24 mAb uniformly stained all Vα24Jα18 transgenic Cα KO liver mononuclear cells with medium MFI, as previously, 6B11 stained three major populations (high, medium, and low MFI). (Figure 2B) This suggests that while Vα24 mAb staining was independent of pairing of the human TCR transgene with the endogenous murine Vβ8, Vβ7, and other partners, 6B11 was able to discriminate between these populations.

Figure 1

Strategy for the generation of clonotypic iNKT-specific antibodies and mAb testing

Figure 2

Invariant TCR-specific mAb specifically react with human iNKT cell Vα24Jα18

We further determined whether 6B11 could be used to specifically identify iNKT cells in tissues by immuno-histochemistry with the Vα24Jα18 transgenic mice and controls (Figure 2C–H). No specific 6B11 staining was observed on paraffin slides (not shown). Wild type mice spleen frozen sections were only stained with mouse CD3 mAb and not 6B11 (Figure 2C–E). However, spleen from Vα24Jα18 transgenic mice specifically stained a similar fraction of cells with 6B11 (Figure 2F–H), but not isotype controls (not shown). Double staining revealed that as expected in the Cα KO background, essentially all 6B11⁺ splenic cells co-stained with mouse CD3 (Figure 2F–H). Therefore, only the human iNKT transgenic spleens contained 6B11-reactive T cells. The 6B11⁺ cells were predominantly located in the splenic T cell zones (peri-arteriolar lymphoid sheath). Thus, cyclic peptides can be used to generate monoclonal antibodies highly specific for human and nonhuman T cells selectively expressing the human invariant Vα24Jα18 transcripts.

6B11 is highly specific for iNKT subsets in PBMC

To evaluate the specificity of 6B11 for iNKT in PBMC, the selectivity of this antibody for the invariant Vα24Jα18 TCR was compared with anti-Vα24 / anti-Vβ11 and human CD1d tetramers loaded with α-GalCer. First, FACS sorting with anti-Vα24/anti-Vβ11, anti-Vα24/6B11, anti- Vβ11/6B11, or hCD1d/CD3 pairings were used to isolate a large panel of clones (Table I). [19] A total of 199 clones were isolated, 187 of which were Vα24⁺. All the clones isolated using anti-Vα24 as part of the pairing reacted with this antibody on expansion, whereas 12/17 isolated with hCD1d tetramers were Vα24⁺. The clones were then evaluated for staining with 6B11, their TCR α-chains sequenced, and CD1d restriction was validated by co-culture with C1R.CD1d or control C1R.neo transfectants. [2, 19] Paring 6B11 with either anti-Vα24 or anti-Vβ11 was highly specific for the generation of invariant and CD1d restricted T cell clones. Only 4/141 clones encoded single amino acid substitutions in the invariant CDR3 loop. These four clones which stained with 6B11, were CD1d-restricted but were found to have substituted a threonine or asparagine for serine at the AV24:AV18 junction; CD1d restriction of this substitution has been noted before. [32] In contrast, 31/41 cells cloned using anti-Vα24/anti-Vβ11 were invariant and CD1d-restricted, whilst all the clones isolated with hCD1d tetramers were CD1d-restricted. Eight of 17 of the clones isolated with the hCD1d tetramer were invariant, and 10/17 6B11 reactive. The two additional 6B11⁺ clones encoded the previously noted threonine and asparagine substitutions. A further two clones in the set selected with hCD1d tetramers were found to be Vα24⁺ and CD1d-restricted, but 6B11(−) negative. Interestingly, sequencing of the TCR α-chain of these clones revealed a serine to glycine substitution (CVVG:DRGST; one of the two sequence differences noted in the CDR3 loops encoded by murine iNKT, which do not stain with 6B11), and a valine to alanine change (CVAS:DRGST). Consistent with these results, staining of PBMC with 6B11 and anti-Vα24 antibodies revealed selective identification of iNKT with very low background staining, (Figure 3A) but very weak staining of the clone harboring the alanine substitution (Figure 3B). As expected, when 6B11/anti-Vα24 staining was compared with α-GalCer-loaded hCD1d tetramer/anti-CD3 staining in a cohort of normal donors, the frequencies of iNKT closely matched that of tetramer staining, but larger and variable numbers of CD1d reactive cells were noted with the tetramer (Figure 3C). Hence, 6B11 is highly selective for the invariant Vα24Jα18 TCR chain and can be used identify and clone iNKT.

Table 1

Incidence of Vα24+, 6B11+, Vβ11+, and CD1d-reactive iNKT clones isolated using anti-Vα24, anti-Vβ11, or 6B11 pairwise combinations, or α-GalCer loaded CD1d tetramers.

Figure 3

Evaluation of PBMC iNKT populations with 6B11 mAb

Stimulation and expansion of iNKT by anti-invariant TCR mAb

The specificity of anti-invariant TCR mAb 6B11 was further demonstrated by showing that it could selectively isolate and stimulate iNKT cells (Figure 4). Initially, total Vα24⁺ T cells were isolated by FACS sorting CD3⁺/ Vα24⁺ T cell populations. These polyclonal T lines were then stimulated with irradiated PBMC and α-GalCer, plate-bound 6B11, or irradiated PBMC and PHA supplemented with IL-2. The relative proportion of cells that were invariant-type Vα24⁺β11⁺ after ten days in culture was determined (Figure 4A). Both α-GalCer and 6B11 treatments selectively and comparably expanded Vα24⁺/Vβ11⁺ T cell populations. Plate bound 6B11 also stimulated a subset of Vβ11-negative Vα24⁺ T cells. These cells all stained with 6B11 suggesting that these cells are iNKT whose TCR α-chain was not paired with Vβ11, whereas α-GalCer appears to favor expansion of the Vα24⁺/Vβ11⁺ iNKT population (Figure 4A; 33). In marked contrast, stimulation of polyclonal Vα24⁺ T cells with PHA resulted in the expansion of T cells that were largely Vα24⁺ Vβ11-negative (Figure 4A), and which did not react with 6B11 or anti-CD161 (data not shown).

Figure 4

Anti-invariant mAb 6B11 is mitogenic and can be used to isolate and expand iNKT

Figure 4B shows that starting with a typical PBMC containing ~ 0.25% iNKT (left), single round 6B11 purification resulted in nearly 90% pure iNKT. This approximately 360-fold enrichment was measured directly ex vivo (center) and was further improved by several weeks 6B11-induced expansion (right.).

Pure iNKT cell lines stimulated with either CD1d⁺ APC or 6B11 secreted 2–3 log units greater amounts of cytokine than those generated using anti-Vα24 alone, comparable to PHA mitogen, unlike either PBMC-derived T cell lines or even Vα24⁺ T cell lines (IL-4 Figure 4C, IFN-γ, not shown). PBMC had little if any detectable CD1d-specific or 6B11-induced cytokine detectable. Thus, 6B11 selectively expands iNKT without an absolute requirement for APC as “feeders,” whereas Vα24 has only relatively modest specificity for iNKT.

Since iNKT are attractive candidates for adoptive cellular transfer for the therapy of cancer and various autoimmune disorders or viral infections, we next devised strategies for ex vivo expansion of subsets of these cells with clinically approved reagents to numbers comparable with previous clinical trials involving T cell transfer. [33] [34] iNKT were isolated using 6B11-biotin and anti-biotin microbeads. Following isolation, various expansion approaches comparing α-GalCer and APC with OKT3 and APC were compared. In addition, the effect of high dose IL-2 (doses used to expand TIL) was compared to conventional IL-2 supplementation. As can be seen from the results of a representative experiment from a prostate cancer donor, the combination of OKT3 (1 ug/ml), IL-2 (100 U/ml) and autologous irradiated APC was optimal, (Figure 5) and OKT3 was selected, as it is FDA approved and feasibility was tested in with a patient consented for leukophoresis for this purpose. Interestingly, pure iNKT lines expanded with OKT3 were relatively biased towards the secretion of both IFN-γ and IL-4 after activation with either α-GalCer or mitogen (PHA), whereas those expanded with α-GalCer were biased towards the secretion of IFN-γ (Figure 5B), suggesting different expansion strategies should be considered depending on the desired phenotype of iNKT to be used for therapy.

Figure 5

Expansion and functional activity of 6B11-selected iNKT at large scale

Detection of human iNKT by immunohistology

Finally, we used 6B11 to stain frozen sections, since no specific staining was observed on human tissue paraffin sections (not shown). Fig. 6 shows that there were many readily detectable iNKT cells (Left panel in blue) in the lung biopsy from a chronic asthmatic patient. As expected, there were more CD4⁺ T cells (Middle panel, red), although a substantial subset of these co-stained with 6B11 (Right panel, purple in overlay) and therefore appeared to be CD4⁺ iNKT. These CD4⁺ iNKT were predominantly located sub-mucosally. Isotype staining was negative (not shown).

Fig. 6

Detection of human iNKT by immunohistology

Discussion

A variety of methods have been used to identify iNKT. Historically, this population was defined by Vα24/CD4-CD8-, Vα24//CD161 or perhaps most precisely by Vα24/Vβ11 staining. These approaches are less than optimal because they include a proportion of non-iNKT, or as is the case with Vα24/CD161 staining, also exclude bona fide iNKT. [5] These earlier methods have been supplanted by staining with CD1d tetramers loaded with α-GalCer or other relevant iNKT antigens. [4] Even when B cell non-specific binding is excluded, CD1d tetramer staining alone invariably includes non-Vα24 T cells and in some subjects the fraction of variant CD1d-restricted T cells can be upwards of 30% of cells. (Table 1, Figure 3C) [28, 29] The biological function of such variant CD1d-restricted T cells is poorly understood. There is good evidence that variant populations are selected by, and recognizes self-antigens processed from different cellular compartments. [29] [30] Even co-expression of Vα24 and Vβ11 does not completely overlap with CD1d-tetramer reactivity in humans, and can be complicated by competition between CD1d tetramers and anti-TCR mAbs or their often bulky chromophores (e.g. anti-Vα24-PE). [28, 29] Concurrent incubation with anti-Vα24 has been used to block tetramer binding to iNKT, and thus identify non-invariant CD1d-restricted populations. [28, 29] Previous estimates of the relative contribution of iNKT subset to the total population of CD1d-restricted T cells has been limited by the lack of a comprehensive set of self antigens, and the use of various strains of mice with iNKT-related gene deletions. [35] However, the introduction of MHC deletions are known to induce major perturbations in lymphocyte homeostasis, and in particular, MHC class II-null mice have an expanded repertoire of variant CD1d-restricted T cells. [36] Hence, the ability to specifically identify, manipulate, and expand iNKT will likely be important. Toward this end we developed antibodies specific for human iNKT by modeling the invariant TCR CDR3 region using a cyclic peptide as an immunogen, one of which, 6B11, fulfilled these criteria.

As the accurate detection of iNKT is the first step in their analysis, we suggest that a more precise phenotypic definition of iNKT than use of Vα24 and / or Vβ11 should be based on the invariant TCR α chain rearrangement (Vα24Jα18), as detected by 6B11. Therefore, alone or in combination with anti-Vα24, anti-Vβ11, or anti-CD3, 6B11 is an excellent criterion for such analysis of human iNKT. Functional high avidity binding of α-GalCer loaded_CD1d tetramers provides a similar but not identical identification: all clones so obtained were CD1d-restricted but a minority were Vα24-negative, unlike with 6B11, all of which were CD1d-restricted and Vα24⁺. The 6B11 antibody was cloned from a large number of peptide-reactive hybridomas generated using a novel approach of immunization with cyclic peptides predicted to define particular loop structures within the target protein of interest. The specificity of 6B11 was defined by binding to the cyclic peptide used as antigen, reactivity with Jurkat cells transfected to express the iNKT TCR, reactivity to T cells derived from a mouse that expressed a human invariant AV24AJ18 TCR α-chain, and selection for and activation of iNKT clones and lines in vitro. Although raised in CD1d KO mice, 6B11 did not react with murine iNKT (or other murine) cells (data not shown), but does react with iNKT of Rhesus macaques, non-human primates [37]. To our knowledge, this antibody is the only anti-CDR3 idiotype-specific mAb available for a TCR of known specificity. Thus, this novel antibody can be used to specifically identify, enumerate [38], and as we show here to localize in tissues, isolate, and expand authentic iNKT for experimental, diagnostic, and therapeutic studies of human and nonhuman primate iNKT. The availability of multiple reagents active in various assays to identify and for manipulating iNKT will enable understanding their role in different patient population subsets (e.g. patients with various cancers (47), adult and pediatric acute versus chronic asthmatics, untreated or on various treatments; 48–53) and their various tissues, as well as and intervening therapeutically. Given that several approaches to exploit iNKT have entered clinical trials [39,54], development of such a selective reagent seems timely.

Material and Methods

Isolation of invariant TCR CDR3-loop specific antibodies

In order to raise antibodies against the human invariant Vα24-Jα18 TCR a cyclic peptide representing the predicted CDR3 loop of the invariant TCR-α sequence (CH2COCVVSDRGSTLGRLAD-C (Thioether linkage to CH2)-G-COOH) was modeled using previous TCR crystal structures as templates. [40] [41] The peptide was cyclized in situ using a thioether linkage, followed by de-protection of the NH2-terminal Cys residue and release from the resin for purification (SynPep, Inc., Dublin, CA). The peptide was of predicted MW = 1749.0 verified by Mass. Spec. and 92.8% pure by RP-HPLC. The strategy of releasing the cyclic peptide with a free sulfhydryl on the N-terminal Cys was designed to take advantage of the fact that the N-terminal Cys encoded by the AV24 element is conserved in all mammalian TCR α-chains since it forms a required Ig-fold domain in the TCR structure. Thus, when the cyclic peptide was coupled to N-ethylmaleimide-activated KLH for immunization, BSA for boosting, and to ovalbumin to screen hybridomas, the putative CDR3 loop would be exposed as an accessible epitope. Imject^tm-activated KLH, BSA, or OVA coupling was performed as recommended by the manufacturer (Pierce, Rockford, IL), (129 × C57BL/6) F2 CD1d KO mice [42] were immunized i.p. and s.c. with invariant peptide-KLH and CFA, boosted 4–6 weeks later with invariant peptide-BSA and IFA, and re-boosted i.v. 10–20 days later with either invariant peptide-BSA or DN2.D6 iNKT clone 4–5 days prior to hybridoma fusion. [2] Following fusion with SP2/0 myeloma by conventional means, hybridomas were screened by ELISA on invariant peptide-ovalbumin and positives counter screened against ovalbumin alone.

Screening for invariant TCR-reactive antibodies

Hybridomas secreting antibodies reactive with invariant peptide were screened for reactivity against the DN2.D6 and other iNKT clones and for reactivity against control unrelated T cell clones by indirect FACS with anti-IgG FITC as described. [2] [32] [43] Briefly, T cells were suspended in FACS buffer (PBS, FBS 1% and NaN3 0.1%) in a 96 well plate. Non-specific binding was blocked by pre-incubating with 5% human serum for 15 mins. Antibodies were added to cell suspensions for 20 minutes. Cells were then washed with FACS buffer, incubated with anti-mIg (H&L) FITC (Pierce), re-washed, and analyzed by FACScan (Becton Dickinson), using CellQuest Software.

Two monoclonal antibodies in particular were carried further since they clearly reacted specifically with all iNKT tested. These were 6B11 (IgG₁) and 3A6 (IgM). Antibodies specifically reactive with iNKT clones were tested for reactivity against PBMC and polyclonal iNKT lines.

In Vitro Expansion of CD1d-reactive iNKT

Clones and lines of iNKT were generated and maintained as described. [19] [43] [44] PBMC were isolated from normal donor whole blood using Ficoll-Paque (Amersham-Pharmacia, Uppsala, Sweden). Polyclonal iNKT lines were obtained using anti-invariant mAb 6B11 or 6B11-biotin conjugate and anti-Ig or anti-biotin microbeads as appropriate on columns up to LS size with various Macs magnets depending on scale up to an AutoMacs or CliniMacs essentially according to manufacturer’s recommendation.

Vα24 positive T cells were high speed FACS-sorted (‘MoFlo’, Cytomation, Boulder, CO) from PBMC, using PE or FITC-conjugated or un-conjugated anti-Vα24 monoclonal antibody (Coulter, Miami, FL) and goat anti-mouse IgG (H+L) FITC (KPL, Gaithersburg, Maryland). Autologous APC were prepared using corresponding Vα24- cells and irradiation (3000 Rads). Vα24⁺ cells were co-cultured in 96 well plates with autologous APC in the presence of α-GalCer (50ng/ml; KRN-7000, kindly provided by Kirin Brewery Co., Gunma, Japan) and recombinant IL-2 (100 U/ml; NBRMP, NIH, Bethesda, MD). Culture supernatants were removed at 48hrs for cytokine ELISA. Anti-invariant mAb 6B11 was also used to sort cells by MoFlo as for Vα24 and by magnetic beads (anti-mouse IgG microbeads, Miltenyi) and these cells subjected to polyclonal or clonal expansion with PHA, anti-CD3, or anti-invariant mAb itself (plate bound) as indicated.

Further phenotypic analysis of invariant antibody or iNKT cell lines at ≥ 15 days of expansion in vitro was performed by multi-color flow cytometry, but otherwise as above using FACScan (BD). Antibodies used were anti-Vα24 PE, anti-Vβ11 FITC, anti-CD8β-PE (Coulter). Other mAb, including anti-CD3-Cych, CD161-PE, CD4-Cych, were all obtained from BD-PharMingen (LaJolla, CA). Alexafluor488-labeled human CD1d tetramers were generated and used as described. [4] [45] TCR-negative Jurkat subline was transfected to transiently express iNKT Vα24Jα18 and Vβ11 TCR, as previously described. [31] Vα24Jα18 transgenic mice and their analysis have been described. [46]

Measurement of Cytokine Production and CD1d reactivity

For quantitative estimation of cytokine production by invariant antibody enriched invariant T cells in response to CD1d, a restriction assay was performed. [19] [2] Replicate 1×10⁵ /well invariant containing T cell populations were co-cultured with equal numbers of CD1d transfected or mock transfected C1R in 96 well plates with RPMI 1640, FBS 10%, IL-2 20U/ml and PMA 1ng/ml or α- GalCer (100ng/ml). Cellular response to CD1d was blocked using anti-CD1d antibody 51.1 at a concentration of 10µg/ml as described. Supernatants were collected from between 4 and 24 hr. for IL-4 and IFN-γ cytokine ELISA. Released cytokine levels were determined by ELISA with matched antibody pairs in relation to recombinant human cytokine standards (PharMingen; Endogen, Inc. Cambridge, MA) and converted to nanograms or picograms/ml using SoftMax (Molecular Devices Corp; Sunnyvale, CA). ELISA detection ranges were for IFN-γ and IL-4 was 20 – 10,000 pg/ml.

Immunohistochemistry

Spleens from C57Bl6 WT and human Vα24Jα18 × Jα18^−/− mice were frozen in OCT and sections fixed, permeabilized, and then double-stained with PE-labeled anti-CD3 and FITC-labeled 6B11 mAb. Stained sections (5 to 10 per staining) were analyzed on a confocal laser microscope (MRC 1024 Bio-Rad), and images acquired by Bio-Rad (Hercules, CA) Software Laser Sharp 2000. The human Vα24Jα18 × Jα18^−/− mice have 5 – 6 fold less T cells than WT mice (46), hence the reduced number of splenic cells stained by anti-CD3 PE.

During flexible fiberoptic bronchoscopy, up to three endobronchial biopsy samples were obtained through IRB-approved protocol from a patient with severe asthma. Biopsy samples were processed by standard frozen methods. Two color immunohistology was performed as shown. Briefly, the frozen sections were stained with PE-conjugated CD4 (red) and FITC-conjugated 6B11 mAb (blue) and analyzed by confocal laser scanning microscopy.

Acknowledgements

For help modeling the invariant TCR CDR3 loop, we particularly thank Dr. M. Yaffe, Massachusetts Institute of Technology. We thank the DIBIT San Raffaele Scientific Institute Histology Core for immuno-staining. For advice, discussions, reagents, and/or suggestions, we wish to thank Drs. G. Beltz, M. Brenner, and K. LeClair. Dr. Brenner also kindly provided the TCR expression system and recipient TCR-negative Jurkat subline. We thank Kirin Ltd., Japan, for kindly providing α-GalCer. Supported by NIH grants CA89567 and DK066917 (MAE), AI42955 and AI45051, a Harvard Center for Human Cell Therapy pilot grant, and by the Hershey Family Prostate Cancer Research Fund (SPB), U19 AI046130-06 & JDRF (SBW), the Italian Association for Cancer Research (AIRC) (GC).

Abbreviations


DN	CD4/CD8 double negative

NKT	Natural Killer T cells

Footnotes

Conflict of Interest:

The authors declare they have no conflicts of interest.

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